Nano-Pharmaceuticals: Principles and Applications Vol.1

Environmental Chemistry for a Sustainable World 46
Vinod Kumar Yata

Shivendu Ranjan
Nandita Dasgupta
Eric Lichtfouse Editors
Nano-
pharmaceuticals:
Principles
and Applications
Vol.1
Environmental Chemistry for a Sustainable
World
Volume 46
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Vinod Kumar Yata • Shivendu Ranjan
Nandita Dasgupta • Eric Lichtfouse
Editors
Nanopharmaceuticals:
Principles and Applications
Vol. 1
Editors
Vinod Kumar Yata Shivendu Ranjan
Animal Biotechnology Centre Faculty of Engineering and Built
National Dairy Research Institute Environment
Karnal, India University of Johannesburg
Johannesburg, South Africa
Nandita Dasgupta
Department of Biotechnology Eric Lichtfouse
Institute of Engineering and Technology CNRS, IRD, INRAE, Coll
Lucknow, Uttar Pradesh, India France, CEREGE
Aix-Marseille University
Aix-en-Provence, France
ISSN 2213-7114 ISSN 2213-7122 (electronic)

Environmental Chemistry for a Sustainable World
ISBN 978-3-030-44924-7 ISBN 978-3-030-44925-4 (eBook)
https://doi.org/10.1007/978-3-030-44925-4
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature
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Preface
Nanomaterials have a special place in pharmaceutical industry due to their size and
ease of penetrability into the mammalian cells. The integration of pharmaceutical
drugs and therapeutic biomolecules with nanomaterials will lead to the develop-
ment of novel nanopharmaceuticals for enhanced therapeutic applications. This
book, Nanopharmaceuticals: Principles and Applications, is intended to provide a
regular update on applications of nanopharmaceuticals along with general
fundamentals.
Chapter 1 presents the comprehensive description of basic principles, method-
ologies, similarities and differences of liposomes and phytosomes. It also focuses
on implications of these nanocarriers in CNS disorder and presents the various con-
cepts of brain delivery in great detail.
Chapter 2 begins with the detailed classification of nanoinonized drug particles,
polymeric nanoparticles and hydrophobic nanoparticles. This is followed by a
description of applications of polymeric nanoparticles. It concludes with the bio-
logical, technical and study-design challenges of nanopharmaceuticals.
Chapter 3 is dedicated to supermagentic iron oxide nanoparticles for the diagno-
sis of brain, breast, gastric, ovarian, liver, colorectal, lung and pancreatic cancers. It
begins with brief introduction to magnetic resonance imaging and ends with future
prospective of iron oxide nanoparticles in cancer detection.
Chapter 4 tackles the DNA nanostructures and its applications in bimolecular
delivery. It also focuses on detailed description of smart DNA nanostructures and
briefly discusses “computational sequence design for DNA nanostructures” at the
end of the chapter.
Chapter 5 includes the toxicity and application of different types of ionic liquids
for humans and environment and also describes characteristics, applications and
regulatory gaps of nanoparticle-ionic liquid combined systems.
Chapter 6 gives a brief introduction about the skin delivery and a detailed discus-
sion about the different types of nanocarriers such as micelles, microemulsions,
nanoemulsions and polymeric and lipid-based nanoparticles. It also covers the
safety issues, clinical benefits, ecotoxicity and regulatory framework of
nanopharmaceuticals.
v
vi Preface
Chapter 7 consists of two parts: the first part deals with detailed classification of
swelling, responsive, mechanical behaviour of synthetic gels, and the second part
discusses the therapeutic, diagnostic and biosensor applications of gel-based nano-
pharmaceuticals. This chapter also deals with the challenges of nanogels in above-
mentioned applications.
Chapter 8 focuses on microfluidics-based lab-on-chip technologies for drug
screening applications. It also provides detailed description of microfabrication
techniques apart from cell- and organ-based drug screening techniques.
Chapter 9 presents innovative and insightful information on synthesis of carbon,
metal nanoparticles and semiconductor nanocrystals. It also describes the surface
functionalization and targeting capabilities of nanoparticles in detail.
All the chapters in this book address the impact of nanoformulations on thera-
peutic and/or diagnostic purposes. Each chapter provides the basic principles to
state-of-the-art developments of nanopharmaceuticals with extensive references.
Karnal, India Vinod Kumar Yata

Johannesburg, South Africa Shivendu Ranjan
Lucknow, India Nandita Dasgupta
Aix-en-Provence, France Eric Lichtfouse
Contents
1 Liposomes vs Phytosomes: Principles, Methodologies,

and Therapeutic Applications with Emphasis on CNS Disorders �� 1
Hafsa Ahmad, Abhishek Arya, Satish Agrawal,
and Anil Kumar Dwivedi
2 Applications of Nanopharmaceuticals
in Delivery and Targeting�� 73
Mohamed Abbas Ibrahim and Ahmed A. H. Abdellatif
3 Applications of Iron Oxide Nanoparticles
in the Magnetic Resonance Imaging for the Cancer Diagnosis �� 115
Kanwal Akhtar, Yasir Javed, Muhammad Imran Akhtar,
and Naveed A. Shad
4 DNA-Based Nanopharmaceuticals �� 159
V. Dilna, Chinnu Sabu, and K. Pramod
5 An Overview on Ionic Liquids: A New Frontier
for Nanopharmaceuticals�� 181
Tânia Santos de Almeida, Rita Caparica, Ana Júlio,
and Catarina Pinto Reis
6 Therapeutic Implications of Nanopharmaceuticals
in Skin Delivery�� 205
Ana Henriques Mota, Ana Santos-Rebelo, António José Almeida,
7 Biomimetic and Synthetic Gels
for Nanopharmaceutical Applications�� 273
Busra Yildiz, Sezer Ozenler, Muge Yucel, Umit Hakan Yildiz,
and Ahu Arslan Yildiz
vii
viii Contents
8 On-Chip Drug Screening Technologies

for Nanopharmaceutical and Nanomedicine Applications�� 311
Rabia Onbas, Rumeysa Bilginer, and Ahu Arslan Yildiz
9 Synthesis of Some Bioactive Nanomaterials
and Applications of Various Nanoconjugates
for Targeted Therapeutic Applications�� 347
Sabyasachi Chakrabortty, Sunil Kumar Vimal,
and Sanjib Bhattacharya
Index�� 377
About the Editors
Dr. Vinod Kumar Yata is an Interdisciplinary Researcher working in the National

Dairy Research Institute, Karnal, India. Previously, he worked as an Assistant
Professor in the Department of Biotechnology, Dr. B.R. Ambedkar National Institute
of Technology Jalandhar, Punjab, India. He received his PhD in Biotechnology
from Indian Institute of Technology Guwahati. He specializes in interdisciplinary
research which includes nanotechnology, microfluidics, biotechnology, cancer biol-
ogy, and bioinformatics. He has developed a microfluidic device for the separation
of live and motile spermatozoa from cattle semen samples. He opened up a new
avenue to prodrug enzyme therapy by introducing the nanocarriers for the delivery
of non-mammalian prodrug-activating enzymes. He elucidated the structural fea-
tures and binding interactions of several biomolecules by in silico methods. He has
published several research papers in peer-reviewed international journals and pre-
sented papers in several international conferences.
ix
x About the Editors
Dr. Shivendu Ranjan has completed his BTech and PhD in Biotechnology from

VIT University, Vellore, India, and has expertise in Nano(bio)technology. He was
elected as a Fellow (FLS) of the oldest active biological society started in 1778, The
Linnean Society (London), and elected as Fellow of Bose Scientific Society (FBSS).
In 2018 he was elected as Fellow of Indian Chemical Society (FICS) – a society
founded in 1924. He has also been elected as Fellow (FIETA) of the Indian
Engineering Teachers Association. Currently, he is Senior Research Associate at the
Faculty of Engineering and Built Environment, University of Johannesburg,
Johannesburg, South Africa. Recently he has accepted the role of Strategic Head,
Research and Development at Ennoble IP, Noida, India. He is also Visiting Faculty
at the National Institute of Pharmaceutical Education and Research-R (NIPER-R),
Lucknow. He is Vice President, Indian Chemical Society North Branch. Earlier, he
has worked as Scientist at DST-Centre for Policy Research, Lucknow, supported by
Ministry of Science and Technology, Government of India. He was also Head,
Research and Technology Development at E-Spin Nanotech Pvt. Ltd., SIDBI
Incubation Center, Indian Institute of Technology, Kanpur, India. After joining
E-Spin Nanotech, IIT Kanpur, he has successfully developed prototypes for many
products, has applied one patent and has taken lead in the DSIR Certification for
R&D Unit of E-Spin Nanotech Pvt Ltd. He is also Advisor for many companies,
such as Eckovation Solutions Pvt Ltd. (IIT Delhi–based start-up), Chaperon Biotech
Pvt Ltd (IIT Kanpur–based start up), Kyntox Biotech India Pvt Ltd., and Xcellogen
Biotech Pvt Ltd. Dr. Shivendu is also reviewer of Iran National Science Foundation
(INSF), Tehran, Iran, and jury at Venture Cup, Denmark, for the past 3 consecutive
years. He had founded and drafted the concept for the first edition of the “VIT Bio
Summit” in 2012, and the same has been continued till date by the university. He is
Associate Editor of Environmental Chemistry Letters (Springer journal of 4.6
impact factor), Section Editor of Pharmaceutical Nanotechnology (Bentham
Science, UAE), Editor of Journal of the Indian Chemical Society and editorial board
member of Biotechnology and Biotechnological Equipment (Taylor and Francis,
USA). He is Advisory Board Member in Elsevier to provide feedback on the new
prototypes of Elsevier, Netherlands. He serves as Executive Editor of a journal in
About the Editors xi
iMed Press, USA, and also as an editorial board member and referee for reputed
international peer-reviewed journals. He has published several scientific articles as
well as books and has h-index of 21. He has bagged several awards and recognition
from several national as well as international organizations.
Dr. Nandita Dasgupta has completed her BTech and PhD from VIT University,
Vellore, India, and is Elected Fellow (FBSS) of Bose Science Society. She has
major working experience in micro-/nanoscience and currently works as Assistant
Professor in the Department of Biotechnology, Institute of Engineering and
Technology, Lucknow, India. Earlier at LV Prasad Eye Institute, Bhubaneswar,
India, she has worked on mesenchymal stem cell-derived exosomes for the treat-
ment of uveitis. She has exposure of working at university, research institutes, and
industries including VIT University, Vellore, Tamil Nadu, India; CSIR-Central Food
Technological Research Institute, Mysore, India; and Uttar Pradesh Drug &
Pharmaceutical Co. Ltd., Lucknow, India; and Indian Institute of Food Processing
Technology (IIFPT), Thanjavur, Ministry of Food Processing Industries, Government
of India. At IIFPT, Thanjavur, she was involved in a project funded by a leading
pharmaceutical company, Dr. Reddy’s Laboratories, and has successfully engi-
neered micro-vehicles for model drug molecules. Her areas of interest include
micro-/nanomaterial fabrication and its applications in various fields – medicine,
food, environment, and biomedical agriculture.
She has published 13 edited books and 1 authored book with Springer,
Switzerland, and 2 with CRC Press, USA. She has finished a contract of three book
volumes with Elsevier, one with Wiley, two book volumes with CRC Press, and one
with RSC (UK). She has authored many chapters and also published many scientific
articles in international peer-reviewed journals. She has received the Certificate for
“Outstanding Contribution” in Reviewing from Elsevier, Netherlands. She has also
been nominated for advisory panel for Elsevier Inc., Netherlands. She is the
Associate Editor of Environmental Chemistry Letters – a Springer journal of 3.2
xii About the Editors
impact factor – and also serves as Editorial Board Member and Referee for reputed
international peer-reviewed journals. She has received several awards and recogni-
tions from different national and international organizations.
Dr. Eric Lichtfouse, PhD, born in 1960, is an Environmental Chemist working at

the University of Aix-Marseille, France. He has invented carbon-13 dating, a method

allowing to measure the relative age and turnover of molecular organic compounds
occurring in different temporal pools of any complex media. He is teaching scien-
tific writing and communication and has published the book Scientific Writing for
Impact Factor Journals, which includes a new tool – the micro-article – to identify
the novelty of research results. He is Founder and Chief Editor of scientific journals
and series in environmental chemistry and agriculture. He has founded the European
Association of Chemistry and the Environment. He received the Analytical
Chemistry Prize by the French Chemical Society, the Grand Prize of the Universities
of Nancy and Metz, and the Journal Citation Award by the Essential Indicators.
Contributors
Ahmed A. H. Abdellatif Department of Pharmaceutics and Industrial pharmacy,

Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt
Department of Pharmaceutics, College of Pharmacy, Qassim University, Buraidah,
Kingdom of Saudi Arabia
Satish Agrawal Division of Pharmaceutics & Pharmacokinetics, CSIR-Central
Drug Research Institute, Lucknow, UP, India
Hafsa Ahmad Division of Pharmacognosy & Ethnopharmacology, CSIR-National
Botanical Research Institute, Lucknow, UP, India
Kanwal Akhtar Magnetic Materials Laboratory, Department of Physics,
University of Agriculture, Faisalabad, Pakistan
Muhammad Imran Akhtar Radiology Department, Allied Hospital, Punjab
Medical College, Faisalabad, Pakistan
António José Almeida iMED.ULisboa, Research Institute for Medicines,
Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal
Abhishek Arya Division of Pharmaceutics & Pharmacokinetics, CSIR-Central
Drug Research Institute, Lucknow, UP, India
Sanjib Bhattacharya Department of Pharmaceutical Science, Southwest
University, Chongqing Shi, China
Rumeysa Bilginer Department of Bioengineering, Izmir Institute of Technology
(IZTECH), Izmir, Turkey
Rita Caparica Department of Biomedical Sciences, University of Alcalá,
Madrid, Spain
xiii
xiv Contributors
Sabyasachi Chakrabortty Max-Planck-Institute for Polymer Research,

Mainz, Germany
Department of Chemistry, SRM University, AP – Amaravati, Guntur, Andhra
Pradesh, India
Tânia Santos de Almeida CBIOS-Universidade Lusófona’s Research Center for
Biosciences & Health Technologies, Lisboa, Portugal
V. Dilna College of Pharmaceutical Sciences, Government Medical College,
Kozhikode, Kerala, India
Anil Kumar Dwivedi Division of Pharmaceutics & Pharmacokinetics, CSIR-
Central Drug Research Institute, Lucknow, UP, India
Mohamed Abbas Ibrahim Kayyali Chair for Pharmaceutical Industries,
Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh,
Kingdom Saudi Arabia
Department of Pharmaceutics and Industrial pharmacy, Faculty of Pharmacy,
Al-Azhar University, Assiut, Egypt
Yasir Javed Magnetic Materials Laboratory, Department of Physics, University of
Agriculture, Faisalabad, Pakistan
Ana Júlio Department of Biomedical Sciences, University of Alcalá, Madrid, Spain
Ana Henriques Mota iMED.ULisboa, Research Institute for Medicines, Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
Rabia Onbas Department of Bioengineering, Izmir Institute of Technology
Sezer Ozenler Department of Chemistry, Izmir Institute of Technology (IZTECH),
Izmir, Turkey
K. Pramod College of Pharmaceutical Sciences, Government Medical College,
Ana Santos-Rebelo Department of Biomedical Sciences, Faculty of Pharmacy,
Universidad de Alcalá, Alcalá de Henares, Spain
Catarina Pinto Reis iMED.ULisboa, Research Institute for Medicines, Faculdade
de Farmácia, Universidade de Lisboa, Lisboa, Portugal
IBEB, Biophysics and Biomedical Engineering, Faculdade de Ciências,
Universidade de Lisboa, Lisboa, Portugal
Chinnu Sabu College of Pharmaceutical Sciences, Government Medical College,
Naveed A. Shad Department of Physics, Government College University
Faisalabad, Faisalabad, Pakistan
Contributors xv
Sunil Kumar Vimal International Institutes for Integrative Sleep Medicine (WPI-

IIIS), University of Tsukuba, Tsukuba, Ibaraki, Japan
Department of Pharmaceutical Science, Southwest University, Chongqing
Shi, China
Ahu Arslan Yildiz Department of Bioengineering, Izmir Institute of Technology
Busra Yildiz Department of Chemistry, Izmir Institute of Technology (IZTECH),
Izmir, Turkey
Umit Hakan Yildiz Department of Chemistry, Izmir Institute of Technology
Muge Yucel Department of Bioengineering, Izmir Institute of Technology
Chapter 1
Liposomes vs Phytosomes: Principles,
Methodologies, and Therapeutic
Applications with Emphasis on CNS
Disorders
Hafsa Ahmad, Abhishek Arya, Satish Agrawal, and Anil Kumar Dwivedi
Contents
1.1 I ntroduction: General Concepts 2
1.2 Liposome: Background 3
1.2.1 Liposome Discovery and Description 3
1.2.2 Classification of Liposomes 4
1.2.3 Composition of Liposomes 6
1.3 Preparation Methods for Liposomes 9
1.3.1 Hydration (by Passive Loading) 9
1.3.2 Sizing Stage 13
1.3.3 Removal of Non-encapsulated Material 13
1.4 Liposomes: Strategies and Applications 14
1.4.1 Formulation Strategies 14
1.4.2 Therapeutic and Clinical Applications 19
1.5 Phytosome: Background 27
1.5.1 Phytosome: Background, Discovery, and Description 27
1.5.2 Properties and Characterization of Phytosomes 28
1.6 Preparation Methods for Phytosomes 28
1.7 Therapeutic Applications of Phytosomes 30
1.7.1 Neutraceutical, Health Food, and Cosmeceutical Applications 30
1.7.2 Clinical Applications 31
1.8 Differences Between Liposomes and Phytosomes 36
1.9 Advantages 38
H. Ahmad
Division of Pharmacognosy & Ethnopharmacology, CSIR-National Botanical
Research Institute, Lucknow, UP, India
A. Arya · S. Agrawal · A. K. Dwivedi (*)
Division of Pharmaceutics & Pharmacokinetics, CSIR-Central Drug Research Institute,
Lucknow, UP, India
© The Editor(s) (if applicable) and The Author(s), under exclusive license to 1
Springer Nature Switzerland AG 2021
V. K. Yata et al. (eds.), Nanopharmaceuticals: Principles and Applications Vol. 1,
Environmental Chemistry for a Sustainable World 46,
https://doi.org/10.1007/978-3-030-44925-4_1
2 H. Ahmad et al.
1.10 C NS-Based Therapies: Challenges and Interventions 38

1.10.1 Drug Delivery to the Brain 38
1.10.2 Liposomal Interventions Implied in CNS Therapies 44
1.10.3 Phytosome Strategies for Disorders of the Brain and CNS 46
1.11 Conclusions 57
References 58
Abstract Lipid solubility and molecular size are the major limiting factors for any
molecule to get absorbed across biological membrane systems. Formulation strate-
gies that address these concerns are of enormous benefit in eliciting the intended
response from a therapy. Liposomes and phytosomes are two very versatile lipid-
based delivery systems that cater to a wide spectrum of therapeutic applications on
account of their composition and structural properties.
This chapter was conceptualized with an aim to present liposomes and phyto-
somes on a common interface with special focus on their implications in CNS dis-
orders besides several other applications. It presents a detailed account on their
classification, composition, salient properties, and advantages, also describing the
basic differences between them. Further the methods of preparation for liposomes
and phytosomes along with their clinical and therapeutic applications have been
discussed in great detail. Neurological diseases impose a huge burden of illness and
have shown an increased incidence due to an increase in the average life expectancy
in the global scenario. We have reviewed recent advances in the liposomal and
phytosomal interventions that have been investigated for various neurological
disorders.
Keywords Liposome · Cationic · Targeted · Phytosome · Phospholipid complexes

· Bioavailability · Blood–brain barrier · Parkinson’s disease · Alzheimer’s disease
· Stroke
1.1 Introduction: General Concepts
The method of drug delivery can have a significant effect on its efficacy. New con-
cepts that allow modification of pharmacodynamics, pharmacokinetics, immunoge-
nicity, non-specific toxicity, bio-recognition, and efficacy of drugs gave impetus to
new strategies in drug design. These new strategies involving interdisciplinary
approaches like combination of pharmaceutics, polymer science, bioconjugate
chemistry, and molecular biology are termed as drug delivery systems (DDS).
Various drug delivery systems aim at minimizing drug loss due to degradation, min-
imizing side effects, and enhancing the bioavailability and therapeutic efficacy of
drugs (Kaparissides et al. 2006; Kulkarni et al. 2011).
1 Liposomes vs Phytosomes: Principles, Methodologies, and Therapeutic Applications… 3
Nanotechnology has emerged as a revolutionary tool capable of controlling and

manipulating the structures at molecular level. Some of the novel nanoparticulate
carriers include microcapsules, microparticles, cells, cell ghosts, liposomes, and
micelles. These carriers can be modified to be slowly degradable and stimuli-reac-
tive (pH- or temperature-sensitive) or targeted (antibody conjugation). The ability
of a drug-loaded system to be directed to act at a particular site of interest is referred
to as drug targeting. Two major mechanisms involved in it are (a) passive, e.g., pref-
erential accumulation of chemotherapeutics in solid tumors due to increased vascu-
lar permeability of tumor tissues as compared to healthy tissues, and (b) active, e.g.,
drug carriers utilizing surface functionalization with ligands that are selectively rec-
ognized by receptors on the surface of the cells of interest (Kaparissides et al. 2006;
Ranghar et al. 2014).
Nanoparticulate-based drug delivery has garnered immense attraction over the
last two decades. Among a wide variety of these drug delivery strategies being cur-
rently investigated, lipid-based delivery systems have emerged as novel carriers of
choice primarily due to their versatility and biocompatibility. Lipid-based nanocar-
riers have the advantage of being tailored and customized for a variety of require-
ments based on safety, efficacy, stability, toxicity issues, cost considerations, type of
disease, and administration routes. Lipoidal delivery was traditionally attempted to
improve solubility of drugs with poor aqueous solubility (BCS Classes II and IV
drugs). However it is now not limited to solubility issues and has been used to pro-
duce commercially viable formulations of drugs, vaccines, and biological and neu-
traceuticals for oral, topical, and parenteral delivery. Lipid delivery also protects the
bioactives from biological degradation or transformation thus leading to enhanced
potency. They also modify the biodistribution of a drug or bioactive thus resulting
in reduced toxicity (Kulkarni et al. 2011; Attama et al. 2012).
The past few decades have observed emergence of many lipid delivery systems
like solid lipid nanoparticles, liposomes, nanostructured lipid carriers, lipid drug
conjugates, nanoemulsions, liquid crystals, niosomes, phytosomes, and transfer-
somes (Attama et al. 2012). Two important lipid-based delivery systems, namely,
liposomes and phytosomes, would be discussed in this chapter based on their com-
position, structural similarities and differences, methods of preparation, therapeutic
applications, and their role in CNS-based therapies.
1.2 Liposome: Background
1.2.1 Liposome Discovery and Description
The first report of discovery of liposome has been established by a British hema-
tologist Dr. A.D. Bangham and his co-workers based at the Babraham Institute, in
Cambridge in 1961. They discovered it while testing their new electron microscope
on addition of a negative stain to dry phospholipids. At this time it was evidenced
4 H. Ahmad et al.
that the cell membrane has a bilayer lipid structure and pictures taken back then
were the first real evidences of discovery of liposomes. The term “liposome” is
derived from Greek words lipos meaning fat and soma meaning body. Thus lipo-
somes are tiny bubbles (concentric vesicles) where a lipid bimembrane encapsulates
an aqueous volume. This bilayered membrane is usually made up of phospholipids.
Phospholipids are amphipathic molecules possessing a hydrophilic head and a
hydrophobic tail. The head is water loving and the long hydrocarbon tail is repelled
by water. Phospholipids, upon hydration, spontaneously form bilayer membrane
vesicles which are termed as liposomes (Bangham et al. 1974; Thassu et al. 2007;
Dua et al. 2012; Wilczewska et al. 2012).
1.2.2 Classification of Liposomes
There are several bases for liposome classifications. The number of layers signifies
the lamellarity of a liposome. Diameter and lamellarity forms the most popular and
conventional basis for classification of liposomes (Table 1.1). Based on the surface
charge determined by zeta potential, liposomes can be classified as cationic, anionic,
or neutral. The lipids used, and the preparation method adopted for, can greatly
influence the size and lamellarity of liposomes. Method of preparation forms
another basis of liposome classification (Table 1.2). Additionally liposomes with
unique properties can also be engineered to cater to defined applications, such as
target cell specificity, reduced environmental sensitivity, long systemic circulation
time, and defined pH. Such liposomes can be tailor made by the appropriate choice
of lipid composition and surface modification. Thus liposomes can also be classified
on the basis of composition and applications (Fig. 1.1) (Amarnath Sharma and
Sharma 1997; Thassu et al. 2007; Kulkarni et al. 2011).
Table 1.1 Classification of liposomes based on size and lamellarity

Type of vesicles Size (nm)
UV Unilamellar vesicles 20–1000
SUV Small unilamellar vesicles 20–100
MUV Medium-sized unilamellar vesicles 100–500
LUV Large unilamellar vesicles 100–1000
GUV Giant unilamellar vesicles >1000
MLV Multilamellar large vesicles 200–3000
OLV Oligolamellar vesicles 100–1000
Multivesicular vesicles 200–3000
Double liposomes >1000
Table 1.2 Classification of REV – reverse phase evaporation method yields single or
liposomes on the basis of oligolamellar vesicles
method of preparation
MLV/REV- reverse phase evaporation method for
producing multilamellar vesicles
SPLV – stable plurilamellar vesicles
FATMLV – frozen and thawed multilamellar large
vesicles
FUV – vesicles obtained by fusion technique
VET – extrusion method obtained by extrusion technique
FPV – vesicles formed by French press method
DRV – vesicles formed by dehydration–rehydration
technique
BSV – bubblesomes
Fig. 1.1 Classification of liposomes based on composition and applications

6 H. Ahmad et al.
1.2.3 Composition of Liposomes
A variety of ingredients can be used for preparing liposomes; however the two raw
materials that architectures a basic liposomal construct are phospholipids and ste-
rols. Certain polymeric materials and other agents might also be used in preparation
of liposomes (Table 1.3).
Types of Phospholipids
Largely phospholipids can be of the following five categories:
Phospholipids from Natural Sources
Most commonly employed lipids known as phosphatidylcholine (PC) along with

phsphotidylinositol (PI), and sphingomyelin (SPM) are obtained from egg yolks
and soybeans. Egg PC’s and soy PC’s differ in their types of acyl chains.
Table 1.3 Ingredients used for liposome preparation

Natural phospholipids: Synthetic phospholipids:
Phosphatidylcholine 1, 2-dilauroyl-sn-glycero-3-
Phosphatidylserine phosphocholine (DLPC)
Phosphatidylethanolamine 1, 2-dioleoyl-sn-glycero-3-
Phosphatidylinositol [phospho-L-serine] (sodium salt)
(DOPS)
Dipalmitoylphosphatidylcholine
Distearoylphosphatidylcholine
Dipalmitoylphosphatidylserine
Dipalmitoylphosphatidylglycerol
1,2-Dilauroyl-sn-glycero-3-
phosphocholine (DLPC)
Unsaturated phospholipids: Charge-inducing lipids:
1-ptearoyl-2-linoleoyl-sn-glycero-3-[phospho-L-serine] Dioctadecyldimethylammonium
(sodium salt) bromide/chloride (DODAB/C)
Dioleoylphosphatidylcholine Dioleoyl trimethylammonium
propane (DOTAP)
Sphingolipids: Glycosphingolipids:
Sphingomyelin Gangliosides
Sterols: Polymeric material:
Cholesterol Lipids conjugated to diene,
methacrylate, and thiol group
Miscellaneous:
Stearylamine and dicetylphosphates
Polyglycerol and polyethoxylated mono and dialkyl amphiphiles
Modified Natural Phospholipids

Natural PC’s are prone to oxidation due to their degree of unsaturation. These natu-
ral PC’s can be catalytically hydrogenated either partially or completely and the
modified PC’s so formed usually have low iodine values due to reduced number of
unsaturated C=C bonds.
Semisynthetic Phospholipids
The unsaturation of natural PC’s makes them susceptible to oxidation which
adversely affects the shelf life of liposomes and differences in their acyl chains
results in variation in batch consistency and leads to stability issues. The original
acyl chains of natural PC’s can be removed and replaced by certain other defined
acyl chains resulting in formation of semisynthetic lipids. An enzyme phospholi-
pase A2 may be used to cut the acyl chain at C2 position of glycerol when replace-
ment of C2 acyl chain is required.
Fully Synthetic Phospholipids

Phospholipids can be synthesized by known chemical pathways (Eibl and
Woolley 1986).
Phospholipids with Non-natural Head Groups

Certain modified phospholipids can be designed to cater to different clinical require-
ments. The residence of a liposome in circulation can be considerably enhanced by
attaching PEG (polyethylene glycol) chains to lipid bilayer. To achieve active tar-
geting, ligands like peptides or antibodies are attached for receptor recognition
(Allen et al. 1991; Kulkarni et al. 2011).
Description of Specific Phospholipids
Some of the important phospholipids include phosphatidylcholine (PC), phosphati-

dylethanolamine (PE), phosphatidylglycerols (PG), phosphatidylserine (PS), phos-
phatidic acid (PA), sphingomyelin (SM), and lysophospholipids. PC can be derived
from natural as well as synthetic sources. PC also known as lecithin is amphipathic
in nature possesses neutral charge and is chemically inert. It has a hydrophobic acyl
hydrocarbon chain linked to a hydrophilic polar head via a glycerol bridge. Lecithins
are primarily extracted from egg yolk and soybean and less commonly from bovine
heart and spinal cord. Mammalian lecithins have higher degree of saturation as
compared to plant based lecithins. PE possesses a head group similar to PC. Here
hydrogen’s are directly attached to the nitrogen of ethanolamine which allows inter-
actions through hydrogen bonding of the adjacent molecules in the membrane. The
amino group is protonated at low or neutral pH resulting in a neutral molecule that
8 H. Ahmad et al.
preferentially forms hexagonal II phase inverted micellar structures above the phase
transition temperature rather than forming lamellar structures (Chrai et al. 2002;
Ahmad et al. 2017).
PG can be isolated from natural sources and can also be prepared semi-syntheti-
cally by using phospholipase D. It bears a permanent negative charge over the nor-
mal physiological pH range. PS is linked to phosphate via hydroxyl group leaving
the carboxyl and amino functions both free and ionized to form a neutral zwitterion.
Due to the charge on phosphate, the net charge on the head is negative. Membranes
that contain PS have shown marked sensitivity to calcium which interacts directly
with the carboxyl group on the head, thus making the PS molecule to aggregate
within the membrane. Phase separation, packing irregularities, inter-liposomal
aggregation and fusion are some of the problems encountered with PS (Chrai et al.
2002; Attama et al. 2012).
PA possesses a strong negative charge due to lack of substitution on the phos-
phate group. When PA is dispersed in water, its dispersions have shown pH values
of 2–3. Rapid neutralization of these dispersions with acids results in formation of
unilamellar vesicles due to membrane reorganization under the influence of electro-
static forces. SM is neutral in nature and possesses the same phosphocholine groups
as in PC; however the packing is comparatively tighter than PC due to additional
hydrogen bonding. It can be found in varying amounts in the plasma membranes of
erythrocytes of mammalian species; however it completely replaces PC in sheep
erythrocytes (Chrai et al. 2002). Lysophospholipids are small bioactive lipid mole-
cules with a single carbon chain and a polar head group. They may be further of two
types based on their backbone structure (Afergan et al. 2008), lysosphingolipids
with sphingoid base backbone (Agarwal et al. 2013) and lysoglycerophospholipids
with glycerol backbone (Zu Heringdorf 2008).
Sterols (Cholesterol)
Cholesterol is another important cell membrane component besides phospholipid,

however it does not form bilayers itself but dissolves readily instead in the phospho-
lipid bilayer. It has approximately half the cross-sectional area of PC. Incorporation
of cholesterol in liposomes imparts them a degree of rigidness and also alters their
intra-vesicle interactions and fluidity. The cholesterol molecule fills in the free
spaces formed due to the kink in the unsaturated PC chain. Liposomes formed with
cholesterol, therefore, have the capability to sustain shear stress to a greater extent.
Additionally it also prevents the leakage of the entrapped drug/solute/bioactive and
retains them thereby reducing the serum-induced instability. However, too much of
it can result in decreased membrane permeability of entrapped therapeutic agents
and adversely affect drug targeting. For optimal drug targeting via liposomes, it is
essential that the liposome carrier eventually becomes permeable and releases the
drug at the desired site; but at the same time it requires high stability in the blood
stream. Thus use of cholesterol in liposomes at an optimum level is advisable to
confer the desired rigidity without negatively affecting the carrier’s permeability
(Melzak et al. 2012; Ahmad et al. 2016c).
1.3 Preparation Methods for Liposomes
Liposomes are formed spontaneously upon hydration of phospholipids. Few more

steps are involved to modify and control the size and lamellarity of liposomes so
formed. The scale of preparation and factors like drug encapsulation efficiency gov-
ern the methods of preparation adopted for preparing liposomes (Thassu et al.
2007). A general scheme for preparation of liposome involves the following stages:
(a) drying of lipids from organic solvent(s), (b) dispersing them in an aqueous
medium, (c) purification, and (d) final analysis of the liposomes. The most impor-
tant steps across this scheme are (a) hydration of lipids, (b) sizing of liposomes, and
(c) removal of non-encapsulated drug from the formed liposomes. Some of the
methods might combine the hydration and sizing steps (Kulkarni et al. 2011;
Akbarzadeh et al. 2013). Liposome preparation involves either passive loading or
active loading of ingredients. When the entrapped agents are incorporated either
before or during the process of preparation, it accounts for passive loading tech-
nique. Whereas in active loading, the therapeutic agent or drug is incorporated into
intact vesicles or preformed liposomes. Active loading is also known as remote
loading and is useful for specific compounds which have ionizable groups and
exhibit both aqueous and lipid solubility. These amphipathic molecules can be
introduced into preformed liposomes by using a pH gradient and potential differ-
ence across the liposome membrane. Loading of these agents is affected by a differ-
ence in the proton concentration across the liposomal membrane. Remote loading
techniques offer several advantages like high encapsulation, reduced leakage of
active ingredient, minimized drug loss due to diffusion or degradation, and increased
safety and stability (Riaz 1996; Gomez-Hens and Fernandez-Romero 2006; Reza
Mozafari et al. 2008). A schematic representation of liposome preparation illus-
trated the various steps and methods (Fig. 1.2).
1.3.1 Hydration (by Passive Loading)
The hydration step can be affected by a number of methods involving passive load-
ing techniques.
Mechanical Methods
The conventional method for preparing MLVs involved hydration of thin lipid films
(obtained on a glass wall from drying of a solution of lipids in an organic solvent)
by shaking at a temperature higher than the phase transition temperature. The size
reduction could be done by extrusion and sonication methods.
10 H. Ahmad et al.
Fig. 1.2 Schematic representation of liposome preparation methodologies
Sonication
It is the most commonly used method for preparing SUVs. A probe type or bath type
sonicator could be used to sonicate MLVs under a passive environment. However
sonication methods often result in low encapsulation efficiencies, metal pollution
due to probe tip, and degradation of bioactives and occurrence of MLVs along with
SUVs. Probe sonication is usually carried out by keeping the liposome vessel in an
ice bath to overcome the heat generated due to coupling energy of the probe tip. On
the contrary, control of temperature is much easier in the bath sonication method
(Riaz 1996; Kataria et al. 2011).
French Pressure Cell
In this method MLVs are extruded through a small orifice at 20,000 psi at 4 °C. It is
a simple and reproducible method suitable for unstable materials and shows obvious
advantages over sonication methods. The size of liposomes obtained here remains
generally larger from those prepared by sonication techniques. Some demerits of
this method are the difficulty to attain high temperature and the low working vol-
umes (Hamilton and Guo 1984; Mozafari 2005; Song et al. 2011; Zhang 2011).
Freeze-Thawed Liposomes
Rapid freezing followed by slow thawing of SUVs results in formation of LUVs as
the short time of sonication disperses the aggregated materials facilitating the fusion
of SUVs to produce LUVs. Encapsulation of about 20%–30% could be achieved by
this method (Pick 1981; Llu and Yonetani 1994).
Thin Film Hydration/Solvent Evaporation
It is the most commonly used method for preparing MLVs. A thin lipid film is
formed at the bottom of a round bottom flask obtained by evaporation of a solution
of lipids in an organic solvent. The film is hydrated in an aqueous buffer and the
dispersion is vortexed. The active ingredient can be incorporated either in the solu-
tion of lipid in the organic solvent or added in the aqueous medium during hydration
depending upon its solubility. This method can be utilized by hand shaking, non-
shaking, or freeze-drying strategies. The disadvantages however can be low encap-
sulation and internal volumes along with heterogeneity in population size (Bangham
et al. 1965; Bangham et al. 1974; Ahmad et al. 2016c).
Other Methods
Other mechanical methods for liposome preparation can be micro-emulsification,
reconstitution of dried vesicles and membrane extrusion. Conventionally prepared
MLVs can be converted into SUVs by passing through membranes (polycarbonate
filters) with pores of defined diameters under moderate pressure. Liposomes of
small size and uniform size distribution can be prepared by this method. As the
concentric layers of MLVs deform to pass through a pore, the membrane breaks and
again reseals and upon repeated cycles; a liposome population with the mean diam-
eter equivalent to that of the pore diameter is obtained; and thus size heterogeneity
is decreased (Hope et al. 1993; Akbarzadeh et al. 2013).
Methods Based on Replacement of Organic Solvent by Aqueous Media
Ether Injection Method

The substance to be encapsulated is maintained as an aqueous solution at
55 °C–65 °C or under reduced pressure and a solution of lipids dissolved in either
diethyl ether or an ether-methanol mixture is slowly injected into it. Ether is removed
under vacuum resulting in formation of liposomes. Major drawbacks of this method
are the exposure of the active constituents to organic solvents and high temperatures
and the liposomes formed exhibit heterogeneity with variations in size range 70 to
200 nm (Deamer and Bangham 1976; Schieren et al. 1978).
12 H. Ahmad et al.
Ethanol Injection
MLVs are rapidly formed upon injection of a lipid solution of ethanol into an excess
of buffer. However the liposomes formed here are very dilute and display heteroge-
neity in size (30–110 nm). Another disadvantage is the difficulty in complete
removal of ethanol as it tends to form an azeotropic mixture with water and the
susceptibility of biologically active ingredients towards inactivation due to ethanol
(Batzri and Korn 1973).
Reverse Phase Evaporation Method
This is a relatively advanced method of liposome preparation based on creation of
inverted micelles. The liposomes prepared by this technique possess a high aqueous
space-to-lipid ratio and can entrap a large percentage of the aqueous fraction. When
a mixture of a buffer (aqueous) medium containing the substance to be entrapped
and an organic phase containing the amphiphilic molecules are sonicated; inverted
micelles are shaped. Upon gradual elimination of the organic solvent, the inverted
micelles are converted into a viscous gel. The gel state eventually collapses at a
critical point and thereby some inverted micelles get disturbed. A complete bilayer
then surrounds the residual micelles formed by the excess of phospholipids in the
environment. The aqueous volume-to-lipid ratio of liposomes formed by this
method is usually four folds greater than the liposomes created by hand shaking
method (Kataria et al. 2011).
Methods Based on Detergent Removal
Lipids can be solubilized by using detergents at their critical micelle concentrations

(CMC). The micelles become rich in phospholipid and eventually combine to form
LUVs upon detachment of detergents via dialysis. Equilibrium dialysis can be exe-
cuted in dialysis bags engrossed in large detergent free buffers and a commercially
available device like LipoPrep (Diachema AG, Switzerland) can be used for elimi-
nation of detergents by dialysis (Kirby and Gregoriadis 1984; Alpes et al. 1986;
Daemen et al. 1995; Shaheen et al. 2006). Several other methods can also be adopted
for removal of detergents like adsorption or binding of Triton X-100 (detergent) to
beaded organic polystyrene adsorbers like Bio-Beads SM-2 (Bio-Rad Laboratories,
Inc., Hercules, USA) and XAD-2 (SERVA Electrophoresis GmbH, Heidelberg,
Germany) beads or by binding of alkyl glycosides (octyl glucoside) to Amberlite-
XAD-2 beads. Gel permeation chromatography involving Sephadex G-50, Sephadex
G-l 00 (Sigma-Aldrich, MO, USA), Sepharose 2B-6B, and Sephacryl S200-S1000
(General Electric Company, Tehran, Iran) columns can be used for detergent
removal through gel filtration (Akbarzadeh et al. 2013).
Methods Based on Size Transformation and Fusion
LUVs can be formed by when the liposome dispersion is heated to phase transition
temperature resulting in fusion of vesicles. However the liposomes attained here do
not afford reproducibility and uniformity in size distribution.
1.3.2 Sizing Stage
There can be two stages here based on requirement of a special sizing step.
No Special Sizing Step Required
Here the conditions during liposome preparation are controlled in a way to produce
liposomes of defines particle sizes. The size distribution resulting from a high shear
homogenization depends upon the operational pressure.
Special Sizing Step Required
Some methods can be used to manipulate the size and distribution of prepared lipo-
somes. Fractionation by centrifugation can be used for size reduction for small dis-
persion volumes. Size resolution can also be managed on an analytical or
semi-preparative scale by using gel permeation chromatography. Specially designed
extruders can also be used for producing a liposome population with a narrow size
distribution (Riaz 1996; Kataria et al. 2011).
1.3.3 Removal of Non-encapsulated Material
Due to high affinity, lipophilic drugs are completely associated with liposomes;
however for other type of drugs, complete encapsulation cannot be achieved. The
non-encapsulated fraction can lead to physical instability. The non-encapsulated
drug can be removed by ion exchange, gel permeation, dialysis, or ultracentrifuga-
tion methods (Nicolas 1985; Akbarzadeh et al. 2013).
14 H. Ahmad et al.
1.4 Liposomes: Strategies and Applications
1.4.1 Formulation Strategies
Improved Stability
Selection of phospholipids with high phase transition temperatures like phosphati-
dylcholines with long saturated fatty acyl chains (e.g., distearoyl PC, hydrogenated
soy PC) that tend to remain in gel phase at physiological temperature improves the
stability of liposomal entrapment. Furthermore, incorporation of cholesterol
(30–50 mol%) results in improved stability as the cholesterol fills in the spaces in
between PC molecules which leads to formation of a tight lipid bilayer thus restrict-
ing the entry of plasma proteins and reducing RES clearance of liposomes. RES
clearance can also be greatly reduced by preparing sterically stabilized liposomes.
The use of PEG-conjugated lipids (3–10 mol %) such as monomethoxy-PEG
(molecular weight 2000)–distearoyl phosphatidylethanolamine (mPEG2000–
DSPE) has shown to significantly increase the residence of liposomes in circulation
by providing steric hindrance on the surface of the lipid bilayer which slows down
their RES clearance. These PEGylated liposomes with circulation half-lives of up to
2 days have special significance in EPR-mediated antitumor therapies (Thassu
et al. 2007).
pH-Sensitive Liposomes
Liposomes must exhibit sufficient stability before reaching their cellular target, and
upon reaching the target they must be able to release their contents in order to pro-
vide the intended therapeutic benefit. Certain strategies can be adopted to prepare
liposomes that are environmentally sensitive and can stabilize and destabilize
accordingly. pH-sensitive liposomes usually destabilize under the mildly acidic
conditions present in the microenvironment of a solid tumor and in endosomal com-
partments which are the target sites for drug therapies. Dioleoyl phosphatidyletha-
nolamine (DOPE) possessing a conical geometry has been most commonly used for
formulation of pH-sensitive liposomes. Its cone shape favors its transition from
bilayer to HII phase. Weakly acidic amphiphiles (oleic acid or cholesteryl hemisuc-
cinate) that stabilize the bilayer at neutral pH but destabilize at mildly acidic pH are
also used to prepare pH-sensitive liposomes. Certain other lipids like oleyl alcohol
and diolein have also been used in preparation of these liposomes. These pH-sensi-
tive liposomes can effectively facilitate the release of membrane-impermeable
drugs (Thassu et al. 2007; Kulkarni et al. 2011).
Some investigators have attempted to study the mechanism of the pH-sensitive
liposomes for tumor-targeting based on evaluation of small pH-sensitive molecules
like oleic acid, linoleic acid, and cholesteryl hemisuccinate and fundamental lipids
cholesterol and phosphatidylethanolamine. Good pH sensitivity was demonstrated
by liposomes formed of all test molecules as was evident through drug release data.
The pH-responsive release characteristics were exhibited in acidic pH by liposomes
formed of 3 test molecules. Oleic acid pH-sensitive liposomes showed a pH-sensi-
tive point of 5, while cholesteryl hemisuccinate pH-sensitive liposomes could stabi-
lize the lipid bimembrane under neutral conditions and showed better pH response
characteristics under acidic conditions due to the steroidal rigid structure possessed
by cholesteryl hemisuccinate (Fan et al. 2017). pH-sensitive liposomes have
emerged as popular drug carriers been for tumor therapy due to their advantages of
target ability and sustained-release characteristics, and several recent studies have
validated the same (Liang Ju et al. 2017). Observations from some recent findings
have been presented here.
In an investigation by Xu et al. (2016) doxorubicin-cholesteryl hemisuccinate
ion-pair complex was formulated as liposomal systems with pH-responsive proper-
ties. These liposomes showed pH-sensitive drug release and exhibited improved
cytotoxicity against MCF-7 cells (Hang Xu et al. 2017). Another study reported the
preparation of pH-sensitive liposomal formulation for effective delivery of doxoru-
bicin to bone tumors. These liposomes significantly reduced tumor volumes and
minimized cardiac toxicity (dos Santos Ferreira et al. 2017). Li et al. (2017c)
reported that liposomes loaded with 1,5-dihexadecyl N,N-diglutamyl-lysyl-L-
glutamate possessing pH-responsive properties were prepared and immunologically
modified for improved efficacy in breast cancer cells (Tianshu Li et al. 2017c). In a
study by Araújo et al. (2017), it was observed that incorporation of cisplatin into
long circulating pH-sensitive liposomes offered protection against intestinal dam-
age and reduced toxicity (Araújo et al. 2017). Ju et al. (2017) reported the fabrica-
tion of pH-sensitive liposomes composed of N-(3-Aminopropyl)
imidazole-cholesterol (a new pH-sensitive material synthesized by them) and phos-
phatidylcholine. These pH-sensitive liposomes were loaded with curcumin. Results
revealed the controlled release behavior of curcumin and pH responsive properties
at around pH 5 by these liposomes. These curcumin-loaded novel pH-sensitive lipo-
somes also exhibited improved cytotoxicity against EC109 cells (Liang Ju et al.
2017). Another interesting study by Mimi M Yang et al. (2017a) demonstrated that
PEGylated pH-sensitive liposomes loaded with a weakly acidic dinitrobenzamide
mustard prodrug with poor water solubility (SN25860) showed improved anti-pro-
liferative potential against EMT6 mouse mammary carcinoma cell line. These lipo-
somes showed faster and greater clathrin-mediated endocytosis and higher
intracellular drug concentration as compared to the non-pH-responsive liposomes
(Mimi M Yang et al. 2017a).
Cationic Liposomes
The difficulties encountered in DNA encapsulation with conventional liposomes
due to the plasmid size warranted for newer approaches based on PE and cationic
lipids that result in better transfection efficiency. Cationic liposomes were first
explored for genetic transfer in 1980s and were primarily based on formation of
electrostatic complexes with plasmid DNA. The positively charged lipids
16 H. Ahmad et al.
neutralized the negatively charged plasmid to facilitate its delivery. The process
usually involves mixing the cationic lipids with DNA and incorporating then in the
cells thereby forming an aggregate of cationic lipid and DNA. Synthesis of DOTMA
(a popular cationic lipid) by Felgner and group forms the first report of its synthesis
and description. The ionic interactions between the positively charged head groups
of DOTMA with DNA’s negatively charged phosphate groups results in complex
formation with complete transfection for gene transfer. Either alone or combined
with several other neutral lipids, DOTMA forms spontaneous MLVs which upon
sonication can be transformed into SUVs. It has been commercialized (Lipofectin.,
Gibco-BRL, Gaithersburg, MD) as a 1:1 mixture with DOPE and has been widely
used to transfect a wide variety of cells. DOPE is commonly employed as helper
lipid to augment fusogenicity of cationic liposomes. Many cationic lipids have been
synthesized; those with monovalent head groups (like 1,2-dioleoyl-3-trimethylam-
moniumpropane, N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride,
and 3-β-[N-(N′,N′-dimethylaminoethyl)carbamoyl]-cholesterol) are known to form
extended spaghetti-like structures while those with multivalent head groups
(2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,Ndimethyl-1-propanamin-
ium trifluoroacetate) form particles with condensed structure with plasmid DNA
(Thassu et al. 2007; Kulkarni et al. 2011).
In a recent investigation 3 cationic glycolipids with different hydrophobic chains
Malt-DiC12MA, Malt-DiC14MA, and Malt-DiC16MA were prepared by using
maltose as starting material through different synthetic routes. All the liposomes
were able to efficiently bind and compact DNA into nanoparticles with appropriate
size and zeta measurements. High gene transfer efficiency and improved uptake was
observed in Malt-DiC14MA (N/P 8:1) (Bo Li et al. 2017a).
Cationic liposomes are potential carrier systems for targeting and delivering
drugs to solid tumor and tumor tissues. They might pose some drawbacks like
charge-related instability and toxicity via intravenous route. In a recent study by
Haohuan Li et al. (2017b) curcumin-loaded cationic liposomes modified with low
molecular weight heparin were found to show increased intracellular distribution in
the cytoplasm and nuclei and enhanced cytotoxicity as compared to anionic lipo-
somes containing curcumin (Haohuan Li et al. 2017b). Wang et al. (2017) reported
doxorubicin-loaded liposomes modified with cationic polymethacrylate polymer
(Eudragit RL100) displayed improved uptake and antitumor efficacy against doxo-
rubicin resistant (MCF7/adr) cell and an aggressive liver cancer H22 cell (Wenxi
Wang et al. 2017).
Cationic liposomes have been used as potential nanoparticles for vaccine deliv-
ery. They easily form complexes with biomacromolecules, facilitate antigen, and
adjuvant delivery to antigen presenting cells, mediate the cellular uptake of vaccine
components and also trigger antigen cross presentation. A rational design of cat-
ionic liposomes could positively affect the intracellular fate of vaccine and its
immunological performance. It was found in a study that cationic liposomes could
enhance the lysosomal pH in dendritic cells, restrict degradation of antigens, and
promote cross-presentation and cross-priming of CD8+ T-cell responses unlike the
anionic liposomes (Jie Gao et al. 2017). Cationic liposomes can also be used as
suitable vectors for vaccination-based therapies for cancer. An investigation demon-

strated a successful intradermal vaccination via cationic liposomes loaded with
well-defined tumor-specific synthetic long peptides and a TLR3 ligand as adjuvant.
These cationic liposomes could strongly activate functional, antigen-specific CD8+
and CD4+ T cells and exhibited targeted cytotoxicity in vivo (melanoma and HPV-
induced tumors) with high potency (Varypataki et al. 2017).
Targeted Liposomes
Toxicity of drugs is a major concern especially in cancer chemotherapy, and there-
fore drug targeting via liposomes assumes greater significance in order to achieve
reduced toxicity and efficient targeting to disease site sparing the normal cell popu-
lations. A targeting moiety can be incorporated in liposomes to target drugs or thera-
peutic agents to specific cell populations. Targeted liposomes are specifically taken
up by target cells and have proved to be efficient carriers for drug delivery and in
overcoming multidrug resistance. The targeting ligand could be a lipid-anchored
antibody or antibody fragment, folate, transferrin, or carbohydrate. The targeting
moiety could be incorporated into liposomes either during liposome formation by
detergent dialysis or after liposome formation by conjugation to reactive lipids or by
post-insertion of ligands from micelles of lipid-derivatized antibodies (Thassu et al.
2007; Kulkarni et al. 2011).
In case of immunoliposomes, the liposomes are conjugated to an antibody (like
HER2, antitransferrin receptor, anti-CD20, anti-CD19) or an antibody fragment
such as Fab and scFv. Besides antibodies liposomes targeted with transferrin and
folic acid have performed well in tumors that over express these receptors. However
the localization of targeted liposomes in tumors might not be drastically greater than
the non-targeted liposomes since the biodistribution of liposomes is governed by
vascular permeability and EPR effect. Also penetration of liposomes into tumors
(having high interstitial pressure) is argued to be negatively affected because of their
size. Despite these odds, targeted liposomes like anti-HER2 immunoliposomes and
folate receptor-targeted liposomes have demonstrated enhanced antitumor efficacy
over non-targeted liposomes in murine models (Baselga et al. 1998; Goren et al.
2000; Ishida et al. 2001; Hongyan Li and Qian 2002).
Some recent studies focusing on targeted therapies with liposomes are being
discussed here. Recently a targeted therapy with poly (L-Lysine) complexed
EpCAM (epithelial cell adhesion molecule) siRNA immunoliposomes emerged as
a promising adjuvant therapy in EpCAM-positive epithelial cancers. These hybrid
immunoliposomes (Egg PC:DSPE-PEG, 8:2) linked with EpCAM antibody as the
targeting ligand showed an encapsulation efficiency of 86% and showed improved
uptake and regressed tumor volumes in SCID mice (Bhavsar et al. 2017). In another
striking study multifunctional liposomes were designed for improved targeting in
glioblastoma multiforme. This multifunctional delivery system modified with cyclic
RGD (c(RGDyK) that could target integrin αvβ3 overexpressed on the blood–brain
tumor barrier and glioma cells and p-hydroxybenzoic acid (pHA) could target
18 H. Ahmad et al.
dopamine receptors on the blood–brain barrier exhibited strong anti-glioma effi-

ciency (Belhadj et al. 2017).
Nguyen et al. (2017) demonstrated a targeted therapy based on trastuzumab (a
therapeutic monoclonal antibody that selectively recognizes HER2/neu receptor)
for improved antitumor efficiency against breast cancer and overcoming drug resis-
tance. Rapamycin and polypyrrole (a photosensitizer) were co-loaded in liposomes
conjugated with trastuzumab as a strategy for combined chemo-photothermal ther-
apy via targeted therapy. These liposomes could effectively deliver rapamycin and
showed enhanced uptake in BT-474 cells. They exhibited better effects in breast
cancer cells that over expressed HER2/neu receptors as compared with cells not
over expressing these receptors (Nguyen et al. 2017).
Another new strategy for targeting in bladder tumors elaborated upon the use of
a synthetic peptidolipopolymer conjugate that could be incorporated into liposomes
to promote specific binding to the fibronectin matrix that surrounds the bladder
tumor cells and promote cellular internalization of fibronectin-integrin complexes.
These liposomes modified with peptide proved to be useful vehicles for targeted
delivery in vivo in MB49 tumor-bearing mice (Young Lee et al. 2017). In another
targeted liposomal strategy, it was observed that the hybrid albumin liposomes
loaded with chlorambucil exhibited significantly better targeting and enhanced drug
accumulation in B16F10 tumors over non-hybrid liposomes. It was found that the
B16F10 melanoma-bearing mice treated with these hybrid liposomes displayed the
longest median survival time (30 days) across all treatment groups (Quan Zhang
et al. 2017).
Fusogenic Liposomes
Fusogenic liposomes represent a class of unique phospholipid vesicles where the
lipid bilayer exhibits an increased ability to interact with cellular membranes in
their liquid crystalline phase which enables them to release their contents into the
cytoplasm. These liposomes incorporate special lipids displaying increased fluidity
and ability to destabilize the biological membranes or inactivated Sendai virus enve-
lope components and other fusogenic peptides. They can be fabricated by reconsti-
tution of envelope proteins of viruses into liposomes or encapsulation of hemolysins
from bacteria with varying degrees of pH dependence. Reducing and enzymatic
conditions prevailing in the endosomal compartment can also be utilized to facili-
tate transport via fusogenic liposomes. These liposomes are taken up by endocytosis
where the main part of their cargo is degraded in lysosomes before reaching its
destination.
These liposomes have been used for a wide spectrum of applications which
includes but is not limited to delivery of nucleic acids and genetic transfer of
materials, to achieve targeting in cancer therapy and to release active substances
intracellularly. These fusogenic liposomes also target specific organelles by the
use of fluorescent derivatives (Thassu et al. 2007). Recently fusogenic liposomes
were used as carriers for delivery of water soluble proteins to mammalian cells.
Proteins (EGFP, Dendra2, R-phycoerythrin) and peptides (LifeAct-FITC,
NTF2-AlexaFluor488) were delivered with high efficiency into mammalian cell

cytoplasm without any degradation (Kube et al. 2017). These liposomes can serve
as important carriers for delivering therapeutic agents based on RNA or DNA
oligonucleotides like antisense DNA oligonucleotide, siRNA. In a study by
Kunisawa et al. (2005) fusogenic liposomes constructed from ultra violet-inacti-
vated Sendai virus were successfully used for delivery of DNA oligonucleotides
into cell cytoplasm (Kunisawa et al. 2005).
Temperature-Sensitive Liposomes
Hyperthermia can be adopted as a strategy to trigger release of liposomal contents
by employing a lipid composition (dipalmitoylphosphatidylcholine or conjugation
to a thermosensitive polymer) with phase transition temperature greater than
37 °C. Incorporation of lipid-conjugated copolymers of N-isopropylacrylamide and
N-acryloylpyrrolidine can successfully produce temperature-sensitive liposomes
exhibiting phase transition at 40 °C (Kong and Dewhirst 1999; Kono et al. 1999;
Needham and Dewhirst 2001). Temperature-sensitive liposomes can be used to
deliver anticancer drugs which can be released at an elevated temperature in locally
heated tumors resulting in high drug concentration within the tumor tissue and pre-
vention of drug exposure to healthy tissues (Grüll and Langereis 2012). In a study
by Kono et al. (2010), temperature-sensitive liposomes were prepared for doxorubi-
cin delivery based on a thermosensitive block copolymer, copoly (EOEOVE-block-
octadecyl vinyl ether) for tumor-specific chemotherapy. These liposomes modified
with this temperature-sensitive copolymer released doxorubicin above 40 °C and
showed almost complete release at 45 °C within 1 minute. These liposomes were
long circulating and resulted in significant suppression of tumor growth when the
tumor site was heated to 45 °C for 10 min at 6–12 hours after injection thus reflect-
ing an effective tumor specific chemotherapeutic intervention (Kono et al. 2010).
Another study demonstrated that vinorelbine bitartrate-loaded temperature-sensi-
tive liposomes could release the drug quickly at 42 °C and resulted in high inhibi-
tion of tumor growth in a lung tumor model (Hui Zhang et al. 2011).
1.4.2 Therapeutic and Clinical Applications
Cancer Chemotherapy
Most of the anticancer drugs are non-specific and often injurious to normal tissues
and their administration as “free drug” through conventional means further limits
their clinical potential due to limited drug concentration in tumor tissues and system
toxicity because of non-targeted distribution in body tissues. Therefore a strategy
that works on enhancing the drug accumulation and availability in the tumor tissues
with minimal exposure to healthy tissues is desirable for the success of cancer che-
motherapy. Targeted and cancer specific drug delivery and overcoming multidrug
20 H. Ahmad et al.
resistance by liposomes is one such strategy that has emerged in recent time as a
successful intervention. Actively targeted liposomes based on surface modification
with targeting moieties (like transferrin, folate or peptides) specific to the up-regu-
lated receptors on the surface of tumor cells enables improved drug delivery and
enhanced cellular uptake. Some interesting examples have already been discussed
under cationic, pH-sensitive, and targeted interventions. Some newer approaches
include liposomes utilizing small molecule-based tumor-targeting moieties and use
of a combination of targeting moieties due to heterogeneity in receptor expression
(Sangbin Lee et al. 2012; Stapleton et al. 2013; Zhou et al. 2013; Sriraman
et al. 2016).
In one study paclitaxel-loaded liposomes surface modified with D-a-tocopheryl
polyethylene glycol 1000 succinate-triphenylphosphine conjugate (synthesized as a
mitochondrial targeting molecule) were prepared and evaluated in human lung can-
cer A549 cells, drug-resistant lung cancer A549/cDDP cells, and the drug-resistant
lung cancer A549/cDDP cells xenografted nude mice. These small (80 nm) targeted
liposomes demonstrated high cell uptake, increased mitochondrial accumulation,
and triggered cytochrome C release. They could enhance apoptosis through mito-
chondrial signaling pathways and appeared to be a promising approach to treat
drug-resistant lung cancer (Zhou et al. 2013).
Another intervention reported that sialic acid-octadecylamine conjugates
anchored onto pixantrone-loaded liposomes showed the strongest cytotoxicity in
S180-bearing Kunming mice. This strategy was based on the fact that sialic acid is
involved in tumor development, and its receptors are highly expressed on the tumor-
associated macrophages. Thus liposomes containing pixantrone surface modified
with sialic acid–octadecylamine conjugates resulted in killing of tumor-associated
macrophages and high anticancer potency (She et al. 2014).
Sriraman et al. (2016) had demonstrated that doxorubicin-loaded dual targeted
(folic acid and transferrin) PEGylated liposomes showed increased penetration and
cell association in HeLa cells. These dual targeted liposomes showed increased
cytotoxicity in vitro in HeLa and A2780-ADR ovarian carcinoma cell monolayers
and significantly higher tumor growth inhibition in a HeLa xenograft nude mice
model compared to non-targeted liposomes (Sriraman et al. 2016).
Recently tetraiodothyroacetic acid (an antagonist that blocks the binding of thy-
roid hormone to integrin αvβ3) was reported as a new targeting moiety for delivery
of chemotherapeutic agents to tumor sites. Here PEGylated liposomes targeted with
tetraiodothyroacetic acid could successfully deliver edelfosine and provide about
100% protection for up to 50 days in A375 xenografted mice (Sangbin Lee
et al. 2012).
In one study the cellular uptake of curcumin-loaded didecyldimethylammonium
bromide (DDAB)-modified liposomes were investigated and compared with that of
the non-modified liposomes on cervical cancer cells. It was observed that DDAB
formulations showed faster release and higher uptake of curcumin demonstrating
better anticancer effects and apoptosis over DDAB-free liposomes (Saengkrit et al.
2014). These examples illustrate how surface modification with different targeting
moieties or therapeutics could provide for attractive and promising approaches for
architecturing liposomal formulations for delivering chemotherapeutic agents in

different cancers.
Antimicrobial Therapy
Infectious diseases might be intracellular, extracellular, device, or film mediated
and cause huge mortality. The challenges in antimicrobial drug therapy can be met
by the use of novel drug delivery systems. Liposomes have been successfully used
to deliver drugs for treating different infections. Schiffelers et al. (2001) reported
that gentamicin-loaded liposomes composed of partially hydrogenated egg phos-
phatidylcholine, cholesterol, and 1, 2-distearoylsnglycero- 3- phosphoethanol-
amine-N- (polyethylene glycol) showed increased therapeutic action against
Klebsiella pneumoniae (Schiffelers et al. 2001). In one study liposomes made of
1,2-dipalmitoyl-sn-glycero-3-phosphocholine and cholesterol could successfully
deliver polymixin B, and this liposomal delivery resulted in enhanced bioavailabil-
ity, decreased bacterial count in lungs, and reduction in lung injury caused by
Pseudomonas aeruginosa (Omri et al. 2002). Kaur et al. (2008) reported the
enhanced HIV targeting of zidovudine to lymphatics by its liposomal delivery (Kaur
et al. 2008). Literature shows liposomal Amphotericin B could provide strong tar-
geted therapeutic action against Aspergillus fumigatus at the infection sites
(Takemoto et al. 2004). In a recent study it was seen that cationic antimicrobial
peptide when encapsulated within liposomes reduced the cytotoxicity and displayed
enhanced stability and bioactivity against herpes simplex virus 1(Ron-Doitch et al.
2016). Recently a number of plant derived bioactives encapsulated as liposomes
have shown good antimicrobial activity. β-lapachone-loaded liposomes showed
good antibacterial activity against meticillin-resistant Staphylococcus aureus and
improved antifungal properties against Cryptococcus neoformans (IMF Cavalcanti
et al. 2015a). Thymol- and carvacrol-loaded liposomes showed strong antimicrobial
effects against different strains of Staphylococcus aureus or Salmonella enteric
(Engel et al. 2017). D-limonene encapsulated into liposomes was found to be effec-
tive against fruit rotting fungi (Botrytis cinerea and Penicillium chrysogenum) and
illness causing bacteria (Escherichia coli and Listeria monocytogenes) (Umagiliyage
et al. 2017).
Vaccine-Based Interventions
Vaccines have been of enormous benefit to public health by alleviating the burden
of illness due to infectious diseases globally. Liposomal interventions which were
initially used as immunological adjuvants around 1974 have been successfully used
in vaccine-based therapies against several antigens of bacterial, viral, protozoan, or
tumoral origin to impart both humoral and cell-mediated immunity. These liposo-
mal preparations are tolerated well, are non-toxic and biodegradable, and display
low reactogenicity and also improve the therapeutic action of the bioactive agents
by enhancing their solubility and stability. Owing to the advantages they offer, lipo-
some-based adjuvant preparations have been approved for human use and are
22 H. Ahmad et al.
currently being clinically evaluated (Ravendra Garg et al. 2017a; Tengfei Ma
et al. 2016).
Oral vaccination provides limited benefits due to the challenges encountered in
the gastrointestinal route. FITC labeled ovalabumin-loaded liposomes incorporated
in a water-in-oil-in-water double emulsion developed recently provided for an inter-
esting model for oral vaccine delivery (Liau et al. 2015). In a comparative study on
Montanide and squalene emulsions, PLGA nanoparticles, and cationic liposomes, it
was found that the cationic liposomes loaded with SLP-induced the functional anti-
gen-T cells in vivo most efficiently upon subcutaneous vaccination in mice and after
transfer of antigen-specific target cells in immunized mice provided the highest
killing capacity in vivo. Thus cationic liposomes stand out as promising biodegrad-
able candidates for immunotherapy in cancer based on SLP delivery (Varypataki
et al. 2016). Though peptide vaccines delivered by liposomes has also been of sig-
nificance in several other infections like HIV-1 (Apellániz and Nieva 2015).
Another intervention using cationic liposomes described the preparation of man-
nosylated zwitterionic-based cationic liposome as DNA vaccine adjuvant against
HIV. These cationic liposomes formed a tight structure capable of providing protec-
tion against nuclei enzyme degradation following complexation with DNA. These
lipoplexes exhibited strong anti-HIV immune response with reduced toxicity and
elicited a Th1/Th2 mixed immunity. These zwitterionic liposomes emerged as a safe
and effective DNA adjuvant for HIV vaccines overcoming the drawbacks of poor
immunogenicity due to poor presentation to antigen presenting cells and insufficient
antigen expression usually encountered with DNA vaccines (Qiao et al. 2016).
Studies suggested that when dimethyldioctadecylammonium bromide (cationic
lipid) was added to stable neutral liposomes composed of distearoylphosphatidyl-
choline (DSPC) and cholesterol, the liposome size reduced while the protein entrap-
ment increased. Addition of trehalose 6,6-dibehenate (immunomodulator) to neutral
or cationic vesicles did not change their physiochemical properties; however the
immune responses increased considerably in presence of trehalose 6,6-dibehenate.
Cationic liposomes exhibited increased amounts of IFN-γ; however its release
reduced with time; whereas for neutral liposomes with trehalose 6,6-dibehenate, the
initial IFN-γ were comparatively lower than that in cationic liposomes, but the over-
all response could be sustained for a longer period of time (McNeil et al. 2011).
Besides cationic liposomes, metal chelating liposomes have also been used as
platform for development of recombinant vaccines and drug targeting. In a study,
nickel chelating liposomes surface modified with His-tagged protein (rHsp90) as
antigen and non-pyrogenic hydrophobized derivative of muramyl dipeptide (C18-
O-6-norAbuMDP) being incorporated as adjuvant provided both TH1 and TH2
immune responses without any side effects upon intradermal injection in mice and
therefore presenting a promising strategy against candida infections (Mašek et al.
2011). Liposomal interventions have been reported against a number of pathogens
and for several infections. In a study it was observed that stronger immunoglobulin
G responses could be detected in mice vaccinated with liposome encapsulated DNA
over those vaccinated with naked DNA. This response was more pronounced in
mice with pCI-neo plasmids encoding Babesia bovis MSA-2c as compared to
bovine herpesvirus type 1 (BoHV-1) gD (Rodriguez et al. 2013). Ma et al. reported

that liposomal formulation containing recombinant E proteins prepared by reverse-
phase evaporation method provided good protection against duck Tembusu viral
challenge in cherry valley ducks upon intramuscular injection of the same (Tengfei
Ma et al. 2016).
Pulmonary Drug Delivery

Drug delivery by pulmonary route has emerged as a promising noninvasive route for
local as well systemic delivery of several small molecules, genes, and protein and
peptide drugs for treatment of chronic respiratory diseases like asthma, chronic
obstructive pulmonary disease, and even lung cancer. It offers clear advantages over
other routes which includes availability of large absorptive area of lungs, thin
absorption barrier, low enzyme activity, evading hepatic first pass, reduced adverse
effects, direct drug delivery at the site of action, and ease of absorption of drugs with
high molecular weights (peptides and proteins). Pulmonary administration of pep-
tides and protein drugs improves patient compliance and overcomes problems like
tissue invasion encountered with parenteral administration of these drugs. Liposomes
have been widely investigated as drug carriers for controlled pulmonary delivery.
Liposomes serve as an attractive platform for delivering drugs to the lungs because
of their safety, biocompatibility, protection of encapsulated drug against enzymatic
degradation, and ability to achieve selective drug targeting and controlled release
characteristics. Several studies have shown the advantages of surface modification
of liposomes in pulmonary delivery of drugs especially peptide drugs (Chono et al.
2009; Murata et al. 2012; Murata et al. 2014). Murata et al. (2012) reported that
liposomes with surface modification with polyvinyl alcohol with a hydrophobic
anchor and with chitosan oligosaccharide increased and prolonged the therapeutic
efficacy of elcatonin after its pulmonary administration to rats. Further it was seen
that liposomes with chitosan oligosaccharide modification adhered to lung tissues
causing tight junction opening which in turn resulted in enhanced elcatonin absorp-
tion; whereas the liposomes with modification with polyvinyl alcohol with a hydro-
phobic anchor induced long-term retention of the drug in the lung fluid resulting in
its sustained absorption. It was therefore evident here that surface modified lipo-
somes could be used as beneficial strategy for pulmonary administration of peptide
drugs (Murata et al. 2012). Further investigations pertaining to the behavior of the
liposomes modified with polyvinyl alcohol with a hydrophobic anchor in the lung
and other parts of the body were carried out by real-time in vivo imaging tech-
niques. These surface modified liposomes induced long-term lung retention and
reduced the association with alveolar macrophages (NR8383) over the non-modi-
fied liposomes. This resulted in prevention of rapid elimination of the modified
liposomes by macrophages and increased their residence in the lungs to provide
prolonged therapeutic effect (Murata et al. 2014).
Chono et al. (2009) had demonstrated that delivery of insulin as an aerosolized
liposomal preparation composed of dipalmitoyl phosphatidylcholine enhanced its
delivery by opening the epithelial cell space in the pulmonary mucosa without
24 H. Ahmad et al.
harming mucosal cells and lung tissues in rats (Chono et al. 2009). Liposomal pul-
monary delivery has also been attempted as potential therapy against bacterial
infections. In a study 25(OH) D (Vitamin D3 metabolite) encapsulated liposomes
were investigated against Pseudomonas aeruginosa pulmonary infection. Tracheo-
bronchial deposition of these liposomes achieved by jet nebulization caused a sig-
nificant reduction in survival of bacteria (Castoldi et al. 2017).
Pulmonary delivery of drugs via liposomes has also been investigated as an inter-
vention to treat pulmonary arterial hypertension. Nahar et al. (2014) developed
magnetic liposomes for delivery of fasudil. These liposomes showed about three-
fold higher uptake and 40% reduction in proliferation in pulmonary arterial smooth
muscle cells in the presence of a magnetic field. The administration of intra-tracheal
fausidil-loaded magnetic liposomes resulted in a 27-fold increase in the half-life
and about a 14-fold increment in area under the curve, compared to plain fausidil
thus making it a viable strategy for treating pulmonary arterial hypertension (Nahar
et al. 2014).
Another strategy in liposomal pulmonary delivery was the development of chito-
somes. Coating liposomes with chitosan–xanthan gum polyelectrolyte complexes in
different ratios to obtain chitosomes was studied by Manca et al. (2012). They found
that the nebulization and rheological properties of chitosomes were affected by the
weight ratio of chitosan–xanthan gum, and a 1:0.5 (w/w) coating could significantly
improve the rifampicin total mass output and drug deposition in the lower stages of
the impinger (Manca et al. 2012). Yet another recent study showed that liposomes
loaded with curcumin when coated with chitosan or hyaluronan could result in
improved nebulization and enhanced antioxidant properties of curcumin in A549
cells (Manconi et al. 2017).
Ocular Drug Delivery

The eye being well protected by several barriers and defense mechanisms, ocular
drug delivery especially to the retina and choroid becomes a challenging task.
However overcoming these obstacles in ophthalmic drug delivery is rather impor-
tant to treat ocular diseases like diabetic macular edema, senile macular degenera-
tion, posterior uveitis, proliferative vitreoretinopathy, cytomegalovirus infection,
and glaucoma and several other rare genetic diseases. Liposomes were investigated
as carriers for ocular drugs in 1980’s to prolong their duration of action and to
achieve intracellular drug delivery. However controlling drug release from lipo-
somes has been a challenge in ocular drug delivery. Light-activated liposomes have
merged as promising interventions to release the drug at a specific time and at the
specific site in the eye (Lajunen et al. 2016). Use of mucoadhesive materials has
also been investigated as a solution to improve the therapeutic efficacy of ocular
drugs by enhancing their bioavailability. Dong et al. reported that ibuprofen-loaded
liposomes coated with silk fibroin as adhesive excipient exhibited rapid uptake in
human corneal epithelial cells and sustained the release and in vitro corneal perme-
ation of ibuprofen over conventional liposomes (Dong et al. 2015).
Liposomes were investigated as carriers for Distamycin A delivery in ocular

HSV infections. Liposomal Distamycin A showed reduced cytotoxicity and
enhanced uptake in rabbit corneal epithelial cells compared to plain Distamycin A
(Chetoni et al. 2015). Recently deformable liposomes coated with chitosan were
developed as an improvement over conventional liposomes for ophthalmic delivery.
Flurbiprofen-loaded deformable liposomes successfully prolonged the precorneal
retention of the drug also improving its transcorneal penetration and absorption and
produced no ocular damage. A strategy like this could be exploited to produce safe
liposome based interventions as alternatives to conventional eye drops (Hongdan
Chen et al. 2016a). In another study chitosan-coated liposomes have been efficiently
used to deliver timolol maleate. These liposomes showed enhanced mucin adhesion,
increased corneal permeation and retention, and reduced the intraocular pressure
and showed negligible irritant effects when compared to commercial eye drops con-
taining timolol maleate (Tan et al. 2017). Another intervention described proglyco-
somes as promising carriers for delivery of tacrolimus to the eye. Incorporation of
propylene glycol resulted in higher drug encapsulation and prevention of drug leak-
age. Findings better elasticity, revealed prolonged precorneal retention and improved
intraocular drug levels in rabbits could be achieved with proglycosomes compared
to conventional liposomes (Vaidehi Garg et al. 2017b).
Topical Drug Delivery

Liposomes have the ability to deliver drugs for a variety of dermatological condi-
tions based on their similarity with the natural membranes. Recently meglumine
antimoniate-loaded topical liposomal formulation had shown benefits in experi-
mental cutaneous leishmaniasis in BALB/c mice (Kalat et al. 2014). Ultradeformable
liposomes have emerged as potential carriers that improve the drug permeation and
therefore offer benefits in topical drug delivery. In one other study ultradeformable
liposomes loaded with amphotericin B showed profound drug penetration into the
deep epithelial layers and therefore significantly improving its effect in cutaneous
fungal infections and leishmaniasis (Perez et al. 2016). Another finding showed that
5-aminolevulinic acid-loaded cationic ultradeformable liposomes were found to be
effective carriers for topical photodynamic therapy as 5-aminolevulinic acid is
thought to be being converted to photodynamic protoporphyrin preferentially in the
epidermis (Kyung Oh et al. 2011). Another example of successful use of ultrade-
formable liposomal strategy showed that co-encapsulation of resveratrol and 5-fluo-
rouracil improved their anticancer activity on skin cancer cells as compared to both
the drugs in free form and as single entrapped agents. Thus it could be a potential
therapy for non-melanoma skin cancer (Cosco et al. 2015).
Besides other uses, topical drug delivery assumes great significance in vaginal
applications. Liposomes could be used for improved drug delivery to vagina for a
variety of conditions. Chitosan-coated liposomes have been reported for efficient
localized therapy of clotrimazole to the vagina. Findings in this study revealed that
the liposomes coated with chitosan increased the tissue retention and lowered the
penetration of clotrimazole in comparison to the control in pregnant sheep vaginal
26 H. Ahmad et al.
tissue. This could be a platform for designing mucoadhesive liposomes for localized
vaginal therapy to avoid systemic absorption in pregnant populations suffering from
vaginal infections (Jøraholmen et al. 2014). Jøraholmen et al. (2015) also reported
that the antioxidant and anti-inflammatory effects of resveratrol were more pro-
nounced when encapsulated into chitosan-coated liposomes. They emphasized that
this could be explored further to produce clinically acceptable topical resveratrol
formulations against a number of pathogens that cause sexually transmitted diseases
leading to vaginal inflammation and infections (Jøraholmen et al. 2015). In one
other study from this group, it was demonstrated that interferon alpha-2b-loaded
PEGylated liposomes could provide a mucus penetrating effect for localized ther-
apy in human papilloma virus (HPV) vaginal infections. They argued that these
liposomal systems could penetrate mucus, provide a closer contact with epithelium
and reach deeper epithelium to provide improved vaginal delivery of interferon
alpha-2b for antiviral therapy (Jøraholmen et al. 2017).
Diagnostic and Imaging

Recent times have observed the manifold usage of liposomal nanotechnology in
combination with light, sound, and electromagnetic fields being developed for
widespread therapeutic and diagnostic applications, and the same have been vali-
dated through a number of studies.
Liposomes-based near-infrared probes have been designed that provides higher
quantum yield and specific tumor targeting abilities. Portnoy et al. (2011) reported
that the near-infrared probe was designed by using indocyanine green (a near-infra-
red fluorescent molecule) and cetuximab (monoclonal antibody for epidermal
growth factor receptor) being attached to liposomes by passive diffusion. This inter-
vention performed better than free ICG in A431 colon carcinoma cells (Portnoy
et al. 2011). In a similar study, it was observed that PEGylated liposomes incorpo-
rating indocyanine green resolved tissue accumulation in tumor-borne animals with
high sensitivity in two different models of different vascularization by using multi-
spectral optoacoustic tomography. These findings investigated that these PEGylated
liposomes had obvious advantages over gold nanoparticles and organic dyes
(Beziere et al. 2015).
Temperature-sensitive liposomes have also been reported to be useful in mag-
netic resonance-guided drug delivery (Yeo et al. 2014). Several theranostic probes
have been evaluated. Scintigraphic imaging studies revealed that folate-PEGylated
long-circulating and pH-sensitive liposomes loaded with 159Gd showed three times
higher accumulation and uptake and better survival rates in Ehrlich tumor-bearing
mice (Soares et al. 2015).
Liposomes have also been reported to play a significant role in gene delivery.
PEGylated liposomes also known as bubble liposomes composed of different cat-
ionic lipids have been reported to be efficient tools for pDNA and siRNA used in
combination with ultrasound imaging (Endo-Takahashi et al. 2013). They were also
reported to be used in therapeutic microbubbles which have been widely investi-
gated for diagnosis and treatment of cancer (McLaughlan et al. 2017). In a study it
was also demonstrated via optical imaging that liposomes could traffic to the heart
and accumulate into the regions of myocardial injury and thus enable efficient diag-
nosis of myocardial injury along with facilitating drug delivery by acting as a carrier
(Lipinski et al. 2016).
1.5 Phytosome: Background
1.5.1 Phytosome: Background, Discovery, and Description
About 70% of the global population utilizes plant and plant products for their pri-
mary healthcare. Different nations and cultures of the world use them either as sin-
gle herb, combination of herbs, or combination of herb and drugs for their health
promoting benefits. Recent times have seen increased resurgence of plant based
therapies for treatment of a variety of disorders (Jian-Li Gao et al. 2009; Che
et al. 2013).
It has been documented that the potency and biological activity of plant extracts
is lost upon isolation and purification of constituents. Thus the therapeutic activity
presents more significantly in the form of extracts rather than pure compound forms.
However the clinical utility of these extracts remains questionable due to loss of
constituents in gastric environment upon oral administration and their extremely
low bioavailability (Saraf 2010).
Most polar bioactives (polyphenolics) derived from plants are poorly absorbed
either due to their large molecular size, which cannot be absorbed by passive diffu-
sion or due to their poor lipid solubility, thus severely limiting their ability to trans-
port across lipid-rich biological membranes, resulting in their poor bioavailability.
Drug candidates of herbal origin displaying low water solubility lead to poor bio-
availability, high intrasubject/intersubject variability and lack of dose proportional-
ity, and therefore their oral use is limited due to high hydrophobicity. Therefore,
adopting suitable formulation strategies becomes essential to improve the solubility
and bioavailability of such drugs (Mauludin et al. 2009; Kamel and Basha 2013).
These polar phyto-constituents can be converted into lipid-compatible molecular
complexes also known as phospholipid complexes, supramolecular complexes, or
herbosomes. This technology was patented as Phytosome® in the late 80s by Indena
(Milan, Italy), a pharmaceutical and neutraceutical giant for incorporation of stan-
dardized plant extracts and water-soluble bioactives into phospholipids for improv-
ing their bioavailability. Phytosomes possess better bioavailability over conventional
plant extracts due to their ability to cross lipid membranes and eventually reaching
systemic circulation. The term “phytosome” has two components: “phyto” means
plant, while “some” means cell-like. Thus phytosomes are little cell like structures
formed from a combination of lecithin and plant bioactives/extract prepared in an
opportune solvent. Based on their physical-chemical and spectroscopic
28 H. Ahmad et al.
characteristics, these phytosomes or lipid complexes can be considered as novel

entities (Saraf 2010; Semalty et al. 2010; Khan et al. 2013; Alexander et al. 2016).
1.5.2 Properties and Characterization of Phytosomes
Phytosomes are prepared by reaction of dietary phospholipids and the substrate

(plant component) in defined stoichiometric ratio most commonly by solvent evapo-
ration method. Several studies have shown molar ratios 0.5:1 to 1:3; however a
molar ratio of 1:1 or 1:2 of plant principle and phospholipid has been most com-
monly used. Phytosomes are amphipathic entities having defined melting point,
generally soluble in non-polar solvents and having moderate solubility in fats.
Factors that can influence the behavior of phytosomes in biological systems
include but are not limited to size, permeability, composition, percent entrapped
solutes, and the quantity and quality of ingredients used. Phytosomes can therefore
be characterized based on the following parameters: size, size-distribution, shape,
drug content, drug release and spectroscopic and thermo-gravimetric studies.
The size of phytosomes can vary from 50 nm–00 μm. These lipid compatible
complexes usually show spherical shape with rough surface morphology and good
flowability. These phytosomes upon treatment with water assume a micellar shape
resembling a liposome like structures. Most crystalline drugs upon lipid complex-
ation have demonstrated loss of crystallinity and present themselves as molecularly
dispersed or amorphous forms. X-ray diffraction studies have shown that the crys-
talline peaks present in drug-lipid physical mixture usually disappears in the drug-
lipid complexes or phytosomes, and this change in crystallinity is considered
responsible for their improved lipid solubility. Spectroscopic data has revealed that
the major interaction between the lipid and the substrate is due to hydrogen bonding
between the polar head groups of phospholipids and the polar functionalities pres-
ent in the substrate (plant bioactive) (Bombardelli and Mustich 1991; Semalty et al.
2010; Khan et al. 2013).
1.6 Preparation Methods for Phytosomes
Phytosomes are phyto-phospholipid complexes that are prepared by reaction

between 2–3 moles of phospholipids with 1 mole of natural ingredient usually poly-
phenols or plant extracts. However a 1:1 ratio has been most commonly employed.
The lipids used may be lecithin derived from plant or mammalian sources, PC, PE,
or PS in which the acyl group might be different or same and commonly derived
from oleic acid, linoleic acid, stearic acid, or palmitic acid. The ratio of phosphati-
dyl group present in a particular lipid is the most commonly used criteria for selec-
tion of a suitable lipid. PC derived from soybean is popularly employed for
phytosome preparation due to high phosphatidyl content. The natural ingredients
Fig. 1.3 Preparation methodologies for phytosomes
used for phytosome preparation are most popularly plant flavonoids like quercetin,
rutin, kaempferol, luteolin, catechins, etc.
The phospholipid complexation reaction between the herbal constituent and the
phospholipid is carried out in an aprotic solvent like methylene chloride, dioxane,
acetone, or methyl acetate. Solvents with low dielectric constants are solvents of
choice for phytosome preparation. The starting material themselves are insoluble in
solvents like ether, chloroform, or benzene; however they exhibit good solubility in
these solvents upon formation of a true stable complex which is lipophilic in nature
(Jose and Bombardelli 1987; Sharma and Sikarwar 2005; Amin and Bhat 2012;
Khan et al. 2013).
A general scheme involves the following steps: The phospholipids and the botan-
icals are placed in a flask and dissolved in the selected solvent by ultrasonication.
The reaction is carried out at suitable fixed temperature for fixed time duration to get
maximum possible yield and drug entrapment. After complex formation reaction is
complete, the complex can be isolated either by removal of solvent under vacuum,
by precipitation with nonsolvents such as aliphatic hydrocarbons (n-hexane), by
lyophilization or by spray drying (Fig. 1.3). The conventionally used aprotic sol-
vents have been largely replaced by protic solvents like ethanol in recent times.
Some recent investigations have used ethanol, tetrahydrofuran, and dichlorometh-
ane as the reaction medium; however absolute ethanol has been the most widely
30 H. Ahmad et al.
solvent for preparing phytosomes in recent investigations (Moscarella et al. 1993;

Khan et al. 2013; Alexander et al. 2016).
Phytosomes of several phyto-constituents have been reported. Maiti et al. (2006,
2007) reported preparation of naringenin and curcumin phytosomes (Maiti et al.
2006, 2007) and Yanyu et al. (2006) reported preparation of silybin-phospholipid
complexes (Yanyu et al. 2006).
Several researchers have reported the use of anti-solvent precipitation technique
for preparing phytosomes. A phyto phospholipid complex of andrographolide was
prepared by using dichloromethane as the solvent for reaction and n-hexane as the
anti-solvent for precipitation (Maiti et al. 2010). In a study based on a dispersion-
oriented variation of this technique, marsupin phytosomes were prepared where
marsupin was dissolved in water and lecithin was dissolved in diethyl ether by soni-
cation. Marsupin solution was added in a drop wise fashion into the lipid solution
under sonication. The resulting formulation was refrigerated and the final complex
showed a 44% entrapment of marsupin into it (Sikarwar et al. 2008).
Supercritical fluid (SCF) technology has emerged as a novel tool for improving
solubility profiles of drugs and preparing particles in size range 5–2000 nm. There
are various methodologies by which SCF technique can be implemented to produce
phytosomes. Some of them are supercritical anti-solvent method (SAS), compressed
anti-solvent process (PCA), rapid expansion of supercritical solutions (RESS), solu-
tion-enhanced dispersion by supercritical fluids (SEDS), and gas anti-solvent tech-
nique (GAS). In a study, puerarin- phospholipid complexes were prepared by three
conventional methods, namely, solvent evaporation, lyophilization, and microniza-
tion, and two SCF methods, namely, GAS and SEDS. The products obtained by
SCF methods showed better morphological characteristics, loss of crystallinity, and
improved dissolution profile (Ying Li et al. 2008; Khan et al. 2013).
1.7 Therapeutic Applications of Phytosomes
1.7.1 N
eutraceutical, Health Food,
and Cosmeceutical Applications
It has been discussed in Sect. 2.4. Phytosome: Background, Discovery, and

Description that extracts, botanicals, and polyphenols have several nutritional and
therapeutic benefits which makes them suitable for preparation of fortified food
products and neutraceuticals; however they suffer from low bioavailability which
limits their intended effects (Ghanbarzadeh et al. 2016). These botanicals also have
tremendous potential for personal care applications like moisturizing, antiaging,
and sunscreen-based skin therapies but again disadvantages like poor penetration
and compound instability limits their usage (Ganesan and Choi 2016). Phytosomes
though initially developed for pharmaceutical applications has emerged as an attrac-
tive way of delivering botanical-based neutraceutical and could be potentially used
in designing new food products and beverages probably owing to its of composition
and ease of preparation. Formulating polyphenols as phytosomal preparations for
fortified food applications can enhance their stability, absorption, and bioavailabil-
ity that in turn improve their nutritional and therapeutic benefits and offer protection
against environmental degradation during processing and storage. It also provides
protection to the poylphenol from the gastrointestinal tract conditions and also
improves its antioxidant and antimicrobial efficiency (Ghanbarzadeh et al. 2016).
Recently Babazadeh et al. (2017) reported preparation of rutin phytosomes that
could mask the undesirable features of rutin like poor aqueous solubility and bio-
availability to overcome the difficulties in fortification of food products with neutra-
ceutical agents that are water insoluble (Babazadeh et al. 2017). Besides being used
extensively as health foods, phytosomes containing plant extracts and bioactives
have been widely used in cosmeceuticals for skin care and beauty applications.
Recently phytosomes loaded with Citrus aurantium and Glycyrrhiza glabra extracts
were investigated for skin aging studies, and it was found that due to enhanced bio-
availability of the extracts (achieved due to phytosomes) in the cream resulted in
protection of the skin from aging. The bioavailability of these bioactive principles
can further be increased by nanosizing the phytosomes (Ganesan and Choi 2016).
Gold nanoparticle-based phytosomal preparation containing quercetin-enriched
plant extracts have also shown improved benefits (Demir et al. 2014). Photoprotection
against UV damage is yet another major area for phytosomal applications for skin.
Several antioxidants can have beneficial effects in protecting against skin damage
caused by UV rays from sunlight. Recently a new topical formulation based on
phytosomes containing chlorogenic acid exhibited significant protection upon UVA
irradiation post 4 hours of topical application in rat skin as compared to its conven-
tional formulation. This elucidated that phytosome strategy could be successfully
utilized for sun protection by skin topicals based on other natural antioxidants
(Bhattacharyya et al. 2014). Other natural agents like curcumin, resveratrol, silyma-
rin, ascorbic acid, genistein, quercetin, and green tea extracts have also been known
to have photoprotective properties which can be explored by phytosomal strategy
(Saraf and Kaur 2010). Several companies and units like Thorne Research (Dover)
have ventured into phytosome products; however a large chunk of market share has
been captured by Indena (Milan, Italy), a leading company in the identification,
development, and production of active principles derived from plants, for use in
health and personal care products and the first to patent phytosome technology.
Some of its popular phytosome strategies being used in health and personal care
have been presented in Table 1.4.
1.7.2 Clinical Applications
Phytosomes as delivery systems offer interesting applications for a wide range of

clinical uses and allow the active ingredients to elicit their maximal therapeutic
benefit by overcoming the biopharmaceutical barriers. Phytosome strategy has been
Table 1.4 Phytosome formulations in health and personal care
S.No. Phytosome name Plant source Assay Indication
1. BOSEXIL® Boswellia serrata Roxb. Ex ≥25% boswellic acids by HPLC Personal care: soothing,
Colebr. -resin anti-photoaging
2. CASPEROME® Boswellia serrata Roxb. Ex ≥25% boswellic acids by HPLC Health food: joint health
Colebr.- resin
3. CENTELLA SIATICA SELECTED Centella asiatica (L.) ≥30.0% ≤35.0% of selected triterpenes by Health food and personal care:
TRITERPENES PHYTOSOMES® Urbanleaf HPLC Anti-wrinkles, collagen restructurant
4. CURCUVET® Curcuma longa L.rhizome ≥18.0% ≤22.0% of curcuminoids by HPLC Health food
5. GINKGOSELECT® PHYTOSOME® Ginkgo biloba L.leaf ≥7.0% of ginkgoflavonglucosides, ≥2.0% of Health food: cognition and circulation
ginkgoterpenes, ≥ 0.8% of bilobalide, ≥0.8% improver, antioxidant, vasokinetic
of ginkgolides by HPLC≤5 ppm of total Personal care: antioxidant, vasokinetic
ginkgolic acids by HPLC
6. GINKGO BILOBA DIMERIC Ginkgo biloba L.leaf ≥10.0% of total biflavones expressed as Personal care: lipolytic, vasokinetic,
FLAVONOIDS PHYTOSOME® ginkgetin by HPLC phosphodiesterase inhibitor
7. GINKGO BILOBA TERPENES Ginkgo biloba L.leaf ≥30.0% of total ginkgoterpenes ≥ 10.0% of Personal care: Soothing, lenitive
PHYTOSOME® bilobalide ≥ 10.0% of ginkgolides A, B, C, J
by HPLC
8. GINSENG PHYTOSOME® Panax ginseng ≥30.0% ≤40.0% of ginseng typical Health food:
C.A. Meyerroot constituents by gravimetry Adaptogen, tonic
Personal care: Skin elasticity improver
9. GREENSELECT ® PHYTOSOME® Camellia sinensis (L.) ≥19.0% ≤25.0% of polyphenols expressed as Health food: Antioxidant activity,
O. Kuntze – Young leaf (−)-epigallocatechin-3-O-gallate, ≥ 13.0% of weight management
(−)-epigallocatechin-3-O-gallate,≤0.1% of Personal care: Antioxidant activity,
caffeine by HPLC whitening agent
10. 18β-GLYCYRRHETINIC ACID Glycyrrhiza glabra L. root ≥27.0% ≤31.0% of 18β-glycyrrhetinic acid Personal care: Soothing, lenitive
PHYTOSOME® by HPLC
S.No. Phytosome name Plant source Assay Indication
11. HAWTHORN PHYTOSOME® Crataegus spp. flowering ≥3.0% of vitexin-2”-O-rhamnoside by Health food: Cardiovascular health,
top HPLC ≥ 28% ≤34% of hawthorn typical antioxidant
constituents Personal care: Antioxidant
12. LEUCOSELECT® PHYTOSOME® Vitis vinifera L. seed ≥25% ≤30% of proanthocyanidins by GPC Health food: Cardiovascular protector,
UV protector, antioxidant
Personal care: UV protectant,
antioxidant
13. MERVIA® Curcuma longa L.rhizome ≥18.0% ≤22.0% of curcuminoids by HPLC Health food:
Joint health
Personal care: Soothing
14. PROANTHOCYANIDIN A2 Aesculus hippocastanum ≥31.0% ≤37.0% of proanthocyanidin A2by Personal care:
PHYTOSOME ® L.bark HPLC UV protectant, Trophodermic, firming
and oval reshaping agent
15. QUERCEVITA™ Sophora japonica (L.) Total quercetine content 17–22% by HPLC Personal care: Soothing, lenitive, first
Schott ex Endl. aid for skin challenges
16. REXATROL® Polygonum cuspidatum ≥30% of resveratrol by HPLC Health food: Antioxidant, anti-aging,
Sieb. e Zucc.Rhizome Sirt1 modulator
17. SILIPHOS® Silybum marianum (L.) ≥29.7% ≤36.3% of silybin by HPLC Personal care:
Gaertn.Fruit antiwrinkles and retinoic acid-like
activity
18. SILYMARIN PHYTOSOME® Silybum marianum (L.) ≥15.0% ≤20.0% of silybin like substances Health food:
Gaertn.Fruit calculated as silybin by HPLC healthy liver
Personal care: antioxidant, UV
protectant
19. VISNADEX® Ammi visnaga (L.) Lam. ≥10.0% ≤13.0% of visnadin by TLC Personal care: vasokinetic
Umbel without fruits
34 H. Ahmad et al.
used to augment the antioxidant, anti-inflammatory, and cytotoxic properties of

plant-based bioactives. They have also been known to be used in cardiovascular,
hepatoprotective, nootropic, and several other interventions (Semalty et al. 2010).
Some examples presented here would make an interesting read.
Antioxidant and Anti-inflammatory Effects

Hequn Ma et al. (2014a) reported that the oral bioavailability of mangiferin, a potent
antioxidant found in the leaves and stem bark of mango, could be enhanced by 2.3
times when formulated as phytosomes (Hequn Ma et al. 2014a). Furthermore in a
recent study undertaken by Khurana et al. (2017), it was found that bioavailability
could further be improved when mangiferin phytosomes were incorporated into
nanostructured lipid carriers, thus presenting a new carrier system to enhance the
therapeutic efficacy of mangiferin (Khurana et al. 2017). Similarly a delivery sys-
tem based on a solid dispersion containing berberine phytosomes along with d-α-
tocopheryl polyethylene glycol 1000 succinate (TPGS 1000) and SiO2 was reported
to augment the bioavailability and therapeutic benefits of berberine, an isoquinoline
alkaloid (Zhenhai Zhang et al. 2014). In another study phytosomes of baicalin (a
potent antioxidant flavonoid found in rhizomes of Scutellaria baicalensis Georgi)
were incorporated into self emulsifying microemulsion to improve its aqueous solu-
bility and impending biological effects (Wu et al. 2014). Chitosan nanoparticles
modified phospholipids has also been investigated as a carrier for resveratrol, a
potent antioxidant (Jeong et al. 2016). Phytosomes have also been investigated for
role in compounding the anti-inflammatory effects of the bioactive agents. It has
been validated that better anti-inflammatory activity has been exhibited by plant
actives or extracts complexed as phytosomes over their uncomplexed forms. Studies
demonstrated about twofold superior effects of glycyrrhetinic acid and silymarin in
croton oil-induced dermatitis model of inflammation over their free forms (Semalty
et al. 2010). Carbomer-based hydrogels containing 18β-glycyrrhetinic acid phyto-
somes (1%) have also been reported to have improved biopharmaceutical properties
for topical applications owing to the anti-inflammatory properties of
18β-glycyrrhetinic acid complexed as phytosomes (Djekic et al. 2016).
Cardiovascular Applications
Phytosomes loaded with Ginkgo biloba and grape seed extracts were found to exert
greater potency compared to their uncomplexed forms. Ginkgoselect ®
PHYTOSOME® was validated as a cardiotonic, and its efficacy in treating periph-
eral vascular disease (like Raynaud’s disease and intermittent claudication) was
about be 30–60% higher than Ginkgoselect®. Another phytosome intervention,
Leucoselect® PHYTOSOME®, decreased the susceptibility of low-density lipopro-
teins towards oxidation and subsequent oxidative stress in heavy smokers (Semalty
et al. 2010).
Hepatoprotective Activity
A 1:1 phytosome complex of curcumin with soy lecithin was found to provide liver
protection by restoring enzymatic levels of major liver enzymes in a carbon tetra-
chloride rat model. These complexes exhibited improved bioavailability and better
antioxidant activity and provided greater liver protection compared to free curcumin
at same doses (Maiti et al. 2007). In another study, silybin (known to have hepato-
protective properties) phytosomes (Siliphos™) were incorporated into liposomes
by extrusion methods. These phyto-liposomes were observed to be internalized in
human hepatoma cells by fluorescence microscopy indicating their promising appli-
cation in treatment of acute or chronic liver diseases (Angelico et al. 2014).
Protective effects of silybin-phytosome along with curcumin and alpha-R-lipoic
acid were validated against thioacetamide-induced liver cirrhosis in rats showing
the antioxidant and antifibrotic potential of these agents (Shimaa Omar Ali et al.
2014). An interesting study revealed that rosmarinic acid, polyphenol found in
Mentha species, showed improved bioavailability and protected against hepatic
damage when formulated as phytophospholipid complexes. The bioefficacy was
even better in an oil solution containing the rosmarinic acid phospholipid com-
plexes (Jun-Hui Yang et al. 2015). A standardized pomegranate extract containing
punicalagins (30% w/w) when formulated as phytosomes showed increased bio-
availability of punicalagins, compared to that from the extract. These phytosomes
showed improved antioxidant activity and hepatoprotective activity in carbon tetra-
chloride-induced liver damage in rats (Vora et al. 2015). Phospholipid complexes of
apigenin improved its solubility and bioavailability. Significant restoration of liver
function markers were afforded with these apigenin phytosomes as observed in car-
bon tetrachloride rat model. They could profoundly augment the glutathione, super-
oxide dismutase, and catalase levels while reduced the lipid peroxidase levels
(Telange et al. 2016). Phytosomal quercetin was found to show significant protec-
tion to rat liver against carbon tetrachloride-induced hepatotoxicity compared to
free quercetin at equivalent dose (Kexia Zhang et al. 2016b). Phospholipid com-
plexation of silymarin, a well-documented hepatoprotective agent, obtained from
Silybum marianum also known as milk thistle has shown to improve its biopharma-
ceutical properties resulting in higher bioavailability (Maryana et al. 2016).
Wound Healing and Cytotoxicity

Several natural agents with chemopreventive potential like curcumin, silybin, and
silymarin have been investigated as phytosome strategies to inhibit carcinogenicity.
It was validated that 13 g daily dose oral administration of silybin-PHYTOSOME®
was well tolerated in patients with advanced prostate cancer, and this dose was rec-
ommended for the phase II studies. Another silybin phytosome formulation
(IdB1016) was found to demonstrate antitumor effects and potentiation of the thera-
peutic effects of cisplatin. Significant tumor inhibition was observed upon its
repeated administration in mice bearing human ovarian cancer xenografts (Semalty
et al. 2010). Tamoxifen, a known anticancer agent, was formulated as phytosomes
and found to exhibit improved solubility, bioavailability, and efficacy (Jena et al. 2014).
36 H. Ahmad et al.
A saponin phospholipid complex was formulated from saponin obtained from

Panax notoginseng. The saponin-phospholipid complex was found to show strong
cytotoxic effect on 7,12-dimethylbenz (a) anthracene-induced mammary carcino-
genesis in rats, compared to the saponin extract (Kim et al. 2016). Hou et al. (2012)
reported that Mitomycin-C-loaded phytosomes displayed marked cytotoxicity in
H22 cells in vitro and exhibited a dose dependent superior anti-proliferative effect
in vivo compared to free Mitomycin-C (Hou et al. 2012). In a recent study sinigrin
(a glucosinolate) found Brassicaceous plants was formulated as phytosomes and
investigated for its wound healing and cytotoxic effects in A-375 and HaCaT cells.
Singrin phytosomes exhibited pronounced wound healing (complete wound clo-
sure) compared to free sinigrin (71% wound closure) after 42 hours. These phyto-
somes showed minimal toxicity towards HaCaT cells but potent effects towards
A-375 cells. This strategy could be explored as an intervention in cancer wound
healing (Mazumder et al. 2016).
Miscellaneous
Quercetin phospholipid complexes were investigated for treatment of senile macu-
lar degeneration. At 200 μM, these phytosomes were found to markedly activate
Nrf2 nuclear translocation and significantly increase target gene expression (HO-1,
NQO-1, and GCL) by different folds at both mRNA and protein levels. Quercetin
formulated as phytosomes demonstrated greater protection against oxidative-
induced damages in ARPE-19 cells (Xin-Rong Xu et al. 2016). Quercetin phyto-
somes have also been investigated for their estrogenic activity at oral doses of 10
and 50 mg/kg/day for 4 weeks in ovariectomized rat model. Findings revealed supe-
rior effects of quercetin phytosomes over free quercetin in increasing serum cal-
cium, inorganic phosphorus, and glutathione content while reducing serum alkaline
phosphatase, acid phosphatase, malondialdehyde level, tumor necrosis factor-alpha,
and glucose level and improving the lipid profile. Thus it could be used as a success-
ful intervention in hormone replacement therapy (El-Fattah et al. 2017).
1.8 Differences Between Liposomes and Phytosomes
Both liposomes and phytosomes are formed by mixing water-soluble actives with
PC in defined ratios under specific conditions. Both of them are spherical particles
but with different guest localization. A liposome encapsulates a fraction of solvent
into its interior and can have one or more than one concentric layers, while a phyto-
some shows hydrophobic interactions between the polar head of the phospholipids
and the polar bioactive. In a liposome several hundreds of PC molecules surround
and entrap the water soluble compound without any bond formation, whereas phy-
tosomes are 1:1 or 1:2 molecular complexes of PC and the natural ingredient involv-
ing chemical bonds. Phytosomes can be smaller in size than liposomes and exhibit
better absorption and bioavailability over them. Phytosomes have shown superior
applications over liposomes in topical and skin care products.
In a liposome the hydrophilic head of phospholipids align themselves towards

the aqueous compartment, and the hydrophobic tails away from it points towards
the central core and forms a bilayer. Consequently the polar substances are entrapped
in the aqueous compartment while the non-polar substances aggregate in the lipid
section allowing both hydrophilic and lipophilic drugs to be encapsulated. Whereas
in case of phytosomes, the bioactives are anchored to polar heads of PC by hydro-
gen bonding. A cross-sectional representation shows the differences between phyto-
somes and liposomes (Fig. 1.4) also see Table 1.5 (Saraf 2010; Jing Li et al. 2015).
Fig. 1.4 Structural differences between (a) phytosome and (b) liposome
Table 1.5 Differences between liposome and phytosome

Liposome Phytosome
Larger in size Relatively smaller in size
No chemical bonds involved Involvement of chemical bonds
Content of PC much higher (5X) Content of PC relatively less than liposome
Several PC molecules surround a water- PC: Natural ingredient, 1:1 or 1:2
soluble substance
Active principle dissolved in medium or Active principle anchored through the chemical
surrounded by a membrane bond to the polar head of phospholipids
Ingredient present in central part of cavity Solid dispersion of extract/plant bioactive in
with limited chances of molecular phospholipid matrix where the ingredient can be
interaction between surrounding lipid and compared to the integral part of lipid membrane
hydrophilic substances
Bioavailability and absorption Better bioavailability and absorption
comparatively lesser
38 H. Ahmad et al.
1.9 Advantages
Liposomes by virtue of their unique properties can enhance the solubility and bio-
availability of the encapsulated substance, improve the intracellular uptake, and
modify the pharmacokinetics and biodistribution of the substance to give optimum
therapeutic effects. They can improve product performance by improving its in vitro
and in vivo stability, tissue compatibility, and minimizing toxicity. Liposomes have
the advantages of controlled release properties, targeting efficiency and cell affinity.
New-generation liposomes like cationic liposomes, stimuli-responsive liposomes,
stealth (long-circulating) liposomes, and ligand-targeted liposome have further
improved the utility for a wider spectrum of applications. Liposomes have gained
considerable attention in the recent past for incorporating a wide variety of drugs,
vaccines, vitamins, enzymes, gene therapeutics, and herbs and natural products for
applications in immunological, antitumor, and anti-infective therapies.
Phytosomes on account of their amphiphilicity provide the advantages of
improved dissolution in GI fluids and enhanced absorption across lipophilic mem-
branes. They can tremendously augment the bioavailability of drugs with poor
aqueous solubility or low lipid solubility, thus making it a suitable formulation strat-
egy for both kinds of drugs. Phytosomes also enhance the duration of action of a
therapeutic agent and improve its stability. Phytosome strategy has been used popu-
larly for successful delivery of plant bioactives. Several standardized extracts like
Gingko, milk thistle, and Vitis have been formulated as phytosomes (Saraf 2010;
Jing Li et al. 2015).
1.10 CNS-Based Therapies: Challenges and Interventions
1.10.1 Drug Delivery to the Brain

General Concepts
A global increase in longevity in general has resulted in an increased social and

economical burden due to many diseases and CNS conditions like Parkinson’s dis-
ease (PD), Alzheimer’s disease (AD), and stroke (Saraiva et al. 2016). A report by
the World Health Organization suggests that around 1.5 billion people globally suf-
fer from neurological diseases (Vieira and Gamarra 2016). Achieving an effective
therapy for these diseases remains the biggest challenge and currently available
clinical treatments are largely symptomatic and only offer limited outcome improve-
ments, unable to improve the quality of life for survivors and to ease rehabilitation
(Huile Gao 2016; Saraiva et al. 2016). Besides stroke, AD, and PD, brain tumors are
other disorders with significant consequences. Patients diagnosed with glioblas-
toma have only a median survival life span of 14 months post surgical resection,
radiation, and chemotherapy. The ineffectiveness in achieving therapeutic outcomes
for these diseases greatly stems from the inability to administer therapeutically rel-
evant doses of pharmacological agents to diseased areas mainly due to the restricted
assess of these drug molecules across the blood–brain barrier (BBB). Almost all of
the macromolecular therapeutics and about 98% of the small-molecule substances
are unable to gain entry into the brain. Biopharmaceuticals including DNA, SiRNA,
peptide drugs, and antibodies have very low BBB permeability. Besides nutrients,
only small lipophilic molecules (<500 Da) are known to cross the BBB to reach an
efficacious concentration in the brain. Anxiety, depression, and epilepsy are other
conditions that require efficient disposition of therapeutic agents to the brain tissues
for their clinical management but again only limited success has been achieved due
to restrictions imposed by BBB (Kozlovskaya and Stepensky 2013; Huile Gao
2016; Fan Zhang et al. 2016a). Besides the BBB, the blood cerebrospinal barrier,
the blood spinal cord barrier, and the blood retinal barrier are other barriers with
varying degrees of permeability developed by the CNS in order to protect itself
from circulating blood cells, invading pathogens, and other neurotoxic molecules.
However the BBB remains the most exclusive and extensive barrier among all and
assumes the greatest significance for disposition of biologically active substances
against brain diseases. The BBB is altered in some brain pathologies like stroke,
certain brain infections, and neurodegenerative disorders resulting in its increased
permeability allowing the entry of molecules which induce inflammatory responses
and neuronal damage (Saraiva et al. 2016).
The last two decades have seen the emergence of nanotechnology as a tool for
brain drug delivery. Delivery systems in this area have been reported not only in
preclinical studies but are also now being investigated in clinical settings. Vesicle-
based nanoparticles and conjugate based systems are popular formulation choices
along with incorporation of special reagents to achieve increased permeability
across BBB and to gain enhanced brain accumulation. Different strategies like
PEGylation, stimuli sensitivity, and targeting have been used with nanomedicine in
order to achieve increased efficacy. Surface modification by coating with certain
agents or with specific targeting moieties has been used with nanoparticles like
dendrimers, micelles, liposomes, and polymer and inorganic nanoparticles in order
to provide better therapeutic outcomes. Directing diagnostic and pharmacological
agents via nanocarriers has shown evident benefits like improved solubility, phar-
macokinetics, and biodistribution compared to free drug along with protection
against digestion by enzymes, enhanced targeting, and cellular internalization BBB
(Kozlovskaya and Stepensky 2013; Huile Gao 2016; Fan Zhang et al. 2016a).
40 H. Ahmad et al.
Physical Barriers in Brain Targeting
Blood–Brain Barrier
Ehrlich is credited to have discovered the BBB way back in 1885 when he observed
that a dye injected i.v. could stain all organs excluding brain. The BBB is composed
of brain capillary endothelial cells (BCEC) secured by continuous tight junctions
and adherens junctions preventing paracellular transport of molecules from blood to
brain. Besides the BCEC, pericytes, astrocytes, and neuronal cells are other cells
that make up the BBB. The passive diffusion of compounds between the blood and
brain is largely restricted due to a high transendothelial electrical resistance exhib-
ited by the tight junctions. There are certain carriers and transporters that can medi-
ate the uptake of substances to the brain and their extrusion from the brain (Huile
Gao 2016; Saraiva et al. 2016).
Blood–Brain Tumor Barrier (BBTB)
It has been observed that in brain tumors of advanced stages, the BBB is compro-
mised in the core but remains intact in the areas of the brain surrounding the tumor
core. The BBTB showed increased expression of drug efflux pumps (multidrug
resistance associated proteins, breast cancer resistance protein, and P-glycoprotein)
along with a smaller pore size as compared to the blood tumor barriers in the periph-
eral tumors.
Nose to Brain Tumor
The delivery route to the brain via nasal cavity has been well documented. The
respiratory and olfactory regions are the two regions of the nasal cavity responsible
for absorption of substances into blood or brain. The olfactory mucosa pathway
provides the most rapid route for brain drug delivery through the nasal cavity.
However a very small volume of about 25–200 μL can be administered intranasally,
which limits the dose and concentration of the drugs reaching the brain. The absorp-
tion time of a drug is also reduced due to cilial clearance in the nasal cavity, while
drug metabolism and secretion are other factors which further restrict transfer of
drugs into the brain (Huile Gao 2016).
Alteration of BBB in Brain Diseases
The BBB maintains brain homeostasis and protects it from neurotoxic molecules.
However in certain CNS pathologies and neurodegenerative disorders, its structural
integrity is compromised which leads to neuro-inflammation and alteration in its
permeability. This alteration in permeability causes disruption of the tight junctions
which secure the BCEC allowing additional substances and circulating macro-
phages to cross the BBB and infiltrate the CNS. These permeability changes can be
bought about by bacterial lipopolysaccharides or by specific events like traumatic

brain injury and stroke or tumors. In case of chronic neurodegenerative conditions
like the AD or PD, the alteration in BBB permeability can result from reduced
P-glycoprotein expression levels that diminish the capabilities of efflux pumps to
transport back the neurotoxic substances from brain to blood. These modifications
can be used as useful platforms for designing efficient therapeutic strategies for the
management of brain and related disorders (Saraiva et al. 2016; Fan Zhang
et al. 2016a).
Stroke is one of the most debilitating conditions occurring in adulthood and is
caused by occlusion of a blood vessel (ischemic stroke) or due to rupture of a blood
vessel (hemorrhagic stroke) each leading to deprivation of blood supply to the brain.
During the events that occur in stroke, there is BBB opening for a short time (few
minutes to hours) followed by a refractory period before reopening for an extended
time period (hours to days). Although reperfusion is important to control and limit
the injury, it often leads to exacerbation of damage which is termed as reperfusion
injury. The major dysfunction in the BBB during an episode of stroke is the loss and
disruption of the tight junctions. These modalities can be of significance in design-
ing therapeutics for stroke. The leaky BBB vasculature or expression of some recep-
tors on the luminal side of BCEC increases the probability of nanoparticles to cross
the BBB and thus present a promising target for delivering therapeutics to the isch-
emic brain (Ahmad et al. 2017; Saraiva et al. 2016).
AD is a leading cause of dementia and affects more than 35 million people glob-
ally. Impairment of cognitive functions is the major feature observed in AD and
accumulation of beta-amyloid (Aβ) peptide (a neurotoxic 40–42 amino acids pep-
tide) presenting as Aβ plaques or senile plaques is the major pathological hallmark
of the disease, along with brain atrophy, presence of tau filaments, and subsequent
cerebrovascular changes culminating in cerebral amyloid angiopathy (Bana et al.
2014). The dysfunction of BBB in AD has long been a topic of controversy among
researchers, and different hypothesis regarding the same are available. One hypoth-
esis suggests that the reduced clearance of Aβ compromises the BBB and also
causes disruption of tight junctions. Reduced activity of efflux transporters of
BCEC, present on the apical side, might also lead to Aβ accumulation in the brain
parenchyma. Another theory suggests that BBB dysfunction in AD occurs before
accumulation of Aβ and is the cause of neurodegeneration (Saraiva et al. 2016).
PD is neurodegenerative disease characterized by tremor, rigidity, bradykinesia,
and postural instability affecting around ten million people worldwide. It involves
selective degeneration of dopaminergic neurons in the substantia nigra resulting in
dopamine depletion in the striatum. PD also shows existence of α-synuclein and
protein inclusions in neurons termed as Lewy bodies (Javed et al. 2016). An initial
understanding believed that development of PD is independent of BBB dysfunction.
However recent studies have demonstrated that a progressive BBB impairment
could be observed in PD pathologies. It was observed that higher levels of drugs like
verapamil and benserazide were found in brains of PD patients which are unlikely
to cross the BBB in normal situations. Additionally the striatum and substantia nigra
of PD patients exhibited the vascular alterations and cerebral blood flow
42 H. Ahmad et al.
deficiencies that usually result loss of integrity in the BBB, thus validating BBB
dysfunction in PD pathologies. Higher levels of lactoferrin receptors have been
found in the substantia nigra dopaminergic neurons of PD patients, and their upreg-
ulation on the blood–brain vasculature has been implicated in BBB changes. This
modality could be taken advantage of in designing nanoparticles that improve the
drug uptake into the brain and also as a therapeutic agent itself to retard the progres-
sion of PD (Saraiva et al. 2016).
Strategies Useful for Drug Delivery to the Brain
Only about 3–5% of brain-directed bioactives have been able to make it to the mar-
ket despite the huge costs involved in the discovery and design of new entities to
treat brain diseases. Most of these therapeutic agents fail to cross the BBB in vivo.
Therefore strategies and different routes to administer a therapy that results in effi-
cient brain drug delivery to treat various diseases of the brain assume significance.
An overview of various strategies useful in drug delivery to the brain has been
shown in Fig. 1.5. The approaches used for brain drug delivery can be invasive,
pharmacological, or physiological in nature. Off late nanomedicine has generated
an interface for several strategies that facilitate drug transport across the
Fig. 1.5 Strategies useful for drug delivery to the brain

BBB. Nanoparticles, lipid-based vehicle, polymer-based vehicles, and carbon nano-

structures have been widely investigated for their role in drug delivery to the brain
(Soni et al. 2016; Saraiva et al. 2016). The invasive approach involves penetrating
the BBB by directly injecting the drug into the brain via craniotomy for intracere-
broventricular (ICV) infusion and intracerebral drug administration. However this
technique presents many serious reservations against it: one that it transports the
drug into the peripheral blood stream which results in less drug to the targeted tissue
and two that the limited drug distribution achieved through this method results in
loss of the intended therapeutic action due to an enormous intracranial pressure
encountered upon direct drug administration. The pharmacological approach on the
other hand allows free passive movement of drug molecules across the BBB based
on their small molecular size, lipophilic character, and low hydrogen bonding
capacity. Physiological approach is based on receptor-mediated and carrier-medi-
ated drug delivery and can be considered the most advanced approach among the
different approaches used in brain drug delivery. Various receptors like transferrin
and insulin are found in plenty on the BBB and drugs associated to ligands of these
receptors enable their transport to the targeted area. Mimicking the endogenous car-
rier substrate is a requirement for a drug to qualify for transporter-mediated deliv-
ery; however in this case the binding ability and kinetics of the transporter molecule
might limit the drug delivery via physiological approach. Besides, these approaches,
drug delivery in CNS disorders can be achieved by nanotechnological advances as
it allows for restoration of the cytoarchitecture and connection pattern in these dis-
orders (Huile Gao 2016; Soni et al. 2016). Antibodies, peptides and proteins can be
physically or chemically immobilized onto the nanoparticles surface to achieve tar-
geting. They can be modulated in terms of their size, shape, chemistry, hydropho-
bicity, coating, and surface charge to improve their release properties, enhance their
penetration into the BBB, increase their circulation stability, and allow reticuloen-
dothelial escape. These nanoparticles can follow any of the following mechanisms
of transport across the BBB depending upon their physicochemical properties.
(i) They open the tight junctions between endothelial cells or induce local toxic
effect that facilitates a localized permeabilization of the BBB to allow the pen-
etration of the drug either in free form or conjugated with the nanoparticles.
(ii) Pass through endothelial cells via transcytosis.
(iii) First transported through endothelial cells via endocytosis upon which they
release their content into the cell cytoplasm and then exocytosed in the endo-
thelium abluminal side.
(iv) A combination of any of these mechanisms (Saraiva et al. 2016).
44 H. Ahmad et al.
Areas of Concern in Brain-Targeted Delivery Systems
1. The BBB protects the CNS from xenobiotics and toxic substances; delivery sys-
tems on the other hand enable the drug to gain entry across the BBB; however it
also imposes a risk of the potential neurotoxicity caused by nonselective expo-
sure to entire brain leading to untoward side effects.
2. The drug delivery systems designed for brain delivery have developed several
advances over the years in targeting efficiency. However these delivery systems
loaded with drugs also release and distribute the drug to normal tissues, besides
the brain which can lead to adverse effects and also reduce the quantum of drug
available at the target site. Therefore, care must be taken to design dual targeting
systems that can keep the drug bound during delivery and enable rapid release in
the target brain tissues (Banks 2016; Huile Gao 2016).
3. The delivery systems formulated for brain drug delivery should be homogenous
in their targeting efficiency and the lack of this would lead to a less pronounced
therapeutic response. Changes in particle size, surface, and ligand properties can
greatly influence the targeting efficiency of a delivery system. Particle size
affects the clearance, elimination, and BBB permeability.
4. As soon as a nanoparticulate drug carrier is introduced into the biological fluids,
a protein corona formed of serum proteins develops that not only alters the
nanoparticles distribution but also tends to cover the ligand thus imposing restric-
tions over the interaction of the ligand and its target. The influence of this corona
must be evaluated in brain-targeted drug delivery systems. It is unfortunate that
in the current scenario, not many studies have evaluated that (Zhang et al. 2016;
Huile Gao 2016).
5. The off-target effects of brain-directed therapies are another area of concern. The
nanocarriers surface modified with ligands are designed so that these ligands can
interact with specific receptors and transporters expressed on the BBB or dis-
eased cells in the brain. This interaction can be attenuated due to the protein
corona; additionally the presence of ligand-specific receptors and transporters in
other tissues besides the brain could lead to off-target effects. The target cells can
be labeled with exogenous ligand to overcome such off-target effects (Huile Gao
2016; Dong 2018).
1.10.2 Liposomal Interventions Implied in CNS Therapies
Among the different novel carrier systems, liposomes have gained tremendous
attention as promising strategies for brain-targeted therapies. Their most advanta-
geous features include their ability to incorporate and deliver large amounts of drug
and the possibility of surface modification with different ligands (Lai et al. 2013).
Optimizing the most suitable liposome preparation that can cross the BBB can have
significant implications in treating neurological diseases. It has been found that cat-
ionic liposomes have shown advantages over conventional, anionic, or neutral
liposomes for drug delivery to the brain. The probable reason for this could be the
possibility of electrostatic interactions between the positive charge carrying lipo-
somes and the negative charge bearing cellular membranes thereby resulting in an
enhanced uptake via adsorptive-mediated endocytosis. However the major demerit
encountered with the use of cationic liposomes is their nonspecific uptake by periph-
eral tissues and their binding to serum proteins that weakens their surface charge.
Due to these factors, a larger number of these carriers would be required to elicit the
desired therapeutic response which in turn would increase the risk of cytotoxicity.
PEGylated systems and theranostic liposomes have also been successfully used for
brain drug delivery (Vieira and Gamarra 2016). Antibody-conjugated liposomes or
immunoliposomes have also been widely investigated with respect to drug delivery
to the brain. These sterically stabilized liposomes can be modulated in terms of their
organ and tissue distribution by conjugation with an appropriate targeting vector
like modified proteins or antibodies that undergo absorptive-mediated or receptor-
mediated transcytosis through the BBB. Cationized albumin, the OX26 monoclonal
antibody to the rat transferrin receptor, or monoclonal antibodies to the insulin
receptor are some of the examples of vectors used for brain targeting (Schnyder and
Huwyler 2005). Transferrin receptor targeted liposomal interventions have by far
been the most relevant strategies for brain targeting in various diseases as these
receptors are expressed on the BCEC but not on other endothelial cells elsewhere in
the body (Johnsen and Moos 2016). PEG-stabilized liposomes carrying polyethyl-
enimine polyplex of oligodeoxynucleotides remained in circulation for a longer
time and were stable in presence of serum proteins and when targeted with antibody
specific to transferrin receptor displayed significantly greater brain accumulation
(Ko et al. 2009). Bi-ligand liposomal vectors have also shown obvious advantages
over single ligand vectors for gene delivery to brain. In a study the transferrin-poly-
L-arginine bi-ligand liposomes showed greater accumulation in the rat brain com-
pared to single ligand (transferrin) liposomes or liposomes without ligand
attachments. These bi-ligand liposomes also afforded increased expression of
β-galactosidase plasmid in rat brain tissue (Gitanjali Sharma et al. 2013). van Rooy
et al. (2011) investigated the targeting ability of five different ligands, namely,
RI7217, COG133, CRM197, transferrin, and angiopep-2 to target liposomes to the
brain. Among all of them, only CRM197-modified liposomes demonstrated binding
to murine endothelial cells in in vitro conditions, and only the RI7217-modified
liposomes could significantly enhance brain uptake in vivo. It was inferred from this
study that all targeting ligands that find mention for brain targeting do not function
well when coupled to a liposomal carrier in vivo (van Rooy et al. 2011). Investigations
aimed at studying transport of liposomes into the brain have suggested that phago-
cytic cells like the neutrophils and monocytes might be involved in transport of
drugs loaded into liposomes into the brain. In a study serotonin loaded negatively
charged liposomes showed higher brain uptake in vitro and in vivo in rats and rab-
bits and that monocytes were the major cells that enabled transport of intact lipo-
somes into the brain (Afergan et al. 2008). Carrier-mediated transport is yet another
means of liposomal transport across the BBB. Glucose transporter 1 (GLUT1)
expressed on BCEC is considered one of the most efficient carrier-mediated
46 H. Ahmad et al.
Table 1.6 Liposomal preparations for brain-targeted drug delivery currently in market or in
clinical trials
Indication Product details References
Cryptococcal AmBisome and Abelcet® containing Robinson and Nahata (1999)
meningitis amphotericin B and Loyse et al. (2013)
Glioblastoma Doxil® and Myocet® containing doxorubicin Chua et al. (2004), Hau et al.
multiforme (both currently in phase II) (2004), Beier et al. (2009),
Ananda et al. (2011) and Di
Legge et al. (2011)
Lymphomatous Depocyt® containing cytarabine Benesch and Urban (2008)
meningitis
Pediatric brain DaunoXome® containing daunorubicin Lippens (1999), Marina et al.
tumors (currently in phase I) and Caelyx® containing (2002), Wagner et al. (2008)
doxorubicin (currently in phase II)
transporters for drug delivery to brain. It has been reported that specifically designed
glucosyl liposome ligands can cross BBB via GLUT1 with high transfer efficiency,
show enhanced brain targeting and good cycling stability in vivo and our easy to
prepare (Boyi Qu et al. 2014). In a study it was observed that liposomes conjugated
with P-aminophenyl-α-D-mannopyranoside (a mannose analog) onto its surface
showed greater brain uptake by cells over expressing GLUT1 and GLUT3. It was
found that these liposomes could gain entry into the brain by transcytosis of GLUT1
and GLUT3 as a pathway and were concentrated in the cerebellum and cerebral
cortex to a greater extent. This distribution could be attributed to the non-homoge-
neous brain distribution of GLUT1 and GLUT3 (Du et al. 2014). Several liposome-
based products are being currently evaluated under clinical trials, and some have
made to the market for various neurological disorders. A brief description of such
liposomal preparations has been given in Table 1.6. Liposomes have been investi-
gated for treatment of AD, PD, stroke, glioma, epilepsy, multiple sclerosis, depres-
sion, and other CNS pathologies. Some recent interventions in these areas have
been discussed in Table 1.7.
1.10.3 P
hytosome Strategies for Disorders
of the Brain and CNS
Recent times have observed an increasing burden of illness due to neurodegenera-

tive diseases owing to an increased geriatric population statistic; however lack of
cure or non-availability of disease modifying treatments for many of these diseases
requires screening of effective therapies to be urgently addressed. It has been known
that various traditional systems of medicines like the Ayurvedic system, traditional
Table 1.7 Liposomal preparations investigated for CNS pathologies
Therapeutic agent Experimental details Purported CNS action Reference
Alzheimer’s disease
α-Mangostin Liposomes composed of DSPE-PEG2,000 with transferring Targeted liposomes could penetrate the Zhi-Lan Chen
as ligand injected i.v. in rats and evaluated at 0.5, 2, 4, 8, BBB to deliver α-mangostin in rat brain et al. (2016b)
18, and 24 hours
H102 Liposomes composed of EPC, DSPE-PEG2,000, and Could ameliorate spatial memory Zheng et al.
cholesterol administered intranasally to rats impairment in rats (2015)
111
In-VHH-pa2H antibody Liposomes composed of DMPC, cholesterol, and Specific brain delivery of single-domain Rotman et al.
DSPE-PEG2,000 EYPC, cholesterol, and DSPE-PEG2,000 antibody fragments beyond the BBB (2015)
with glutathione as ligand administered intravenously to
mice
14
C and 3H Liposomes composed of N-palmitoyl-d-erythro- Bifunctionalized liposomes could affect Bana et al. (2014)
sphingosylphosphorylcholine and cholesterol using brain Aβ-oligomer aggregation/
mApoE and phosphatidic acid administered i.v. in mice disaggregation in vivo
Lipid-PEG-Curcumin Liposomes evaluated on post-mortem brains samples of Both multifunctional liposomes showed Mourtas et al.
derivative/ curcumin AD patients high affinity for the amyloid deposits and (2014)
derivative with anti-transferrin to could delay Ab1–42 peptide aggregation
antibody
Rivastigmine Intranasal administration of liposomes composed of EPC Targeted liposomes could efficiently Zhen-Zhen Yang
and cholesterol EPC, DSPE-PEG2,000, and cholesterol in deliver rivastigmine to rat brain et al. (2013b)
rats. Cell penetrating peptide used as ligand
Liposomes composed of DPPC and cholesterol Greater inhibition of acetylcholinesterase Mutlu et al.
administered intraperitoneally to mice (2011)
Methoxy-XO4 Intravenous administration of liposomes composed of Liposomes might cross the BBB, since the Tanifum et al.
(4,4′-[(2-methoxy-1,4- DPPC, DSPE-PEG2,000, and cholesterol in mice nanostructures selectively bind to (2012)
phenylene)di-(1E)-2,1- Aβ-plaque deposits to both parenchymal
ethenediyl]bisphenol) plates and cerebral amyloid angiopathy
Galantamine Liposomes composed of phosphatidylglycerol were Promising results in AD treatment based on Weize Li et al.
1 Liposomes vs Phytosomes: Principles, Methodologies, and Therapeutic Applications…
administered intranasally in rats rapid transport of galantamine into brain (2012)

tissues
47
(continued)
Table 1.7 (continued)
48

Quercetin Intranasal administration of liposomes composed of PC, Improved functional outcomes in learning Tong-un et al.
EPC, and cholesterol in rats and memory due to reduced (2010)
acetylcholinesterase activity and lower
levels of oxidative stress
Intranasal administration of liposomes composed of PC, Attenuation in the death of neurons and Phachonpai et al.
EPC, and cholesterol in rats cholinergic neurons in the hippocampus (2010)
Parkinson’s disease
Levodopa/ levodopa prodrugs Intraperitoneal injection of liposomes composed of HSPC, Ligand-targeted liposomes increased the Xiang et al.
DSPE-PEG2,000, and cholesterol with Chlorotoxin as ligand brain drug distribution with significant (2012)
in mice attenuation in serious behavioral disorders
Intraperitoneal injection of liposomes composed of DMPC Improved drug delivery and release for the Di Stefano et al.
and cholesterol in rats brain striatum of anti-PD agents (2004) and Di
Stefano et al.
(2006)
Intraperitoneal injection of liposomes composed of EPC Levodopa-loaded liposomes inhibited Kucherianu et al.
and cholesterol in mice; 7-day treatment akinesia with increased efficiency and (1997)
increased muscle rigidity when compared
to the free drug
Intraperitoneal injection of liposomes composed of EPC Levodopa-loaded liposomes enhanced the Yurasov et al.
and cholesterol in mice; 14-day treatment dopamine levels in the mouse striatum (1997)
Liposomes composed of EPC and cholesterol administered Low-dosed levodopa-loaded liposomes Yurasov et al.
intraperitoneally in mice enhanced dopamine metabolism rate and (1996)
altered the metabolism of signal
phospholipids in the striatum
GDNF/GDNF plasmid Intranasal administration of liposomes composed of Neurotropic action in the brain Migliore et al.
DOPC, cholesterol, and stearylamine in rats for 3–4 weeks (2014)
Intraperitoneal injection of liposomes composed of DPPC, Sustained therapeutic effects are achieved Xia et al. (2008)
DODAB, and DSPE-PEG2,000 in rats; OX26 was the ligand in experimental PD with the formulation
used and treatment given for 8 weeks described here
H. Ahmad et al.
Stroke
ZL006 Liposome conjugated with T7 peptide (T7) and stroke The dual targeted liposomes loaded with Yue Zhao et al.
homing peptide (SHp) ZL006 could transport across BCEC cells (2016a)
and significantly enhanced cellular uptake
and reduced cell apoptosis of excitatory
amino acid stimulated PC-12 cells. These
liposomes were and mostly accumulated in
ischemic region rather than normal cerebral
hemisphere of in MCAO rats
Intravenous administration of liposomes composed of soy Ligand-targeted liposomes for the delivery Zhongyuan Wang
lecithin, DSPE-PEG2,000, and cholesterol; transferring of the ZL006 significantly ameliorated et al. (2015)
peptide used as ligand, 24-hour treatment neurological deficit and reduced infarct
volume induced by ischemia reperfusion
Angiogenic peptides and Liposomes composed of PC, PE, and cholesterol were Liposomes as imaging agents delivered the Hwang et al.
99m
Tc administered to rats via an intra-arterial injection; 7-day angiogenic peptides to the ischemic brain (2015)
treatment hold promise for an effective angiogenic
therapy in stroke
Hb Intravenous administration of liposomes composed of Reduced volume of cerebral infarction by Kaneda et al.
DSPC, DSPE-PEG2,000, and cholesterol in rats; 24-hour liposomal Hb delivery (2014)
treatment
Intravenous administration of liposomes composed of Liposomal Hb provided protection in Kawaguchi et al.
DSPC, DSPE-PEG2,000, and cholesterol in rats; 22-day amygdala SAT in transient whole-brain (2014)
treatment ischemia
Intra-arterial infusion of liposomes composed of DSPC, Reduced injury resulting from decreased Shimbo et al.
DSPE-PEG2,000, and cholesterol in rats; 24-hour treatment effect of MMP9, due to higher production (2014)
of neutrophils
RhB, Gd, and citicoline Liposomes composed of DSPC, DSPE-PEG2,000, and Reduced lesion volumes on treatment with Agulla et al.
cholesterol administered i.v. in rats; anti-HsP72 antibody liposomal citicoline (2014)
49
used as ligand, 7-day treatment

(continued)
50

18F 2-hour treatment with liposomes composed of DSPC, PEGylated liposomes were found to be Fukuta et al.
DSPE-PEG2,000, and cholesterol administered i.v. in rats accumulated in and around the ischemic (2014)
region and provided benefits in reducing
injury
Chrysophanol Liposomes composed of lecithin and cholesterol injected Protection against ischemia-reperfusion Yan et al. (2014)
intraperitoneally in mice, 24-hour treatment injury by reducing either oxidative stress or
apoptosis
Gd and Rhb Liposomes composed of DSPC, DSPE-PEG2,000, DSPE- Direct in vivo MRI-based detection after Deddens et al.
PEG2,000-mal, and cholesterol injected i.v. in mice. stroke was achieved only with ICAM-I- (2013)
Anti-ICAM-1 antibody used as ligand. Treatment regimen: targeted MPIO
6 hours and 1, 3, and 7 days
SOD enzyme Intracarotid injection of liposomes composed of PC, Ligand-targeted liposomes offered Yun et al. (2013)
DSPE-PEG2,000, DSPE-PEG2,000-mal, and cholesterol in protection against I/R injury, levels of
mice. Anti-NRI-receptor antibody used as ligand; 24-hour inflammatory markers decreased, and
treatment improved behavioral outcomes were
observed in vivo
Isopropylidene-shikimic acid Intravenous administration of liposomes composed of Enhanced drug distribution in ischemic Changhai Qu
DSPC and cholesterol in rats; 8-hour treatment. brain and subsequently improved et al. (2013)
therapeutic efficacy
Glioma
Doxorubicin Liposomes modified with tumor-specific pH-responsive Significant antitumor and antiangiogenic Yang Zhao et al.
peptide H7K(R2)2 as a targeting ligand investigated effects could be demonstrated by these (2016b)
in vivo in C6 tumor-bearing mice and in U87-MG pH-sensitive liposomes loaded with
orthotopic tumor-bearing nude mice doxorubicin
Intravenous administration of liposomes composed of PC, Ligand-targeted liposomes could elicit Zong et al. (2014)
DSPE-PEG2,000 DSPE-PEG1,000, and cholesterol in mice; synergistic targeted delivery of doxorubicin
4-hour treatment; TAT-peptide and transferring used as in brain tumors
ligand
Liposomes composed of HSPC, DSPE-PEG2,000, and Enhanced targeted therapeutic effect on Yiyi Yang et al.
H. Ahmad et al.
cholesterol administered to mice by intravenous injection. glioblastomas observed by delivery of (2013a)

Treatment for 55 days; RGERPPR peptide used as ligand doxorubicin from targeted liposomes
Paclitaxel Mice treated with liposomes composed of SPC, DSPE- Selective targeting and enhanced anticancer Liu et al. (2016)
PEG2,000, and cholesterol given i.v.; treatment for >55 days; therapeutic effects could be achieved
cell-penetrating peptides used as ligand
Liposomes composed of SPC, DSPE-PEG2,000, and Effective targeting to cancer stem cells, Shi et al. (2015)
cholesterol administered i.v. in mice. Treatment for more destroying the vasculogenic mimicry
than 60 days; TR peptide used as ligand channels via ligand targeted liposomes
delivering paclitaxel
Liposomes composed of SPC, DSPE-PEG2,000, and Ligand-targeted liposomes for the delivery Liu et al. (2015)
cholesterol administered i.v. in mice. RGD peptide used as of PTX showed an effective targeting
ligand; treatment for 52 days ability for brain-cancer stem cells
7-day treatment in mice via i.v. route by administering Ligand-targeted liposomes for the delivery Li Qin et al.
liposomes composed of SPC, DSPE-PEG2,000, and of PTX were developed and presented high (2014b)
cholesterol; using RGD peptide and transferrin as receptor brain distribution
EPI plus celecoxib Liposomes composed of PC, DSPE-PEG2,000, and Targeted liposomes were able to cross the Rui-Jun Ju et al.
cholesterol administered in mice intravenously; PTD HIV1 BBB and accumulate in the tumor, leading (2016)
peptide used as ligand; treatment for more than 50 days to the destruction of glioma vasculogenic
mimicry channels
Gd-DTPA Liposomes composed of DOPC, DSPE-PEG2,000, and Gd-DTPA-loaded liposomes exhibited a Pacheco-Torres
cholesterol intravenously administered in mice; 60-min pH responsive release of the contrast agent et al. (2015)
treatment
Irinotecan Liposomes composed of DSPC, DSPE-PEG2,000, and Irinotecan-loaded liposomes demonstrated Pin-Yuan Chen
cholesterol administered via vascular and intratumoral good antitumor activity on glioblastomas et al. (2012)
injection by CED; treatment for >70 days. and a higher median survival time of
tumor-bearing mice
asOs siRNA Liposomes composed of DODAP, DSPC, C16 Cer- CTX-enhanced internalization of liposome Costa et al. (2013)
PEG2,000, and cholesterol administered via intravenous carrying nucleic acids into glioma cells
injection in mice; CTX used as ligand
(continued)
51
52
Daunorubicin Liposomes conjugating with p-aminophenyl-α-D- Transport ratio across the BBB model was Ying et al. (2010)
mannopyranoside and transferrin (TF) evaluated in C6 significantly increased, C6 glioma spheroid
glioma-bearing rats in vivo volume ratio was significantly lowered and
the median survival time of tumor
Bearing rats significantly prolonged
compared to free drug
Epilepsy
Resveratrol Liposomes containing resveratrol evaluated on penicillin- Liposomal resveratrol along with Ethemoglu et al.
induced epileptic seizure model in rats resveratrol effectively decreased the spike (2017)
frequency and spike amplitudes than other
treatments
Nimodipine Liposomes containing nimodipine evaluated on Nimodipine-loaded liposomes prevented Isabella Macário
pilocarpine-induced seizures in mice the installation of 100% of the Ferro Cavalcanti
Pilocarpine induced seizures and prevented et al. (2015b)
the death of 100% of the mice treated with
pilocarpine
Curcumin Liposomes containing curcumin evaluated on increasing Liposomal curcumin (25 and 50 mg/kg) Agarwal et al.
current electroshock seizures (ICES) test, significantly increased the seizure (2013)
pentylenetetrazole (PTZ) induced seizures, and status threshold current and latency to myoclonic
epilepticus in mice and generalized seizures in ICES test and
PTZ-induced seizures. It also increased the
latency to the onset and decreased the
seizures duration during status epilepticus
in mice.
H. Ahmad et al.
Amiloride Liposomes loaded with amiloride evaluated on ICES test, Liposomal amiloride produced strong Atif Ali et al.
PTZ-induced seizures and PTZ-induced status epilepticus anticonvulsant effects without inducing (2007)
in mice peripheral toxicity (hyperkalemia) over the
free amiloride
Depression
Trefoil factor 3 (TFF3) Liposomes modified with cyclic RGD(cRGD) peptide that TFF3-loaded cRGD liposomes Jing Qin et al.
has high affinity for integrin receptors of leukocytes demonstrated antidepressant like effects. It (2015)
allowed a persistent release of TFF3 for
12 hours and improved its concentration in
basolateral amygdala regions of the brain
related to depression
Edaravone cRGD liposomes loaded with edaravone evaluated in rats cRGD liposomal edaravone produced Jing Qin et al.
for potential antidepressant-like effects significant antidepressant-like effects in (2014a)
both forced swim and novelty suppressed
feeding test upon a single injection
Anxiety and cognition
Quercetin Male Wistar rats were pretreated with quercetin liposomes Significant improvement in cognitive Tong-un et al.
(20 μg), via right nasal cavity once daily continually for outcomes and densities of survival and (2010)
4 weeks and evaluated for rodent learning and memory by cholinergic neurons could be observed in
Morris water maze test. All rats were sacrificed for the hippocampus.
determining the survival and cholinergic neurons densities
in hippocampus.
(continued)
53
54
Anxiolytic and cognitive-enhancing effects of liposomal Cognition enhancing and anxiolytic effects Priprem et al.
quercetin (oral and intranasal administration at 20 μg/day) were exhibited by both free as well as (2008)
were evaluated in elevated plus maze and Morris water liposomal quercetin; however a lower dose
maze tests and compared to free quercetin and a faster rate were observed with
intranasal liposomes and they were more
effective in delivering quercetin to the CNS
Abbreviations: 14C carbon 14, 18F fluorine-18, 3H hydrogen 3, 99mTc technetium-99 m, AD Alzheimer’s disease, Aβ amyloid beta, BBB blood–brain barrier,
BCEC brain capillary endothelial cells, CED convection-enhanced delivery, Cer ceramide, CNS central nervous system, cRGD cyclic RGD, CTX chlorotoxin,
DMPC dimyristoylphosphatidylcholine, DODAB dioctadecyldimethylammonium bromide, DODAP 1,2-dioleoyl-3-dimethylammonium-propane, DOPC dio-
leoylphosphatidylcholine, DPPC dipalmitoylphosphatidylcholine, DSPC distearoylphosphatidylcholine, DSPE distearoylphosphatidylethanolamine, DTPA
diethylenetriaminepentaacetic acid, EPC egg phosphatidylcholine, EPI epirubicin, EYPC egg-yolk phosphatidylcholine, Gd gadolinium, GDNF glial cell-
derived neurotrophic factor, H102, Hb hemoglobin, HSPC hydrogenated soy phosphatidylcholine, i.v. intravenous, I/R ischemia reperfusion, ICAM-1 intercel-
lular adhesion molecule 1, ICES increasing current electroshock seizures, Mal maleimide, mApoE apolipoprotein E-derived peptide, MCAO middle cerebral
artery occlusion, MMP-9 matrix metalloproteinase-9, MPIO micron-sized iron oxide particles, MRI magnetic resonance imaging, OX26 anti-transferrin recep-
tor antibody, PC phosphatidylcholine, PD Parkinson’s disease, PE phosphatidylethanolamine, PEG polyethylene glycol, PTX paclitaxel, PTZ pentylenetetra-
zole, RhB rhodamine B, SAT Sidman avoidance test, SHp stroke homing peptide, siRNA small interfering RNA, SOD superoxide dismutase enzyme, SPC
sphingosylphosphorylcholine, TF transferrin, TFF3 trefoil factor 3, ZL006 5-(3, 5-dichloro-2-hydroxybenzylamino)-2-hydroxybenzoic acid
H. Ahmad et al.
Chinese medicine, traditional Persian system, or several tribal systems of medicine

have recognized the role of herbs, botanicals, and plant products to be useful in dif-
ferent CNS pathologies like AD, PD, stroke, multiple sclerosis, and schizophrenia
to name a few (Fonseca-Santos et al. 2015; Shojaii et al. 2016; Ghanavati et al.
2016; de Rus Jacquet et al. 2017; Wen-ting Yang et al. 2017b). However to bring
objectivity into these claims effective therapies pertaining to these herbs must be
scientifically evaluated. Artemisia absinthium, Ocimum basilicum, Ginkgo biloba
L, Camellia sinensis, Gastrodia elata, Lavandula officinalis, and oil of O. europaea
are some of the herbs that have been documented to be useful in providing neuro-
protection in stroke (Jivad and Rabiei 2015). Several in vitro and in vivo studies
have demonstrated that standardized extracts of Withania somnifera roots possess
many beneficial neuropharmacological effects (Yenisetti et al. 2015). Resveratrol, a
polyphenol obtained from red wine and found in several plants, has been found to
provide protection in several animal models in AD (Teng Ma et al. 2014b). These
are just a few examples of phyto-constituents and plants that are useful in different
brain and related disorders; enlisting all of them would be beyond the scope of this
chapter. As discussed earlier, solubility and bioavailability of phyto-constituents
and plant extracts are the major limiting factors in achieving effective therapeutic
outcomes and the same holds true in perspective of CNS therapies especially due to
restrictions imposed by the BBB. Phytosomes have emerged as useful carriers of
these polar phyto-constituents and standardized plant extracts for CNS applications.
Phytosomes incorporate these plant components into phospholipids to produce a
product that is better absorbed and is more bioavailable. This strategy has been suc-
cessfully used for various CNS pathologies, and some of the examples would be
discussed here.
Silymarin obtained from Silybum marianum has been found to improved social
recognition and memory function on exposure to ethanol in rat pups (Reid et al.
1999). Silymarin phytosomes have shown prevention of deficits in social memory
function upon in utero exposure to ethanol in male rats. It was also later demon-
strated that silybin phytosomes also showed improvement in functional outcomes in
social memories in the off springs of female of rats fed with 35% ethanol derived
calories during pregnancy (Busby et al. 2002).
Huperazine A is known to be a reversible acetylcholinesterase inhibitor that can
cross the BBB and exhibits high specificity for acetylcholinesterase. It has found to
show benefits in cognitive impairments associated with AD. Cai et al. (2012)
reported the preparation of huperazine A phospholipid complex with a view to
improve its biopharmaceutical properties which was loaded into an injectable
implant system as a controlled release reservoir. In vitro results suggested a signifi-
cant decline in the initial burst release and a prolonged release up to 2 weeks. These
results correlated well with the in vivo pharmacokinetic studies upon a single sub-
cutaneous injection in rabbits (Cai et al. 2012).
Gingko biloba (Ginkgoaceae) an important herb of the traditional Chinese sys-
tem of medicine is also known to be useful in several CNS disorders like cerebral
insufficiency and AD. Its major active principles include diterpene lactones namely
ginkgolides A, B, C, M, and J and bilobalide, biflavones like bilobetin, ginkgetin,
56 H. Ahmad et al.
isoginkgetin, and flavone glycosides including kaempferol, quercitin and isorham-

netin, and organic acids such as 4- hydroxy benzoic acid and shikimic acid. However
extensive GI metabolism upon oral administration resulting in reduced bioavailabil-
ity is the major limiting factor with Gingko extract. Phytosomes of Ginkgo biloba
were formulated and were found to demonstrate antiamnesic and antidepressant
activities in the scopolamine-induced amnesia test and behavioral despair test,
respectively, upon oral administration. There was a significant reduction in pento-
barbitone-induced sleeping time, enhanced motility, alteration in general behavior,
and an inhibition of chlorpromazine-induced blockade of conditioned and uncondi-
tioned responses in rodents (Naik et al. 2006).
Bacopa monnieri L. (Scrophulariaceae) is another herb of the Ayurvedic system
of medicine also known as brahmi and has been found to be endowed with neuro-
protective properties in anxiety and depression. Bacopa phospholipid complex was
found to significantly reverse the cognitive deficits in aged mice compared to bacopa
extract at equivalent dose. The phospholipid complex could maintain the effective
bacopasides concentration for longer period of time in rat serum compared to
bacopa extract. This enhanced absorption of bacopasides could be responsible for
improved antiamnesic effects of bacopa phospholipid complex (Habbu et al. 2013).
Phospholipid complexes of Centella another traditional herb known for its role in
learning and memory were attempted to improve the biopharmaceutical properties
of its phyto-constituents. Studies revealed that the phospholipid complex of stan-
dardized Centella extract afforded better dissolution characteristics and permeation
compared to the extract itself. In vivo studies also showed significant improvement
in spatial learning and memory in aged mice in the Morris water maze test (Saoji
et al. 2016). Centella asiatica leaf extract phytosomes has also been evaluated in a
traumatic brain injury (TBI) model. TBI is a CNS disorder that results from head
trauma. Damage in the nerve membrane phospholipids and reduced protein synthe-
sis of neuregulin-1 due to transcription factor Krox-20 are hallmarks of this disease.
Nerve remyelination is reduced that results in decline in the cognitive functions.
Findings revealed that the Centella extract phytosome was able to significantly
improve nerve cells via Krox-20 activation, neuregulin-1 expression, and phospho-
lipid distribution. It also improved the cognitive functions in rats induced with TBI
as evident from Morris water maze test. However it was found that Centella phyto-
some administered along with citicoline (a neuroprotective agent) produced a higher
increase in phospholipids distribution and gives the fastest time in the cognitive
tests compared to Centella phytosome alone or citicoline alone (Jazmi et al. 2017).
Phospholipid complexes of a standardized fraction of Withania somnifera roots
were evaluated for their protective effects in stroke using the middle cerebral artery
occlusion (MCAO) model in rats. Beneficial effects of this formulation could be
demonstrated by reduced MDA levels, increment in GSH levels, reduced cerebral
infarction, and reduced deficit scores in 1-hour pretreatment and 6 hours posttreat-
ment groups (Ahmad et al. 2016b). Rutin phytosomes were also found to improve
the functional outcomes in cerebral stroke using the same experimental model at a
dose less than half of the effective dose of rutin (Ahmad et al. 2016a).
1.11 Conclusions
An upsurge in the aging population worldwide has led to an increased prevalence of

neurological disorders which is further expected to rise in the coming decade.
Despite huge costs involved in research directed at effective therapies for CNS
pathologies like neurodegeneration, multiple sclerosis, stroke, gliomas, and other
brain-related disorders, the results are rather disappointing. The major obstacle here
is the circumvention of the blood–brain barrier. Liposomes and phytosomes have
emerged as formulation strategies that can have benefits in various brain-related
disorders besides several other pharmaceutical applications that have been described
in this chapter.
Different formulation strategies can be used for achieving different functional
objectives. The use of sterically stabilized liposomes, targeted liposomes, fusogenic
liposomes, cationic liposomes, pH-sensitive, and temperature-sensitive liposomes
are a few examples. Liposomal formulations have been successful in improving the
therapeutic index of both new and known drugs by modifying drug absorption,
reducing toxicity, and enhancing the biological half-life thereby altering the biodis-
tribution. Liposomes are capable of mimicking biological cells and are known to be
biocompatibile and biodegradable systems with limited toxicity. These properties
made them attractive drug delivery systems for antimicrobial and anticancer ther-
apy, in diagnostics and imaging and also for pulmonary, ocular, and topical applica-
tions. Several commercial preparations of liposomes have shown market presence
since 1980s and 1990s. Liposomes have shown great promise in CNS applications.
Cationic, PEGylated, theranostic, and immunoliposomes have been widely investi-
gated for brain drug delivery. Transferrin receptor targeted liposomal interventions
have been used as a relevant strategy for drug delivery to the brain for a variety of
disorders like AD, PD, stroke, glioma, and other diseases. Recent reports suggest
that drug bearing liposomes have shown encouraging results in preclinical studies
and advanced clinical trials and many of these candidates have already been clini-
cally approved. New-generation liposomes working on the principle of a combina-
tion of properties seem to be the foreseeable future in liposome research. Exploiting
a variety of properties like longevity, targetability, stimuli sensitivity, drug release,
co-delivery, and contrast properties in desired combinations can help achieve better
therapeutic efficacy with these new generation liposomes.
Phytosomes were first patented as a technology to incorporate plant extracts and
polar bioactives into phospholipids for enhancing their bioavailability by Indena
(Italy) in the late eighties. Phytosomes have been investigated as health foods and
neutraceuticals and have shown tremendous potential in moisturizing, antiaging,
and sunscreen-based skin therapies. Several phytosomal therapies have been
reported for antioxidant, anti-inflammatory, wound healing, and cytotoxic effects
along with cardiovascular and hepatoprotective applications. Many marketed phy-
tosomal preparations are available for a variety of applications like personal care,
healthcare, or health food. Many investigations have shown the beneficial effects of
phytosome strategy in cerebral insufficiency, AD, dementia, stroke, traumatic brain
58 H. Ahmad et al.
injury, and various other CNS pathologies. Phytosomes are the future of herbal
medicine as they have formed a bridge between conventional and novel delivery
systems and many therapeutic products that cater to different clinical uses can be
expected in the near future.
Based on exhaustive literature coverage and all the pertinent information out-
lined in this chapter, it can be said that liposomes and phytosomes appear to be
promising carrier systems for efficient drug delivery for a variety of therapeutic
applications. An in-depth understanding in the architectural differences and proper-
ties of liposomes and phytosomes could further pave the way for future research in
the area of CNS therapies utilizing these formulation strategies. The authors have
attempted to create a common reference point pertaining to these formulation strate-
gies with special emphasis on their implications in CNS disorders, and this chapter
could serve as important reference material for further research on liposomal and
phytosomal interventions.
Acknowledgments The authors are thankful to the knowledge resource center at CSIR-CDRI,
Lucknow, for accessing the exhaustive literature used in compiling this work, and also to SERB,
New Delhi for funding support to Dr Hafsa Ahmad (PDF/2017/00137).
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Chapter 2
Applications of Nanopharmaceuticals
in Delivery and Targeting
Mohamed Abbas Ibrahim and Ahmed A. H. Abdellatif
Contents
2.1 I ntroduction 74
2.2 G oals of Nanoparticle-Based Drug Delivery Systems 77
2.3 Classification of Nanoparticles 77
2.3.1 Classification of Pharmaceutical Nanoparticles 77
2.4 Applications of Nanoparticles 94
2.4.1 Enhanced Solubility and Dissolution Rate 96
2.4.2 Controlled Drug Release Rates 97
2.5 Nanoparticle Medicinal and Pharmaceutical Applications:
Prospective Clinical Challenges 101
2.5.1 Biological Challenges 102
2.5.2 Technological Challenges 102
2.5.3 Study-Design Challenges 102
2.6 Conclusion 103
References 103
Abstract Nanotechnology became a widespread technology in recent years in sev-

eral medical and pharmaceutical applications. Drug-delivery systems based on
nanoparticle technologies have the prospective industrial revolution that could be
considered as a landmark of future pharmaceutical industries. The major goals in
M. A. Ibrahim
Kayyali Chair for Pharmaceutical Industries, Department of Pharmaceutics,
College of Pharmacy, King Saud University, Riyadh, Kingdom Saudi Arabia
A. A. H. Abdellatif (*)
Department of Pharmaceutics, College of Pharmacy, Qassim University,
Buraidah, Kingdom of Saudi Arabia
e-mail: a.abdellatif@qu.edu.sa; ahmed.a.h.abdellatif@azhar.edu.eg
https://doi.org/10.1007/978-3-030-44925-4_2
74 M. A. Ibrahim and A. A. H. Abdellatif
designing nanoparticles as a delivery system include enhancing bioavailability by

enhancing solubility and dissolution rate, targeting the drug to specific organs, and
controlling drug release rate. Some other nanoparticles illustrate vast promise in the
field of tumor imaging and the early identification of malignant tissue such as fluo-
rescent polymeric nanoparticles. The chapter discusses different classes of pharma-
ceutical nanoparticles including nanonized drug (API) particles, biodegradable
polymeric nanoparticles, and hydrophobic nanoparticles. The nanonized drug (API)
particles are mostly applied for enhancing drug solubility and dissolution rate,
which, in turn, can improve its bioavailability. These nanosized drugs (API) parti-
cles are prepared by either top-down or bottom-up techniques. In addition, the chap-
ter also spots the light on variable polymer classes utilized in polymeric nanoparticles,
including hydrophilic, hydrophobic, and biodegradable polymers. Furthermore,
different manufacturing techniques adopted for the production of polymeric

nanoparticles based on the type of the polymers were described. Also, the nanoniza-
tion techniques of the polymeric nanoparticles are based on physical methods
including primary and multiple emulsion solvent evaporation methods, ionic gela-
tion, spray-drying, supercritical fluid technology, as well as precipitation with a
compressed fluid techniques anti-solvent were clarified. Other polymeric nanopar-
ticle manufacturing techniques are based on chemical synthesis schemes such as
silica nanoparticles of variable internal structures. Furthermore, the polymeric
nanoparticles as targeting system are not only for healthy cells but also diseased
cells. The applications of nanoparticles in drug delivery and targeting, focusing the
conventional and recent methods of preparation, were briefly specified. At the end
of this chapter, final remarks and recommendations on the optimal methods of man-
ufacturing techniques are suggested.
Keywords Nanopharmaceuticals · Drug delivery · Targeting · Nanotechnology ·

Medicinal application · Pharmaceutical applications
2.1 Introduction
Nanotechnology is defined as the art that deals with the creation of materials on an
atomic and molecular scale of 0.1–1000 nm (Labhasetwar and Leslie-Pelecky
2007). Even though nanotechnology is a moderately recent advance in scientific
research, the growth of its fundamental concepts occurred over a long period of time
(Farhatnia et al. 2013). The goals of nanotechnology were beginning with 1986 by
the publication of the book Engines of Creation (Drexler and Minsky 1990).
Recently, the beginnings of marketable applications of nanotechnology started,
while these were limited to bulk applications of nanomaterials (Corot and Warlin
2013). Nanotechnology has gradually included over the long-ago and will continue
to evolve even further in the future. Approximately last 100 years engineers and
scientists were capable to construct devices on a macro-level, but now they
2 Applications of Nanopharmaceuticals in Delivery and Targeting 75
condensed the scale substantially to be capable to fabricate devices on a nano-level

(Oita et al. 2010). Carbon nanotubes, as an example, which are the most construc-
tive material known to human, they can facilitate to increase the efficiency of trans-
mission lines (Di et al. 2016; Li et al. 2016). The green energy area has been the
biggest application of nanotechnology (Xu et al. 2016a). Nanotechnology can also
aid solar cells to turn into the much more efficient system. Nanosolar, a thin-film
solar producer, is using nanotechnology to produce a solar project in Spain that will
create 16,500-megawatt hours per year (Kang et al. 2016; Kim et al. 2016).
There are numerous biomedical applications of nanotechnology, such as drug
targeting and gene therapy (Couvreur 1988; Goppert and Muller 2005; Ma et al.
2012). Colloidal nanoparticles can be synthesized to offer opportunities for the site-
specific delivery of drug after injection into the bloodstream. Nanoparticles can be
used as a targeting agent to different organ sites in the body, for example, lung,
swelling sites, liver, spleen, bone marrow, and tumor cells (Fan et al. 2012; Xu et al.
2012; Yoon et al. 2012). Nanoparticles show huge promising tools in the field of
tumor imaging, drug delivery, and the early detection of normal and malignant tis-
sues (Portney and Ozkan 2006). Nanoparticles do not only have a characteristic size
but also a distinctive surface chemistry. Functional nanoparticles are available by
conjugation throughout electrostatic or by a covalent attachment such as peptides,
proteins, nucleic acids, or small molecules, which can guide the nanoparticles to a
specific site in the body (Sinha et al. 2006).
There are many pharmaceutical applications for nanoparticles. All of these are
with most promising tools including, drug delivery, targeted delivery, and sustained,
prolonged delivery (Abdellatif et al. 2018b). Nanomedicines can be a promising
delivery system to treat many diseases such as AIDS, cancer, tuberculosis, diabetes,
malaria, prion disease, and many other diseases in different trial phases. They have
the main problem that human body recognizes hydrophobic particles as unfamiliar
particles, and they can be quickly taken up by the phagocytic system (MPS); these
can be overcome by surface alteration of nanoparticles (Abdellatif et al. 2018a;
Abdellatif and Tawfeek 2018). They also are used as delivery systems for the pas-
sage of different barriers in the human body by increasing the efficiency of drugs
with poor solubility. Today, the nanotechnology is not only for diseases, it is also for
the formulations in beauty care and the normal body external use which have special
formulations available in the market ( Abdellatif and Tawfeek 2015). Many exam-
ples of the new systems, including vesicles, liposomes, gels, and particles with dif-
ferent moieties that include the encapsulated hydrophobic drugs are available.
Nevertheless, many of those delivery systems are in the investigational step or in the
primary step of the connection with animals (Marks 1996).
Polymeric nanoparticles (PNPs) are NPs that formed of a polymer or copoly-
mer which dispersed in the polymer medium. These NPs are possible of different
shape (platelets, fibers, spherical). Microparticles can be transferred to nanoparti-
cles, but this leads to change in its physical as well as chemical properties (Bobo
et al. 2016; Soica et al. 2016). Polymeric nanoformulations present numerous
advantages more than the traditional pharmaceutical formulations, the major ones
being the specific targeted therapy with very minute doses of active medicaments,
longer duration time circulation in the bloodstream, and higher drug loading
capacity for diagnostic and healing molecules. Furthermore, PNPs can be func-
tionalized so as to act as nanotheranostics within the better frame of tailored medi-
cine (Bobo et al. 2016). A model of a nanopolymer is silicon nanoparticles which
demonstrate quite different properties; their size is 30–1000 nm, and they are more
solid than silicon itself, their solidity being between that of cobalt and diamond
(Harun et al. 2011; Hasan et al. 2016; Kim et al. 2012).
Polymeric nanoparticles have biomedical applications for particular drug deliv-
ery and imaging. It was reported that magnetic nanoparticles embedded in polylac-
tide-co-glycolide matrixes (PLGA-MNPs) were prepared and characterized as a
dual drug delivery and imaging system, Fig. 2.1. These nanoparticles are capable of
Nanoparticle
Multifunctional
Nanoparticles Imaging
Therapy output
PET
Chemotherapy
Fluorescence
MRI
Phototherapy
Photoacustic
Targeting
Gene Therapy
Octreotide
NH2
O NH
H
N
O H
HO S
Antibodys HO
O S
OH
O
O
NH N
Peptides
N O
H
HN NH
N
H H
O
NH2
Fig. 2.1 Schematic diagram illustrating the polymeric nanoparticles and their biomedical applica-
tions as drug delivery and imaging
encapsulating both hydrophilic and hydrophobic drugs (Singh et al. 2011). The
usage of polymeric nanoparticles as drug carriers demonstrated advantages for ther-
apeutic application, including their little toxicity, enhancing solubility, can be evalu-
ated in vitro and in vivo and easily used as targeted tools (Moritz and Geszke-Moritz
2015). Moreover, nanoparticles can be used to attain controlled release and to pro-
tect some sensitive materials, such as protection of the antioxidant activity of the
polyphenols. Polymeric nanoparticles encapsulated with white tea extract based on
poly(epsilon-caprolactone) and alginate was productively performed (Sanna et al.
2015). Cyclosporine A was loaded successfully into cationic Eudragit RS 100
nanoparticles for the ocular appliance (Basaran et al. 2011). Recently, polymeric
nanoparticles can improve the immune response, by targeting antigens to dendritic
cells that have a critical role in promoting immune responses and might be poten-
tially helpful in immunotherapy (Craparo and Bondi 2012). Zinc-loaded polymeric
nanoparticles present a promising advance to selectively enhance zinc in the brain
within a little amount of time (Chhabra et al. 2015).
This chapter demonstrates the dual usable purpose of formulated nanoparticles
toward either, in therapeutics by delivering different hydrophobic or hydrophilic
drugs separately or in mixture and imaging for tumor therapeutics in the upcoming.
2.2 Goals of Nanoparticle-Based Drug Delivery Systems
Nanoparticle-based drug delivery systems are designed so as to achieve a variety of

therapeutic goals including:
a- Improving drug aqueous solubility and dissolution rate and, in turn, improving
its bioavailability
b- Controlling and prolongation of the drug release from its pharmaceutical dos-
age forms
c- Targeting the active pharmaceutical ingredient (API) to the specific site for its
action (as the case of using nanocarriers in cancer therapy). Drug targeting and
lowering clearance can result in increasing its therapeutic index and lowering the
dose required for efficacy (Koo et al. 2005).
2.3 Classification of Nanoparticles
2.3.1 Classification of Pharmaceutical Nanoparticles
Pharmaceutical nanoparticles can be classified according to the composition of the

nanoparticulate system into:
1 - Nanonized drug (API) particles

2- Polymeric nanoparticles:
a- Hydrophilic (hydrogel) nanoparticles
b- Hydrophobic nanoparticles
c- Biodegradable nanoparticles
Nanosized API Particles Instead
The aqueous solubility of drugs plays a principal role in determining its permeabil-
ity and absorption rate through biological membranes. Most of the drugs are classi-
fied as class IV (low permeability and low solubility) according to the
biopharmaceutical classification system (BCS). Therefore, aqueous solubility and
permeability through biological membranes are the controlling factors that govern
both rate and extent of oral absorption of several drugs (Dunne et al. 1999). The
enhancement of the aqueous solubility of poorly soluble drugs was investigated by
several authors, and different approaches were established for enhancing. Some of
these approaches include micronization, salt formation, co-solvency, solid disper-
sion, micellar solubilization, as well as inclusion complexes using cyclodextrins
(Ganesh et al. 2013). Recently, the application of nanoparticles in the fields of drug
delivery gained a great consideration (Jia 2005). Nanotechnology presents essential
means of improving drug accumulation at the targeted sites, which in turn, improves
its biodistribution and pharmacokinetics. Nanonization of drug particles was inves-
tigated as a potential way in improving drug solubility, permeability, and bioavail-
ability (Basha 2017; Jahangirian et al. 2017), Fig. 2.2.
Pharmaceutical
nanoparticles
Polymeric Nanonized drug

nanoparticles particles (APIs)
Hydrophilic
Hydrophobic Biodegradable
(hydrogel nanoparticles)
Fig. 2.2 Classification of pharmaceutical nanoparticles

Milling (grinding)
Top-down
High Pressure
Homogenization
(HPH)
Supercritical fluid
API Nanonizaon
Techniques
Evaporative
precipitation into
aqueous solution
(EPAS)
Boom-up Spray-freezing
into liquid
Rapid expansion
of supercritical
solution (RESS)
Precipitation with
compressed fluid
anti-solvent (PCA)
Fig. 2.3 Different nanonization techniques of API
API Nanonization Techniques
Nanonization of drug particles could be carried out either by “top-down” or “bot-

tom-up” techniques, Fig. 2.3:
Top-Down Techniques
“Top-down” techniques mean breaking down the large drug crystals or particles into
the nanosize either by milling (grinding) or high-pressure homogenization (HPH).
The starting material in top-down nanonization technique is coarse particles that
will be subjected to particle size reduction to be nanosized.
Media Milling Techniques

Media milling is the commonly adopted industrial technique for the manufacture of
drug nanoparticles due its low cost and capacity for rapid production. Several com-
mercial nanoparticle-based pharmaceutical products that are manufactured by
media milling are available. In this technique, the milling container is charged with
milling ball, dispersion media (e.g., water), and API powders along with a suitable
stabilizer (s). The milling balls are rotated at a very high speed to produce strong
shear forces to grind drug powers into nanoparticles (Merisko-Liversidge et al.
2003). The grinding ball media are made of glass, zirconium, steel, or plastic, but
zirconium balls are superior in producing very small nanosized particles. Stabilizers
are added during nanonization by wet milling to minimize crystal growth and pro-
duce stable formulation (Tanaka et al. 2012).
Different parameters can influence and control the physical characteristics of the
produced nanocrystals including the type, size, weight, and number of milling balls,
the ratio of drug to stabilizer, volume of milling solvent, milling cycle, speed and
time, and the temperature of milling bowl (Chen et al. 2011). The difficulty in the
removal of residual milling media from the final product is one of the potential
drawbacks of media milling that results in loss of drug particles due to adhesion to
the inner walls of the milling container (Chen et al. 2011).
High-Pressure Homogenization (HPH)

The high-pressure homogenization (HPH) nanonization method is based on a cavi-
tation method, in which vapor bubbles are generated in the milling liquid with the
application of high pressure (Muller et al. 2011). HPH may be carried out in either
aqueous or non-aqueous milling solvent. Non-aqueous solvents are (e.g., PEG 400)
suitable for water sensitive APIs. A suspension of solid drug and the suitable stabi-
lizer is forced through the narrow gap of a homogenizer at high pressure
(500–2000 bar). The pressure generates disruptive forces such as collision, cavita-
tion, and shearing, which grind large drug crystals to nanoparticles. HPH offers an
excellent choice for producing high-quality drug nanoparticles on an industrial
scale. Very small nanoparticles or nanocrystals can be gained by controlling the
homogenization pressure and speed as well as the number of homogenization cycles
(Keck and Muller 2006).
Bottom-up Techniques
“Bottom-up” techniques, are based on the formation of nanoparticles from molecu-
lar solution. Several representing examples were reported for the bottom-up tech-
niques including precipitation with an anti-solvent compressed fluid, fast expansion
from a solution liquefied gas, liquid by spray-freezing, aqueous solution process by
evaporative precipitation, and supercritical fluid (SCF), Fig. 2.4. Drug nanonization
by top-down technologies (mechanical milling or high-pressure homogenization)
1 2 3
Fig. 2.4 Crystal growth of nanosized particles according to Ostwald ripening theory
suffers from some drawbacks as it needs long nanonization times, results in impuri-
ties, and low flexibility in monitoring particle surface and shapes (de Waard et al.
2011; Gumz et al. 2017).
Comparative to mechanical nanonization techniques, precipitation from solution
can propose superior flexibility for governing the crystalline or amorphous form of
the active pharmaceutical ingredient (API) in addition to achieving high drug load-
ings (Matteucci et al. 2006). The drug is dissolved in an organic solvent and mixed
with an aqueous anti-solvent solution containing a suitable stabilizing surfactant or
polymer to form nanosized particles. One of the bottom-up nanonization procedures
is the spray-freezing into liquid (SFL). The API (in form of a solution, emulsion or
suspension) is atomized through a nozzle into a cryogenic liquid such as argon,
nitrogen, or halocarbon refrigerants. The atomized droplets are forced to pass
through cold halocarbon vapor to solidify and freeze gradually when being in con-
tact with the boiling refrigerant liquid. Thereafter, the nanonized particles are
lyophilized to become free-flowing and dry. Nanoparticles produced by this tech-
nique are highly wettable with the increased surface area but of wide size range and
agglomeration (Bayda et al. 2017; Fan et al. 2015; Xing et al. 2017).
In supercritical fluid (SCF), the API is dissolved in a supercritical fluid such as
carbon dioxide, ethylene, nitrous oxide, or water. The particles size produced by
such procedures have a wide range from nano- to microparticles. However, this
technique is safe and reproducible, in addition to its low cost. Therefore, it is applied
widely by several pharmaceuticals manufacturers to improve the solubility of poorly
water-soluble drugs (Kankala et al. 2017; Kompella 1999), Table 2.1.
Nanoparticles/Nanosuspensions: Stability Issues
Nanonized API particles are subjected to instability problems owing to nucleation

and particle growth. This might be due to the high surface area that results in ther-
modynamically unstable particles and supports their flocculation in form of agglom-
eration and crystal growth a phenomenon is known as “Ostwald ripening,” Fig. 2.4.
This crystal growth prevails mainly in absence of suitable stabilizers. The phenom-
enon of crystal growth of the nanosized API particles during the manufacture and
storage influences its dissolution and, in turn, its bioavailability (Dolenc et al. 2009).
Furthermore, crustal growth is considered as the limiting step in the development of
nanoparticle-based pharmaceutical drug delivery.
To overcome or minimize the stability problem of nanoparticles, stabilizers are
added to the nanosuspension during nanonization procedures. The stabilizers act by
adsorption onto the surfaces of drug nanoparticles and afford a steric or electrostatic
stabilization effect owing to the moieties present in their surfaces causing some
energetically rough surfaces presenting repulsive entropic forces that prevent
coalescence. Stabilization of the nanosized APIs particles by stabilizer can be
achieved either by electrostatic interactions or hydrogen bonding (Wang et al. 2013).
82
Table 2.1 Selected marketed nanoparticle-based pharmaceutical dosage forms that are approved for clinical use or under clinical trials
Product API Dosage form Therapeutic indications Nanonization technique Company
Triglide® Fenofibrate Oral tablets Hypercholesterolemia High-pressure homogenization SkyePharma/Sciele, approved in 2005
Tricor® Fenofibrate Oral tablets Hypercholesterolemia Media milling Elan/Abbott, approved in 2004
Rapamune® Sirolimus Oral tablets Immunosuppression Media milling Elan/Wyeth, approved in 20
Emend® Aprepitant Oral capsule Antiemetics Media milling Elan/Merck, approved in 2003
Megace ES® Megestrol Oral suspension Antianorexia, cachexia Media milling Elan/Par Pharmaceuticals, approved
in 2005
Gris-PEG® Griseofulvin Oral tablets Antifungal Bottom-up, coprecipitation Novartis
Cesamet® Nabilone Oral capsule Antiemetic Bottom-up, coprecipitation Lilly
M. A. Ibrahim and A. A. H. Abdellatif
Mechanistically, the stabilizers that are adapted to stabilize nanoparticles act by

adsorbing onto the nanosized drug surface and result in a steric or electrostatic sta-
bilizing action because of the presence of hydrophobic moieties in the stabilizers’
molecules. This means that the stabilizer covers the surface of the nanoparticles
during the nanosuspension preparation by adsorbing at their surfaces. The continu-
ous thermal motion that takes place for the chain molecules adsorbed on the surface
causes a dynamically rough surface that inhibits nanoparticles aggregation or
coalescence using repulsive forces (Rachmawati et al. 2013). In addition, hydrogen
bonding between stabilizer and nanoparticle surfaces as well as electrostatic inter-
actions may form which can extra strengthen the adsorption. Hydrogen bonds
formed on the surface of nanoparticles causes a strong electrostatic repulsion
between the charged nanoparticle surface and the totally ionized functional groups
of the stabilizer (Rachmawati et al. 2013).
Pluronic F127 is a surfactant used in stabilizing nanosuspension and nanoparti-
cle. It is considered as one of the safe and least toxic commercial block copolymers
(Xiong et al. 2005). Pluronic F 127 is used extensively as a pharmaceutical excipi-
ent in a wide range of pharmaceutical manufacturing of low-molecular-weight
drugs and proteins as well. Moreover, Pluronic F 127 acts by steric stabilization for
ensuring the stability of nanonized drug particle during nanonization procedures. It
keeps the drug crystallinity unchanged because it does not usually destroy the crys-
talline drug structure; this is in contrast to other stabilizing surfactants of low-
molecular-weight such as sodium dodecyl sulfate (Choi et al. 2005). Other
surfactants act as stabilizers for nanoparticulate systems by forming micelles that
entrap a small number of dissolved drug molecules.
In case of the top-down nanosuspension preparation techniques, the drug particle
size decreases usually to a steady state value with time depending on both stabilizer
nature and concentration added. In addition, the stability of nanosuspensions, as
well as their robustness, are mainly controlled by controlling different formulation
and process parameters (Chiang et al. 2011). Therefore, the stabilization of these
produced nanosuspensions by the aid of stabilizers is a precarious parameter. So,
the choice of the suitable steric and electrostatic stabilizers at suitable weight ratios
is an important issue in controlling nanoparticles’ stability during nanosuspension
formulation (Chiang et al. 2011).
Another stabilizing polymer can be used as a secondary or auxiliary stabilizer to
add more stabilizing effect to the nanosuspension. Physical stability of nitrendipine
nanosuspensions was noticeably enhanced upon using modified chitosan (cationic
polysaccharide) (Delehanty et al. 2009). The stability of the chitosan-modified
nanosuspensions was enhanced meaningfully after storage at 30 °C for 24 days in
comparison to the nitrendipine nanosuspensions stabilized by PVA. The stabilizing
effect of chitosan was contributed to the electrostatic repulsion and steric stabiliza-
tion caused by chitosan cationic nature (Delehanty et al. 2009). It was concluded
that the improved electrostatic repulsion prohibited the growth of particle size. Also,
chitosan adds a steric stabilization effect due to its deposition on the surface of the
nanosuspensions and, in turn, increases the stability of particle size of the prepared
nanosuspensions more and more.
Polymeric Nanoparticles
Nanoparticle-based pharmaceutical formulations such as polymeric nanoparticles

(either nanospheres or nanocapsules), liposomes, solid lipid nanoparticles, and
nanoemulsions have been extensively used as novel drug delivery systems.
Nanosized drug delivery carriers can efficiently shield unstable APIs from degrada-
tion, denaturation, or any stability problem and, in turn, minimize the adverse reac-
tions of such drugs by producing targeting to a specific site or controlled release.
Polymeric nanoparticles have revealed potential in both systemic and topical drug
delivery. The main areas of the pharmaceutical applications of polymeric nanocar-
riers are to (Rangari and Ravikumar 2015):
a- Protect the APIs from chemical and physical degradation
b- Control and prolong drug release
c- Target API to a specific site of action, thereby reducing systemic adverse
reactions
Polymeric materials should process drug delivery characteristics to be good nano-
carriers candidates. Polymers should be biocompatible (non-toxic, non-antigenic).
Also, the polymeric matrix should reserve the encapsulated or entrapped API par-
ticles until its site or time of action. In addition, nanocarriers should not be harmful
to body cell or tissues.
Polymeric nanoparticles are classified according to the nature of the polymer to:
1 - Hydrophilic polymeric nanoparticles
2- Hydrophobic polymeric nanoparticles
3- Biodegradable polymeric nanoparticles
Hydrophilic Polymeric Nanoparticles (Hydrogel Nanoparticles)
In recent years considerable attention was gained toward hydrogel nanoparticles as

they are considered as one of the most promising nanoparticulate drug delivery
systems, due to their properties combining the hydrogel system attributes (as hydro-
philicity and very high-water content) with a nanoparticle (very small size) (Hamidi
et al. 2008). The affinity of hydrogel nanoparticles to absorb water is owing to the
presence of hydrophilic groups such as –OH, –CONH–, –CONH2–, and –SO3H in
the forming polymer chains (Owens 3rd and Peppas 2006).
Both natural and synthetic polymers are used for the preparation of hydrogel
nanoparticulate systems. Chitosan and alginate are examples of the natural poly-
mers that have been studied widely for preparation of hydrogel nanoparticles. Also,
as examples of synthetic polymer-based hydrogel nanoparticles, poly(vinyl alco-
hol), poly(ethylene oxide), poly(ethyleneimine), poly(vinyl pyrrolidone), and poly-
N-isopropylacrylamide have been investigated. The release mechanism of the
incorporated drug from hydrogel nanoparticles is rather multifaceted and is con-
trolled by three key factors: drug diffusion, hydrogel matrix swelling, and chemical
reactivity of the drug/matrix.
pH and temperature-sensitive hydrogel nanoparticles of sizes up to 50 nm diam-

eter loaded with marker compound FITC-dextran (mol wt. 19.3 kD) were prepared
in the aqueous core of reverse micellar droplets and were dispersed in aqueous buf-
fer (Sahoo et al. 1998). These hydrogel nanoparticles composed of copolymers of
acrylic acid and vinylpyrrolidone cross-linked with NN´ methylene bis-acrylamide
(MBA). The results showed that these nanoparticles exhibited high entrapment effi-
ciency and are easy to be re-dissolved in buffer without aggregation. The results
revealed also that the in vitro release of FITC-dextran from nanoparticles was
dependent upon medium pH and temperature. The release was retarded in acidic
medium and increased substantially as the medium pH was increased and was also
increased with the increase of temperature.
Missirlis et al. (2006) described the preparation of stable, doxorubicin-loaded
hydrogel nanoparticles, composed of poly(ethylene glycol) and poloxamer 407
(Pluronic® F127), by inverse emulsion photopolymerization. They investigated the
feasibility of this technique for small hydrophobic drugs. They were able success-
fully to encapsulate doxorubicin through hydrophobic interactions, taking advan-
tage of particle nanoarchitecture. In vitro, doxorubicin release studies showed a
very low burst (approximately 10% at 37 °C) and sustained, diffusional release for
more than 1 week. In addition, drug encapsulation noticeably delayed and mini-
mized doxorubicin degradation.
The clinical trials of paclitaxel and other taxanes (as anticancer agents) have
been limited by their highly hydrophobic characteristics. New nanoparticle-based
drug delivery system has been developed that is based on binding paclitaxel with to
form hydrogel nanoparticles of 130-nm size free from any kind of solvent. These
hydrogel nanoparticles were superior to an equitoxic dose of standard paclitaxel
with a meaningfully low incidence of toxicities in a large, international, randomized
phase III trial. This hydrogel nanoparticle-based formula can give the oncologist
several effective treatment options for patients (Miele et al. 2009). The Food and
Drug Administration (FDA) on September 6, 2013, approved this formula contain-
ing paclitaxel-loaded albumin-stabilized nanoparticle formulation (Abraxane® for
injectable suspension, made by Abraxis BioScience, LLC, a wholly owned subsid-
iary of Celgene Corporation) the first-line treatment of patients with metastatic
adenocarcinoma of the pancreas (U.S. Food and Drug Administration).
Biodegradable Polymeric Nanoparticles
Biodegradable polymeric nanoparticles have concerned great attention as a novel

drug carrier because of their long half-life and high entrapment efficiency (Katas
et al. 2012). In addition, the polymeric biodegradable nanoparticles have comprised
the site-specific targeting and have the ability to permeate through the skin due to
their very small particle size.
Starch Nanoparticles
Starch is natural polysaccharide polymer used as a polymeric matrix in nanoparti-
cle-based drug delivery. It functions as storage materials in plants. Starch is com-
posed chemically of repetitive units of glucopyranose in an alpha D-(1, 4) linkage.
Upon hydrolysis, starch produces glucose (monosaccharide). Starch is widely used
in pharmaceutical manufacture as co-polymer and excipient in immediate and con-
trolled drug formulations, as drug carriers, in tissue engineering scaffolds as hydro-
gels and as solubility enhancers (Santander-Ortega et al. 2010).
The prospective of starch nanoparticles prepared by emulsification-diffusion as a
transdermal drug delivery system (TDDS) was investigated (Santander-Ortega et al.
2010). Starch-based nanoparticles were found to enable drug delivery without inter-
ference to the skin’s integrity. Both modified and unmodified maize starch were
adopted as polymeric materials to prepare nanoparticles. Modified starch nanopar-
ticles were found to be non-toxic. Flufenamic acid, caffeine, and testosterone were
used as model drugs in starch-based nanoparticle transdermal delivery.
Chitosan Nanoparticles
Due to its exclusive physical, chemical, and biological properties, chitosan is con-
sidered as an attractive polymer because of its broad areas in industrial and pharma-
ceutical applications. Chitosan is a biodegradable polymer used for several
biomedical applications due to its biodegradability, low immunogenicity, low-toxic-
ity, and biocompatibility. In addition, chitosan also processes antimicrobial activity,
which obviously expects a massive potential for future development (Vaghari
et al. 2013).
Chitosan nanoparticles are considered as promising drug delivery carriers for
wide classes of drugs particularly hydrophobic API is in cancer drug delivery appli-
cation (Rajan and Raj 2013). The small size and large surface area to volume ratio
of chitosan nanoparticles mark them superior in drug delivery of anticancer drugs.
Manufacturing Techniques
Several manufacturing techniques are used to prepare chitosan-loaded nanoparticle-
based on the desired shape and particle size. The main preparation methods used for
the preparation of chitosan nanoparticles are emulsion cross-linking, emulsion-
droplet coalescence, coacervation/precipitation, ionotropic gelation, reverse
micelles, template polymerization, and self-assembly polyelectrolytes (Malmiri
et al. 2012). The most extensively used methods are ionotropic gelation and self-
assembling polyelectrolytes. These nanoparticle preparation techniques present
several formulation advantages as the organic solvents and high shear are avoided
in these procedures. In addition, these methods are simple and mild preparation
method and can be applied for a broad range of drugs including macromolecules,
proteins, and anticancer agents.
Chitosan nanoparticles have wide pharmaceutical and biomedical applications,

one of which are related to colon targeted drug delivery (metronidazole), cancer
therapy (paclitaxel), gene delivery (multidrug resistance gene, MDR), mucosal
delivery (insulin), topical and ophthalmic delivery (flurbiprofen) (Rajan and Raj
2013; Y. Yang et al. 2009).
In gene delivery applications, chitosan nanoparticles can ionically interact with
the negatively charged DNA and form polyelectrolyte complexes. These complexes
enable DNA to be better shielded against nuclease degradation, and this can result
in enhanced DNA transfection efficiency (Agnihotri et al. 2004). The serious block-
age of conventional cancer therapy comprises high toxicity of most anticancer
agents’ due to the unselective distribution of chemotherapeutic agents towards dis-
ease and healthy cells following systemic administration. In addition, due to the low
aqueous solubility of anticancer agents, organic solvents or detergents were added,
resulting in undesirable adverse reactions as venous irritation and respiratory dis-
tress. Therefore, encapsulation of large doses of anticancer drugs using chitosan
nanoparticles is essential for successful cancer therapy (Makwana et al. 2015).
Moreover, chitosan nanoparticles are positively charged. Therefore, they will pro-
cess selective adsorption and neutralizing effects on the surface of the tumor cell.
Polylactic Acid (PLA) and Polylactic-co-glycolic Acid (PLGA) Nanoparticles

The physiological α-hydroxy acids, lactic acid and glycolic acid, are the building
blocks of PLA and PLGA (Fig. 2.5) representing the final products resulting from
complete polymer degradation. In contrast to lactic acid and glycolic acid and their
oligomers which are water-soluble with a molecular weight of approx. 1000
(Bastioli 2005), PLA and PLGA are only soluble in organic solvents, such as dichlo-
romethane, chloroform, acetone, and ethyl acetate. Both polymers are usually syn-
thesized under anhydrous conditions by ring-opening polymerization from cyclic
Fig. 2.5 Structure of

(A) PLA and (B) PLGA,
where R = H or CH3
dilactide and diglycolide using a catalyst such as stannous-ethyl hexanoate (Bastioli

2005). As lactic acid contains a chiral center, poly(L-lactic acid), poly(D-lactic
acid), and poly(D, L-lactic acid) with different D/L ratios may be synthesized which
display different degrees of crystallinity. For the design of drug delivery devices,
usually, the racemic poly(D, L-lactic acid) is used. PLGA is assumed to be mainly
a random copolymer whereby the lactide/glycolide ratio affects its degradation rate
(Tawfeek et al. 2017).
The co-encapsulation strategy with polymeric nanoparticles could be a promis-
ing advance in enhancing dissolution in oral, ocular, and cellular delivery for dis-
ease therapy (Abdellatif and Tawfeek 2015). It was reported that orally administrable
PLGA-NPs co-encapsulated with tamoxifen and quercetin have an approximately
three- to fivefold increase in oral bioavailability as compared to the free drugs.
Parallel, considerably higher tumor inhibition was observed in case of the prepared
formulation in contrast to the respective free drug(s) and their combination (Jain
et al. 2013). SR13668 has been formulated in stable poly(lactic-co-glycolic acid)
nanoparticles through flash nanoprecipitation which showed higher oral bioavail-
ability of SR13668 when compared with the Labrasol (R) formulation in a mouse
model (Shen et al. 2012). Also, an anticancer drug (SR13668) [2,10-dicarbethoxy-
6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole] has been verified effective in can-
cer treatment, but the restricted bioavailability has slowed down its clinical transla-
tion. Moreover, polymeric nanoparticles considered as promising carrier systems
for improving the ocular bioavailability of drugs (Alonso 2001). Furthermore, poly-
meric nanoparticles not only used for enhancing bioavailability but also for sus-
tained drug delivery (Gupta et al. 2011).
The degradation profile of both PLGA and PLA follows the principle of bulk
erosion, i.e., water diffusing into the polymer matrix are faster than polymer hydro-
lysis, leading to homogeneous polymer degradation all over the polymer matrix.
Polymer bulk erosion results in an accumulation of degradation products inside
polymer matrix until a critical degree of degradation is reached, leading to a spon-
taneous increase in permeability (Göpferich 1997).
The drug delivery based on PLA or PLGA nanoparticles should have the ability
to deliver the loaded API within the desired period of time, concentration, and bio-
distribution to achieve the required therapeutic function. Pharmacokinetics and bio-
distribution and of both PLA and PLGA track a nonlinear and dose-dependent
profile (Göpferich 1997).
Several investigators successfully prepared PLA and PLGA nanoparticles by apply-
ing microparticle preparation techniques for manufacturing PLA and PLGA
nanoparticles (nanospheres and nanocapsules) by using a small dispersed phase
ratio and stirring rate. The most commonly used technique for the manufacturing
these polymeric nanoparticles is the emulsification or solvent evaporation method.
This technique is mainly applied for encapsulating hydrophobic drugs. The double
or multiple emulsion/solvent evaporation techniques are a modification of this
method which considered as a favored procedure for encapsulating hydrophilic

APIs and proteins as well (Göpferich 1997). PLA and PLGA nanoparticles can also
be prepared using nanoprecipitation methods. In brief, organic solutions of polymer
and drug are added to an aqueous solution containing a suitable stabilizer (e.g.,
Pluronic F68), and evaporation of the organic phase takes place at appropriate tem-
peratures and reduced pressures resulting in precipitation of polymer and drug in
form of nanoparticles (Xie et al. 2017). Another technique utilized for the manufac-
ture of PLA and PLGA nanoparticles is the salting out in which a water-in-oil emul-
sion is firstly formed by dispersing the organic phase (usually acetone) containing
the polymer and salt (e.g., magnesium acetate tetrahydrate) and stabilizer. Water is
then added to this organic dispersion until the volume is adequate to diffuse organic
phase into the water, resulting in nanoparticle formulations (Konan et al. 2003).
Nanoparticulate systems based on PLA and PLGA have been utilized to develop
protein and peptide-based nanomedicines, nano-vaccines, and genes for in vivo
delivery systems (Kumari et al. 2010).
Poly-є-caprolactone (PCL) Nanoparticles

Due to its biocompatibility and biodegradability, PCL nanoparticles were widely
investigated for drug delivery and tissue engineering applications in several formu-
lations. The properties of PCL nanoparticles depend also on polymer nature and the
organic solvent which used similarly to microspheres.
The simplest nanoparticle preparation techniques for PCL nanoparticles are the
emulsion solvent evaporation and diffusion solvent evaporation, because of the
higher entrapment efficiency values that could be obtained (Dash and Konkimalla
2012). Other methods can be used for the preparation of PCL nanocapsules such as
the interfacial disposition method, in which where another oil phase is added to
complete repositioning of the polymer at the interface. Also, dialysis method has its
own advantage of simplicity and production of uniformly sized particles. The
method of preparation and processing condition was reported to have strong influ-
ence on mean particle size and size distribution. Zeta potential of the prepared PCL
nanoparticles is affected by the addition of surfactant during the preparation proce-
dures. It increases in the presence of a surfactant, and it was found that surfactant
influences particle size, surface properties, and release rate of the incorporated
drugs in nanoparticles. The most commonly used surfactants in PCL nanoparticle
formulation are PVA, poloxamer, and sodium cholate (Dash and Konkimalla 2012).
Various classes of APIs were incorporated in PCL nanoparticles for targeted, con-
trolled release drug delivery, and improved bioavailability, for example, insulin
(Damge et al. 2007), dexamethasone acetate (Ruy et al. 2003), doxorubicin and
paclitaxel (Pulkkinen et al. 2009), and ellagic acid (Sonaje et al. 2007).
Hydrophobic Nanoparticles
Solid Lipid Nanoparticles (SLN)

The effective application of nanoparticles for drug delivery depends on the ability
of nanoparticle-based delivery systems to penetrate through variable anatomical
barriers, controlling the release of the entrapped APIs and their stability during stor-
age in the nanosized state. The insufficiency of biologically safe polymers with
regulatory approval and their high cost restricted the widespread clinical and medi-
cal application of nanoparticles (Scheffel et al. 1972). To avoid these drawbacks of
polymeric nanoparticles, lipids are utilized as an alternative nanocarrier, principally
for hydrophobic drugs. These lipid-based nanoparticles are described as solid lipid
nanoparticles (SLNs) (Jumaa and Müller 2000). SLNs are considered as submicron-
ized lipid emulsions where the liquid lipid (oil) has been replaced by a solid lipid.
SLN possess exclusive properties that put SLN as attractive and potential drug
delivery systems to improve the performance of pharmaceuticals, nutraceuticals,
and other materials. These properties include small size, large surface area, high
drug loading and the interaction of phases at the interfaces (Cavalli et al. 1993).
More formulation advantages could be achieved by using SLNs as drug delivery
systems including their physical stability, protection of incorporated degradable
APIs, sustaining drug release, and exceptional tolerability (Sarathchandiran 2012).
SLN-based formulations have been developed and characterized for both in vitro
and in vivo for different routes of application (parenteral, oral, dermal, ocular, pul-
monary, rectal) (Gokce et al. 2012; Khameneh et al. 2015).
However, some formulation and drug delivery disadvantages were reported for
SLN such as the low drug loading efficiency and expulsion of the drug because of
polymeric transition during storage, in addition to the relatively high-water level of
the dispersions. The loading efficiency of the drug in SLN is dependent upon its
solubility in the lipophilic phase during its melting, composition of the lipid matrix,
and the crystalline state of the lipophilic matrix (Mukherjee et al. 2009). Several
preparation methods were adopted for the manufacture of SLN including the use of
homogenization techniques (either high shear, hot or cold homogenization), ultra-
sonication or high-speed homogenization, solvent emulsification/evaporation,
emulsion-based methods, spray-drying method, and double emulsion method.
The furthermost commonly selected excipients for the preparation of SLN-based
formulations are dietary oils composed of medium long-chain triglycerides (coco-
nut or palm seed oil) or long chain triglycerides (corn, olive, peanut, or soybean oils,
including hydrogenated soybean or vegetable oils) and lipid-soluble solvents (poly-
ethylene glycol 400, ethanol, propylene glycol, glycerin). In addition, different
pharmaceutically and biologically acceptable surfactants are routinely used such as
Cremophore® EL, RH40 or RH60, polysorbate 20 or 80, and many others (Mukherjee
et al. 2009). Solid lipid nanoparticles (SLN) have appeared as an important upcom-
ing drug delivery system with potential applications in drug delivery, cosmetics,
research, clinical medicine, and other sciences.
SLN are used as adjuvants for vaccination to improve the immune response.
Effective adjuvants are required for the safer new subunit vaccines because they are
less effective in immunization. Therefore, new developments in the adjuvant area

are the emulsion systems, which are oil-in-water emulsions that degrade fast in the
body fluid. The lipophilic constituents of SLNs will be more slowly degraded result-
ing in a prolonged long-term exposure to the immune system (Parhi and Suresh
2012). SLN-based antituberculosis drugs (as rifampicin, isoniazid, pyrazinamide)
were able to minimize the dose frequency and enhance patient compliance
(Sarathchandiran 2012). These anti-tuberculosis drugs can be loaded by using the
emulsion solvent diffusion technique.
Various drugs can be loaded to SLNs to be used for topical delivery such as flur-
biprofen (Maia et al. 2002). Thereafter, flurbiprofen-loaded SLN gel for topical
application presents a prospective drug delivery advantage of delivering the drug
directly to the site of action, which will result in higher tissue concentrations of
flurbiprofen. Vitamin A-loaded solid lipid nanoparticles can be also prepared from
glyceryl behenate for the improvement of drug penetration with the prolonged
release (Jenning et al. 2000).
Different classes of anticancer agents have been encapsulated in
SLN. Incorporation of the chemotherapeutic agents into SLN was found to improve
their efficacy and simultaneously minimized their associated side effects. The
important features of SLN that make them a suitable carrier for chemotherapeutic
drugs are the improved stability of drugs, encapsulation of chemotherapeutic agents
of diversified physicochemical properties, enhanced drug efficacy, improved phar-
macokinetics, and less in vivo toxicity. For example, tamoxifen has been loaded to
SLN to sustain the release of drug after IV administration in breast cancer.
Tamoxifen-loaded SLN resulted in achieving reasonable tumor targeting of the drug
(Abdelwahed et al. 2006).
Different preparation methods are used for the manufacture of SLN including
microemulsion method (Khurana et al. 2013), ultrasound and high-pressure homog-
enization (HPH) methods (Khameneh et al. 2015), micro-emulsion method (Zhang
et al. 2015), solvent injection method (Bikkad et al. 2013), and solvent diffusion
method (Han et al. 2014).
Silica Nanoparticles
Silica nanoparticles resemble an important class of the hydrophobic nanoparticles
due to their wide range of industrial applications and the easy manufacturing meth-
ods. The application of silica nanoparticles extends to include catalysis, pigments,
pharmacy, electronic and thin film substrates, electronic and thermal insulators, and
humidity sensors (Giesche 1994). Indeed, the quality of silica nanoparticle-based
product is highly dependent on their core nature, methods of manufacture, and the
size and size distribution of these particles. Recently, a considerable attention has
been paid to core-mesoporous shell architectures nanoparticles (either hollow, solid,
or rattle types). This is due to their applications in drug delivery, catalysis, water
treatment, and protein separation. The core material is often constituted from either
magnetic, metal, or quantum dots nanoparticles, while the mesoporous shells are
made from silica or carbon. Mesoporous nanoparticles are developing for their drug
delivery benefits especially for controlling and targeting purposes. Mesoporous sil-
ica nanoparticles (MSNs) are considered now as auspicious and novel drug delivery
vehicle because of their exceptional mesoporous architectures that provide a certain
level of chemical stability of the loaded APIs, biocompatibility, and surface func-
tionality, in addition to their low toxicity and high drug loading capacity (Brannon-
Peppas 1995).
Silica (SiO) is widely exist in the environment, but in comparison to other metal
oxides like titanium and iron oxides,it has superior biocompatibility (Kresge et al.
1992). The unique properties of the mesoporous form of silica could be summarized
in their high loading capacities of therapeutically active agents along with subse-
quently controlled release patterns. Silica-based mesoporous nanoparticles are more
stable to external response such as degradation and mechanical stress (because of
the strong SiO bond) as compared to other nanoparticulate drug delivery forms as
niosomes, liposomes, and dendrimers, which minimize the need to the addition of
external stabilizers during the synthesis procedures (Kwon et al. 2013). The meso-
porous structure-related properties (as pore size and porosity) can be adjusted so as
to accommodate the size and type of drugs.
Applications of Silica Nanoparticles
a- Detoxication
Mesoporous silica nanoparticles were widely used in adsorption of toxic mole-
cules and drug delivery system due to their high surface area, low toxicity, and
selective adsorption of substance (Vallet-Regi et al. 2012).
b- Paracellular Insulin Delivery
Microfabricated mesoporous silica nanoparticles were used for the paracellular
insulin delivery across an intestinal Caco-2 cell monolayer; this was one of the early
trials of drug delivery using silica nanoparticles. Cellular uptake is mediated by the
active endocytosis pathway as mesoporous silica nanoparticle endocytosis is inhib-
ited by lowering in temperature to 4 °C that is incubation with metabolic inhibitors.
Therefore, functionalization of the external surface of mesoporous silica nanopar-
ticles with groups in which cells express specific receptors like folic acid. This will
alter the cellular zeta potential and, in turn, will enhance the cellular uptake effi-
ciency (Balas et al. 2006).
c- Targeted Drug Delivery
Mesoporous silica nanoparticles can be functionalized for the purpose of tar-
geted therapies in which these nanoparticulate systems are used to block the growth
and spread of cancer by interfering directly with specific molecules involved in
growth and progression of the tumor. Also, these nanoparticulate systems can act
indirectly by stimulating the immune system to recognize and destroy cancer cells
(Colilla et al. 2010).
Drug targeting to a specific site is an important and highly attractive issue in drug
delivery as a mean for the pharmacologically active agents to instinctively differen-
tiate the site of disease. This can result in a reduction of the drug administration
frequencies, dose and also minimize the drug adverse reaction and toxic side effects
during circulation. Matsumura and Maeda postulated that passive accumulation of
mesoporous silica nanoparticles in tumor tissue could be comprehended by the
enhanced permeability and retention (EPR) effect (Matsumura and Maeda 1986).
They showed that the differential tissue localization of macromolecules, and parti-
cles of certain sizes, might be attributed to the tumor nature and microenvironment,
the comparatively slow elimination rate, and reduced lymphatic drainage.
Controlling particle size, surface charge, or hydrophobicity can facilitate the effi-
ciency of the EPR effect.
d- Controlled Drug Delivery
Double mesoporous core-shell silica spheres (DMCSS) were loaded with thymo-
quinone, which (a potential novel anticancer drug). Thymoquinone showed con-
trolled release behavior from the pores of DMCSS. Drug uptake within DMCSS
was 81 wt.% for. Furthermore, DMCSS loaded with thymoquinone was more effec-
tive in inducing cancer cell apoptosis than uncontained thymoquinone, because of
the slow release of the drug from the mesoporous structure (El-Toni et al. 2012).
Mesoporous shell formation on silica-coated magnetic nanoparticles loaded with
the anticancer drug, docetaxel, was prepared using different synthesis conditions
(El-Toni et al. 2013). The data showed that the encapsulation efficiency of docetaxel
into MCMSS samples prepared with 0.87 and 0.8 g/mL ethanol is ca. 27.54,
and 33.8 wt % respectively. Regarding in vitro docetaxel release for MCMSS sam-
ples, docetaxel was found to exhibit burst releases from the sample prepared by high
ethanol content which shows superior surface area and pore volume; however, the
release rate becomes slower over a long release time more than 168 h. The very slow
release of docetaxel from its-loaded MCMSS was attributed to the adsorption of some
portion of docetaxel molecules which takes place on the outer surface of the mesopo-
rous silica shell, even after acid washing, rather than inside their inner mesochannels.
On the other hand, MCMSS-3 showed slower release, which suggests that most of the
docetaxel molecules were trapped inside the inner cavity of mesochannels.
e- Water Purification
Mesoporous silica nanoparticles functionalized with organic functional groups
have been investigated as the proper adsorbents for the removal of environmental
pollutant due to owing high surface area, large adsorption capacity, and are easy to
be modified with other functional groups (i.e., amino functionalization) (Najafi
et al. 2011). Recently efforts were increased towards the design and synthesis of
core-mesoporous shell-based nanoparticulate systems (including hollow, solid, and
rattle-type core-mesoporous shell) to be used in water treatment. El-Toni et al.
(2014) studied the effect of amino-functionalized hollow core-mesoporous shell
silica spheres (NH2-HCMSSs) and the synthesis parameters on the removal of
heavy metal cations from polluted water. The obtained data revealed that controlling
synthetic parameters resulted in a considerable increase of the nanoparticles’ sur-

face area as well as their pore volume from 319.44 to 718.024 m2/g and 3.309 × 10−1
to 1.190 cm3/g, respectively. In addition, the heavy metal absorption capacity of the
prepared HCMSSs samples was enhanced to be 194.4, 190.5 and 193 mg/g for
Pb(II), Cd(II), and Zn(II) metal cations, respectively.
Gold Nanoparticles
Gold nanoparticles (AuNPs) are considered as one of the most studied nanotechnol-
ogy-based system nanoparticles, because these nanoparticles have potential appli-
cations as drug delivery carriers, radiosensitizers, and photothermal agents, contrast
agents, in addition to their promising potential in cancer therapy. Several types of
AuNPs are now well investigated for their properties and applications including col-
loidal gold, silica-gold nanoshells, gold-gold sulfide nanoparticles, and gold
nanorods (Pillai 2014).
Surface coating with polyethylene glycol stabilizes gold nanoparticles, thereaf-
ter; anticancer agents can be attached to the PEGylated nanoparticles. Due to their
exclusive physicochemical characteristics such as optical properties and activity to
be bound to amino and thiol groups, various biomedical applications can be obtained
by functionalizing the surfaces of AuNPs. For example, tumor-specific ligands as
transferrin, folic acid, monoclonal antibodies, and tumor necrosis factor have been
attached to the coated surface of gold nanoparticles combined with an anticancer
drug for targeted delivery to the tumor (Pillai 2014). Aurimmune (Cytimmune
Sciences, Rockville, MD) is a 27 nm gold nanoparticle coated with thiolated PEG
and attached to recombinant human tumor necrosis factor α (TNF-α; cytotoxic
immunomodulatory agent). Before gold nanoparticles (autoimmune), trials have
been done to use TNF-α in satisfactory doses for attaining anticancer response, but
these trials were not successful because of the drug dose-limiting toxicity. However,
upon using autoimmune, no dose-dependent toxicity was detected even when the
dose was as high as 500–600 microgram/m2 of TNF-α was given to patients with
solid tumors.
Silica nanoparticles coated with a thin layer of gold, called AuroShell
(Nanospectra Bioscience Inc., Houston, Texas), are another example of gold
nanoparticles applications in clinical trials. When this gold-coated silica nanoparti-
cle is irradiated with near-infrared rays from a laser source, photothermal therapy of
both head and neck cancer can be possible, because of the efficiency of gold
nanoshells in converting the incident light in to eat than nanoparticles, which is
known as AuroLase therapy (Shao et al. 2013).
2.4 Applications of Nanoparticles
Nanotechnology is considered recently as a promising approach prospective to

advance well-established drug delivery systems and to generate new formula with
novel desired characteristics and functions to provide a wide range of
pharmaceutical and medicinal applications. Nanotechnology has many applications

in life sciences research; particularly at the cell level resembles the stage for a revo-
lution for healthcare. The expected potential pharmaceutical and medical applica-
tions of nanotechnology can be achieved principally in disease diagnosis and
imaging, analysis, drug monitoring, and therapeutics. Moreover, the accessibility to
more prolonged drug delivery systems is considered as a great scientific interest and
gives anticipation for cancer therapy and in minimization of dose fluctuations espe-
cially in chronic diseases (Logothetidis 2006).
Novel nanomaterials also act as drug-delivery and drug targeting systems. The
small sizes of these nanoparticle-based drug delivery systems results in a phenom-
enon that they are not recognized by the human body, and in turn, they migrate
through cell membranes below a critical size range and are able to pass and develop
nanoscale carriers that can transport highly potential pharmaceutically active ingre-
dients (APIs) precisely to their targeted sites (Suri et al. 2007). Recently, nanopar-
ticles have evolved as one of the most promising candidates for gene delivery. The
extremely small size of nanoparticles (i.e., at least one dimension less than 100 nm)
enables the nanoparticles to achieve better tissue penetration and targeting (Angell
et al. 2016; Wong et al. 2017). Nanoparticles are described as colloidal dispersions
or solid particles having a size in the range of 10–1000 nm. The drug may be
entrapped, dissolved, encapsulated, or attached to a nanoparticle polymeric matrix.
Therefore, different nanoparticulate systems can be obtained such as nanoparticles,
nanospheres, or nanocapsules. In case nanocapsules (reservoir-type), the drug (core)
is localized to a cavity surrounded by a unique polymeric matrix, while in nano-
spheres (monolithic type), the drug is physically and homogeneously dispersed in
the polymeric matrix (Florence 2007; Henderson et al. 2014), Fig. 2.6.
Polymeric nanoparticles have been considered as successful delivery systems
due to numerous reasons such as efficient stability that may aid drug penetration,
ease formulation, and availability of scaling up the formulation process (Abdel-
Mottaleb et al. 2015; Lazzari et al. 2012; Motwani et al. 2006). There are many
Drug
(core)
< 1000 nm
Polymeric matrix (coat)
Nanospheres (monolithic type) Nanocapsules (reservoir type)
Fig. 2.6 Types of polymeric nanoparticles

biomedical applications of nanotechnology, including drug targeting, controlling

drug release, improving dissolution rate and bioavailability (Couvreur 1988;
Goppert and Muller 2005; Ma et al. 2012).
2.4.1 Enhanced Solubility and Dissolution Rate
Polymeric nanoparticles have many applications for enhancing dissolution rate and
bioavailability. Nanosized drugs are more soluble compared to usual drugs due to
the particle size of them (Ekanem et al. 2015; Saade et al. 2016). The dissolution
rate of poorly water-soluble drugs is more affected by particle size and surface area
of the drug particles. Nanosized particles may possibly demonstrate improved dis-
solution rate and saturation solubility for the reason that of the vapor pressure effect
(Adibkia et al. 2011). There are two common methods of formulating nanosized
particles. One of the two ways is, to begin with, a large substance then it’s breaking
into smaller particles using motorized or chemical method (Chen et al. 2011). The
other advance is to produce the material from molecular type via chemical pro-
cesses, allowing for the precursor particles to grow up in preferred nanosized par-
ticles known as a bottom-up approach (Farghaly Aly, Abou-Taleb et al. 2019).
Solubilization and dissolution rate improvement is generally used to increase the
bioavailability of drugs with poor water solubility. Self-emulsification and micelliza-
tion are ways for particle size reduction as nanosized particles and nanosuspensions
(Beg et al. 2011). The drug can be entrapped, encapsulated, or emotionally involved
to a nanoparticle matrix depending upon the technique of preparation (Tawfeek
et al. 2018, Abdellatif et al. 2020).
API Nanocrystals
The dissolution rate of drugs can be improved when drug solubilized as nanocrys-
tals. Nanocrystals can be formulated by nanoprecipitation, high-pressure homoge-
nization, wet milling, and spray-drying. The nanocrystals increase diffusion of the
drug into the skin. Drug nanocrystals are unlike the nanoparticles composed of a
matrix and an incorporated drug (Dizaj et al. 2015). Recently, the drug precipitated
as nanocrystals have rapidly evolved into a promising drug delivery strategy
(Rescignano et al. 2015). There are many products have been marketed such as
nanocrystals of sirolimus, aprepitant, fenofibrate, megestrol acetate, and paliperi-
done palmitate. Sirolimus nanocrystals (Rapamune®) were marketed in two formu-
lations, as oral suspensions and as a tablet (Dizaj et al. 2015). Rapamune
aprepitanttablets showed a 21–27% improved bioavailability compared to Rapamune
itself (Junghanns and Muller 2008).
API Nanosuspensions
Nanosuspension method is one of the promising methods for enhancing the dissolu-
tion rate and bioavailability of poorly water-soluble drugs (Mohammed et al. 2019).
A nanosuspension not only can improve the reduced solubility and bioavailability
of poorly water-soluble drugs but also could change the pharmacokinetic profiles of
drugs and thus enhance their safety and efficacy (Adibkia et al. 2007). The formula-
tions could also be freeze-dried and further formulated into typical dosage forms
such as capsules and tablets for oral use (Sigfridsson et al. 2011).
2.4.2 Controlled Drug Release Rates
Polymeric nanocapsules are carrier systems that present advantages including mod-
ifies in the release profiles of active compounds and their transport to the site of
action, reduced losses, and low toxicity in the surroundings and humans (Jung et al.
2004; Kulhari et al. 2014; Zhao et al. 2016). Polymeric nanoparticles are considered
important device in controlled-release systems using non-degradable polymers,
degradable, and biodegradable polymers (Aryal et al. 2013; Hu et al. 2015; Kamaly
et al. 2016). These polymers are the ideal choice for the development of polymeric
drug delivery formulations. The terms biodegradable, bio-absorbable, bio-elim-
inable, and bio-erodible are frequently used to illustrate polymers such as PLGA
and PLA as defined by the IUPAC (Vert et al. 2012).
Reducing Dose Frequency and Toxic Reactions
Carbendazim and tebuconazole were loaded in the nanoparticles which showed

decreased in their toxicity and the release profile (Campos et al. 2015). Oral sus-
tained release polymeric nanoparticles of nateglinide showed reduce dosing fre-
quency, diminish side effects, and enhance bioavailability. Nateglinide-loaded poly
E-caprolactone nanoparticles were formulated by emulsion solvent evaporation
with ultrasonication technique (Kaleemuddin and Srinivas 2013). A new approach
for producing biodegradable nanoparticles for sustained nucleic acid release is
obtained, where the nanoparticles were formed by precipitating a water-in-oil
microemulsion in supercritical CO2 (Ge et al. 2010). Nanoparticles loaded with
5-fluorouracil was investigated as a probable means to sustain the release of this
drug. 5-fluorouracil-loaded nanoparticles could be readily incorporated into a
hydrogel-based delivery system to offer sustained drug release for transepithelial
drug-delivery uses (McCarron et al. 2000).
Targeted Drug Delivery Systems
The delivery of polymeric nanoparticles has received major attention in the field of
drug targeting research. This specific mode of action leads to an accumulation of
drugs at the target site. This should reduce side effects and increase drug delivery
efficacy to the target site (Patel 1999; Rufini et al. 2006; Taniyama et al. 2005).
Targeting of nanoparticles can eventually be significantly improved when they are
ligand modified. These nanoparticles can then be used to deliver a certain number
of drugs to specific sites in the human body, have a long blood circulation time, and
are stable in the blood circulation, allowing them to reach the specific target
sites (Abdellatif, Aldalaen et al. 2018).
Up to date the enhancement of drug delivery in terms of a more controlled body
distribution to decrease side effects was focused. Different innovative drug carrier
systems in the micro- and nanometer size range were produced to overcome these
problems (Lee et al. 2010; Mogosanu et al. 2016). In theory, drug targeting follows
different mechanisms including passive targeting and active targeting.
Passive targeting of nanoparticles means the success of drug to be directly circu-
lated in the bloodstream. This is carried out by cloaking the nanoparticle with some
kind of coating materials. Numerous substances can achieve this, such as polyethyl-
ene glycol (PEG) (Costantino and Boraschi 2012; Elzoghby et al. 2016; Lee et al.
2010; Mogosanu et al. 2016). By coating with PEG to the surface of the nanoparti-
cle, it becomes hydrophilic (Abdellatif, Dalia Farag A et al. 2018). Then water mol-
ecules can bind to the oxygen molecules on PEG via hydrogen bonding, which
yields a film of hydration around the nanoparticles makes the substance antiphago-
cytic (Im et al. 2016; Szczepanowicz et al. 2016). The mechanism of passive target-
ing of nanoparticles depends on the size which should be between 10 and 100
nanometers. These sizes are established to circulate systemically for longer periods
of time. The duration of nanoparticles within circulation is modulated by its
exchanges with the environment and can be modified by altering the size, particle
figure, and surface characteristics (Makwana et al. 2015; Xu et al. 2015).
Active targeting of drug-loaded nanoparticles improves the property of passive
targeting to formulate nanoparticles for specific target sites. The active targeting can
target exclusively diseased tissue in the body by distinguishing the nature of recep-
tors on the cell for which the drug will be targeted to (Kolhatkar et al. 2011). This
kind of active targeting was found to be successful when utilizing nanoparticles for
cell-specific receptor targeting (He et al. 2016). The octreotide and somatostatin
were conjugated to the nanoparticle to target different cells that express somatosta-
tin receptor-mediated endocytosis mechanisms on their surface. This means that
active targeting was found to enhance uptake, as compared to non-conjugated
nanoparticles (Abdellatif and Tawfeek 2015; Abdellatif et al. 2016). Vapreotide was
conjugated to Qdots for active targeting somatostatin receptor in blood
cells (Abdellatif et al. 2018b). Moreover, the cetuximab was coated with octreotide
for enhanced solubility and targeting of somatostatin recepors expressed in colorec-
tal cancer cells (Abdellatif, Ibrahim et al. 2020)
Additionally, nanoparticles have the facility to be activated by a trigger that is

precise to the target site, such as using supplies that are pH reactive. Most of the
body has a consistent, neutral pH (Galvin et al. 2012). Nevertheless, some parts of
the body are more acidic in nature than others, and, thus, nanoparticles can obtain
the advantage of this ability by releasing the drug when it reaches a specific pH
(Noyhouzer et al. 2016).
By formulating drug-loaded nanoparticles, both passive and active targeting has
the advantage to overcome a conventional drug delivery associated drawbacks. It is
capable to circulate all over the body for an extended period of time until it is suc-
cessfully reached to its target throughout utilizing of cell-specific ligands, magnetic
positioning, or pH reactive materials (Yang et al. 2015). Active targeting is capable
also to be achieved through peptide-based drug targeting system (He et al. 2012).
Cellular Uptake Induction
The cellular uptake of nanoparticles may be induced by many factors such as size,
shape and surface properties of the particles (Choi et al. 2010; Pang et al. 2002; Xin
et al. 2012; Xu et al. 2016b; Yang et al. 2011). There are different types of endocy-
tosis for particles by cells including phagocytosis, macropinocytosis, clathrin-medi-
ated endocytosis, caveolae-mediated endocytosis and clathrin-caveolae-independent
endocytosis (Conner and Schmid 2003; Mayor and Pagano 2007; Verma et al.
2008). The process by which cells engulf big particles such as bacteria is named
phagocytosis. Krepetic et al., displayed the cellular uptake of 22.6, 34.2, and
43.4 nm sized NPs through phagocytosis in murine macrophages (Krpetic et al.
2010). The process by which cells engulf liquid and solutes is named macropinocy-
tosis (Rossman et al. 2012; Swanson and Watts 1995). The most important mecha-
nism for receptor-mediated uptake is called clathrin-mediated endocytosis, Fig. 2.7.
There are numerous examples of receptor-mediated endocytosis which figured by a
ligand binding to its receptor. Differently sized (14, 50, and 74 nm) coated AuNPs
spheres or rods were taken up into Hela cells via a receptor-mediated endocytosis
(clathrin-dependent) (Chithrani et al. 2006). The internalization of folate conjugated
to PEG-coated-Qdots was confirmed via receptor-mediated internalization in cells
express folate receptors (Song et al. 2009).
When small flask-like shaped plasma membrane invaginates to engulf nutrients
or solute (i.e. cholesterol uptake, solute transport, tumor suppression), it is called
caveolae-mediated endocytosis (Hao et al. 2012). The internalization mechanism of
the small-sized app. 4.5 nm AuNPs by living HeLa cells showed that the delivery of
nanoparticles was inhibited when the cholesterol was depleted with methyl-cyclo-
dextrin. Sucrose did not interrupt endocytosis indicating that the caveolae-mediated
endocytosis is the preferred pathway for the intracellular delivery of small-sized
AuNPs (Hao et al. 2012). Clathrin- and caveolin-independent endocytosis are still
largely unexplained. It is only described in a few examples, such as the recovery of
membrane proteins in neurons or the internalization of the IL-2 receptor on lympho-
cytes, Figs. 2.7 and 2.8 (Conner and Schmid 2003; Mayor and Pagano 2007).
Pinocytosis
Macropinocytosis
Phagocytosis
Clathrin- Caveolin- Clathrin- and
mediated mediated caveolin-
endocytosis endocytosis independent
endocytosis
Fig. 2.7 Schematic diagram for endocytic pathways differ with regard to the size of the endocytic
vesicle, the nature of the cargo (ligands, receptors, and lipids) and the mechanism of vesicle
formation
Fig. 2.8 Clathrin-mediated endocytosis (1) Ligand binds to a specific receptor. (2) Invagination of
the cell membrane, clustering of the ligand-receptor complexes and (3) formation of clathrin-
coated pits. After pinching off of the cell membrane the ligand-receptor complexes are sequestered
in clathrin-coated vesicles (4). Clathrin depolymerizes and proton influx acidifies the early endo-
somes (pH ∼ 6) (5). Several early endosomes can fuse to build late endosomes (pH ∼ 5–6) from
which the receptors can be recycled after the release of the ligand (7) or fuse with lysosomes
(pH ∼ 5–5.5) (8) leading to degradation
Nanoparticles could help to investigate these routes of uptake due to their small size,
narrow size distribution, easy detectability, and ligand specificity.
Receptor-mediated endocytosis is an important example of a specific internaliza-
tion mechanism following ligand binding to their receptor (Thomsen et al. 2012;
Wang et al. 2012; Zhang et al. 2012). It allows for an import of extracellular mole-
cules for around 1000-fold increase of the intracellular concentration of
macromolecules (Chen et al. 2008; Delehanty et al. 2009; Kelf et al. 2010). In the
process of endocytosis, the plasma membrane is engulfed inwards by specialized
membrane micro-domains forming either clathrin or caveolin-coated pits (Kelf
et al. 2010).
Studies show that, the attachment of PEG on the surface of particles reduces the
non-specific internalization of particles by cells (Gopee et al. 2009; Kim et al. 2010;
Owens 3rd and Peppas 2006; Poulose et al. 2012; Zhao et al. 2010). This is because
the PEG-layer supports a steric hindrance during interaction with surfaces. Also,
direct attachment of nanoparticles to the cell membrane is obstructed, as well as
the no protein adsorption on the surface of NPs that would otherwise enhance inter-
nalization. However, the specific uptake of nanoparticles via surface receptors can
be increased either by direct interactions between coated particles and receptors or
via ligands attached to nanoparticles (Hild Breunig and Goepferich 2008; Kelf et al.
2010; Osaki et al. 2004).
Polymeric nanoparticles can be prepared to provide opportunities for the site-
specific delivery of medication after injection into the circulation. Nanoparticles can
be used to target different organ sites in the body, such as the lung, inflammation
sites, liver, spleen, bone marrow, and tumors (Fan et al. 2012; Xu et al. 2012; Yoon
et al. 2012). Nanoparticles show enormous promise in the field of tumor imaging,
drug delivery, and the early identification of malignant tissue (Portney and Ozkan
2006). Moreover, polymeric nanoparticles could target different ocular sites such as
the cornea, retina, and choroid by surficial applications and intravitreal injection
(Zhou et al. 2013). Polymeric micelles in nanosuspension formulated with N,
N-methylene bis-acrylamide (MBA) showed rapid treatment and higher anti-inflam-
matory activity for a longer duration when compared to an aqueous suspension of
the same drug (Rafie et al. 2010).
2.5 N
anoparticle Medicinal and Pharmaceutical
Applications: Prospective Clinical Challenges
As the application of nanoparticle-based drug delivery systems developed exten-

sively, the FDA supplies regulatory systems for their pharmaceutical trials.
Nanomedicine products manufacturers should provide the FDA with data accumu-
lated from preclinical studies on animals, human, or cell culture before clinical tri-
als are launched. The researchers in the preclinical studies, determine the
biochemical, pharmacological, and toxicological effects of nanoparticle-based drug
delivery systems. In addition to the safe human dose, the FDA approves the starting
of phases I, II, and III. If the nanoparticle-based drug delivery system is then safe,
the manufacturer can market the drug in the United States, and during marketing,
data must be gathered from the clinical data for any adverse reactions (McNamee
2014). The FDA has added regulatory guidelines for nanoparticle products and they
are treated as new drugs, and such guidelines were reported on 2007: “FDA’s
authority over products subject to premarket authorization is comprehensive and

provides FDA with the ability to obtain detailed scientific information needed to
assess the safety and as applicable, effectiveness of products, including relevant
effects of nanoscale materials” (FDA 2007).
Nanoparticle-based drug delivery systems and medicinal devices are expected to
face series of challenges during their clinical application. These expected chal-
lenges may be due to the biological and technological changes and study design
as well.
2.5.1 Biological Challenges
The biological challenges that may encounter during applications of nanoparticle-

based drug delivery systems and devices that may limit their success and efficacy,
including the modification of nanoparticle biodistribution or controlling rate of
nanoparticle passage through the biological barriers and into target cells. A variety
of the approved and clinically investigated nanoparticle-based drug delivery sys-
tems are either PEGylated or PEG-terminated. Pegylation of nanoparticles surfaces
minimizes the interaction of these nanoparticles with immune cells and delay their
clearance by immune cells (Moghimi et al. 2001). Therefore, nanoparticles can
reside in circulation for longer periods of time resulting in increasing the possibility
of reaching and entering target sites, e.g., targeting tumors by enhanced permeation
retention (EPR) effect (Golombek et al. 2018).
2.5.2 Technological Challenges
Scaled-up of formulation and synthesis of nanoparticle-based drug delivery sys-

tems, optimization, and predictions of their performance will be a crucial issue in
safeguarding the clinical achievement of upcoming nanoparticle-based formula-
tions (Anselmo and Mitragotri 2016). Characterization procedure, choice of equip-
ment, robust formulation and stability are addressed as four essential prerequisites
for the production of formulations and the scale up of drug nanocrystals (Srivalli
and Mishra 2015).
2.5.3 Study-Design Challenges
The approval and design of the clinical study in humans in another important issue
in regard for evaluating the clinical efficacy of nanoparticle-based drug delivery
systems and new formulations. Definitely, study size and the timing of nanoparticle
therapies in a treatment regimen can influence how results from clinical studies are
perceived. As such, clinical results greatly influence future nanoparticle clinical

studies; special attention must be given to ensure that clinical trials are designed to
extract the most information regarding nanoparticle interactions, fate, and function
while still testing key hypotheses (Hassani et al. 2013).
2.6 Conclusion
Nanonized drug (API) particles, biodegradable polymeric nanoparticles, and hydro-

phobic nanoparticles stated here are some of the best applicants for the development
of more professional nanoparticle-based drug delivery vehicles. Most of the devel-
oped nanoparticles guided to resources with lower toxicity, high biocompatibility,
enhancing drug solubility and dissolution rate. Further, these delivery systems pro-
tect drug molecules from rapid degradation. All successfully reported method of
nanonization techniques are established either based on physical methods including
primary and multiple emulsion solvent evaporation methods, ionic gelation, spray-
drying, supercritical fluid technology, as well as precipitation with a compressed
fluid techniques anti-solvent or on chemical synthesis schemes such as silica
nanoparticles of variable internal structures. Moreover, these nanonized systems
play the main role to reach site-specific drug delivery compared to conservative dos-
age forms due to their benefits in site specificity and stability. In addition, the sur-
face properties play an essential role in targeting the active drug molecule to its
specific site with the minimal dose and reduced dosing frequency. Nevertheless, the
final product or the procedure is used to make such effective and targeted nanopar-
ticles; it is perfect that much more effort is required to develop a highly safe NPs for
medical applications.
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Chapter 3
Applications of Iron Oxide Nanoparticles
in the Magnetic Resonance Imaging
for the Cancer Diagnosis
Kanwal Akhtar, Yasir Javed, Muhammad Imran Akhtar, and Naveed A. Shad
Contents
3.2 M RI as Diagnostic Technique 119
3.3 D esign Considerations of IONPS as Contrast Agents for Tumor Targeting 121
3.3.1 Surface Modification of IONPs Through Organic and Inorganic Materials 125
3.4 Role of IONPs in Cancer Detection as Contrast Agents 129
3.5 IONPs-Mediated MRI Diagnosis of Different Cancer Types 131
3.5.1 Brain Cancer Detection 132
3.5.2 Breast Cancer Detection 134
3.5.3 Gastric Cancer Detection 134
3.5.4 Ovarian Cancer Detection 136
3.5.5 Liver Cancer Detection 136
3.5.6 Colorectal Cancer Detection 138
3.5.7 Lung Cancer Detection 140
3.5.8 Pancreatic Cancer Detection 140
3.6 Conclusion and Perspective with Future Outlook 141
References 144
Abstract Magnetic resonance imaging (MRI) is considered as one of the most

powerful tools for diagnostic purposes, which provides the detailed compositional
and structural information of tumors with enhanced spatial resolution. Contrast
agents having molecular specificities are being introduced as diagnostic probes
which alter the relaxation times of local protons and lead towards binding of s pecific
K. Akhtar · Y. Javed (*)

Magnetic Materials Laboratory, Department of Physics, University of Agriculture,
Faisalabad, Pakistan
M. I. Akhtar
Radiology Department, Allied Hospital, Punjab Medical College, Faisalabad, Pakistan
N. A. Shad
Department of Physics, Government College University Faisalabad, Faisalabad, Pakistan
https://doi.org/10.1007/978-3-030-44925-4_3
116 K. Akhtar et al.
contrast agents with extracellular matrix components or cell surface receptors. Iron
oxide nanoparticles (IONPs) are considered as most compatible contrast agents in
MRI because of their relaxation rates for both transverse and longitudinal compo-
nents for tumor imaging. For cancer detection, IONPs have been greatly employed
due to low side effects, inhibiting metastasis and reduce multidrug resistance.
Exogenously administrated IONPs allow specific compartment enhancement of
tumors and improve imaging ability of interstitial volume and functional blood. This
chapter will deal with the general MRI principle, synthesis protocols, and contrast
mechanisms and then use these nanoparticles for detection of different types of can-
cer. For increasing specificity and imaging sensitivity, improved synthesis methods
render the existing probes for diagnostic applications even at lower concentrations.
Keywords Iron oxide nanoparticles · Biomedical application · Magnetic

resonance imaging · Magnetic properties · Contrast agents · Cancer detection ·
Tumor targeting · Toxicity · Relaxivity values · Medical imaging
3.1 Introduction
Although significant cancer survivals have been noted from different cancer types
over the last three decades, still 1,658,370 cancer patients and 589,430 causalities
have been reported in 2015 in United States. In tumor fatality, this continuous and
notable decrement is greatly affiliated to prevention, early detection, and therapeu-
tic approaches (Siegel et al. 2015). Detection of cancer at early stages is challenging
before metastasis cancer cells appear because clinical symptoms show up at later
stages. Therefore, minimal noninvasive methods like MRI are currently used for
detection of tumors at early stages. In this aspect, many IONPs-based contrast
agents are used for sensitive imaging to detect tumors at early stage (Bakhtiary et al.
2016). Until now, approved IONPs through Food and Drug Administration (FDA)
have been extensively used as contrast imaging agents in MRI and for treatments of
iron deficiency and drug carriers in clinical and preclinical experiments (Fütterer
et al. 2013). IONPs are used increasingly in clinical practice with broad and imme-
diate implications in anemia treatments, cancer therapy, cancer diagnosis, and their
effects on tumor microenvironments (Corot et al. 2006; Li 2014; Neuwelt et al.
2007; Weissleder et al. 2014; Zanganeh et al. 2016). In addition to this, researchers
are also working to combine two different therapies in a single system due to inef-
ficiency of one therapy. For this, gold is also an important candidate due to its plas-
monic properties which are being evaluated for so-called photothermal therapy. In
this technique, gold nanoparticles can be excited by light of suitable wavelength
especially with laser or near-infrared light. Hybrids or core shell structures of iron
oxide and gold can be used in this way for both types of therapies, i.e., magnetic
hyperthermia and photothermal (Hoskins et al. 2012).
MRI is a promising noninvasive imaging methodology which provides deep tis-
sue information inside the body. MRI is categorized into two classes: longitudinal
relaxation-weighted images (T1 contrast) and transverse relaxation-weighted images
3 Applications of Iron Oxide Nanoparticles in the Magnetic Resonance Imaging… 117
(T2 contrast) based on relaxation process (Javed et al. 2017; Jun et al. 2008b). In
MRI, contrast agents are used for improved visualization of the internal body struc-
ture. IONPs with the diameter more than 10 nm are usually preferred to be used as
negative contrast agents, while diameter less than 5 nm is currently emerged as posi-
tive contrast agents. When size is less than 5 nm, magnetization along longitudinal
axis is suppressed which results in enhancement of transverse component of relax-
ivity and consequently positive contrast. Diagnosis with treatment of deadly disease
like cancer is very challenging due to traditionally limited use of clinical approaches
such as chemotherapy, surgery, and radiotherapy (Alvarez-Berríos et al. 2016;
Nguyen et al. 2014; Tolentino et al. 2011).Therapeutic effects of noninvasive meth-
ods are highly desirable due to time monitoring effects for optimizing therapeutic
strategies. Due to intrinsic properties such as strong magnetizing ability, superpara-
magnetic IONPs exhibit strong superparamagnetic behavior which makes them
promising candidate as contrast agent (Chen et al. 2006; Ifediba and Moore 2012;
Medarova et al. 2016). MRI is used to detect early cancer stage owing to its enhanced
abilities to distinguish healthy tissues from malignant ones based on different com-
positions in tissues (Ifediba and Moore 2012; Stevens et al. 2005).
In tumor microenvironment, locally and systematically administrated IONPs are
incorporated with tumor-associated macrophages. This effect is widely used for
malignant tumor detection, immune cancer therapies (Ansari et al. 2014; Daldrup-
Link and Coussens 2012; Klenk et al. 2014), and shuttling IONPs for conjugated
therapeutic drugs in tumors (Miller et al. 2015; Vinogradov et al. 2014).
Superparamagnetic IONPs have many applications in medicine field and have rapid
development emphasis on their use in MRI as contrast agents (Gupta and Gupta
2005; Karakatsanis et al. 2016). Positive contrast agents are preferred frequently for
diagnostic practice, but toxicity issues and high mobility in vascular system shorten
their presence. These drawbacks motivate researchers to develop ultra-small super-
paramagnetic IONPs-based contrast agents. IONPs act as negative contrast agent
(via outer sphere mechanism) because of their enhanced proton relaxivity values
and high magnetic moments. To achieve negative contrast, IONPs can be used to
decrease relaxation time T2. IONPs are employed as contrast agents because they do
not cause any major toxicity issues in human (LaConte et al. 2005; Shan et al. 2016).
Currently available MRI agents are based on IONPs because of their low toxicity.
Non-targeted IONPs-based contrast agents do not effectively differentiate normal tis-
sues from cancer tissues. But targeted IONPs that are equipped with special moieties
can recognize affected tissues from normal ones (Jung and Jacobs 1995; Yigit et al.
2013). Different types of cancer develop multidrug resistance during traditional thera-
pies. It is one of the major impediments to the success of cancer diagnosis and treat-
ment, which results when a disease becomes tolerant to specific pharmaceutical
treatment. Initially, many types of cancer are susceptible to the chemotherapy.
However, through different mechanisms, resistance is being developed. Two molecu-
lar pumps named as p-glycoprotein and multidrug resistance-associated proteins in
tumor cell membrane alter the drug accumulation level, and cells become resistant to
the anti-cancerous drugs (Li et al. 2013; Morales et al. 2003).
Due to higher cellular uptake, IONPs are being used to create difference between
infected and healthy tissues in tumors. Contrast agents having two imaging capa-
bilities make them promising candidate in biomedical preclinical trials for cancer
diagnosis (Laurent et al. 2014; Yoo et al. 2014). Contrast agents in tumoral uptake
are validated by imaging through noninvasive MRI. The oligonucleotide therapeutic
component may have double standard small interfering RNA (siRNA) molecule to
suppress disease causing mRNAs or antisense to inhibit overexpressed microRNA
(miRNA) in cancer (Fig. 3.1). Controlling irregular gene expressions is considered
as most important implications for cancer therapeutics (Ghosh et al. 2014). Clinical
management for cancer treatment reflects well-balanced relation between toxicity
and efficacy. Combination therapies clearly improve progression time (as compared
to sequential monotherapy that results in increased toxicity) and response time
(Mahmood and Weissleder 2003; Na et al. 2009).
Large-scale use of IONPs-based contrast agents in MRI is still challenging and
needs to be fulfilled due to some current limitations of inefficiency and detection
sensitivity of tissue specificity. IONPs with diameter range less than 4 nm are used
preferably for long circulation in blood (Babes et al. 1999; Wang et al. 2001).
Targeting
agent
Diagnostic
imaging
Biocompatible polymer
coating
Fig. 3.1 Multifunctional nanoparticles for MRI contrast and treatment

Transverse relaxation time (T2) depends strongly on magnetic moments which are
related to volume and size control of NPs directly. For optimal results, better control
over surface ligands distribution is delicately required. Lack of single molecular
level targeting and inaccurate quantification for cellular disorders are also other
limitations of IONPs used for diagnostic purposes (Bulte and Kraitchman 2004;
Wang 2011). Therefore, specific design strategies are required to overcome these
limitations and enable IONPs to recognize special surface moieties for targeting
specific cell organelles. Clinically, IONPs should address with magnetic core mate-
rials and ligands for their effective pharmacokinetics profiles. This review summa-
rizes superparamagnetic IONPS as contrast agent in MRI for imaging and early
detection of major cancer types.
3.2 MRI as Diagnostic Technique
Noninvasive imaging techniques are considered as more powerful tool that not only
use early identification of lesions but also provide complete coverage with repetitive
measurements, which are not possible with typical invasive biopsy techniques. In
imaging voxel, low level of cell receptor concentration strongly restricts our choice
for high-sensitive imaging modalities for detection. Therefore, many imaging tech-
niques are used such as single-photon emission computed tomography, positron
emission tomography (PET), and MRI. Among these mentioned techniques, MRI is
preferred as clinically used diagnostic imaging technique (Artemov et al. 2003;
Wang et al. 2016). Magnetic imaging is considered as in vivo analysis with com-
plete characterization of biological phenomena even at cellular/molecular levels.
MRI does not depend on simple reflection/transmission of energy. Highest resolu-
tion that can be achieved in MRI is in the range of magnitude of the wavelength used
in whole process. Underlying biology opens a new arena for scientists that lead
towards development of novel agents in imaging technologies with modification in
signal amplification strategies. Through MRI techniques, early detection becomes
possible through specific image targeting. Magnetic resonance images are used to
represent tumor boundaries (Lurie 2016; Tari 2017).
To image the specified molecules through sensitive and high-resolution MRI,
many factors are important that must be met: (1) accessibility to high-affinity probes
to get reasonable pharmacodynamics, (2) high-affinity probe’s ability to get over
biological delivery barriers (such as cell membrane, interstitial, and vascular), and
(3) use of biological/chemical strategies (Huang et al. 2012; Lurie 2016). MRI is
becoming competitive and popular molecular imaging modality due to its detailed
anatomical detail and high spatial resolution. To make MRI a more sensitive tech-
nique, contrast agents having high relaxivity values are required (Geraldes and
Laurent 2009; Huang et al. 2016). Iron concentration has been directly related with
relaxation time (T2) measured with MRI. During imaging experiments, MRI relax-
ivity measurements are used to determine specified amount of IONPs that have been
accumulated in the tumors experiments (Eghbali et al. 2016; Jeener 2002)
(Table 3.1).
Table 3.1 Characteristics of IONPs explored for medical imaging experiments

Targeted contrast Experimental
media Biological target condition Pharmacophore Results
Magnetic iron oxide Inflammation In vivo Antibodies In vivo
nanoparticles accumulation
conjugated with (with no control
polyclonal human of magnetic iron
immunoglobulin G oxide
(Jun et al. 2008a; nanoparticles
Mornet et al. 2004) uptake) in
inflammation
USPIO-monoclonal In colon carcinoma, In vivo Monoclonal Specific binding
antibody (610) surface antigen on In vitro antibodies of monoclonal
(Corot et al. 2006; cell line antibody coated
Laurent et al. 2008) USPIO on tumors
for in vivo and
in vivo analysis
Arabinogalactan- Asialoglycoprotein In vivo Arabinogalactan In vivo: high
coated ultra-small receptor on In vitro tumor contrast in
superparamagnetic hepatocytes liver cancer
iron oxide particles patients
(Corot et al. 2006; In vitro: primary
Lee et al. 2015; Prata tumor diagnosis
et al. 2006)
USPIO-C2 (Laurent Phosphatidylserine In vivo Synaptotagmin I Apoptotic cell
et al. 2008; Zhao of apoptotic cells In vitro Protein detection in
et al. 2001) tumors by in vivo
and in vitro
analysis
USPIO-peptide uMUC-1 In vivo Peptide USPIO-EPPT
(EPPT) (Corot et al. In vitro accumulation by
2006; Moore et al. in vivo and
2004) in vitro means on
adenocarcinoma
tumors
USPIO-polyethylene Folate receptor In vitro Folic acid Internalization of
glycol-folate (Burtea (FR) USPIO-PEG-
et al. 2016; Laurent folate in BT-20
et al. 2008; Richard cancer cells
et al. 2016a; Zhang
and Zhang 2005)
USPIO-folate Folate receptor In vivo Folic acid In vitro
(Lameijer et al. In vitro internalization
2013; Li et al. 2004; and binding on
Sonvico et al. 2005) FR +-KB tumor
cells
3.3 D
esign Considerations of IONPS as Contrast Agents
for Tumor Targeting
Contrast agents based on iron oxide exhibit tremendous effects for detection of dif-
ferent diseases due to their unique properties at the nanoscale that result in remark-
able interaction of mononuclear phagocytic cells, biological barriers, blood protein,
macrophage in tissues, cancer cells, etc. These interactions are strongly determined
with physiochemical surface properties and size of the magnetic NPs. So IONPs of
different morphologies and functionalities have been employed as contrast agents in
diagnostic methodologies (Javed et al. 2014; Lartigue et al. 2013) (Fig. 3.2).
Therefore, size distribution, magnetic susceptibility, surface physiochemical prop-
erties, and hydrodynamic size of these IONPs are considered as most important
parameters for their applications in MRI. Hydrodynamic size is a more prominent
parameter than size of IONPs in dry state for in vivo applications, because human
body is a water-loaded biological system. That is why iron oxides as contrast agents
are preferred over other magnetic material in noninvasive diagnostic techniques like
MRI (Qiao et al. 2009).
Considering hydrodynamic size criterion, contrast agents based on IONPs are
broadly classified into small particle iron oxide (hydrodynamic size greater than
40 nm) and ultra-small particle iron oxide (hydrodynamic size less than 40 nm)
(Wang et al. 2013). Small-sized IONPs are taken quickly by reticuloendothelial
system that eventually accumulated in the spleen or liver to purify blood. Therefore,
small-sized IONPs have been widely used for tumor/lesion detection in the liver,
whereas ultra-small IONPs do not accumulate in reticuloendothelial system and
lead towards longer blood circulation time. Ultra-small IONPs with size less than
Ligand Exchange with hydrophilic ligands
Metal Precursor
Hydrophobic Shell
NP
Surfactant NP
Synthesis Protocols
Solvent - Co-precipitation
- Water - Thermal decomposition NP
- Polyol - Hydrothermal
- Oxalic acid etc. - Sol gel etc.
Encapsulation with organic or inorganic shell

- Organic Shell
- - Amphiphilic polymer
- - Polyacrylic acid (PAA)
- - Polyethylene Glycol (PEG) etc.
- Inorganic Shell
- - Gold
- - Gadolinium etc.
Fig. 3.2 Steps involve for the synthesis of biocompatible iron oxide nanoparticles
10 nm, when injected intravenously, can produce hypo-intense signals with accu-
mulation in lymph nodes contrary to pre-contrast stage, where metastatic nodes
with less macrophage appear iso-intense. For this reason, ultra-small IONPs are
used potentially for diagnosis in lymph node metastases (Javed et al. 2017). These
nanoparticles are also used for in vivo breast cancer detection (Harisinghani et al.
2003; Jae-Hyun et al. 2007) (Table 3.2).
With reduction in size of materials near single magnetic domain, a phenomenon
called superparamagnetism emerged. Superparamagnetic materials show no reten-
tivity values unlike ferromagnetic counterparts after the removal of external mag-
netic field. At room temperature, IONPs (magnetite and maghemite) with core
diameter (approximately 20 nm) become superparamagnetic (Frey et al. 2009; Teja
and Koh 2009). Superparamagnetic IONPs are widely used in many applications
including magnetic cell sorting (Groman et al. 2007; Schellenberger et al. 2004),
call labelling (Song et al. 2005), tissue repair (Gupta and Gupta 2005), and cancer
treatment (Sonvico et al. 2005; Wu et al. 2007). Among all these applications, MRI
is one of the most fascinating applications of superparamagnetic IONPs as contrast
agents for diagnosis of cancer (Li et al. 2008a; Teja and Koh 2009). For this purpose
IONPs with high control on surface characteristics, size, shape, and magnetic prop-
erties are required (Dos Santos Coelho et al. 2008; Xu and Teja 2006). IONPs are
preferred over other traditional contrast agents due to their relatively low cytotoxic-
ity, improved delineation margins of tumors with low sensitivity (Landmark et al.
2008), long-lasting enhancing contrast agents, and high magnetic signal strength
(Corot et al. 2006). The following synthesis protocols are used widely and divided
into gas phase and chemical phase methods.
Gas Phase Method Gas phase method used for the synthesis of IONPs depends on
disproportionation, thermal decomposition, reduction, oxidation, or other reaction
involved in gas phase to form precipitates from solid products. For example, in
chemical vapor deposition (CVD) method, a gas stream continuously delivers to
precursors by gas delivery system in reaction chamber where at high temperature
(greater than 900 °C) vacuum is maintained. Due to reactions taking place inside
heated reaction chamber, NPs or clusters of NPs are formed (Atchudan et al. 2015).
Aggregation and growth of particles started at outlet of the reaction chamber due to
rapid expansion of two phase gas stream. For structural and compositional modifi-
cations of the synthesized NPs, they are subjected to heat treatment in different
types of highly purified gas streams (Khalil et al. 2017). To deposit iron oxide
through halide reaction (i.e., iron trichloride in water at 80–100 °C), CVD process
is usually employed. At low pressure (less than 1 Torr) and temperature ranges
(300–800 °C), metallo-organics can be used as precursor to start the reaction in
CVD process (Atchudan et al. 2015; Ho et al. 2011). Through thermal decomposi-
tion, iron oxide thin films were obtained with iron trifluoroacetylacetonate and acet-
ylacetonate at temperature ranges (300 °C, 400–500 °C), respectively. Some other
precursors such as tris(tbutyl-3-oxo-butanoato) Fe(III) and tris(2,2,6,6-
tetramethyl-3,5-heptadionato) Fe(III) are also reported. Park et al. used Fe(II) dihy-
dride complexes H2Fe[P(CH3)3]4 to deposit magnetite thin films in oxygen presence
Table 3.2 Commercially available ultra-small IONPs with physiochemical features (Corot et al. 2006; Daldrup-Link et al. 2011; Iv et al. 2015)
Size of Blood Blood
Hydrodynamic crystal core half-life (in half-life r1 relaxivity r2 relaxivity
Coating (reference) Name size (nm) (nm) human) (rodents) (mM−1 s−1) (mM−1 s−1)
Carboxydextran (Mishra et al. 2016; Ferumoxytol 28–32 6.4–7.2 10–14 h 67 min 38 83
Nosrati et al. 2017; Ulbrich et al.
2016)
Dextran T-10 (Liu et al. 2016; Weissig Ferumoxtran-10 20–50 5.8–6.2 >24 h 2–3 h 23 53
and Guzman-Villanueva 2015)
Carboxydextran (Aryal et al. 2013; Iv Ferucarbotran 20 <5 6 h 35 min 24 60
et al. 2015) C
3 Applications of Iron Oxide Nanoparticles in the Magnetic Resonance Imaging…
123
at 300 °C (Park et al. 2006). Low-pressure CVD can be used to achieve ferric
dipivaloylmethanate as surfactant for magnetite direct growth. These films after oxi-
dation were changed into maghemite (Baaziz et al. 2014; Salas et al. 2012).
Liquid Phase Method Liquid phase methods provide better yields and easy sur-
face treatments and are considered most effective. Among other aqueous solutions,
coprecipitation is most often applied. This method is carried out by mixing ferrous
and ferric ions in 2:1 molar ratio, which is a highly basic solution at elevated room
temperature. Shape and size of the IONPs depend on several reaction parameters,
e.g., ratio of ferrous and ferric ions, pH value, and ionic strength of medium. This
synthesis technique can affect critically the chemical and physical properties of
IONPs (Teja and Koh 2009). Usually, nanostructured materials have smaller satura-
tion magnetization values as compared to bulk materials, provided no alteration in
ionic configuration. Reported saturation magnetization values of IONPs vary in the
range of 30–80 emu/g smaller as compared to the Ms = 30–89 emu/g value of bulk
material. Under ambient conditions, Fe3O4 NPs are not much stable and dissolved
easily in an acidic media or oxidized Fe2O3. To avoid the oxidation issues, prepara-
tion of Fe3O4 is done under anaerobic conditions. To synthesize Fe2O3, annealing
treatment under the presence of oxygen or oxidation is done (Cushing et al. 2004).
However, oxidation is not considered as much influencing factor because of its own
chemical reactivity/stability in an acidic or alkaline environment. This technique
results particles with wide size distribution. Sugimoto et al. prepared the spherical-
shaped magnetite NPs with a diameter range of 30 to 100 nm by reaction of oxidant
(nitrate ions), Fe(II), and base in an aqueous solution (Sugimoto and Matijević
1980). To obtain the homogeneous spherical NPs of maghemite or magnetite, mix-
ture of ferric and ferric hydroxides reacted in aqueous solutions. Size and phase of
NPs depend upon solution pH, cationic concentration, and counter ions present in
the solution. By adjusting the ionic strength and pH of solution, mean size of the
NPs can also be controlled (Gupta et al. 2007).
Sol-gel is another progressively used liquid phase method for the synthesis of
metal oxide NPs because it is cost-effective and provides high purity and good
homogeneity. For modification of precursors, this method requires low temperature.
For magnetite synthesis, metal organic precursors can be used. To achieve high
crystallinity and uniformity, other techniques can be employed, but many toxic
reagents and complex synthetic steps are involved (Xu et al. 2007). Sol-gel method
usually involves many inorganic species with hydrolysis and condensation reac-
tions. Reactive groups released alcohol molecule at the start of chemical reactions
from water molecule. NPs are formed with polycondensation and polyaddition
reactions. Kinetics of hydrolysis and condensation is influenced by pH, solvent
nature/type, concentration of precursor, and temperature (Jitianu et al. 2006).
Thermal decomposition is used to synthesize highly crystalline and monodisperse
NPs to avoid many limitations. Thermal decomposition usually offers good control
over growth and nucleation mechanisms through two different routes. One route
involves controlled heating of compounds for nuclei formation in surfactant solution,
whereas organometallic compounds are directly injected in hot surfactant solution in

other route, which results in nuclei formation (Sun and Zeng 2002; Woo et al. 2004).
3.3.1 S
urface Modification of IONPs Through Organic
and Inorganic Materials
In this section, various strategies and recent advancement for surface functionaliza-
tion of IONPs through organic and inorganic materials are discussed. Stability of
ferrofluids is most important for preparation and storage purposes. Organic materi-
als are effectively employed to modify the IONPs surface during/after synthesis
protocols to avoid the agglomeration. Inorganic material is also emerged as a poten-
tial candidate for coating of IONPs, which on one way provide stability and other
way add its additional properties to the new system.
Surface Functionalization of IONPs Through Organic Materials IONPs pos-
sess hydrophobic surfaces with enhanced surface-to-volume ratio and without
proper coatings at the surface of NPs; hydrophobic interactions lead towards large
cluster formation due to aggregation of these NPs. Additionally, use of magnetic
IONPs in biomedical applications requires biomolecules to be employed for bio-
compatibility. Generally iron oxide is considered as core, and combine structure can
be categorized into shell-core-shell, matrix and core-shell. Core can be of any type
of IONPs (i.e., maghemite or magnetite) in these structures (Wu et al. 2008). Shells
may be composed of any kind of organic materials, shell–core, and mosaic core or
iron oxide cores of organic shells. In last type, IONPs are connected to the organic
core through chemical bonds, whereas mosaic structure is composed of organic-
based shell layers coated with IONPs. Among different types of matrices, polymers
are mostly used materials. Organic molecules provide ensembled reactive func-
tional groups such as carboxyl groups, aldehyde groups, amino groups, and hydroxyl
groups. These groups can link actively to the bio-substances (i.e., DNA, enzyme,
and antibody) for their role in biomedical applications. Organic macromolecules,
small molecules, biological molecules, and polymers can functionalize the corre-
sponding properties of IONPs (Gupta and Gupta 2005). In this section, different
coating molecules have been discussed.
Polymers In the recent past, functionalization of IONPs through polymers has

gained much attention due to the advantage of strong repulsive forces which bal-
anced the van der Waals forces of attraction and magnetic forces acting on NPs.
Additionally, polymer coatings also provide high potential in several applications.
That is why surface modification of IONPs through polymer coatings enhances the
chemical and physical properties. Careful activation and passivation of the poly-
mers and their reaction conditions may result in formation of NPs with desired and
tailored properties (Gupta and Wells 2004; Raviv et al. 2003). Polymer materials
used for coating purposes can be classified in to natural and synthetic polymers, and
few are listed in the Table 3.3.
Table 3.3 Advantages of macro-organic molecules functionalized with IONPS

Polymers
class Coating material Advantages References
Synthetic Poly(lactide Enhances the biodegradability, Chen et al. (2008) and
polymer acid) biocompatibility, and low toxic Gómez-Lopera et al.
(2006)
Synthetic Poly(ethylene Improves water solubility and Mondini et al. (2008)
polymer glycol) (PEG) hydrophobicity, enhances blood and Paul et al. (2004)
circulation and biocompatibility
Synthetic Poly(vinyl Increases monodispersibility, prevents Chastellain et al.
polymer alcohol) aggregation (2004) and Pardoe
et al. (2001)
Synthetic Polyacrylic acid Improves bioconjugation, Arbab et al. (2003) and
polymer biocompatibility, and stability Shan et al. (2003)
Synthetic Alginate Enhances biocompatibility and stability Ma et al. (2008) and
polymer Morales et al. (2008)
Natural Gelatin Used as hydrophilic emulsifier and Gaihre et al. (2008)
polymer gelling agent with enhanced and Olsen et al. (2003)
biocompatibility
Natural Starch Improves biocompatibility and used as Kim et al. (2003)
polymer target drug delivery and MRI
Natural Dextran Improves biocompatibility, blood Berry et al. (2003),
polymer circulation, and stability; enables the Guin and Manorama
polar optimum interactions with the (2008), and Zhang
surfaces of IONPs et al. (2004)
Natural Chitosan Hydrophilic, nontoxic, alkaline, Li et al. (2008b, c)
polymer biocompatible
Surfactants and Small Molecules Surfactants or small molecules which are used
to modify the surface characteristics of IONPs can be classified into three major
classes: amphiphilic, oil soluble, and water soluble. Oil-soluble class usually con-
tains molecules with weak attraction towards solvent environment, hydrophobic
groups (i.e., alkyl phenol (branched/ linear n = 6–10) and fatty acid) (Sousa et al.
2001), whereas water-soluble class contains chemical groups with strong attraction
towards the solvent environment, generally hydrophilic groups (i.e., polyol, ammo-
nium salt, and lysine). Amphiphilic class is combination of both hydrophobic and
hydrophilic chemical groups. Among these three classes, oil-soluble class is pre-
ferred to control aggregation of IONPs and enhance the stability (Bourlinos et al.
2002; Sahoo et al. 2005). Oleic acid is used widely for synthesis of ferrite NPs
because of formation of protective dense monolayer which results in the formation
of monodisperse and uniform NPs. Shaoo et al. reported magnetite NPs with aver-
age diameter range of 4–8 nm by hexadecyl phosphonate, dihexadecyl phosphate,
lauric acid, dodecyl phosphonate, and oleic acid. Phosphate and alkyl phosphonates
can be used for the formation of more stable dispersions of magnetite-based NPs.
Organic molecule-based ligands form quasi-bilayered structures where the primary
layer bonded to the NPs surface strongly (Sahoo et al. 2005).
Biological Molecules Many biological molecules including antibody, avidin,

polypeptide, biotin, and proteins may bound directly/indirectly through chemical
coupling to the IONPs surface via functional groups to prepare target-specific NPs.
These biological molecules greatly enhance the biocompatibility of magnetic NPs
which assist effective separation of DNA, biochemical products, cells, proteins, etc.
(Mikhaylova et al. 2004). Zhang et al. synthesized Fe3O4 magnetic NPs of size
200 nm coated with human serum albumin (HSA) through microemulsion method.
They used such coated NPs as 188Re-labelled radioisotope carrier and inquired the
optimized labelled condition with 188Re (Chunfu et al. 2004). Lee et al. prepared
c-Fe2O3 NPs conjugated with oligonucleotides. They first prepared carboxyl groups
attached at the surface of water-soluble NPs and modified streptavidin protein at the
c-Fe2O3 NPs surface through 1-ethyl-3-(3-dimrthylaminopropyl) carbodiimide
hydrochloride (EDC) as reagent (Lee et al. 2006).
Surface Functionalization of IONPs Through Inorganic Materials Significant

work has been done to synthesize the functionalized IONPs with inorganic materi-
als such as sulfides, metals/nonmetals, silica, and metal oxides. However, simulta-
neous control over their biocompatibility, magnetic properties, shape, surface
structure, and size is still challenging. Functionalization through inorganic materials
enhances greatly antioxidative properties of bared IONPs and its scope in various
biomedical applications including bioseparation, catalysis, and biolabelling
(Salgueiriño-Maceira et al. 2006). If core of NPs is assumed to be of iron oxide,
then the structure of IONPs coated with inorganic materials can be classified into
five major types: shell–core, mosaic, core–shell, dumbbell, and shella core–shellb
structures. In core shell composites, structure of NPs is the two-layered, e.g.,
Fe3O4@Au NPs include gold shell and magnetite core. Colloidal superparamag-
netic particles offer many attractive possibilities in biodetection or bioseparation
(Tartaj et al. 2004). With the reduction in dimensions of IONPs, reactivity of these
particles promisingly increases, that is why small-sized particles will show fast bio-
degradation when exposed to biological microenvironment. Hence, matrix-dispersed
IONPs can be prepared in a wide variety of oxidation states and increase the size of
bared IONPs. Mosaic structures can be prepared with iron oxide in hollow silica
spheres, and core–shell structures can be prepared through iron oxide that connects
their inner layers (Bruce et al. 2004). Shella–core–shellb composites can be synthe-
sized through layer-by-layer technology and can reduce the abovementioned limita-
tions. Shella is composed of metal NPs, quantum dots, or polymers, whereas shellb
may be of different or same types of functional material. These types of composites
are tremendously used in variety of applications. Dumbbell structures can be
obtained through iron oxide epitaxial growth on the inorganic materials to obtain
the bifunctional NPs composites. Magnetic properties can be combined with some
other properties through entrapped magnetic IONPs in some suitable inorganic
compound layers (Ma et al. 2006). Here, we will elaborate some recent and typical
examples of composite NPs, their feasible methods, and role in biomedical
applications.
Silica For synthesis of IONPs, silica coatings have been used most commonly. This
coating prevents aggregation of NPs, and interparticle interactions additionally pro-
vide more control over shell thickness, stability, biocompatibility, size tunability,
and hydrophilicity. This coating acts as a bridge for binding many ligands and bio-
logical molecules at the surface of NPs for their use in many biomedical applica-
tions. Ashtari et al. used effective methods for ssDNA target recovery based on
silica-coated modified amino Fe3O4 NPs (Ashtari et al. 2005). However, sol-gel
method, stober method, and aerosol pyrolysis are promisingly used methods for sil-
ica coatings on IONPs. Silica coating in general changes the magnetic properties and
particles size of composite NPs. Thickness of silica coating in the range of 5–200 nm
can be easily tuned by changing the ratio of tetraethoxysilane and concentration of
ammonia in water. Coating can be produced at the surface of IONPs in aqueous
environment through hydroxyl groups by using sol-gel and stober method which
showed low aggregation and better dispersions/dimensional stability/tunable struc-
ture. For the extended functions of silica-coated IONPs, other optical materials and
quantum dots can be added (Ashtari et al. 2005). Ma et al. synthesized core–shell of
FexOy@SiO2 NPs through modified sol-gel and stober methods. For the isolation of
magnetic core, silica-coated superparamagnetic IONPs were preferably used. For
that purpose, dye molecules are doped inside silica-based second shell, which
enhance the photostability and provide advanced versatile surface functionalities.
Results revealed the decreased saturation magnetization values up to 35 emu/g with
silica-coated shell thickness (10–15 nm). Decrease in coercivity and blocking tem-
perature was also reported (Ma et al. 2006). Lu et al. synthesize silica-coated IONPs
through sol-gel process, where the shell thickness was controlled through precursor
amount added to 2-propanol solution. They incorporated fluorescent dyes through
sol-gel method in silica shells which bind covalently with organic compounds. These
multifunctional NPs simultaneously manipulated for external magnetic field and
conventional fluorescence microscopy. Synthesis of NPs with tailored thickness and
uniform silica shells was reported using microemulsion/water in oil emulsion meth-
ods (Lu et al. 2002). Rojas et al. reported silica-coated IONPs with 4.5 nm radii
prepared through thermal decomposition process. Different thickness of silica shell
with varying reaction time and precursor amount in the range of (0.5–19 nm) was
prepared. Abovementioned example indicates potential of silica shell on IONPs, that
is why it is employed in many commercial formulations (Smith et al. 2006).
Metal/Nonmetals Control growth of nonmetal/single metal has tremendous effect

on IONPs applications. Gold passivates the magnetic NPs surface to avoid any type
of oxidation. Silver, carbon, or gold metal coating revealed reduce saturation mag-
netization, but the platinum, palladium, copper, and cobalt showed opposite behav-
ior due to magnetic susceptibility values of different metals. Diameter of metallic/
nonmetallic on IONPs surface can be tailored by repeating coating steps and con-
trolling reduction conditions. Functionalized IONPs through different types of met-
als can be obtained by direct reduction of metal ions present on the IONPs surface
(Yu et al. 2005). Mandal et al. separated the silver- and gold-coated Fe3O4 NPs by
direct reduction of Ag and Au, respectively, to achieve more stabilized magnetic
NPs with size range of 18–30 nm. They reported the reduced saturation magnetiza-
tion value (38 emu/g) of gold-coated NPs as compared to Fe3O4 bulk NPs (92 emu/g).
Wu et al. reported gold-coated Fe3O4 NPs through sonolysis of solution of gold ions
and modified amino Fe3O4 NPs with addition of reducing agent. Reported diameter
and saturation magnetization values of composite NPs were 30 nm and 63 emu/g,
respectively. They reported reduced value up to 0.03% as compared to the saturation
magnetization value of bulk material (Mandal et al. 2005).
3.4 Role of IONPs in Cancer Detection as Contrast Agents
In diagnostic imaging, positive contrast agents are developed rapidly. However, in

available contrast agents, substantially existing issues of toxicity and high mobility
play major role in shortening their life cycle in vascular system. These drawbacks
motivate scientists to develop ultra-small superparamagnetic IONPs as T1 contrast
agents. The inner-sphere relaxation mechanism is induced in positive (T1) contrast
agents after the interaction of water protons with high-spin 5d transition metal ions
(Mn or Fe) or 7f Gd ions. Application of superparamagnetic IONPs in medical field
finds it rapidly developing with the focus on contrast agents for MRI. T1 contrast is
mostly opted in current diagnostic practices; however, the contrast agents (i.e., gad-
olinium) being used poised high toxicity issues and have shorter life cycle in the
body. These limitations of gadolinium contrast agents urged the scientists to develop
T1 contrast agents based on ultra-small superparamagnetic IONPs. However, super-
paramagnetic IONPs improve proton relaxation time primarily via outer sphere
mechanism and hence present negative contrast imaging (Corot et al. 2006).
In the beginning, several contrast agents based on non-targeted IONPs have been
evaluated clinically. However, efficacy of these non-targeted contrast agents to
accumulate in tumor sites is very low. Targeted delivery improves MRI sensitivity.
Synthesis and development of targeted IONPs for diagnosis of specified metastases/
cancer types are very feasible. Next-generation contrast agents based on IONPs
were developed by conjugating with specific tumor targeting moieties and coatings
with suitable materials (organic or inorganic) (Bakhtiary et al. 2016).
Superparamagnetic properties of IONPs provide high-contrast enhancement in
comparison with conventional Gd-based paramagnetic contrast agents in MRI. In
blood plasma, use of superparamagnetic IONPs as contrast agent results in biofuel-
ing of those nanoparticles that produce aggregation. Superparamagnetic behavior of
aggregated superparamagnetic IONPs is greatly reduced (Liu et al. 2011). Yushuang
et al. studied the effectiveness and strength of pH-responsive superparamagnetic
IONPs with different sizes (64, 82, 103, and 121 nm). Nanoclusters with pH-
responsive NPs of size 64 nm showed effective tumor imaging in MRI. Reported 1/
T2 values were 40 mM−1 s−1 for 64 nm, 77.3 mM−1 s−1 for 82 nm, 99.6 mM−1 s−1 for
129.4 nm, and 194.1 mM−1 s−1 for 121 nm-sized NPs. Results confirmed that assem-
bled superparamagnetic IONPs had increase significantly 1/T2 contrast and showed
shortened T2-enhanced contrast capabilities (Wei et al. 2017) (Table 3.4).
130
Table 3.4 Characteristics of clinically marked/under investigated IONPs-based contrast agents

Relaxometric
Coating Name Application Company Hydrodynamic size (nm) properties (mM−1 s−1)
Citrate (Nosrati et al. 2017; VSOP-C184 Cellular labelling for Ferro Pharma 7 r1 = 14
Taupitz et al. 2004) blood pool agents r2 = 33.4
Carboxydextran (Simon et al. Ferucarbotran SHU-555A Cellular labelling for Schering 60 r1 = 9.7
2006b) liver imaging r2 = 189
Copolymers of sulfonated Ferristene Abdoscan Oral GI imaging GE Healthcare 3500 –
styrene-divinylbenzene (Lee
and Hyeon 2012; Li et al.
2005, 2007)
Pegylated starch (Daldrup- Feruglose NC100150 Blood pool agents GE Healthcare 20 –
Link et al. 2003)
Carboxydextran (Ittrich et al. SHU-555C Cellular labelling for Schering 21 r1 = 10.7
2013) blood pool agents r2 = 38
Dextran T10, T1 (Corot et al. Ferumoxtran-10 Cellular labelling for Guerbet Advanced 15–30 r2 = 65
2006; Simon et al. 2006a) AMI-227 blood pool agents Magnetics
BMS-180549
Silicon (Maccioni 2010) Ferumoxsil Oral GI imaging Guerbet Advanced 300 –
AMI-121 Magnetics
Dextran T10 (Arena et al. Ferumoxides AMI-25 Liver imaging Guerbet Advanced 120–180 r1 = 10.1
2016; Li et al. 2005; Raynal Magnetics r2 = 120
et al. 2004)
Carboxylmethyl-dextran (Li Ferumoxytol code 7228 Macrophage imaging/ Advanced 30 r1 = 15
et al. 2005) cellular labelling for magnetics r2 = 89
blood pool agents
K. Akhtar et al.
Before injection 40 min after 1 day after Before Injection 30 Min After Injection 3 Days After Injection
90(ms)
80
muJ591:MIC6
Treatment
70
60
50
Control
PBS
40
30
Fig. 3.3 Negative contrast spin echo images of a mice show tumors pointed out with circles before
and after (40 min and 1 day) injection of muJ591:MIC6 and PBS. (Abraham et al. 2017)
In recent past, Abraham et al. employed contrast agents based on superparamag-

netic IONPs that result in shortening of relaxation time (T2) of prostate cancer xeno-
grafts in MRI. Contrast agents were engineered by conjugating deimmunized
antibody (muJ591) with Molday ION Carboxyl-6 (MIC6) to target prostate-specific
membrane antigen (PSMA). These contrast agents were injected into mice intrave-
nously, and effects were compared to phosphate-buffered saline (PBS) and MIC6.
3T MRI scanner was used to get values of T2 relaxation time (Fig. 3.3). Contrast
agents based on muJ591:MIC6 conjugate were used to detect and target PSMA-
positive cancer cells that deceases signal growth, relaxation time, and tumor growth
(Abraham et al. 2017).
3.5 I ONPs-Mediated MRI Diagnosis of Different

Cancer Types
There are over 100 types of cancer affecting the human beings. Smoking, alcohol
consumption, and many factors induce deadly effects which finally can emerge in
the form of cancer. In cancer, a normal cell can transform into infected cell due to
the abnormality in regular cell growth and differentiation. There are many ways
through which cancer cells can be destroyed, but still early detection of malignant
cell is challenging.
3.5.1 Brain Cancer Detection
Among brain cancers in human, glioblastoma multiforme (GBM) is considered as

most lethal, aggressive, and prevalent with poor prognosis. It has been limited in
specified region due to poor infiltrating delineation of tumor margins. To diagnose
and treat GBM, immune therapies are becoming a promising research avenue. For
the stimulation of immune system, amphotericin B plays major role in suppressing
growth of brain tumor initiating cells. Ultra-small IONPs contrast agents are being
extensively utilized for malignant tumor tracking. These contrast agents are used in
MRI for detection of increased infiltration of monocyte in brain tumors due to
amphotericin B that acts as an indicator for drug response as reported in literature
(Yang et al. 2016). Superparamagnetic IONPs have been promisingly used as nega-
tive contrast agents for imaging and detection of malignant tumors. For potential
diagnostic applications, gold superparamagnetic IONPs-incorporated micelles
coated with polymers of polyethylene glycol-polycaprolactone have been studied in
the literature. These micelles with high r2 value of 221.92 mM−1 s−1 and high field
strength of 1.4 T have been reported (Fig. 3.5). Components based on gold nanopar-
ticles (d = 1.9 nm) were used to ionize radiations to sensitize tumor cells, while
components based on iron oxide (d = 15 nm) were leveraged as contrast agents for
imaging of those malignant tumors (Sun et al. 2016). Multifunctional nanoparticles
were fabricated with superparamagnetic IONPs core and multifunctional PEG/PEI/
polysorbate 80 (Ps 80) composed shell encapsulated with doxorubicin (DOX@Ps
80-IONPs) having Dh = 58 nm and saturation magnetization (24.1 emug−1). Real-
time monitoring of DOX@Ps 80-IONPs were done with MRI as reported in litera-
ture (Xu et al. 2016).
Due to anti-inflammatory, antioxidative, and anti-metastatic properties of func-
tionalized iron oxide, nanoparticles with caffeic acid (γFe2O3@CA NPs) were
investigated for in vitro detection of brain cancer cell line U87-MG (Fig. 3.4). These
nanoparticles were intravenously injected into U87 glioblastoma-bearing mice. In
cancerous tissues, negative contrast enhancement on 11.7 Tesla with high transverse
relaxivity value equal to 375 mM−1 s−1 suggests high T2 contrast effects were
observed specifically in MRI image (Richard et al. 2016b).
IONPs were conjugated with heat shock protein (Hsp70) that enhances the abil-
ity of tumor targeting through MRI for malignant brain tumor. Conjugated IONPs
with Hsp70 possess relaxivity values of negative T2 MRI contrast (Shevtsov et al.
2015). Lactoferrin-conjugated IONPs with hydrodynamic diameter of 75 nm, trans-
verse relaxivity of about 5.6 mM−1 s−1, and saturation magnetization of 51 emu/g
were used as effective contrast agents for brain glioma diagnosis (Xie et al. 2011).
IONPs conjugated with arginine–glycine–aspartate peptides with high transverse
relaxivity value of about 187 mM−1 s−1 were used clinically to detect glioblastoma
at early stages (Zhang et al. 2012) (Fig. 3.5).
Fig. 3.4 (a) Schematic of action miRNA in brain cancer cell with nanosystems proficient to cross
blood–brain barrier. (b) MR negative contrast images of the mouse brain before and at other times
of post-incubation of respective γFe2O3@CA NPs. Tumor is pointed here with red arrow. (c) Size
distribution of γFe2O3@CA NPs. (d) Change in contrast between tumor and contralateral over
time. [Reprinted with permission from (Richard et al. 2016b). Copyright (2016) American
Chemical Society]
Fig. 3.5 MR images of C6 cells with control, only C6 cells; SPION, superparamagnetic iron oxide
NPs with C6 cells, and Lf-SPION, lactoferrin-coated iron oxide with C6 cells (left side upper).
Iron uptake by C6 cells incubated with iron oxide and lactoferrin-iron oxide (left side lower). In
vivo MR images acquired after administration of iron oxide (right side upper row) and lactoferrin-
iron oxide (left side lower row). [Reproduced with permission from (Xie et al. 2011)]
3.5.2 Breast Cancer Detection
In European countries, breast cancer emerged as fatal cause of death among women.
Progress for early breast cancer detection through different imaging methods like
PET, SPECT, ultrasound, MRI, and optical imaging has developed (Linge et al.
2014; Martin et al. 2010; Masotti et al. 2009). For MRI detection of cancer, dextran-
functionalized IONPs were assembled with bombesin to produce targeted contrast
agents. These nanoparticles with size 6.0 ± 0.5 nm and p < 0.05 have saturation
magnetization 29.2 emug−1. Favorable behavior of these nanoparticles builds them
an attractive candidate for early stage breast cancer detection (Jafari et al. 2015).
IONPs were tightly packed inside liposome core to obtained enhanced MRI contrast
for breast cancer tumor detection (Zhang et al. 2014). Ultra-small superparamag-
netic IONPs were employed for detection and assessment of auxiliary lymph node
metastases in breast cancer patients (Harada et al. 2007). IONPs were used as poten-
tial candidate for metastatic cancer cells tracking. In cancer cells, retention of these
IONPs allows long-term in vivo tracking. These IONPs were used for investigation
of breast cancer at early stages (Economopoulos et al. 2013).
3.5.3 Gastric Cancer Detection
Gastric cancer is the fourth commonly occurring cancer that leads towards death
worldwide. At advanced stages, it is difficult to cure gastric cancer. In clinical prac-
tice, early detection along with tumor staging, surgical resection planning, and prog-
nosis is important (Leake et al. 2012; Yang 2006). Gastric cancer is a type of
localized tumor, but locoregional metastasis is the most significant negative
prognostic factor. Diagnosis of gastric cancer consists of tumor imaging, early

detection with endoscopy/biomarkers, and mobilized tumor cells that belong to gas-
tric cancer (Imano et al. 2012; Ishigami et al. 2010). Contrast agents based on IONPs
are widely used for locoregional imaging with early detection of gastric cancer
(Baetke et al. 2015; Zhang and Zhao 2014). Superparamagnetic IONPs generally
named as ferumoxides and ferucarbotran (Resovist) are clinically approved, specifi-
cally used to diagnose gastric cancer (Bakhtiary et al. 2016). In patients with gastric
cancer, location and presence of lymph node metastases are essential. For detection
of lymph node, reliable diagnostic technique along with contrast agents (size and
shape) is adequate. Ferumoxtran-10 is a lymphatic contrast agent that is highly effi-
cient for lymph node metastases detection. Enhanced MRI was evaluated with feru-
moxtran-10 for early stage detection of gastric cancer (Harada et al. 2007; Tatsumi
et al. 2006).
SiO2-coated superparamagnetic IONPs were labelled as anti-CD146 monoclonal
antibody with near-infrared fluorescence dye. These prepared nanoparticles in the
form of core–shell (iron oxide core diameter 15 nm and SiO2 shell thickness 10 nm)
presented 110.57 mM−1 s−1 value for T2 relaxivity coefficient. These particles were
used as contrast agents for gastric cancer detection at early stages (Wang et al.
2015a). Ultra-small superparamagnetic IONPs were used for regional lymph node
metastases detection in patients suffering with gastric cancer (Fig. 3.6) (Tatsumi
et al. 2006; Wang et al. 2015a) (Table 3.5).
Fig. 3.6 Confocal microscopic images of iron oxide–silica complexes. Binding affinity studies of
nanoparticles on MKN45 cells. (a) 800ZW-iron oxide@dense silica-YY146 targeting group, (b)
YY146 blocking, (c) 800ZW-iron oxide@dense silica. Strong cell membrane staining was recog-
nized for targeting group (a), and background levels were observed in blocking (b) and control (c).
[Reproduced with permission from (Wang et al. 2015a)]
Abbreviations: DAPI 4′,6-diamidino-2-phenylindole; CD146 target tumor; 800ZW near-
infrared fluorescence (NIRF) dye; YY146 monoclonal antibody for CD146
Table 3.5 Contrast agents based on IONPs for gastric cancer detection
Nanoparticles Targeting strategy Imaging modalities(reference)
SPIO NPs Passive targeting MRI (Tatsumi et al. 2006)
Dextran IONPs Trastuzumab MRI (Chen et al. 2009)
PEG-coated Fe3O4 nanoparticles MiRNA-16 MRI (Sun et al. 2014)
PEG-g-PEI-SPION CD44v6 SiRNA MRI (Chen et al. 2012, 2013)
SiO2-coated IONPs Anti-CD146 Mab MRI (Wang et al. 2015a)
3.5.4 Ovarian Cancer Detection
Ovarian cancer is a major diagnosed gynecologic malignancy. Major cause of death

in ovarian cancer is late diagnosis because of unavailability of non-specific/mini-
mal/non-reliable biomarkers (Moyle et al. 2010; Ozols et al. 2004; Sato and Itamochi
2012). In ovarian cancer cases, first choice is debulking surgery. In most cases, even
after surgery, ovarian cancer recurs and becomes more drug resistive (Konner et al.
2011; Morgan et al. 2012; Ozols et al. 2004). Folic acid-based IONPs (Fe3O4) were
used in MRI as negative (T2) contrast agent for accurate and early ovarian cancer
detection in an intraperitoneal xenograft tumor model. Folic acid-functionalized
Fe3O4 nanoparticles with average size (9.2 ± 1.7 nm), mild cytotoxicity, and high
values of r2 475.92 mM−1 s−1 make magnetic susceptibility and permeability effects
more sensitive. This facilitates in targeting malignant tissues (Zhang et al. 2016b).
3.5.5 Liver Cancer Detection
About 7000 cases of human hepatocellular carcinoma (HCC) are diagnosed every
year. For improving poor survival of patients suffering from HCC, early detection
of malignant/premalignant lesions is very essential (Llovet et al. 2008; Skelton et al.
2007; Weissleder 2006). Among clinically available cancer diagnostic techniques,
MRI is preferred due to lack of ionizing radiations and 3D maps with high-resolution
features. To enhance contrast effects and differentiation between tissues, shorten
relaxation parameters of water are required (Thorek et al. 2006). Improved charac-
terization and depiction of hepatic lesions are possible by using specific MRI con-
trast agents because of physiological properties of the liver. Superparamagnetic
IONPs are sequestered and opsonized with phagocytic Kupffer cells in normal
reticuloendothelial system. In Kupffer cells, phagocytosed IONPs produce relax-
ation effects (T1 and T2) in liver parenchyma, while malignant tumors retain no
signal change. Kupffer cells results in increased contrast for lesion detection (Ward
et al. 2003).
In reticuloendothelial cells, superparamagnetic IONPs are actively distributed

according to phagocytic activity, which causes rapid dephasing of nearest proton
spins due to local field inhomogeneities. It results in shortening of transverse relax-
ation times (T2). In metastases liver cancer, these contrast agents are not absorbed
due to lack of reticuloendothelial cells. Therefore, these IONPs produce stronger
decrease in intensity of signal (negative contrast) of MRI in tissues (Corot et al.
2006). For liver MR imaging, two formulations of superparamagnetic iron oxide
have been clinically approved, i.e., dextran-coated Ferumoxides and carboxydextran-
coated ferucarbotran. Several other formulations for imaging applications in humans
such as feruglose (Taupitz et al. 2004) and citrate-coated IONPs have also been
reported (Kellar et al. 2000). Contrast agents based on dextran-coated IONPs and
ferumoxides are used for hepatic tumors detection through tissue-specific MRI
(Paley et al. 2000; Ward et al. 2000).
Coprecipitated ultra-small superparamagnetic IONPs conjugated with human-
ized antibody SM5-1 mAB were selectively demonstrated as targeted contrast
agents for in vivo detection of hepatoma because of high bio-stability with small
size, improved magnetism, and targeting moieties. These nanoparticles with core
diameter 8–10 nm and hydrodynamic diameter of 25.3 ± 2.1 nm were potentially
used for detection of binding protein (SM5–1) that caused malignant tumors (Kou
et al. 2008). Dextran-/carboxydextran-coated IONPs showed significant differences
in relaxivity times that substantially enhanced their detectability and pretherapeutic
evaluation of hepatic metastases through MRI (Miyata et al. 2006; Tanimoto and
Kuribayashi 2006). Contrast agents based on stabilized IONPs with alginate were
used for detection of liver cancer. Investigation of efficacy, before and after injection
of IONPs-alginate for primary hepatocellular carcinoma detection in rats and xeno-
graft liver cancer in rabbits, was done (Fig. 3.7). Reported relaxivity values for
SPIO-alginate were 7.86 ± 0.20 and 281.2 ± 26.4 mM−1 s−1 (Ma et al. 2008).
Multifunctional superparamagnetic IONPs loaded with doxorubicin (YCC-
DOX) were also used for early liver cancer detection (Maeng et al. 2010).
Multifunctional novel Gadolinium incorporated polymeric nanoparticles
were simultaneously used with anti-vascular endothelial growth factor (VEGF) and
Fig. 3.7 MR T2 signal of liver cancer in rabbit at different time (a) 0 min (b) 10 min (c) 30 min
(d) 60 min after IV administration. After 60 min, enhanced contrast can be observed between
tumor and liver parenchyma. [Reproduced with permission from (Ma et al. 2008)]
gadolinium-diethylenetriamine penta-acetic acid (Gd-DTPA) as modified cotrast

agents. Reported relaxivity (r1, r2) values for contrast agents were 18.394 mM−1 s−1
and 24.863 mM−1 s−1, respectively (Liu et al. 2011). Hybrid Fe3O4–Au nanoparticles
fabricated at room temperature were also used to detect HCC at early stages (Zhao
et al. 2015).
3.5.6 Colorectal Cancer Detection
In the United States, second major cancer-related mortality and third most common
cause of neoplasm is colorectal cancer. Detectable tumors have been removed with
surgeries at earlier stages, but residual micrometastases cause relapse. At stage I, if
disease is limited till mucosa, reoccurrence rate will be less than 3%. If the disease
is spread to lymph node (stage III) regionally, reoccurrence rate will be greater than
50%. Overall, 30% patients face reoccurrence regionally/locally, and 50% surgi-
cally treated patients face relapse, while 70% patients face reoccurrence at distant
sites especially at the lungs or liver (Meyerhardt and Mayer 2005; O’Connell et al.
2004). So there is a clinical need for detection of such diseases through image-based
detection techniques. IONPs and its core–shell-based contrast agents are more com-
patible and considered suitable for MRI (Fortina et al. 2007; Frick et al. 2005; Qiang
et al. 2006). Two key stages of colorectal cancer are tumor stage (T stage) and
regional nodal metastasis stage (N-stage) (Beets-Tan and Beets 2004). With MRI,
first stage rectal cancer detection is a normal practice because it is used to diagnose
the rectal wall laminar structure. The detection of N staging in the patients is more
difficult with imaging modality. For N staging, MRI is used in combination with
different techniques such as PET and computed tomography (Dewhurst et al. 2012;
Rollvén et al. 2013; Uçar et al. 2013).
MRI is considered more accurate than other diagnostic techniques. For T stag-
ing, phased array coil with thin section MRI is widely used. MRI not only used to
diagnose rectal wall laminar structure but also give detailed relationship of malig-
nant tumor with surrounding organs and mesorectal fascia. High-resolution MRI is
used for locally advanced colon cancers (Bipat et al. 2004; Edelman and Weiser
2008). MRI is recommended for T staging by American College of Radiology. For
rectal cancer detection, 3 Tesla MRI is performed with pelvic phased array coil as
reported in the literature. For primary rectal evaluation, T2-weighted high-resolution
imaging is the key sequence (Kaur et al. 2012; Vliegen et al. 2005).
Low-signal intensity of negative contrast imaging is used to visualize rectal wall
mucosa and muscularis propria, while high intensity is used for visualization of
submucosa and mesorectum. Initially, many studies have been reported for T stag-
ing MR imaging with high accuracy, but those results were not reproduced widely.
For tumor infiltration, many limitations with difficulties in distinguishing early
stage tumors have been faced (Beets-Tan and Beets 2004; Feng et al. 2014).
In patients of colorectal cancer, MRI has been used for prediction of lymph node
involvement. For identification of normal and metastatic lymph nodes, method of
uptake for ultra-small IONPs is proposed (Fig. 3.8). These NPs were used as con-
trast agents in three-dimensional negative contrast imaging for diagnosis of short-/
long-axis diameter, estimated percentages, and border irregularities (Koh et al.
2004). Kim et al. reported diagnostic accuracy of iron oxide up to 87.5% and 88.8%
without any statistical significance (Kim et al. 2010).
Fig. 3.8 In vivo MR imaging of nude mice with tumor after IV administration of lectin–iron
oxide@gold nanoparticles. Images were taken at (a) preinjection (b) 6 h, (c) 12 h, (d) 24 h postin-
jection. Red circle is indicating tumor location. [Reproduced with permission from (He et al. 2014)
copyright (2014) American Chemical Society]
Ultra-small IONPs-based contrast agents were used for detection of rectal cancer
at early stages in patients allergic to gadolinium-based contrast agents (Lahaye et al.
2008). Max et al. used ultra-small IONPs as MRI contrast agents with dextran-
coated low molecular weight iron oxide for primary rectal cancer detection. Max
et al. reported lymph node diameter for short and long axis (3.6 mm and 4.7 mm),
while range variations observed were 2–9 nm and 2–13 nm, respectively (Lahaye
et al. 2008).
3.5.7 Lung Cancer Detection
Major reason of death in patients with lung cancer is tumor metastasis. With distant
metastasis, 30% of patients suffering with lung cancer have been diagnosed, while
distant metastases occurred in 50–60% of patients during treatments, and 80–90%
of patients with lung cancer died with metastases (Bernards and Weinberg 2002;
Siegel et al. 2011). To improve efficacy of diagnosis and treatments, specific and
sensitive detection of metastasis is required. In patients with lung cancer, differen-
tial expression of tumor-specific proteins has been reported (Bharali and Mousa
2010; Wang et al. 2015b). With differential expression of these specified tumor pro-
teins, metastasis can be specifically and sensitively detected. To detect lung cancer,
MRI is considered as one of the promising diagnostic techniques at molecular level
(Jaffer and Weissleder 2005).
For effective and sensitive metastasis detection in lung cancer patients, super-
paramagnetic IONPs were functionalized with oleic acid and carboxymethyl dex-
tran used in immuno-imaging through MRI (Fig. 3.9). Prepared nanoparticles were
conjugated in monoclonal antibody (anti-CD44v6) of mouse. Contrast agents based
on these immune IONPs are efficiently used due to high saturation magnetization
and transversal relaxation values (194.2 and 126.4 mM−1 s−1) for targeting diagnosis
of lung tumor (Wang et al. 2013).
3.5.8 Pancreatic Cancer Detection
Pancreatic ductal adenocarcinoma is considered as one of more dangerous malig-

nancies due to resistance, aggressiveness, and late presentation against current treat-
ments (Kimple et al. 2012; Zhang et al. 2016a). Physiochemical properties along
with feasibility of PEG-PAsp (polyethylene glycol-polyaspartic acid)-coated mag-
netite IONPs were used as contrast agents for pancreatic cancer detection at early
stages. Relaxivity (r2) values for PEG-PAsp were 138 mM−1 s−1. PEG-PAsp signifi-
cantly exhibit negative contrast agent in T2-weighted images in tumor region when
combined with TGF-β inhibitor (Kumagai et al. 2009) (Table 3.6).
SPION-OA-CMD
SPION-OA
0.11
0.10 SPION-OA-CMD R2=126.4 mM-1s-1
0.09 SPION-OA R2=194.2 mM-1s-1
0.08
0.0125mM 0.5mM
0.07
1/T2 (ms-1)
0.06
0.05
0.04
0.03
0.02
0.01
0.00
0.0 0.1 0.2 0.3 0.4 0.5

Fe concentration (mM)
Fig. 3.9 MRI sensitivity measurements of iron oxide with oleic amine and iron oxide with oleic
amine and carboxymethyl dextran. T2-weighted MR images (left) and linear fitting of 1/T2 of two
nanoparticles systems at different iron concentrations. [Reproduced with permission from (Wan
et al. 2016)]
3.6 Conclusion and Perspective with Future Outlook
Clinically, targeted IONPs possess great potential towards diagnosis of cancer with
acceptable safety margins, but only few platforms are available for successful appli-
cation in cancer treatments. Whole field of tumor detection with targeted imaging
suffers from lack of versatile and specific tumor markers/target moieties/imaging
contrasts. Most biomarker- and nanoparticle-based contrast agents with high sensi-
tivity can be fabricated from functional targeting ligands. But the main limitation is
complex techniques to fabricate nanoparticles with narrow size distribution and spe-
cific morphology. Unexpectedly, many IONPs-based contrast agents overexpressed
a diverse panel of tumors. Second major limitation is adverse effects linked with
these fabricated nanoparticles. Researchers should make comprehensive progress to
solve these limitations in clinical practice.
Although, toxicity of IONPs is extensively studied, but still complete picture of
the scene is not very clear. This parameter is very important because it will decide
about the fate of the IONPs in vivo. IONPs possess many detrimental effects includ-
ing genotoxicity, inflammation, and intracellular interference signaling. Many
mechanisms of these NPs can induce toxic effects in the body, but major problem is
the generation of reactive oxygen species in excess amount. Generation of reactive
142
Table 3.6 IONPs-based imaging for different cancer diagnosis modified from literature
NP core Hydrodynamic
NP coating (reference) composition Loaded drug size Cell/in vivo model Targeting ligand Application
PEG (Khandhar et al. Iron oxide None 10 nm J774 macrophages None IONPs T1 contrast
2017; Tromsdorf et al. agent (in vivo
2009; Xiong et al. 2017) analysis)
Dextran (Devaraj et al. F-iron oxide None 30 nm BALB/c mice None Trimodal MRI
2009; Thurber et al. composite contrast agent (in vivo
2010) analysis)
None (Bates et al. 2014; Iron oxide None 35 nm DUI145 cells Specific membrane T2 contrast agent
Chan et al. 2017; Wei (HTB-81) anti-prostate antigen (prostate cancer cell
et al. 2017) LNCaP cells J591 antibody targeting)
(CRL-1740)
Dextran (Calcagno and Bismuth iron None 98 nm Liver carcinoma cells None T2 MRI contrast
Fayad 2017; Naha et al. oxide (Hep G2) agent (in vivo murine
2014; Unterweger et al. FibroblastsBJ5ta model)
2017) Mice C57BL/6 J
Chitosan (Fang et al. Iron oxide Temozolomide 50 nm Glioblastoma cells Chlorotoxin Drug delivery for
2015; Laurent et al. (U-118 MG) glioblastoma brain
2017) C57BL/6 J mice cancer (in vitro
analysis)
Chitosan (Kievit et al. Iron oxide Suppressing siRNA 40 nm Ependymoma cells None Brain tumor detection
2015; Woźniak et al. apurinic endonuclease (Res196) (in vitro analysis)
2017; Zhu et al. 2017) Medulloblastoma cells
PEG (UW228–1)
PEI
PEG (Wang et al. 2017; Iron oxide Chlorin e6 100 nm Murine breast cancer None Breast cancer in
Yoo et al. 2017) (4 T1) cell murine model
K. Akhtar et al.
(continued)
NP core Hydrodynamic
NP coating (reference) composition Loaded drug size Cell/in vivo model Targeting ligand Application
PEI (Cai et al. 2017; Iron oxide Targeting human 100 nm PC3 cells None Inhibit growth of liver
Karimzadeh et al. 2017; siRNA telomerase MCF-7 cells cancer (in vivo and
Li et al. 2014) reverse transcriptase Hep G2 cells in vitro analysis)
SKOV-3 cells
With Hep G2 BALB/c
mice in xenograft
tumors
APTES (Nafiujjaman Iron oxide Pheophorbide A 15 nm Epithelial cancer (KB) None Imaging of epithelial
et al. 2015; Wu et al. cells cancer (in vitro
2017) analysis)
PEG (Huang et al. 2017; Gold–iron None 25 nm Colorectal cancer cell Antibody (A33 Targeting colorectal
Kirui et al. 2010) oxide (SW1222 and HT 29) scFv) cancer (in vivo
composites analysis)
3 Applications of Iron Oxide Nanoparticles in the Magnetic Resonance Imaging…
143
oxygen species can be minimized through functionalization of IONPs by different

organic and inorganic materials.
Enhanced contrast properties have been observed for morphologies other than
spherical such as with nanocubes or nanoflowers and by doping with other transi-
tion metals. IONPs are considered highly biocompatible, when acute cytotoxic
effects are absent, because these particles induce no effect on cell homeostasis. Due
to high bioavailability, optimum dose of IONPs is required to avoid the toxicity
issues. Therefore, detailed understanding on the developmental, long-term effects,
bioavailability, and reproductive toxicity of IONPs is still ambiguous. Moreover,
standard protocols should be established for the quantification and physiochemical
characterization of IONPs in vivo.
In addition, many IONP formulations have been examined and commercialized
for different cancer treatments; T2-based IONPs contrast agents could not replace
the already marketed gadolinium-based positive contrast agent because of concerns
over artifacts present in the NPs, large size, and longer circulation time. Now
researchers have started exploration of positive contrast properties of IONPs to
make these NPs more useful. Another important link that remained unexplored is
the understanding of complete life cycle and processing of IONPs. There are few
studies reported that showed processing of IONPs with some specific coating. More
detailed analysis about IONPs fate with different polymer coating is required. More
studies are needed to focus on fabrication techniques to develop nanoparticle-based
systems/contrast agents for cancer detection.
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Chapter 4
DNA-Based Nanopharmaceuticals
V. Dilna, Chinnu Sabu, and K. Pramod
Contents
4.2 D NA Nanostructures 161
4.2.1 DNA–Polymer Assembly 164
4.3 DNA Origami 165
4.3.1 Smart Structures by DNA Origami Approach 166
4.3.2 DNA Origami as Template for Biomolecule Delivery 172
4.3.3 Structural Modifications in DNA Origami Structures 172
4.3.4 Aptamer-Conjugated DNA Origami Structures 174
4.3.5 Computational Sequence Design 174
4.4 Conclusion 174
References 175
Abstract Nanopharmaceuticals are systems in which therapeutic drug delivery

works at the nanoscale. Even though several such systems are developed, most of
them possess significant drawbacks. Some noted drawbacks include immunogenic-
ity, cytotoxicity, accessibility to the target tissue, in vivo stability, biocompatibility,
and effort in synthesizing procedure composed of complicated chemical reactions
or techniques. Therefore, studies concerned with the fabrication of nanostructures
based on biomolecules gained wide interest. The unique characteristics of biomol-
ecules like DNA enable them to self-assemble and develop into variable nanostruc-
tures having tremendous applications. In this review, we focus on nanostructures
fabricated from DNA. Their biocompatibility, structural stability, and unique recog-
nition sites make them most suitable building blocks for the development of smart
nanostructures. The first DNA-based nanostructure was a stick cube with a motif-
based design. Incorporation of materials like polymers, development of newer tech-
nique like DNA origami, and the possibility of further modifications in the developed
V. Dilna · C. Sabu · K. Pramod (*)

College of Pharmaceutical Sciences, Government Medical College, Kozhikode, Kerala, India
https://doi.org/10.1007/978-3-030-44925-4_4
160 V. Dilna et al.
structures enable its high utility. In this review, we discuss concepts and applications
of DNA nanostructures, DNA–polymer assembly, DNA origami technique, and
structural modifications of DNA nanostructures.
Keywords Self-assembly · DNA · Single-stranded · Origami · caDNAnano ·

Scaffold · DNA bricks · Staple strand · Holiday junction · Aptamer
4.1 Introduction
Self-assembly of certain materials provides a new direction to nanotechnology

which can be described as designing of nanostructures from its own components by
interaction or rearrangement between them without an external impulse (Donald
2005). The specificity of nucleotide sequence and base pairing following Watson
and crick rule makes the DNA molecule an ultimate material for self-assembly pro-
cess for a wide range of applications. DNA self-assembly concept is instigated by
Seeman in 1982 which is a fascinating approach for researchers (Seeman 1982).
DNA-based self-assembly is achieved by bottom-up fabrication which is a robust
and biomimetic strategy for developing a variety of custom-shaped 2D and 3D
nanostructures. Self-assembled DNA nanostructures are based on several approaches
including self-assembly of the medium-sized oligonucleotide into the finite-sized
structure, oligomerization of building blocks that developed from medium-sized
oligonucleotide to finite-sized structure, 2D and 3D structure fabrication from short
strands or DNA-brick, and DNA origami technique (Gradisar and Jerala 2014).
DNA origami technique was first familiarized by Paul Rothemund in 2006. He
constructed a smiley face and several other structures from a mixture of long single-
stranded scaffold DNA from bacteriophage and a large number of short staple
strands by annealing in particular buffer (Rothemund 2005). The Rothemund’s
technique for self-assembled DNA nanostructures is emerging as a new arena for
nanopharmaceuticals. DNA origami technique can be exploited for designing more
complex and sophisticated self-addressable nanostructure compared to DNA tile-
based technique which deals with small and simple structures (Zadegan and Norton
2012). Various attempts are reported to fabricate different shapes using biomimetic
genetic material DNA. Open-cage DNA origami, origami box, DNA nanotube, tet-
rahedral DNA origami, icosahedral DNA origami, and wireframe DNA origami are
a few to mention. They are particularly useful for the delivery of cytotoxic agents
like doxorubicin, daunorubicin, CpG, and siRNA, in addition to their numerous
other biomedical applications (Douglas et al. 2009a, b; Glaser et al. 2016; Ko et al.
2008; Kocabey et al. 2015; Kumar et al. 2016; Lee et al. 2012; Schüller et al. 2011;
Zadegan and Norton 2012).
DNA origami is a nanoscale folding of DNA into the desired shape by utilizing
computer software caDNAnano for designing staple strand which is an open-source
software and can be exploited for designing correct sequence for staple strand to
establish preferred shape (Holliday 1964). This computer-aided design (CAD)
4 DNA-Based Nanopharmaceuticals 161
software enables easy access to more complex sophisticated structures and dimin-
ishes the error-prone effect. The DNA origami structure is a smart drug delivery
vehicle. It also has tremendous applications in various fields such as cancer therapy,
prodrug medication, and enzyme replacement therapy.
4.2 DNA Nanostructures
DNA, a polynucleotide polymer, possesses an inherent role in the fabrication of

nanoscale structures. According to Watson and Crick model, DNA is a double helix
having antiparallel strands composed of highly specific base pair. This specificity
makes DNA a fundamental tool for nanofabrication since it becomes more predict-
able in nature. Even though DNA is a genetic material, programmed self-assembling
capacity and biocompatibility enable DNA as a promising vector for drug delivery.
Moreover, DNA molecule is devoid of any cytotoxicity and immunogenicity which
are essential requisites of an efficient carrier for drug delivery. Hydrogen bond
between base pairs and aromatic π–π stacking between adjacent bases confer more
stability to DNA helix. Hence, structural DNA nanotechnology has a great impact
in the research field which encompasses strategies like hybridization, stably
branched DNA, and appropriate sequence design (Seeman 2010). The structural
DNA nanotechnology comprises the designing of DNA-based artificial structures
and its utilization in technologies in which stick cube was the first structure design
(Chen and Seeman 1991).
Among the various material nanostructures, more efficient ones are those of
DNA since they are homogeneous in size, composition, and surface chemistry. DX
(double crossover – two helices are linked), TX (triple crossover – three helixes are
linked), PX (two helices are joined by strands crossing over all the possible places),
and JX2 (PX itself except two crossover in the middle) are the prominent motifs
involved in structural DNA nanotechnology (Seeman 2003). Among these, DX is
the fundamental building block for programmed self-assembly of DNA (Boulais
et al. 2017). Multiple crossover motifs are also investigated. But its junction flexibil-
ity made the fabrication of complex structures more difficult. The DNA tiles includ-
ing DX, TX, PX, and JX2 are the building blocks for DNA nanostructures by
self-assembly into 2D lattices (Lapique and Benenson 2017).
In 1964, Robin Holiday introduced a DNA-based Holiday junction which is
cross-shaped structure intermediate in genetic recombination (Seeman and
Kallenbatch 1983). The Holiday junctions are unstable due to its sequence asym-
metry. The different architectures can be created based on the stable junction ana-
logs to Holiday junction. These stable junctions were known as immobile junctions
and are composed of hexadecameric strand developed from the nucleotide sequences
which are thermodynamically equivalent to RNA helix. The hexadecameric strand
composed of 13 overlapping segments of length 4 and each of the segments is
known as criton. The Holiday junctions are interconnected as tile and form 2D and
3D DNA lattices. The strands in holiday possess sticky ends known as pads. 2D and
3D DNA lattices are formed by self-assembly of a group of DNA tiles through
162 V. Dilna et al.
Table 4.1 Applications of DNA nanostructures in bioimaging and drug delivery

Nanostructure Description Application Reference
Reconfigurable Protease-caged nanovault with Control enzyme– Grossi et al.
DNA nanovault reversible opening/closing substrate interaction (2017)
Icosahedral DNA Light-induced release of Targeted delivery of Veetil et al.
nanocapsules dehydroepiandrosterone from DHEA into neuronal (2017)
targeted DNA nanocapsules cell
Bioinspired pH-responsive shedding of polymeric Targeted delivery of Sun et al.
nanococoons shell of nanocapsules occurs doxorubicin into (2014a)
triggering the enzyme activity and cancer cell
subsequent drug release
DNA dendrimer Aptamer-tethered, doxorubicin- CpG motif carrier Zhang et al.
loaded structure (2015)
DNA tweezer Arms of tweezers modified with Actuation of activity Liu et al.
G6PDH and cofactor NAD+ of enzyme/cofactor (2013)
pair
DNA Nanorobot loaded with combination Controlled release of Douglas
nanostructure- of antibody fragment and controlled payloads et al. (2012)
based logic gate by aptamer-encoded logic gate
nanorobot
Wireframe DNA Virus-inspired PEGylated lipid Enhancement of Perrault
nanooctahedron bilayer-enveloped structure in vivo stability et al. (2014)
DNA nanoparticle Prostate cancer membrane antigen Targeted doxorubicin Zhang et al.
(PSMA)-targeted aptamer and delivery (2017)
pH-sensitive spacer-coded,
doxorubicin-loaded DNA
nanoparticle
DNA nanotrain Aptamer-tethered DNA nanotrain Targeted transport of Zhua et al.
chemotherapeutic (2013)
agent
DNA origami Antibody as payload Targeted delivery of Katsnelson
nanorobot payloads to cancer (2012)
cell
hybridization of pads. Double crossover (DX) and triple crossover (TX) tiles create
these lattices; multiple crossover can create more sophisticated structure (Peterson
and Heemstra 2015). Thus, structural DNA nanotechnology creates motifs that can
be further assembled to more sophisticated complex nanostructures. The interaction
between these motifs and sticky-end cohesions leads to the construction of self-
assembled structure.
The self-assembled DNA nanostructures are much desirable in the case of tar-
geted drug delivery systems as well as early diagnosis, especially in the case of
cancer (see Table 4.1). The easy programmable nature of DNA has a great impact
on control over density and spatial orientation of ligand like folic acid for targeting
efficiency. The stability of DNA nanostructure is limited when the ligand used is
cationic due to ionic interaction between the oppositely charged molecule and sub-
sequent aggregation (Albert et al. 2017). One of the fascinating DNA self-assembled
nanostructures is bio-inspired nanococoon for the targeted delivery of chemothera-
peutic agent doxorubicin to tumor site in response to pH. It is a self-degradable drug
delivery vehicle encompassing acid-responsive DNAse I capsule embedded in DNA
nanoclew. In the acidic environment of cancer, shedding of polymeric shell of nano-
capsules occurs, triggering the enzyme activity and self-degradation of acid-
responsive cocoon-like structure provoking the release of drug doxorubicin at
targeted site (Averick et al. 2011).
Nanoparticles for tumor targeting must possess suitable size with the purpose of
reducing clearance and retention in the tumor site. But the long-term persistence of
nanoparticles without biodegradation leads to chronic toxicity. In the past, research-
ers focused on engineering flexibility of inorganic nanoparticles, but their non-
biodegradable nature is still a major concern. Mixing the functionalized nanoparticles
with linker DNA in a hybridizing buffer accomplishes the DNA–nanoparticle
assembly, and it is possible to further encrust it with additional ligands like PEG to
modify interaction towards the cells and tissues. The superstructure shows reduced
macrophage uptake due to its large size as compared to naked core or satellite
nanoparticle (Chou et al. 2014). DNA-driven self-assembly of functionalized
nanoparticles into binary heterogeneous three-dimensional superlattices can be car-
ried out using plasmonic (gold), magnetic (iron oxide, FeO), luminescent (quantum
dots), and catalytic (palladium) nanoparticles. DNA functionalization of this
nanoparticle is achieved by passing through carboxylic group grafting, streptavidin
conjugation, and biotinylated DNA attachment. Carboxylic group grafting is accom-
plished by ligand exchange process for hydrophilic nanoparticles and amphiphilic
polymer attachment for hydrophobic nanoparticles. For example, poly-N-vinyl-2-
pyrrolidone (PVP) in PVP-capped palladium nanoparticles (hydrophilic nanoparti-
cles) is exchanged with 11-mercaptoundecanoic acid. An amphiphilic polymer like
lipid-PEG-carboxylic acid is attached to hydrophobic nanoparticles, FeO, and
quantum dots for carboxylic group grafting. Streptavidin conjugation is imple-
mented through amide bond formation by exploiting carbodiimide (EDC) chemis-
try. The DNA functionalization of nanoparticle is finally achieved by using
biotinylated DNA utilizing the strong affinity between biotin and streptavidin which
is previously conjugated on nanoparticle. The DNA-aided functionalized nanopar-
ticle assembly ensures tunable interparticle distance and rich phase behavior. The
magnetic nanoparticle FeO alone in solution exhibits weakly ordered phase known
as F phase. When it is added along with the plasmonic nanoparticle, a conversion
into D phase occurs which is a binary FeO–Au phase, and these phase behavior
kinetics depend on DNA length (Zhang et al. 2013).
Another reported nanostructure is composed of designer-specific three-
dimensional nanomold and nanoparticle seed that grows to fill the cavity. The nano-
mold is a DNA barrel with two DNA lids, and the nanoparticle seed consists of gold
and silver triangular and cuboid nanoparticles. The nanoparticles are anchored in its
cavity by annealing, and the lids are attached to form a box-like cuboidal structure.
164 V. Dilna et al.
The structure possesses remarkable applications in areas such as biosensing and

nanophotonics (Sun et al. 2014b).
4.2.1 DNA–Polymer Assembly
The unmodified polynucleotide is less biostable and has poor intracellular delivery
and limited access to the specific target. This compelled the synthesis of DNA–
polymer conjugate in which conjugation of the emerging field, polymer science,
and unique properties of DNA has occurred (Kedracki et al. 2013). The DNA–poly-
mer conjugate could be utilized in drug delivery, cellular imaging, and materials
science. The synthesis of DNA–polymer conjugate is mainly by solution coupling
and solid–phase synthesis. The solution coupling method focuses on the formation
of a new bond between the polymer sequence and nucleotide sequence which are
separately synthesized and purified (Singh et al. 2010). This involves amide bond,
disulfide bond, and Michael addition reaction. But its use is limited due to poor
yield (Kachalova et al. 2004). The solid–phase method avoids complicated chemi-
cal reactions and purification steps, and it is carried out utilizing a simple tool like
syringe filter (Kwak and Herrmann 2011). Similar to conjugation of DNA with
polymers, the combination of DNA with materials like metals and semiconductors
at nanoscale level is a fascinating approach for establishing newer techniques
(Kundu et al. 2017). The DNA–polymer conjugation is further discussed in sections
on bioconjugates, click conjugation, and DNA block copolymer.
Bioconjugates
Conjugation of polymers with biomolecules is possible in order to extend their

applications. The most suitable material for bioconjugate fabrication is DNA–inor-
ganic polymer composite. The DNA–polymer hybrid nanoparticle carrying plasmid
DNA can exhibit remarkable transfection efficiency and low cytotoxicity (Schnitzler
and Herrmann 2012).
The phospholipid-based vesicular structure for drug delivery is an amazing
approach. Now this concept extends to the construction of a vesicular DNA-based
structure and is termed as DNAsomes. The superb structure is composed of a hydro-
phobic membrane encapsulating the cargo and a DNA shell. The structure can be
repaired for on-demand cargo delivery by certain modifications. DNAsomes formed
from self-assembly of DNA-phenyleneethynylene hybrid amphiphile are a pH-
responsive drug delivery vehicle (Averick et al. 2014).
Click Conjugation
A biomolecule can be modified using organic chemical reactions. Among these,

click reactions are useful in conjugation chemistry. A form of controlled dissembled
DNA nanostructure is formed from the conjugation of DNA to star polymer by click
reaction. The formed nanoparticle can be assembled to higher-order architecture by
hybridization. The major drawback of click reactant is the possibility of susceptibil-
ity to react with the biologically abundant functional groups. The conjugation of
DNA to star polymers is based on most efficient click reaction using copper-
catalyzed azide–alkyne cycloaddition (CuAAC) where there is no remarkable cross-
reaction of azide and alkyne group with a biological functional group (Grossi
et al. 2017)
DNA Block Copolymer (DNABCp)
Block copolymers has a great impact on nanoelectronics, nanoscience, and bio-

medicine ever since the involvement of biomolecules has started in synthesis of
block copolymers. When a single polymer is combined with the different polymer
segments, it causes distortion of polymer property. Designing of the supramolecular
structure using DNA block copolymer (DNABCp) is a fascinating approach for
researchers. One of the excellent examples for such a structure is DNABCp micellar
structure comprising hydrophobic polymer core and a DNA corona having a broad
spectrum of applications, viz., drug delivery vehicle, a template for self-assembly of
viral capsid and in nanoelectronics (Veetil et al. 2017). One strategy to create
DNABCp is a separate synthesis of polymer and DNA strand and their conjugation
by covalent reaction (b–t). The conjugation of two such bulky molecules is a chal-
lenging process. Moreover, the purification step is more difficult in this approach.
Thus, an initiator-mediated method (b–f) is useful to synthesize DNABCp which is
a monomer addition-based reaction in contrast to conjugation of the incompatible
large molecule in b–t strategy. In the b–f method, the activator radicals formed from
atom transfer radical polymerization (ATRP)-mediated polymer growth occurs on
the solid support like gold nanoparticles through DNA initiator hybrid (Zhang
et al. 2015).
4.3 DNA Origami
The elegant art of paper folding is called origami. It is concerned with the protocol
for the construction of complex 3D structure from 2D paper. The concept of DNA-
based origami instead of paper was first exploited by Paul Rothemund in 2006. It
was a remarkable event in DNA nanotechnology that lead to fabrication of DNA-
based structures having a wide range of application. DNA origami is nanoscale
folding of DNA by long scaffold DNA strand and short staple strand. The first DNA
166 V. Dilna et al.
origami was fabricated from long single-stranded scaffold viral DNA derived from
bacteriophage M13mp18 composed of 7249 nucleotide sequences with 100 s short
staple strand. Therein, staple strand pulls the long scaffold strand into the desired
shape like a smiley face and several other shapes in one pot reaction by rapid heat-
ing followed by slow cooling. The correct ingredient ratio is a prerequisite factor to
create more sophisticated structure. The scaffold strand must be a long single-
stranded structure with the absence of a secondary structure. So more appropriate
scaffold is M13 (Liu et al. 2013). Base composition of DNA strand has a great
impact on its self-gathering into the discrete structure, among which GC base com-
position has great influence. Mammalian expression vector pEGFP N-1 is an exam-
ple of scaffold strand composed of more GC composition as compared to M13
scaffold, and its fabrication to the desired structure is more complex. The incidence
of lower GC composition is also inappropriate for DNA origami approach. Thus,
the optimization of base composition used as the scaffold is a requisite step to
design target structure (Douglas et al. 2012). Similar to base composition, the salt
concentration in the buffer for self-assembly is also critical to compromise the nega-
tive charge of the phosphate group of DNA. Beyond a limit, it causes an appearance
of the presence of unstructured particles or precipitation of any component in the
medium. To mention, the structure cylindrical 24-helix bundles dispersed in liquid
crystalline disodium cromoglycate (DSCG) solution are a suitable example. The
24-helix bundles are fabricated from 7560 nucleotide long scaffold strands and
approximate 200 synthetic staple strands. In this case, the salt concentration is a
more crucial factor as excess salt precipitates DSCG. Beyond the critical concentra-
tion of magnesium, DSCG agglomerates and nematic (N) and columnar (M) phase
of liquid crystal disappear (Perrault and Shih 2014).
It is possible to obtain different shapes for DNA-based nanostructures prepared
by a self-assembly method. Among the different shapes prepared, triangle DNA
origami shows better drug loading efficiency. The interaction between intercalated
doxorubicin and the structure in response to acidic pH enables efficient targeted
delivery of drug (Zhang et al. 2017). Open-cage pyramidal DNA loaded with doxo-
rubicin has demonstrated twofold higher cytotoxicity than free doxorubicin (Zhua
et al. 2013). Single nucleosome can be efficiently integrated into a DNA origami
nanocaliper. A nucleosome must be coordinated into the origami by means of two
connection focuses, one on each arm, so that the nucleosome structure changes are
coupled to the nanocaliper angle. The connection approach utilized depends on a
biotin−neutravidin−biotin linkage (Fig. 4.1) (Le et al. 2016).
4.3.1 Smart Structures by DNA Origami Approach
Different smart structures were developed based on DNA origami approach exhibit-
ing a wide range of application (see Table 4.2). The pioneering structures are
reviewed in the following sections (Fig. 4.2).
Fig. 4.1 Schematic representation of assembly and integration of nucleosome into a DNA origami
nanocaliper. (Adapted with permission from Le et al. (2016); Copyright (2016) American Chemical
Society)
DNA Nanotubes
Two versions of DNA nanotube design are nanotube (S Nano) and twisted nanotube
(T Nano). The twisted version is composed of more nucleotide (8634 nt compared
to S Nano which is 7560 nt) since it is fabricated from S Nano by base pair addition.
The T Nano exhibits slower release of the drug (Kumar et al. 2016). More function-
alized nanostructures composed of DNA nanotubes covalently modified with cyto-
chrome 3 (Cy3) and folate exhibit dual functions of imaging and enhanced cellular
uptake. Compound Cy3 shows red fluorescence responsible for imaging, while
folate increases cellular uptake owing to the presence of overexpressed folate recep-
tors in cancer cell (Kuzuya et al. 2015). Supramolecular self-assembled bundles of
DNA nanotubes are designed based on depletion force by crowding agent
poly(ethylene glycol) (PEG) from single-stranded oligonucleotide with a length of
42 nucleotides (Jiang et al. 2015).
DNA Tetrahedron
The self-assembled tetrahedron is an architecture for the drug delivery into the
drug-resistant cancer cell (Fig. 4.3) (Ke et al. 2009). Reconfigurable DNA tetrahe-
dron is obtained by mixing 4 strands in 15 mM MgCl2. Here, the reconfigurable
motif is a hairpin loop. The hairpin loop opened by addition of fuel strand at 3′ end
of the strand, which lacks involvement in the tetrahedral formation and hybridizes
with both stem and loop, results in an increase in the length of the edge. The original
closed hairpin loop is obtained by sequential addition of fuel and antifuel strand.
168 V. Dilna et al.
Table 4.2 Smart structures by DNA origami approach and their applications
Smart
structures Description Application Reference
DNA Labeled with Cy3 probe and Imaging of tumor cells Ko et al.
nanotube modification with folate (2008)
receptor
DNA Labeled with Cy3 and Cy5 Optical imaging in internalization Walsh et al.
tetrahedron by HEK cells (2011)
Modification with NIR emitter Near-infrared (NIR) fluorescence Jiang et al.
DyLight 755, radioactive imaging and single-photon (2016)
isotope99mTc, and tumor- emission computed tomography
targeting folic acid (SPECT)
Conjugation with cetuximab Delivery of doxorubicin Setyawati
et al. (2016)
Modified with biotin Delivery of ruthenium Chang et al.
polypyridyl complexes (RuPOP) (2011)
into HepG2 cancer cells
Self-assembly with six ssDNA Targeted delivery of siRNA Lee et al.
in vivo (2012)
DNA Coupled with Cy5 and aptamer Cell imaging and delivery of Li et al.
nanoflowers chemotherapeutic drugs for (2015)
cancer treatment
DNA Encapsulated with fluorescein Determines the pH changes Modi et al.
icosahedron isothiocyanate in vivo (2009)
Conjugation of the aptamer Delivery of doxorubicin Chang et al.
with MUC-1 sequence (2011)
DNA box Modification with lactose Nanocontainer for nanocargos in Andersen
controlled drug delivery et al. (2009)
DNA Truncated octahedral nanocage Enhances stability in biological Vindigni
octahedra functionalized with single fluid and its uptake is detected by et al. (2016)
biotin biotin conjugation
DNA Thiol–DNA-modified gold Biosensing and nanophotonics Ma et al.
dendrimer nanoparticle (2016)
Large DNA Folding of large lambda/M13 Biomedical research and Marchi
origami hybrid scaffold by chip-derived nanoelectronics et al. (2014)
staple strand
When the antifuel strand displaces fuel strand, it leads to change in length, estab-
lishing originally closed strand (Subramanian et al. 2010).
DNA Nanoflowers
Several smart structures are created based on programmable self-assembly of poly-

nucleotide material, DNA. Most of these structures are produced on the basis of
Watson and Crick base pairing rule in between the DNA building blocks and are
more susceptible to nuclease degradation. In contrast to this, nanoflower is devel-
oped by a liquid crystallization technique and dense packaging of DNA building
Fig. 4.2 Schematic
representation of DNA
self-assembled 3D
nanoarchitectures: (a)
DNA cube, (b) DNA
tetrahedron, and (c) DNA
octahedron. (Adapted with
permission from Lin et al.
(2009); Copyright (2009)
American Chemical
Society)
(a) (b) ~55nm

16
15
14 16
13 15
12 14
11 13
10 12
9 11
8 10
7 9
5
6
7
8 ~53nm
4 6
3 5
2 4
1 3
2
1
Fig. 4.3 Schematic representation of (a) 3D model of tetrahedron and (b) 2D model indicated
with strand tracing with the cage opened. (Adapted with permission from Ke et al. (2009);
Copyright (2009) American Chemical Society)
block, which are developed from rolling circle replication (RCR). Due to its dense
packaging of building blocks, more functional moieties like aptamer, imaging agent,
and a drug may be encapsulated; the structure enables its efficient use in cell target-
ing, intracellular imaging, and drug delivery (Lv et al. 2015).
DNA Icosahedra
Mechanical stability of DNA origami structures is improved if its fabrication is

based on tensegrity principle which means geometrical integrity upon tension (Ke
et al. 2009). Icosahedron is the smallest closed structure characterized by high rigid-
ity, symmetry, and resemblance to spherical viral capsid protein. To develop such an
advanced structure, the creation of five-point star tile is the first step. It is a one-pot
reaction in which five-point star tile further assembles to icosahedra by sticky-end
cohesion containing G–C base pairs. Five-base long single-stranded DNA into ico-
sahedra by a tile-based method is in contrast to polyhedra designed by Paul
170 V. Dilna et al.
Rothemund utilizing several staple strands to pull the long single-stranded DNA
into the desired shape (Zhao et al. 2012).
DNA Origami Box
Self-assembled DNA origami box with the controllable lid of DNA keys was
designed by utilizing the scaffold DNA strand from self-assembled M13 bacterio-
phage to form six interconnected sheets and staple strand which folds the sheets by
bridging the edges (Katsnelson 2012). Gold nanoparticle encapsulating DNA ori-
gami box is fabricated through sequences which consist of the scaffold and staple
strands that form open motif structure; one of the six faces is attached with function-
alized gold nanoparticle with thiol-modified DNA strand, and edges are joined by
joint strands and hinge strands resulting in a closed motif structure (Rothenmund
2006). 3D DNA origami box of 42 × 36 × 36 nm3, designed by computational meth-
ods, exhibits significant serum stability (Andersen et al. 2009). Thus, modified 3D
DNA origami box is a promising approach to enhance the stability of such nano-
structure and could be exploited in several applications like molecular structure
determination, as nanoscale ruler for single-molecule imaging and in genomic
applications (Areddy 2011; Martin and Dietz 2012; Steinhart et al. 2009; Sun and
Gu 2015).
DNA Octahedra
Various nanoarchitectures like plasmonic nanoparticle clusters could be prepared

using DNA origami. Gold nanoparticles functionalized with ssDNA (sticky ends)
which is complementary to an octahedral frame having chiro-optical activity have
been designed by exploiting the novel technique of DNA origami using caDNAnano
software package for the construction of octahedral-based nanocluster (Tian et al.
2015). Even though several smart structures were designed, their stability in bio-
logical fluid is a great challenge. Pristine DNA octahedral cage exhibiting enhanced
stability in biological fluid, cellular uptake, and efficient cell binding is possible.
These octahedral nanocages utilize the oxidized low-density lipoprotein receptor-1
(LOX-1) for targeting efficiency. It is a receptor associated with tumor and cardio-
vascular disease (Vindigni et al. 2016). The entry of DNA nanocages into the cell
can be depicted with a truncated DNA octahedral cage with a biotin molecule on
one edge of the structure (Fig. 4.4).
Fig. 4.4 Schematic
representation of truncated
octahedral cage with a
biotin covalently bound to
one strand of a double
helix. (Adapted with
permission from Vindigni
et al. (2016); Copyright
(2016) American Chemical
Society)
DNA Origami Dendrimers
Dendrimers are highly branched, star-shaped macromolecule with nanometer-scale

dimension. DNA origami dendrimer with gold nanoparticle (AuNP) has tremen-
dous applications in biosensing. Here, DNA origami dendrimer is developed from
four hairpin strands and target DNA. Self-assembled DNA with metal like gold
nanoparticles are widely utilized in nanophotonics and biosensing by exploiting the
interparticle distance-based optical property of such particles. AuNP exhibits spe-
cific color change during their aggregation and redispersion based on the principle
of interparticle plasmon coupling. DNA origami dendrimer by coupling exponential
hairpin assembly and modified AuNP results in enzyme-free chain-branching reac-
tion establishing a large, uniform, and well-ordered nonporous structure, and these
structures are talented to serve as an ultrafilter in nanofluidics and nanosorting.
Moreover, it is enabled for nucleic acid detection with a detection limit of 25 pM
within 15 min. The aggregation of AuNP is prevented by repulsion between
nanoparticles by using thiol–DNA-modified AuNP which substantially increase
negative charge (Ma et al. 2016).
Large DNA Origami
Large-sized DNA origami can be developed by means of several tiles instead of

staple strands in Rothemund’s technique of DNA origami (Ma et al. 2015). A large
number of chemically distinct species are necessary for developing DNA origami
structures that lead to numerous errors during assembling to the nanostructure.
Fabrication of structures using coupling between DNA-binding protein (RecA) and
DNA has circumvented this limitation of DNA origami technique. This novel tech-
nique aids researchers to develop more large and sophisticated structures. RecA
172 V. Dilna et al.
binds to one end of DNA and extends to the entire length of DNA structure by an
attractive force exerted on another side (Marchi et al. 2014).
4.3.2 DNA Origami as Template for Biomolecule Delivery
Several studies regarding DNA origami approach as smart drug delivery vehicle
have been reported. In addition to the drug, they can be utilized for the delivery of
molecule-like cytosine–phosphate–guanine (CpG) and SiRNA. CpG sequence is
efficiently recognized by Toll-like receptor 9 (TLR 9) on endosome, and consequent
release of cytokine derivatives like interleukin 6 (IL– 6) and interleukin 12p70
(IL–12p70) facilitates immune cell stimulation. The programmed DNA origami
structures can be explored for such immunological applications by decorating with
sequences like CpG. The hollow 30-helix DNA origami tube decorated with 62
CpG sequences is fabricated from single-stranded DNA origami scaffold M13mp18
and 227 staple oligonucleotides (Schüller et al. 2011). DNA nanostructure also acts
as a platform for vaccine delivery by self-assembling the antigen and as a CpG
adjuvant into DNA nanostructure (Liu et al. 2012). The DNA origami structures like
DNA tetrahedral nanoparticle are capable of delivering biomolecules like siRNA
which can silence the target gene in the tumor (Lee et al. 2005). The DNA nano-
structure can be utilized for enhancing the conductance by using synthetic pore like
DNA origami porin in lipid membrane (Gopfrich et al. 2016).
A triangle DNA origami functionalized with the MUC-1 aptamer can stack
doxorubicin and convey AuNRs to restrain the development of safe bosom disease
cells. MUC-1 aptamers reach out from the altered target staple strands in the com-
posed district of the DNA origami. Furthermore, the helpful medication DOX can
be stacked onto the DNA origami structure by means of intercalation between base
sets (Fig. 4.5) (Song et al. 2017).
4.3.3 Structural Modifications in DNA Origami Structures
The DNA nanostructures are most often fabricated by programmable self-assembly

of tightly cross-linked DNA helices. Thus, numerous complex shapes are engi-
neered from various degrees of twist and curve at the nanoscale by DNA helices.
The twist and curve of DNA at various degrees happen when targeted insertion or
deletions of base pairs develop into wireframe beach ball or square-toothed gear-
like structures (Dietz et al. 2009). The most auspicious design strategies of DNA
origami 3D structures are associated with its complex curvature in 3D space. The
Fig. 4.5 Schematic representation of the functionalized DNA origami nanostructure in doxorubi-
cin delivery. (Adapted from Song et al. (2017) with permission of The Royal Society of Chemistry)
in-plane curvature structures generate from a concentric ring of DNA, and out-of-
plane curvature is from shifting of crossover points between DNA double helices.
DNA nanostructures like nanoflask, ellipsoidal shell, 3D spherical shell, and 2D
arrangement of a concentric ring can be fabricated based on this design principle
curvature (Han et al. 2011). An additional strategy for developing DNA-based com-
plex nanostructure is designing of DNA gridiron in which DNA helices and four-
arm junctions are used as vertices and edges, respectively. These structures range
from 2D array, multilayer, to 3D structures and curved objects (Han et al. 2013; Han
2017). These structures can be further modified to extend their application.
Ligand conjugation is a promising approach for structural modifications in DNA
origami structures. Structural modification of DNA origami leads to its utilization in
a wide range of applications. Such modification creates a difference in their surface
properties and improves their cellular uptake. For example, modified DNA origami
nanostructures by noncovalent interaction with designed intercalator acridine
derivatized with aside chain containing esterified fatty acid alter the surface proper-
ties of DNA origami nanostructures consequently altering cellular uptake (Brglez
et al. 2015). Cellular delivery of DNA origami is improved by encrusting it with
174 V. Dilna et al.
purified cowpea chlorotic mottle virus capsid protein. This approach achieved
13-fold higher cellular delivery than uncoated DNA origami (Mikkila et al. 2014).
4.3.4 Aptamer-Conjugated DNA Origami Structures
Aptamer-conjugated DNA origami structures show increased cellular internaliza-

tion (Chang et al. 2011). Aptamer is a single-stranded DNA possessing antibody-
like activity for targeting function and is capable of interacting with signals like ATP
and pH. The ATP aptamer conjugate hybridizes with complementary scaffold strand
resulting in double-stranded DNA. Upon stimulation by the increased amount of
ATP in the intracellular environment, the hybridized DNA becomes single-stranded
and subsequently results in drug release (Sun and Gu 2016). Chemical modification
of DNA nanostructures with carbohydrate increases its cellular uptake by compro-
mising the multiple negative charges on DNA nanostructure (Sut et al. 2016). Folate
receptor is a specialized protein that overexpresses on the cancer cell. Therefore, its
detection is a promising approach for cancer detection, and the DNA origami
approach can be utilized for such purposes. The hybridization between folate-
tethered DNA origami and DNA-functionalized gold nanoparticle produces a color
change that gives the result regarding detection of folate receptors overexpressed on
cancer cell surface. In addition to this, folate provides terminal protection of DNA
against exonuclease (Zhu et al. 2015).
4.3.5 Computational Sequence Design
DAEDALUS (DNA Origami Sequence Design Algorithm for User-defined

Structures) makes designing of DNA origami-based 3D geometry simple by input-
ting CAD (computer-aided design) file in the format of PLY (polygon file format)
(Schiffels et al. 2017). caDNAnano is an open software that can be utilized for
designing certain arbitrary shape with minimum error-prone effect, and it is
platform-independent software. It is available from http://cadnano.org and its source
code available under MIT license (Veneziano et al. 2016).
4.4 Conclusion
DNA, the basic unit of genetic material, can be used to fabricate nanostructures by
self- assembly process. The self-assembled DNA nanostructures and DNA-polymer
hybrid nanoparticles are well suited as drug delivery vehicles. The fabrication of
DNA–polymer hybrid is a complex process involving organic reactions with several
inorganic materials. In contrast to this, the novel technique DNA origami is simpler
and avoids the use of toxic inorganic materials. The DNA origami technique is
known for designing more sophisticated and complex structures like DNA origami
box, DNA tetrahedron, DNA octahedra, and DNA icosahedra. These structures can
be further self-gathered to form more complicated structures. These structures are
capable of targeted delivery of biomolecules like CpG, SiRNA, and anticancer
drugs such as doxorubicin. Structural modification of DNA origami structures can
be exploited to extend their applications including enhancement of cellular uptake
and targeted drug delivery. The DNA origami technique may be susceptible to an
error during sequence design, and it has been solved by developing software like
caDNAnano.
It is concluded that the DNA-based nanopharmaceuticals are the excellent choice
for efficient therapeutic drug delivery. The major challenge in the use of DNA-based
nanopharmaceuticals is the access of drug to specific tissue due to the same negative
charge of DNA and biological membrane which make difficulty in cellular uptake.
But it is successfully overcome by encrusting the structure with a molecule like
PEG which improves interaction between the structure and biological membranes.
Alternatively, it can be conjugated with polymers such as hydrophilic carbohydrates
and synthetic polymers. The major advantage of incorporation of polymers into
DNA composite is stabilization through masking of exonuclease acting site of DNA.
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Chapter 5
An Overview on Ionic Liquids: A New
Frontier for Nanopharmaceuticals
Tânia Santos de Almeida, Rita Caparica, Ana Júlio, and Catarina Pinto Reis
Contents
5.2 I onic Liquids 183
5.2.1 Synthesis and Purification 184
5.2.2 Classification of Ionic Liquids 185
5.2.3 Toxicity for Humans and the Environment 188
5.2.4 Applicability in Drug Delivery Systems 190
5.3 Nanopharmaceuticals and Ionic Liquids 191
5.3.1 Applications of Nanoparticles and Ionic Liquid Combined Systems 192
5.3.2 Regulatory Gaps 197
5.4 Conclusions 197
References 198
Abstract Background: The pharmaceutical and cosmetic industries are constantly

trying to develop new and more efficient delivery systems, as well as new functional
ingredients, to overcome drawbacks, such as poor drug solubility, loading and
release, as well as reduced stability. These challenges, namely, low drug solubility
in water, usually lead to an insufficient systemic exposure and, consequently, low
drug bioavailability.
T. S. de Almeida
CBIOS-Universidade Lusófona’s Research Center for Biosciences & Health Technologies,
Lisboa, Portugal
e-mail: tania.almeida@ulusofona.pt
R. Caparica · A. Júlio
Department of Biomedical Sciences, University of Alcalá, Madrid, Spain
e-mail: rita.caparica@ulusofona.pt; ana.julio@ulusofona.pt
C. P. Reis (*)
iMED.ULisboa, Research Institute for Medicines, Faculdade de Farmácia,
e-mail: catarinareis@ff.ulisboa.pt
https://doi.org/10.1007/978-3-030-44925-4_5
182 T. S. de Almeida et al.
Several applications of ionic liquids have been widely studied due to their valu-
able physicochemical properties. In consequence of this broad applicability, ionic
liquids have recently been studied in the pharmaceutical field, particularly as func-
tional excipients to improve the efficiency of drug delivery systems. Despite the
potential utility of ionic liquids in drug delivery, concerns about their toxicity have
been raised, since some of these salts are more toxic than certain organic solvents.
Hence, studies concerning the safety of ionic liquids are fundamental.
Nanoparticles have also been studied to improve the efficiency of drug delivery.
The main interest in nanosystems is due to the many advantages these systems may
confer, particularly in terms of drug safety and efficiency.
In consequence of the valuable characteristics that both ionic liquids and
nanoparticles hold, there has been an emergent interest in the synergetic effects aris-
ing from the combination of ionic liquids and nanoparticles. This approach may be
key to develop more stable systems, with improved performance. However, for the
preparation of these ionic liquid–nanoparticle systems, environmentally friendly
procedures need to be developed, and the toxicity of the prepared systems needs to
be carefully assessed.
Major advances: Herein, we reviewed the main characteristics and applicabilities
of ionic liquids, as well as the impact of the synthesis and use of these salts on
humans and the environment. Moreover, we also discuss recent developments in the
use of ionic liquids in drug delivery. Finally, the prospective combination of ionic
liquids and nanosystems towards drug delivery is also explored. In this matter, sev-
eral studies are presented, showing that the combination of ionic liquids with
nanoparticles to produce more efficient delivery systems has great potential. It is
shown that ionic liquids improve the chemical and thermal stability of the particles
and may change their size and morphology. Finally, some concerns about the com-
bination of ionic liquids with nanoparticles, such as regulation gaps and the research
needed for the widespread application of these combined systems in pharmaceuti-
cals, are also addressed.
Keywords Ionic liquids · Toxicity · Environment · Drug delivery systems ·

Nanopharmaceuticals · Ionic liquid and nanoparticle combined systems ·
Enhanced drug solubility and loading · Improved performance and stability · Size
and morphology control · Regulatory considerations
5.1 Introduction
The pharmaceutical and cosmetic industries spend a great amount of their financial
resources in the development of new and more efficient drug delivery systems to
overcome drawbacks, such as poor drug solubility, loading and release, systemic
side effects, and reduced stability of the developed delivery systems. Hence, several
strategies have been made to formulate new and more efficient delivery systems,
like the development of controlled release systems, such as nanopharmaceuticals,
since they may prevent drug degradation and improve drug transportation,
5 An Overview on Ionic Liquids: A New Frontier for Nanopharmaceuticals 183
distribution, and release (Reis et al. 2006b; Bawa 2010; Tiwari et al. 2012; Weissig
et al. 2014; Safari and Zarnegar 2014; Hare et al. 2017). Some efforts have also been
made to find new functional ingredients that may be useful to surpass the referred
challenges. In this context, some studies have already shown the relevance of incor-
porating ionic liquids in drug delivery systems to improve drug solubility, loading,
permeation, and stability. Additionally, due to intermolecular interactions between
ionic liquids and nanoparticles, ionic liquids may improve the physicochemical
properties of nanoparticles. Namely, ionic liquids may confer chemical protection
and have influence on the morphology, pore, and particle size of the nanoparticles
(He and Alexandridis 2016). Hence, combining ionic liquids and nanoparticles may
be a valuable strategy to improve the stability and efficiency of nanopharmaceuti-
cals. Also, the possibility to modulate the properties of ionic liquid–nanoparticle
systems is a key advantage that may lead to a higher performance of the new com-
bined drug delivery systems and needs further investment and understanding from
the scientific community.
Subsequently, the evaluation of the synergies arising from the combination of
ionic liquids with nanoparticles is an emergent topic and may be crucial to the
development of new and more efficient drug delivery systems.
5.2 Ionic Liquids
Ionic liquids are organic salts which are liquid at temperatures below 100 °C or, in
some cases, liquids at room temperature (Ferraz et al. 2011; Dobler et al. 2012;
Kubota et al. 2016). They are composed entirely of ions, an organic cation, and an
anion that can be inorganic or organic (Torimoto et al. 2010; Dobler et al. 2012;
Gouveia et al. 2014; Zhang et al. 2016a).
These viscous liquids (Gouveia et al. 2014), with pure ionic character, have
numerous valuable properties, such as the ability to dissolve organic, inorganic, and
polymeric materials (Dobler et al. 2012). Also, these salts are recyclable, nonvola-
tile, and nonflammable and, consequently, are classified as green solvents (Mizuuchi
et al. 2008; Ghandi 2014; Balk et al. 2015). Ionic liquids are also known to have
high ionic conductivity and are usually considered highly polar, due to their ionic
nature. It is also known that ionic liquids have high thermal and chemical stability
(Moniruzzaman et al. 2010a) and negligible vapor pressures (Dobler et al. 2012;
Balk et al. 2015). In particular, ionic liquids are very well known for their properties
as solvents, since many compounds are fairly soluble in ionic liquids and the solu-
bility properties of these salts resemble those of polar organic solvents (Rantwijk
and Sheldon 2007). However, the possibility to deliberately modify the anions and
cations that constitute these ionic compounds (Moniruzzaman et al. 2010a, b; Balk
et al. 2015) is probably the most important property, since it makes the ionic liquids
highly susceptible to modifications (Moniruzzaman et al. 2010a). The high predis-
position to structural alterations offers high flexibility and variability (Balk et al.
2015), since it allows to synthesize ionic liquids with specific physicochemical
properties, towards a particular application (Mitkare et al. 2013). In fact, it has been
described that the number of possible combinations, when preparing ionic liquids,
is about 1018, and consequently ionic liquids, particularly room temperature ionic
liquids, have been termed as “designer solvents” (Balk et al. 2015). Although ionic
liquids are well known by the scientific community (Handy 2011), in the last
decades, they have been the object of more studies, due to the advantageous proper-
ties of these salts which allow them to be used in the most diverse areas (Khupse and
Kumar 2010; Ghandi 2014; Egorova et al. 2017). Consequently, the interest in the
synthesis of ionic liquids has increased along the years.
5.2.1 Synthesis and Purification
When developing new strategies for the preparation of ionic liquids, various factors
become quite relevant such as the efficiency of the developed synthetic procedures,
the presence of impurities, as well as the toxicity and biodegradation of the prepared
ionic liquids. In general, the synthesis of ionic liquids is frequently divided into two
main methods, metathesis and acid–base neutralization (Ferraz et al. 2015b). The
metathesis reaction, which consists in an exchange of partners between cations and
anions, is usually used for the preparation of the so-called traditional ionic liquids.
This reaction uses cations such as alkylimidazolium to prepare halide-free ionic
liquids from halide salts, with chloride and bromide salts being the most used
(Wasserscheid and Welton 2008). However, these metathesis reactions present some
disadvantages such as the possibility of contamination with small amounts of halide
ions that may react with solute materials. Moreover, the limited diversity of the salts
commercially available represents another limitation of the metathesis reactions.
On the other hand, the acid–base neutralization reactions involve equimolar mix-
ing of tertiary amines with halide acids or some organic acids and avoid contamina-
tion problems existent in the metathesis reactions (Ferraz et al. 2015b). By using
stronger acids and stronger bases, the proton transfer process may be improved.
These neutralization reactions, of a Brønsted base with a Brønsted acid, afford
protic ionic liquids (Shamsuri and Abdullah 2010). Aprotic ionic liquids are obtained
by quaternization of imidazoles, alkylamines, or phosphines, using alkyl halides
and other alkylating agents and through metathesis reactions (Ozokwelu et al. 2017).
Reactions involving the anion counterpart of the ionic liquids may also be valu-
able when attempting to modify the properties of the synthesized ionic liquids. In
fact, to obtain new ionic liquids, the addition of various metal salts, to obtain differ-
ent Lewis acidic ionic liquids, as well as metathesis reactions involving the anion,
may be used (Kuzmina and Hallett 2016).
Another relevant element to consider in the preparation of ionic liquids is the
purity of the prepared salts since it may greatly influence the physicochemical prop-
erties of the ionic liquids, lead to environmental impact, and restrict the applicabil-
ity of these salts. In fact, during the synthesis of ionic liquids, substantial amounts
of solvents are used, and therefore, the existence of impurities is impossible to avoid
(Ren et al. 2010). The impurities most commonly found in ionic liquids are water
and organic solvents (Holbrey et al. 2002; Ren et al. 2010; Oommen et al. 2017),
such as methanol, ethanol, benzene, acetonitrile, ethyl acetate, and dichloromethane
(Ren et al. 2010). The presence of residual amounts of water or organic solvents
influences not only the purity but also the physicochemical properties of the pre-
pared ionic liquids (Holbrey et al. 2002; Ren et al. 2010). For example, the presence
of water can modify properties such as viscosity, density, and conductivity of ionic
liquids (Stoppa et al. 2009; Zhu et al. 2009; Ren et al. 2010) or even inactivate the
catalysis in ionic liquids (Holbrey et al. 2002; Ren et al. 2010). Also, the low vapor
pressure of ionic liquids prevents their purification by distillation, but this technique
may allow the separation of volatile impurities from the ionic liquid. The most com-
monly used method for the removal of water or organic solvents is by heating the
ionic liquids at (60–120 °C) under vacuum for several hours (12–48 h). This process
presents some disadvantages such as the process time, which is at least 12 h; the
presence of small amounts of water, even after 24 h of process (Ren et al. 2010); and
the impossibility of some ionic liquids, such as many protic ionic liquids, to be
subjected to this procedure (Oommen et al. 2017). There are still other methods and
techniques used in the purification of ionic liquids, such as zone melting, that allows
to separate ionic liquid components from impurities by exploiting the differences in
their molecular shape and the use of sorbents, such as activated carbon, silica, and
alumina, particularly useful for the removal of colored impurities (Oommen et al.
2017). However, it is usually best to eliminate impurities, as much as possible, at the
beginning of the synthesis and to use preparation procedures that give rise to the
least possible side products. In fact, before use, all starting materials and solvents
used should be dried (Wasserscheid and Welton 2008).
Furthermore, when considering the applicability of ionic liquids, besides the
synthesis and purity of the chosen ionic liquids, it is also equally vital to know their
toxicity and environmental impact since different classes of ionic liquids may have
different toxicities.
5.2.2 Classification of Ionic Liquids
Ionic liquids are generally classified into three generations (Fig. 5.1), which differ
according to their structure and chemical properties (Hough et al. 2007; Egorova
et al. 2017). The first generation of ionic liquids is formed by the dialkylimidazo-
lium and alkylpyridinium cations with metal halide anions, and they are sensitive
to air and water. On the other hand, the second generation is stable to air and water
and is usually composed by the dialkylimidazolium, alkylpyridinium, ammonium,
and phosphonium cations and tetrafluoroborate and hexafluorophosphate anions.
Finally, the third generation is formed by biodegradable and natural ions or by ions
with known biological activities and is consequently very interesting due to the
possible application of these ionic liquids in pharmaceutics and in areas such as
ecology and biology. Ionic liquids prepared from choline and amino acids are
Fig. 5.1 Evolution of ionic liquids (ILs) generations through time with focus on the properties,
advantages, and disadvantages of each generation
among the most common in the third generation (Hough et al. 2007; Egorova
et al. 2017).
Regarding the synthesis and structure of ionic liquids, they may also be divided
in protic and aprotic ionic liquids. The protic ionic liquids, which contain an acidic
proton, are formed by proton transfer from a Brønsted acid to a Brønsted base
(Ghandi 2014), while aprotic ionic liquids do not contain an acidic proton and
require synthetic strategies different from the simple acid–base reactions used to
obtain protic ionic liquids (Wasserscheid and Welton 2008).
Protic ionic liquids are more recent, easier to prepare, and less toxic (Menne
et al. 2013) when compared with aprotic ionic liquids. Also, protic ionic liquids usu-
ally have lower melting points and have higher conductivity and fluidity, when com-
pared with aprotic ionic liquids. However, the ionicity of protic ionic liquids may be
limited when compared to aprotic ionic liquids, since protic ionic liquids may form
a hydrogen bond network (Noda et al. 2003; Fumino et al. 2009; Miran et al. 2011).
Moreover, ionic liquids have also been classified into four main categories
(Fig. 5.2), in terms of the cation that constitutes them, either dialkylimidazolium,
N-alkyl-pyridinium, phosphonium, or alkylammonium (Ghandi 2014; Santos de
Almeida et al. 2017a).
The imidazolium-based ionic liquids are the most studied because they are sta-
ble, within reductive and oxidative conditions, and also because of the low viscosity
and easy synthesis of these ionic liquids (Ghandi 2014; Santos de Almeida et al.
2017a). Several applications of these ionic liquids have been described, such as
solvents, catalysts (Santos de Almeida et al. 2017a), or, more recently, solubility
promoters (Dobler et al. 2012; Santos de Almeida et al. 2017b). However, the use of
Fig. 5.2 Four main categories of ionic liquids (ILs) in terms of the contained cations: dialkylimid-
azolium, N-alkylpyridinium, phosphonium, and alkylammonium
imidazolium-based ionic liquids requires caution, since they have been described as
toxic, depending on the size of the alkyl chain attached to the imidazolium cation
(Romero et al. 2008; Santos de Almeida et al. 2017b). Furthermore, for imidazolium-
based ionic liquids, it should also be considered the fact that unexpected secondary
reactions may occur in basic medium, leading to N-heterocyclic carbenes (Ghandi
2014; Santos de Almeida et al. 2017a).
In terms of the pyridinium-based ionic liquids, they are more recent than the
previously described ionic liquids, and therefore, studies concerning the stability,
reactivity, and catalytic role in organic synthesis of these ionic liquids continue to
be developed (Ghandi 2014). These ionic liquids have been successfully used as
solvents in various well-known organic reactions, such as the Friedel–Crafts
(Snelders and Dyson 2011; Ghandi 2014; Santos de Almeida et al. 2017a) and
Grignard reactions (Ghandi 2014; Santos de Almeida et al. 2017a). Pyridinium-
based ionic liquids have also been used as catalysts in organic reactions (Tharun
et al. 2014) and towards the synthesis of some pharmaceutical compounds (Pajuste
et al. 2011). On the other hand, further studies demonstrate that pyridinium-based
ionic liquids have a negative effect on the rate of some Diels–Alder reactions
(Khupse and Kumar 2011) and poor regioselectivity in palladium-catalyzed telom-

erization of butadiene with methanol (Ghandi 2014; Santos de Almeida et al. 2017a).
The phosphonium-based ionic liquids are more recent when compared to imid-
azolium- and pyridinium-based ionic liquids and thermally more stable than imid-
azolium- and ammonium-based salts. The higher thermal stability makes the use of
phosphonium-based ionic liquids suitable for reactions at temperatures above
100 °C (Dake et al. 2009). They have also been used as catalysts and solvents in
various reactions (Cao and Zhu 2009; Fan et al. 2012).
Finally, the quaternary ammonium-based ionic liquids are among the more
recently studied, and compared with the previously depicted ionic liquids, they are
described as having lower toxicity (Melo et al. 2013; Gouveia et al. 2014). Regarding
their applications, the ammonium-based ionic liquids have been used, as electro-
lytes, due to the low viscosity and melting point of these compounds (Khupse and
Kumar 2010; Melo et al. 2013). Ammonium-based ionic liquids have also been used
industrially due to the surface activity, bioactivity, and high antimicrobial activity of
these salts (Pernak et al. 2006; Melo et al. 2013) as well as to promote, simultane-
ously, the topical drug delivery and antibiotic activity (Zakrewsky et al. 2014).
Ammonium-based ionic liquids have also been referred to as greener alternatives to
the solvents commonly used in the pharmaceutical industry (Melo et al. 2013).
In general, ionic liquids have been widely used as catalysts and reaction media,
in separations and extractions, as electrolytes, and also as lubricants and propellants
(Balk et al. 2015). However, it is in the pharmaceutical and medical fields that the
applications of ionic liquids have attracted special attention and where the knowl-
edge of the toxicity of these materials gains a major importance.
5.2.3 Toxicity for Humans and the Environment
Nowadays, the use of organic compounds by the industry, such as volatile organic
compounds, has become a growing concern (Thuy Pham et al. 2010; Handy 2011).
The toxicity of these volatile organic compounds to process operators and to the
environment, as well as the volatile and flammable nature, encourages industries to
find alternatives, less toxic and, therefore, to identify solvents and processes that are
“environmentally friendly” (Thuy Pham et al. 2010).
Ionic liquids have been suggested as a “green” alternative to the traditional
organic solvents, mostly because of the negligible vapor pressure of these com-
pounds (Thuy Pham et al. 2010; Handy 2011). Furthermore, it is also known that
ionic liquids, as solvents, dissolve and favor the transformation of organic, inor-
ganic, and polymeric materials allowing a more efficient use of renewable resources
or residual materials (Cevasco and Chiappe 2014). For instance, ionic liquids have
been used in the dissolution of cellulose and biopolymers, to develop new process-
ing strategies in materials sciences and fuels, for the recycling of waste plastics by
the depolymerization of non-natural polymers, in the recovery of rare Earth ele-
ments, in the extraction of metals from waste, and to capture CO2, and other gases,
produced by the burning of fossil fuels and other industrial activities (Cevasco and
Chiappe 2014).
When using ionic liquids and to guarantee the elimination of potential environ-
mental hazards, other issues need to be considered such as the degradation of ionic
liquids and the solubility of these salts in water, which may represent a medium
through which the ionic liquids may be released into the environment (Thuy Pham
et al. 2010). Hence, the applicability of ionic liquids, although very promising, must
always be carefully considered, since it is fundamental to evaluate the impact of
these compounds on humans and the environment. Bearing this impact in mind and
although much is still to be done, several studies considering the toxicity of these
salts have already been developed (Frade and Afonso 2010; Thuy Pham et al. 2010;
Petkovic et al. 2011; Amde et al. 2015). In result, although some ionic liquids have
been referred to as “green” and “environmentally friendly” (Khupse and Kumar
2010), some are considered more toxic than conventional solvents (Garcia et al.
2005). In fact, it is known that some of these salts, such as the imidazole-based ionic
liquids, have been described as more toxic than some of the volatile organic com-
pounds used in the industry (Garcia et al. 2005). It is also well known that the size
of the cations’ alkyl chain is related to an increase in the toxicity of these ionic liq-
uids (Romero et al. 2008; Santos de Almeida et al. 2017b).
Several toxicity studies performed in aquatic (Couling et al. 2006; Wells and
Coombe 2006) and terrestrial (Matzke et al. 2007) environments, as well as on
human, rat, and mice cell lines (Ranke et al. 2007; Frade and Afonso 2010), showed
an increase in toxicity with the size of the cation alkyl chain. Additionally, a recent
study, performed on porcine ear skin, also showed that the size of the alkyl chain, in
imidazole-based ionic liquids, increases the permeation of these ionic liquids
through the skin. Results revealed that 1-hexyl-3-methylimidazolium bromide
showed a permeation percentage through the skin higher than 80% (Júlio et al.
2017), thus showing the impact that the toxicity of these ionic liquids may have
towards humans, particularly when incorporated in delivery systems. It is also
known that the toxicity of ionic liquids is mostly affected not only by the length of
the cation side chain but also by the number of longer alkyl chains (Frade and
Afonso 2010).
Moreover, even though the cations present seem to have a higher impact on the
toxicity of ionic liquids, especially phosphonium and imidazolium cations, the
anions present, particularly lipophilic and unstable anions, may have a meaningful
influence on the overall toxicity (Matzke et al. 2007; Ventura et al. 2012). Although
the information about anions’ contribution to the toxicity of ionic liquids is less
known, a recent study evaluated the human dermal toxicity of various ionic liquids
for several representative anions. Results revealed that bis(trifluoromethylsulfonyl)
imide-based ionic liquids showed significant toxicity in human keratinocyte and
fibroblast cell line, similar to the toxicity of xylene, a toxic organic solvent (Hwang
et al. 2017), confirming that the type of anion present may also considerably impact
the toxicity of ionic liquids.
Thus, some ionic liquids are very far from the so-called “environmentally
friendly” classification, and caution needs to be taken when using this denomination.
On a more promising note, it has been described that some structural transforma-
tions, such as the incorporation of functional polar groups in the alkyl chain (Thuy
Pham et al. 2010), or the substitution of the alkyl group with a hydrogen atom (Pretti
et al. 2009), may reduce the toxicity of the prepared ionic liquids or even increase
the biodegradation of these compounds. These possibilities prove, once again, that
tailoring these salts, accordingly to a certain property, is a major advantage that
should continue to be used to develop new and eco-friendly ionic liquids. In fact, it
has been recently described in the synthesis of room temperature ionic liquids, pre-
pared from the biocompounds, such as choline and amino acids, that showed low
toxicity for humans and to the environment (Gouveia et al. 2014). These ionic liq-
uids may represent a promising and more eco-friendly class of materials with vast
applicability in many different areas, such as health and particularly in drug delivery.
5.2.4 Applicability in Drug Delivery Systems
Because ionic liquids may be tailored towards a specific property, they may be
incorporated in water, oils, or hydroalcoholic solutions, depending upon the ions
present, which convey upon them the possibility to be used in different mediums
and to be included in diverse drug delivery systems, towards various goals relevant
to the pharmaceutical field (Moniruzzaman et al. 2010a, b, Dobler et al. 2012, 2015,
Santos de Almeida et al. 2015, 2017b; Zhang et al. 2016b). In fact, ionic liquids have
been used to extract bioactive compounds from plants (Tang et al. 2012) or nonste-
roidal anti-inflammatory drugs from aqueous streams (Marrucho et al. 2014; Álvarez
et al. 2015). Ionic liquids have also been used as highly selective sorbent coatings
for analyte-specific extractions in solid-phase microextractions (Ho et al. 2011).
Additionally, there has been a growing interest in ionic liquids as stabilizers of pro-
teins and enzymes (Fujita et al. 2005; Rantwijk and Sheldon 2007; Micaêlo and
Soares 2008; Chen et al. 2010; Constatinescu et al. 2010; Marrucho et al. 2014;
Álvarez et al. 2015; Ghosh et al. 2015; Nandi et al. 2017; Sprenger et al. 2017).
Although proteins have a pharmaceutical potential, their instability is a limitation
for their use in therapeutics. In this matter, ionic liquids have been applied to enhance
the solubility and stability of proteins (Fujita et al. 2005), and some ionic liquids
have shown to suppress protein aggregation, regardless of the protein stabilizing–
destabilizing effect (Constatinescu et al. 2010). Also enzymes seem to tolerate some
ionic liquids in higher concentrations when compared to water-miscible molecular
solvents (Rantwijk and Sheldon 2007). Moreover, ionic liquids have been studied as
functional excipients, namely, to enhance solubility sparingly soluble or insoluble
drugs (Forte et al. 2012; Santos et al. 2013; Marrucho et al. 2014; Santos de Almeida
et al. 2015, 2017b) and to promote skin penetration, due to the ionic character of
these salts and the capacity to act as surfactants (Dobler et al. 2012, 2015).
More recently, there has been a growing interest in the use of active pharmaceuti-
cal ingredients to synthetize ionic liquids with biological activity, the so-called
pharmaceutical ingredient ionic liquids. These salts may present great potential,
provided the performed modifications do not impact negatively the properties of the
active (Ferraz et al. 2015a). Also, the preparation of these pharmaceutical ingredient
ionic liquids may help to overcome some drawbacks, concerning permeability, bio-
availability, polymorphism, and low solubility in water (Hough et al. 2007;
Stoimenovski et al. 2010; Shamshina et al. 2013; Marrucho et al. 2014), but it is still
necessary to consider the associated risks in developing a liquid form of a drug
(Shamshina et al. 2013).
Even though there has been an increased interest in the various applications of
ionic liquids in the pharmaceutical and medical fields, it is vital to consider the tox-
icity and more importantly if these salts are useful at non-toxic concentrations,
where cell viability is maintained.
Some studies have shown that certain types of ionic liquids, such as those derived
from ammonium ionic liquids, are considered less toxic to humans and the environ-
ment (Gouveia et al. 2014) and may be used as alternative solvents in the manufac-
ture of pharmaceuticals (Melo et al. 2013). More recently, it has been shown that
choline- and amino acid-based ionic liquids may be suitable as functional ingredi-
ents since at non-toxic concentrations, they allow an increase in drug solubility and
loading while maintaining the stability of the prepared formulations (Santos de
Almeida et al. 2017b).
Although several studies continue to show the potential utility of ionic liquids in
the pharmaceutical field, such as solubility enhancers, the lack of information con-
cerning the environmental impact of these salts continues to represent a major bar-
rier to their use by industry.
5.3 Nanopharmaceuticals and Ionic Liquids
Many pharmaceutical companies continue to pursue different strategies to over-

come low drug solubility and loading. Among the many strategies attempted
included the structural modification of the active pharmaceutical ingredients (pro-
drug) or using complex formulations like solid dispersions, nanosuspensions, com-
plexation, and crystal engineering or nanoparticles. Indeed, the concept of
nanoparticles for the design of new drug delivery systems offered unique opportuni-
ties for diagnosis and therapy of several diseases (Reis et al. 2006a; Saji et al. 2010).
Based on the current progress, nanoparticles can be engineered to provide opportu-
nities for the site-specific delivery of drugs (Ferraz et al. 2011; Shamshina et al.
2013) and to improve the bioavailability of numerous therapeutic agents with bio-
medical applications (Reis et al. 2008; Elkhodiry et al. 2016; Zazo et al. 2016;
Rancoule et al. 2016).
As seen in Fig. 5.3, there are several nano-based systems for drug delivery such
as quantum dots, carbon nanotubes, fullerenes, liposomes, and other lipidic systems
such as ethosomes, solid lipid nanoparticles, nanostructured lipid carriers, phyto-
somes, transfersomes, micelles, metallic nanoparticles, such as gold and silver, or
mesoporous silica systems, ceramic-based nanoparticles, mesoporous dendrimers,
and polymeric nanoparticles (Santos de Almeida et al. 2017a). Several methods
Fig. 5.3 Representation of several organic and inorganic nano-based delivery systems used in
drug delivery
have been applied to obtain the smallest, most stable, and more efficient systems to
carry the drug to the target tissue.
More recently, in attempt to obtain more efficient nanosystems and considering
the described properties of both ionic liquids and nanoparticles, there has been a
growing interest in combining the valuable properties of both these materials.
5.3.1 A
pplications of Nanoparticles and Ionic Liquid
Combined Systems
Ionic liquid and Nanoparticle combined systems present extraordinary properties

(Ueno and Watanabe 2011; Prabhu Charan et al. 2014; He and Alexandridis 2016).
These mixed systems strongly depend upon the balance between intra- and intermo-
lecular interactions (He and Alexandridis 2016), and the ionic liquids may be used
as a protective layer around the nanoparticles, improving the chemical and thermal
stability of the particles. Moreover, since ionic liquids may be designed accordingly
to a specific property, they may be prepared to stabilize the colloidal particles (Ueno
and Watanabe 2011). In fact, studies showed that iridium or gold nanoparticles were
stabilized with ionic liquids and less agglomeration was observed (Itoh et al. 2004;
Kim et al. 2006; Huang et al. 2016).
Ionic liquids also facilitated the synthesis of nanoparticles from highly polar
starting materials under ambient and water-poor or anhydrous conditions
(Zahmakıran and Ozkar 2011; Huang et al. 2016). Gold nanoparticles were synthe-
tized using N-(2-hydroethyl)-N-methyl morpholinium tetrafluoroborate, and alco-
hol ionic liquids acted as reducing agents and stabilizers leading to the formation of
monodisperse and size-selective metal nanoparticles (Kim et al. 2006).
The conjugation of ionic liquids and nanoparticles for the development of bio-
sensors has also been studied. Biosensors have been the object of numerous studies
due to their higher selectivity and simplicity, compared to traditional analytical pro-
cedures. In fact, recently, Zappi et al. (Zappi et al. 2017) have performed a modifica-
tion of a working electrode to construct different biosensing platforms, by
immobilizing three different enzymes: lipase from Candida rugosa, glucose oxi-
dase from Aspergillus niger, and alcohol dehydrogenase from Saccharomyces cere-
visiae. Choline-glycine, choline-serine, choline-phenylalanine, choline-histidine,
and three nanomaterials (graphene, gold nanoparticles, multiwalled carbon nano-
tubes) were biologically friendly. Of all the studied combinations, the best modifi-
cation resulted in a glassy carbon-multiwalled nanotube with choline-phenylalanine.
All biosensors showed good features, particularly analytical parameters such as
sensitivity, limit of detection, and linearity range.
Ionic liquids can also change nanoparticle morphology (Zhou and Antonietti
2003; Xu et al. 2008; Xia et al. 2009; Esmaili and Habibi-Yangjeh 2011; Imanishi
et al. 2011; Ji et al. 2011; Kowsari and Ghezelbash 2012; Liu et al. 2012; Rajiv
Gandhi et al. 2013; Rabieh and Bagheri 2014). The use of ionic liquids in shape-
controlled microwave-assisted synthesis of nanoparticles has also been reported,
providing excellent microwave-absorbing agents (Xu et al. 2008; Xia et al. 2009; Ji
et al. 2011; Tong et al. 2013).
Furthermore, ionic liquids may be used to modulate particle surface, and the
grafted ionic liquids can render nanoparticles stably dispersed in their media and
improve the stability over time. This fact was observed in gold nanoparticles with
ionic liquids based on the imidazolium cation (Itoh et al. 2004). Results indicated
that hexafluorophosphoric acid acts as an efficient phase transfer agent for the
1-modified gold nanoparticles allowing the solubilization of the particles in the
ionic liquid phase. Several examples of surface modification are described in the
literature (Wolfram et al. 2015; Mahmoodi et al. 2016; Padmanabhan et al. 2016).
One example of surface manipulation is related to the properties of polymeric
nanoparticles which were improved by Field Emission Scanning Electron
Microscopy, using hydrophilic ionic liquids (Takahashi et al. 2015). Another exam-
ple includes the change of the surface and mechanical properties of polymeric mem-
branes, such as chitosan, after using ionic liquids (Otvagina et al. 2016).
The combined systems may be formed by covalently bonding ionic liquids onto
the nanoparticles’ surface or by incorporating nanoparticles into preformed struc-
tures or emulsions containing ionic liquids (He and Alexandridis 2016). The inter-
molecular interactions of nanoparticles and ionic liquids were extensively accessed
in nanoparticles with 5-fluorouracil. These soft delivery systems based on lyotropic
liquid crystals were prepared and characterized, in bulk and as disperse
nanoparticles, to be potentially used as drug delivery systems of 5-fluorouracil

(Astolfi et al. 2017). In this study, it was shown that encapsulation of 5-fluorouracil
in the studied lipid matrices does not impact the particular phase structure observed,
unless electrostatic interactions were involved. Those cubosomes containing 5-fluo-
rouracil were successfully prepared and had particle size of 215 nm and narrow
particle size distribution (polydispersity index of 0.2). Despite the low entrapment
efficiency values (25%), 5-fluorouracil-loaded cubosomes significantly enhanced
the cytotoxic activity of the drug against breast cancer cells, MDA-MB-231 cells.
Ionic liquids may also be chemically bonded onto nanoparticles’ surfaces, gen-
erating the so-called supported ionic liquid nanoparticles, providing an upgrade in
terms of dispersibility and thermostability (Selvam et al. 2012).
Ionic liquids have also been applied in the preparation of porous crystalline
nanomaterials. In this case, TiO2 nanoparticles were prepared using N,N-bis[2-
methylbutyl] imidazolium hexafluorophosphate, since it has a symmetrical struc-
ture with two branched alkyl groups on the imidazolium ring (Meng et al. 2010).
Those nanoparticles, sizing around 50 nm, were applied as adsorbent of biomacro-
molecules because they have a relatively large pore size (0.04 versus 0.28 cm3/g)
and surface area (5.2 versus 68.3 m2/g). Similarly, core shell silica nanoparticles
were functionalized with N-methylimidazole to extract sulfonylurea herbicides
from water samples (Lerma-García et al. 2013).
In a different perspective, ionic liquids may also act as initiators of polymeriza-
tion for nanoparticle synthesis. In a previous study, stannous 2-ethylhexanoate was
applied during the polymerization process of poly(lactic-co-glycolic acid) nanopar-
ticles for drug delivery (Ebrahimnezhad et al. 2013). The studied drug, silibinin, a
polyphenolic flavonoid, has a very low water solubility. An increase in its bioavail-
ability was observed, leading to a downregulation of telomerase gene expression in
cancer cells.
Additionally, the size of nanoparticles influences the performance of the parti-
cles, and the use of ionic liquids has shown to be relevant in terms of size control
(Moniruzzaman et al. 2010a; Wang et al. 2013; Prabhu Charan et al. 2014; He and
Alexandridis 2016). Copper-based systems with ionic liquids have also been pre-
pared, and the incorporation of ionic liquids led to a particle size reduction, as well
as relatively greater diffusion coefficient and electrical conductivity (Chen et al.
2009). Recently, many other electrodes have been designed for the analysis of bio-
logical and food compounds (Bavandpour et al. 2015; Cheraghi et al. 2016, 2017;
Karimi-Maleh et al. 2016; Sheikhshoaie et al. 2017).
Polyionic liquids, a subclass of polyelectrolytes, are considered multitask per-
formers in catalysis and material chemistry (Yuan et al. 2013; Zhang et al. 2016b).
These polyionic liquids share properties from ionic liquids and polymers, and they
are good stabilizers for nanoparticles’ dispersion; consequently, they may be used
in the synthesis of nanoparticles (He and Alexandridis 2016) to address the limit of
precise control over structural complexity and ordering in polymer nanoparticles
(Zhang et al. 2016b).
When considering the applications of ionic liquid–nanoparticle combined sys-
tems in the pharmaceutical area, some studies have already shown the applicability
of ionic liquids in microemulsion carrier systems (Moniruzzaman et al. 2010a, b;

Goindi et al. 2014). In particular, ionic liquid-in-oil microemulsions were applied as
drug carriers for low water-soluble drugs like acyclovir, methotrexate, 1-[(5-(p-
nitrophenyl)furfurylidene)amino]hydantoin sodium (dantrolene sodium)
(Moniruzzaman et al. 2010a), and 5-fluorouracil (Goindi et al. 2014). Moniruzzaman
and coworkers (Moniruzzaman et al. 2010a) prepared nanometer-sized ionic liquid
droplets in isopropyl myristate with a blend of non-ionic surfactants like Tween-80®
and Span-20®, using a set of ionic liquids as a dispersed phase. Results showed
enhanced solubilization of the abovementioned drugs in the prepared ionic liquid
microemulsions. The same group had also prepared a similar blend of non-ionic
surfactants, with isopropyl myristate as an oil phase and the ionic liquid dimethyl-
imidazolium dimethylphosphate as pseudophase, to increase the solubility of acy-
clovir (Moniruzzaman et al. 2010b). Small particles were produced (smaller than
34 nm), and skin studies using hairless micropig skin showed that no transport of
free acyclovir was observed in Franz cell model. With the ionic liquid microemul-
sions, skin penetration of acyclovir was improved. Another ionic liquid-in-oil
microemulsions, with 5-fluorouracil, was developed for skin delivery (Goindi et al.
2014). This system was prepared using isopropyl myristate, Tween 80®/Span 20®,
and the imidazole-based ionic liquid 1-butyl-3-methylimidazolium bromide. The
solubility of 5-fluorouracil was 2.6 times higher in 1-butyl-3-methylimidazolium
bromide than in aqueous solution. Furthermore, small and monodisperse droplets
were formed (less than 11.5 nm and polydispersity index lower than 0.141), and the
ex vivo results showed an improvement in 5-fluorouracil permeation using animal
models when compared to both aqueous solution and water-in-oil microemulsions.
In addition, the in vivo studies using dimethylbenz(a)anthracene/12-O-
tetradecanoylphorbol- 13-acetate-induced mice skin carcinogenesis model con-
firmed no side effects such as erythema and irritation. The histopathological analysis
also supported the previous observation. Moreover, this study also confirmed that
the ionic liquid-in-oil microemulsions reduced tumor volume.
Analogously for skin delivery, an ionic liquid-in-water microemulsion with
etodolac was also prepared using the ionic liquids 1-butyl-3-methylimidazolium
hexafluorophosphate, Tween 80® as surfactant, and ethanol as co-surfactant (Goindi
et al. 2015). Ex vivo permeation studies using rat skin showed that the ionic liquid-
in-water-based microemulsions lead to a higher drug permeation when compared to
the oil-in-water microemulsions and to the oily solution of etodolac. Results showed
that the ionic liquid-in-water microemulsions containing the etodolac drug were
more effective in controlling in vivo inflammation than the oily solution, ionic
liquid-in-water microemulsions, and marketed formulation of etodolac.
In another study, microemulsions with ionic liquids were developed and charac-
terized for topical delivery of dencichine (Wang et al. 2018). The incorporation of
two imidazolium ionic liquids, 1-hydroxyethyl-3-methylimidazolium chloride and
1-butyl-3-methylimidazolium dodecanesulfate, into the aqueous and surfactant
phases, respectively, allowed an outstanding enhancement on skin permeation. The
results from in vitro skin permeation assay suggested a strong enhancement (ten-
fold) on the topical delivery of the drug for the formulation with ionic liquids. The
nanocarrier, containing ionic liquids, reduced the skin barrier properties by disrupt-
ing the regular and compact arrangements of corneocytes and moderating the sur-
face properties of the stratum corneum. The in vivo pharmacodynamic evaluation
indicated the significant hemostatic activity of the drug by the topical application of
the vehicle. Additionally, the formulation showed minor cell toxicity and skin
irritation.
The influence of these ionic liquids on the skin penetration and antimicrobial
properties of the drug was also investigated in an emulsion gel of 4-hydroxybenzoic
acid propyl ester, caffeine, and testosterone (Dobler et al. 2015). In this study, the
addition of hydrophilic ionic liquids, imidazolium- and pyridinium-based, leads to
a strong decrease of the viscosity of the emulsion gel containing SEPINEO™ P 600,
but with no influence in hydroxyethylcellulose gel, and all formulations were stable
over storage time (3 months). No phase separation or significant changes in viscos-
ity, droplet size, or pH were observed during the same period. After a 24 h period,
the skin penetration of caffeine was twofold higher compared to the formulation
without hexylpyridinium chloride, but no skin penetration was observed for the
lipophilic drug (testosterone).
In another area such as tissue engineering, polycaprolactone particles have been
developed as an injectable cell delivery system by using trioctylmethylammonium
chloride, a room temperature ionic liquid, and camphene (Kim et al. 2016). These
authors aimed to develop a neural cell delivery scaffold for neuron tissue engineer-
ing, and results were very promising. They showed that camphene with trioctyl-
methylammonium chloride weight ratio considerably influenced the sphere and
pore size, the pore structure, as well as the surface morphologies. Authors suggested
that the prepared microspheres may possibly be applied.
For oral delivery, another formulation based on long-chain lipids was freshly
prepared as homogeneous mixtures of lipids, surfactant, and cosolvent (Sahbaz
et al. 2017). Self-emulsifying drug delivery systems with ionic liquids (didecyldi-
methylammonium, butyldodecyldimethylammonium, or didecyldimethylammo-
nium salts, among others) were formed directly from tolfenamic acid, meclofenamic
acid, diclofenac, and ibuprofen by pairing with lipophilic counterions. In vivo
administration of these self-emulsifying drug delivery systems led to a reduced
maximum plasma concentration, and the time period of exposure extended up to
48 h post-dose.
Environmentally, the combination of ionic liquids and nanoparticles has also
been studied. It is known that industrial effluents contain many hazardous sub-
stances such as nitrobenzene or 4-nitrophenol that are discharged from industrial
processes through water sources, potentially leading to negative health harsh effects.
The use of advanced oxidation techniques exploiting nanomaterials is a cost-
effective, fast, and proper method, towards the degradation of aromatic nitro com-
pounds, that may serve as an alternative to overcome these events. Additionally,
ultrarapid catalytic degradation of 4-nitrophenol was done with ionic liquid recover-
able and reusable ibuprofen derived silver nanoparticles, which had a size distribu-
tion in the range of 12.5 ± 1.5 nm (Hassan et al. 2017). The prepared nanoparticles,
when stored at room temperature, were highly stable for more than 2 months and
showed outstanding ultrarapid catalytic activity for the complete degradation of

toxic 4-nitrophenol into non-toxic 4-aminophenol within 40 s.
Hence, studies to date reveal a promising potential in the combination of ionic
liquids and nanosystems, though much is still to be done. In fact, although the for-
mulation of ionic liquids with nanosystems combined materials may represent a
major progress in drug delivery, caution needs to be taken, and more studies need be
done to assess some relevant questions such as the impact of these systems on
humans and the environment, as well as to better understand the interactions
between ionic liquids and the nanosystems.
5.3.2 Regulatory Gaps
The pharmaceutical industry is facing a growing pressure on several levels such as

a wide range of environmental issues, increasing cost of healthcare systems, major
losses of revenue owing to patent expirations, lengthening of drug-development
cycles, and more demanding regulatory requirements. In the case of ionic liquids,
the most important question is how ionic liquid and nanopharmaceutical combined
systems will be defined and regulated. Regulatory authorities should clearly define
and understand ionic liquids, providing a scientific classification system for these
salts as well as techniques for proving the structure and purity of these compounds.
For clinical translation, the delivery mechanisms and all steps such as demonstra-
tion of shelf life should be also required. Many of the problems facing isolated ionic
liquids are shared with nanotechnology; these are related to toxicity, relative
expense, and a need for terminology. Using nanopharmaceuticals already approved
by the regulatory authority in the design of these new ionic liquid and nanoparticle
combined systems may be a good starting point, because then success will encour-
age further investment.
5.4 Conclusions
This chapter illustrates the considerable potential of ionic liquids, particularly with
respect to the applicability of these salts in the pharmaceutical field and their com-
bination with nanopharmaceuticals and also on how promising, but little explored,
the combination between nanopharmaceuticals and ionic liquids is.
The synthesis, purification, classification, and toxicity of ionic liquids are con-
sidered herein to help in understanding how these salts may help to improve the
efficiency of drug delivery systems, particularly those involving
nanopharmaceuticals.
It becomes clear that ionic liquids may be valuable ingredients to incorporate and
improve the performance of several drug delivery systems in consequence of the
structural and functional flexibility of these compounds. However, the impact of
ionic liquids on the environment is a valid concern, and recent efforts have been
done on understanding the toxicity of these materials and in developing new and
less toxic ionic liquids. In terms of considering the toxicity of ionic liquids, several
studies evaluating the applicability of these compounds in the health area have been
developed, where the concern with toxicity is a focus.
More recently, in consequence of the known prized properties of both ionic liq-
uids and nanoparticles, new systems combining those materials have been devel-
oped. Most of the examples described here, concerning the combination of
nanoparticles and ionic liquids, are still “proofs-of-concept,” and no significant
efforts have been made to bring them from the laboratory to an industrial scale. In
fact, the development of nanopharmaceutical applications using ionic liquid-based
methodologies requires a deep understanding of the molecular and macroscopic
properties of ionic liquids. Even though the conventional pharmaceutical industry is
generally based on solids and conventional dosage forms like tablets and capsules,
the use of ionic liquids should be pushed forward because it might recycle many of
the drugs that have been put on hold owing to limited bioavailability, thus bringing
a new market value for those products.
Acknowledgments Ana Júlio would like to thank the Foundation for Science and Technology,
I.P., who financed her work under the project UDI/DTP/04567/2016.
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Chapter 6
Therapeutic Implications
of Nanopharmaceuticals in Skin Delivery
Ana Henriques Mota, Ana Santos-Rebelo, António José Almeida,

Contents
6.1 Introduction 206
6.1.1 Skin Delivery 208
6.2 Nanotechnology 209
6.3 Nanopharmaceuticals 210
6.3.1 Conventional Therapeutics Versus Nanopharmaceuticals
for Skin Application 210
6.3.2 Physicochemical Characteristics of Nanocarriers and Their Role 211
6.3.3 Influence of Nanoparticles in the Skin 212
6.3.4 Type of Nanopharmaceuticals and Nanocarriers 212
6.3.5 Safety Issues of Nanomedicine 250
6.3.6 Toxicity 255
6.3.7 Regulatory Framework for Nanopharmaceuticals 256
6.4 Conclusion 257
References 257
A. H. Mota · A. J. Almeida
A. Santos-Rebelo
Department of Biomedical Sciences, Faculty of Pharmacy, Universidad de Alcalá,
Alcalá de Henares, Spain
C. P. Reis (*)
e-mail: catarinareis@ff.ulisboa.pt
https://doi.org/10.1007/978-3-030-44925-4_6
206 A. H. Mota et al.
Abstract Background: The delivery of drugs or actives through the skin provides a
convenient route of administration because it is noninvasive and can typically be
self-administered. In skin delivery, different strategies have been developed to
enhance the rate and extent of drug transport across the skin. These strategies consist
in particulate carriers such as nanoparticles generally made of polymers and lipids
where some advantages are solubility improvement of poorly water-soluble drugs,
increase of skin permeation through different mechanisms, and ability to modify
drug pharmacokinetics. Besides polymeric- and lipidic-, metallic-based nanoparti-
cles like silver nanoparticles are recently used as nanocarriers for skin delivery due
to their strong and broad-spectrum antimicrobial characteristics. All encapsulant
materials (polymers, lipids, and metals) and solvents used in the drug delivery are
approved by Food and Drug Administration (FDA) and European Medicines Agency
(EMA) (as biocompatible and biodegradable materials). Major advances: In this
chapter, a compilation of the type and the therapeutic implications of nanotechnol-
ogy applied to the skin in pharmaceutical area as well as safety issues, ecotoxicity
concerns, and regulatory framework of different nanoparticles for skin delivery over
the last 40 years was described.
Keywords Skin delivery · Conventional therapeutics · Nanopharmaceuticals ·

Nanocarriers · Lipidic-based nanoparticles · Polymeric-based nanoparticles ·
Metallic-based nanoparticles · Magnetic-based nanoparticles · Surfactant-based
nanoformulations · Inorganic and organic nanoparticles
6.1 Introduction
The skin, as an interface between the organism and the external environment, plays
a major role in protecting and supporting the life it encloses (Barry 2001; Brown
et al. 2006; van der Maaden et al. 2012; Gupta et al. 2012; Alexander et al. 2012;
Marwah et al. 2016).
The skin structure presents three layers: epidermis (viable epidermis, 130–180 μm
thick, with stratum corneum (SC), stratum granulosum (SG), stratum spinosum
(SS), and stratum basale (SB)), dermis (2000 μm thick), and hypodermis (Fig. 6.1).
Moreover, in the epidermis of palms of hands and soles also exists stratum lucidum
(SL) (Brown et al. 2006; van der Maaden et al. 2012; Gupta et al. 2012; Alexander
et al. 2012; Andrews et al. 2013; Singh et al. 2015; Bahamonde-Norambuena et al.
2015; Marwah et al. 2016; Mota et al. 2017).
The barrier function could be affected by different factors in SC lipids: extracel-
lular localization, the amount of lipid, elongated and tortuous pathway, organization
into lamellar membrane structures, hydrophobic composition, a correct molar ratio
(≈1:1:1, of three key lipids – ceramides, cholesterol, and free fatty acids), and the
unique molecular structures (as acylceramides) (Trotta et al. 2002; Prausnitz and
Langer 2008). However, the loss of barrier function does not involve changes in the
6 Therapeutic Implications of Nanopharmaceuticals in Skin Delivery 207
Fig. 6.1 Schematic representation of the skin’s structure
Table 6.1 Parameters of the skin that influence drug permeability

Thickness Permeation rate (mg/ Permeability
Skin region (μm) cm2/h) coefficient Diffusivity (cm2/s×1010)
Abdomen 15.00 0.34 2.27 × 102 6.00
Volar forearm 16.00 0.31 1.94 × 102 5.90
Back 10.50 0.29 2.76 × 102 3.50
Forehead 13.00 0.85 6.54 × 102 12.90
Back of the 49.00 0.56 1.14 × 102 32.30
hand
Palm 400.00 1.14 2.85 × 103 535.00
proliferation and stratified organization of the keratinocytes, but it has been associ-
ated with ultrastructural anomalies in the upper granular layer, suggesting a pertur-
bance of the assembly or extrusion of lamellar bodies (Epp et al. 2007). The
parameters as thickness, permeation rate, permeation coefficient, and diffusivity are
different below the skin region for the drug permeability of SC, as presented in
Table 6.1 (Marwah et al. 2016).
Thus, skin disorders and skin diseases are emerging and affecting millions of
people every day, mainly in developing countries. Skin diseases can be caused by
numerous infectious pathogens or inflammatory situations, leading to significant
therapeutic challenges. These problems are associated and they depend on the type
of pathogens involved, the integrity of skin layers and respective structures, the
underlying medical condition of the patient. Besides infectious diseases, other
pathologies also exist such as tumors, inflamed skin, chronic inflammatory skin
disorders (psoriasis), sensitive skin (allergic contact dermatitis, atopic dermatitis,
seborrheic dermatitis, or rosacea), etc., which may occur when the skin function is
altered (Gupta et al. 2012; Dawid-Pać 2013; Misery et al. 2014; Mota et al. 2017).
6.1.1 Skin Delivery
The skin is a potential candidate for drug delivery, which has been studied since the
1960s, but it is still a complex barrier (Nounou et al. 2008; Jones and Moss 2010;
Uchechi et al. 2014; Singh et al. 2015; Katikaneni 2015; Bahamonde-Norambuena
et al. 2015; Mota et al. 2017). Drugs can penetrate the SC by intercellular, transcel-
lular (paracellular), transappendageal, and transfollicular routes (Nounou et al.
2008; Singh et al. 2015; Yukuyama et al. 2016; Marwah et al. 2016; Abrego et al.
2016; Mota et al. 2017). In this case, SC is biologically responsible for the limita-
tion in the penetration of drugs or other compounds across the skin (Alexander
et al. 2012).
Cosmetics and drugs can be applied into the skin for different layer targets, as
cosmetics for skin decoration, barriers or repellents, sunscreens, antimicrobials and
antiseptics, or they may maintain the active ingredient onto the surface of the
skin such as corticosteroids, anesthetics and antipsoriatics may be applied in the
skin, between others (Brown et al. 2006; Trommer and Neubert 2006). In skin deliv-
ery, it is important to consider several parameters like anatomical features (skin type
and actual skin condition), drug properties (particle size, lipophilicity, and protein
binding capacity), and formulation characteristics (vehicle composition, rheological
properties, among others) (Trommer and Neubert 2006).
Transepidermal route includes transcellular and intracellular routes. The trans-
cellular route consists in passage through keratinocytes and lipids (Trommer and
Neubert 2006; Marwah et al. 2016), being the shortest pathway for drug penetra-
tion, although it may present a significant resistance to permeation due to cross
lipophilic and hydrophilic characteristics. The intercellular (or paracellular) route
allows the permeant to overcome the SC by permeating between the corneocytes
(Trommer and Neubert 2006). The transappendageal route occurs when the drug
penetrates through the pores present at hair follicles, glands, and sweat ducts; how-
ever, this route only represents 0.1% of the total human skin surface (Trommer and
Neubert 2006; Marwah et al. 2016). The follicular penetration has been revealed as
an important pathway for the administration of topical drugs, allowing up to a cer-
tain extent the penetration of drugs formulated in large particles, such as in the case
of UV protective agents in sunscreens (Trommer and Neubert 2006).
The advantages of skin delivery are the possibility to achieve a targeted delivery,
lower systemic exposure, and lower toxicity (being an alternative to oral and intra-
venous routes). It is also easy to use and non-invasive, extremely commodious and
user-friendly (which increase the compliance, avoiding painful injections). It may
be possible to reduce medical costs (Prausnitz and Langer 2008; Kulkarni et al.
2011; van der Maaden et al. 2012; Alexander et al. 2012; Andrews et al. 2013;
Giannos 2015; Marwah et al. 2016; Hamed et al. 2016). Furthermore, this route
allows the drug permeation across the skin and into the systemic circulation, avoid-
ing the first-pass effect where the molecules of active drug can be converted to inac-
tive molecules or to molecules with side effects. This route may decrease the dose
frequency and ultimately, improve the bioavailability. It is also considered a suitable
route for unconscious or vomiting patient (Prausnitz and Langer 2008; Kulkarni et
al. 2011; van der Maaden et al. 2012; Alexander et al. 2012; Andrews et al. 2013;
Giannos 2015; Marwah et al. 2016; Hamed et al. 2016). However, this route pres-
ents some limitations such as the range of molecular weight of the drugs (<1000 Da),
affinity for lipophilic and hydrophilic phases, low melting point, and charge of
drugs (Uchechi et al. 2014; Katikaneni 2015; Mota et al. 2017). Those drugs may
present other characteristics, such as high potency, short half-life, and no irritation
(Uchechi et al. 2014; Mota et al. 2017). A molecular weight <500 Da with the aim
of ensuring ease of diffusion across the SC (solute diffusitivity is inversely related
to its size) is necessary. Drugs or actives need to have a sufficient aqueous and lipid
solubility with a Log P (octanol/water) of between 1 and 3 for the permeant to suc-
cessfully penetrate the SC and its underlying aqueous layers for systemic delivery
to occur. In skin delivery, intra and intervariabilities are associated with the perme-
ability of intact or diseased skin.
In addition, conventional topical preparations present low percutaneous penetra-
tion (by the barrier function of SC) (Hamishehkar et al. 2013).
In skin delivery, nanoparticles (NPs) can be engineered for topical, dermal or
transdermal delivery for different purposes. Several studies have been demonstrated
that nanoparticles with sizes <50 nm are distributed through lymphatic drainage
into draining lymph nodes where are stimulated the antigen-presenting cells and
lymphocytes (Elsabahy and Wooley 2013).
6.2 Nanotechnology
Pharmaceutical nanotechnology aims to manipulate pharmaceutical compounds

into molecular structures (smaller than 1000 nm) and the development of suitable
carrier (Spuch et al. 2012; Nassiri Koopaei and Abdollahi 2016). This domain is
important in development of new therapeutics helping in overcome major problems
associated with classical formulations as low solubility (an essential factor for drug
effectiveness) or other factors such increase the surface area and the dissolution rate
by reducing the particle size; improve the stability and shelf life; improve the
absorption of insoluble compounds and macromolecules enables to improve the
rates of bioavailability and release; reduce the amount of dose required and by
increase of the safety by reduction of side effects; provide sustained-release profiles
(>24 h) improving patient compliance with drug regimens); combine with other
compounds like as ligands, for targeting drug delivery, among others (Lien and Wu
2008; Jain 2008; Jong and Borm 2008; Singh and Lillard 2009; Spuch et al. 2012;
Leyva-Gómez et al. 2015; Mota et al. 2017). Particularly in skin delivery, pharma-
ceutical nanotechnology can produce different carriers systems such as
lipidic-based NPs (vesicular systems: liposomes, niosomes, microemulsions, nano-

emulsions), polymeric-based NPs (nanocapsules, nanospheres, and dendrimers),
metallic-based NPs (gold and silver NPs), and inorganic and organic NPs (carbon
nanotubes and fullerenes) for topical or transdermal delivery or both (Nounou et al.
2008; Duangjit et al. 2014; Uchechi et al. 2014; Huang et al. 2016; Nassiri Koopaei
and Abdollahi 2016; Mota et al. 2017).
6.3 Nanopharmaceuticals
The definition of nanopharmaceuticals is still not consensual. In 2000, nanopharma-

ceuticals were defined by the National Institutes of Health (NIH) in the National
Nanotechnology Initiative (NNI) as the understanding and control of substances
with dimensions between 1 and 100 nm with therapeutic potential (Parboosing et al.
2012; Weissig et al. 2014). On the other hand, Rivera et al. defined nanopharmaceu-
ticals as pharmaceuticals engineered on the nanoscale, i.e., pharmaceuticals in
which the nanomaterial plays a therapeutic role or adds additional functionality.
Reis et al. like many other others define the term “nanoparticle for drug delivery” as
particles with sizes smaller than 1000 nm. To conclude the classification of nano-
pharmaceuticals two criteria were established: nanoengineering has a major role in
the manufacturing process and the nanomaterials have to be essential for the thera-
peutic activity or have to give additional and unique properties to the active com-
pounds or drugs (Weissig et al. 2014).
6.3.1 C
onventional Therapeutics Versus Nanopharmaceuticals
for Skin Application
In the 1940–1950s, the first understanding of the role of formulation base for the
efficacy of a transdermal preparation was introduced, and in the 1970s, the transder-
mal drug delivery was also introduced followed by the new age of nanodermatol-
ogy, and science has revolutionized since then (Mousavi et al. 2017).
Conventional therapeutics are in general locally applied. The term “conven-
tional” in this review refers to systems based on passive permeation through the SC,
rather than the disruption of the same (Gupta et al. 2012). The traditional therapies
act on the outer surface of the skin (Gupta et al. 2012). In this way, the conventional
topical skin treatments include ointments or creams. Those formulations have sev-
eral disadvantages such as highly concentrated layer of active ingredient, several
applications or application for several days, greasiness and stickiness associated
with application, and low efficiency of delivery system with possible toxic reactions
(irritation or allergic reactions) (Khan et al. 2011; Gupta et al. 2012).
With the aim to minimize the fluctuations of drug level in the body and to increase
drug penetration across the skin layers and transport rate through the epidermis, it is
possible to use penetration enhancers (propylene glycol and oleic acid, among oth-
ers) or novel drug delivery systems (Khan et al. 2011; Gupta et al. 2012). However,
concerning the penetration enhancers, these compounds may increase unwanted
effects (irritative and other toxic side effects) (Gupta et al. 2012).
Moreover, to overcome the disadvantages of conventional therapeutics, other
strategies have been developed like physical techniques (iontophoresis, sonophore-
sis (by ultrasound), microneddle, and others), chemical techniques (prodrugs, salt
formation, and others), and formulation techniques (by the use of nanocarriers)
(Jones and Moss 2010; Vitorino et al. 2014; Singh et al. 2015). The nanocarriers
design give a control release of drug or active compound to the affected region pre-
senting a localized effect by the creation of skin reservoirs. These nanocarriers can
also sustain the release or enhance the uptake (Gupta et al. 2012). For these systems,
the dose required to have a therapeutic effect is generally minimized (Singh et al.
2015). It was in 1995 that the first nano-drug was FDA-approved (Cevc and Vierl
2010; Mousavi et al. 2017).
6.3.2 P
hysicochemical Characteristics of Nanocarriers
and Their Role
Nanocarriers present some physicochemical characteristics. The particle size has

influence the cellular uptake, intracellular trafficking pathways, biodistribution,
strength and type of immune response, inflammatory responses, and in vivo assays
of nanocarriers (Elsabahy and Wooley 2013). NPs smaller than 100 nm are hardly
recognized by the immune system being easily taken up by the cells. An in vitro
study with very small silica NPs showed a higher induction of the TNF-α produc-
tion when compared with larger NPs (Elsabahy and Wooley 2013). Interaction with
cells depends on the material used to make nanocarriers, and the reaction of the cells
is different when interacting with lipids (phospholipids) or with metals (Jong and
Borm 2008). The hydrophobicity affects the immunotoxicity of nanocarriers. For
example, it was described an increase of immune response in association with
poly(Ɛ-caprolactone) and poly(lactic-co-glycolic acid) NPs, PCL and PLGA NPs.
In this way, it was concluded that higher hydrophobicity is associated with a greater
immune response for nanocarriers (Elsabahy and Wooley 2013). Other important
parameters are the half-life time in the circulation, the stability in blood, and the
surface charge for penetration through the blood vessel epithelium and targeted cel-
lular membrane (Epple et al. 2010). The skin acts as a negatively charged mem-
brane. In consequence, it is expected that the presence of charge on the nanocarrier’s
surface influences the diffusion drug across the skin, and the positively charged
nanocarriers present a strong interaction with cells and a better drug permeability
and prolonged pharmaceutical activity (Gupta et al. 2012). The composition is also
important. The nanoemulsions, nanocarrier vesicles, NPs, solid lipid particles,

micelles, and dendrimers are some colloids that are considered for (trans)dermal
drug delivery (Cevc and Vierl 2010). By these parameters, it is important to study
size distribution, polydispersity index (PdI), zeta potential, pH, surface and struc-
ture, stability, chemical interactions, encapsulation efficiency (EE), cytotoxicity and
efficacy.
6.3.3 Influence of Nanoparticles in the Skin
NPs have a high affinity for cellular membrane mainly due to electrostatic interac-
tions (Uchechi et al. 2014). The attachment of NPs to cell membrane depends on the
surface charge of the particles (Uchechi et al. 2014; Mota et al. 2017). The cells
present a negative charge at their surface, which is due to the presence of sulfated
proteoglycan molecules and the interactions between cells, and NPs should be more
probable when NPs present a positive charge (promoting the transdermal perme-
ation), due to electrostatic interaction neutralizing and binding of the membrane or
by endocytosis for cellular uptake (Uchechi et al. 2014; Mota et al. 2017). However,
the composition of the skin might have a significant influence on the final surface
charge of the skin. Cellular uptake and intracellular distribution in lysosomes, mito-
chondria, and cytoplasm of NPs can be changed by different surface properties
of NPs.
Polymer charge density also has a significant impact on membrane permeability,
i.e., having more density, the transport of dye molecule across the membrane would
be easier (Uchechi et al. 2014).
Moreover, the coating process of NPs generally increases the crossing ability
through the endothelial cell layer by three- to four-fold when compared to uncoated
NPs (Uchechi et al. 2014). However, coated NPs with neutral particles generally do
not change their permeation characteristics across the endothelial cell monolayer
(Uchechi et al. 2014).
Nowadays, several studies have indicated the potential ability of NPs to cross the
barrier function of the SC, and this capacity depends on the type of skin, surface
area, and exposure time. Other NPs-related factors that play a role in skin penetra-
tion of NPs are their type and concentration and physicochemical parameters (size,
surface polarity, physical state of the NPs, and the vehicle (and their respective
nature) (Labouta et al. 2011).
6.3.4 Type of Nanopharmaceuticals and Nanocarriers
In the last years the nanoscience and nanotechnology have gain an important role of
research in medicine and the development and production of improved biocompat-
ible materials (with reduction of the toxicity and thus, improving safety) is crucial.
Therefore, it is needed a profound knowledge on drug incorporation and release,

formulation stability and shelf life, biocompatibility, biodistribution, targeting and
functionality. Nanopharmaceutical formulations include emulsions, lipidic-based
NPs, PEGylated proteins, polymeric-based NPs, polypeptides, aptamers, nanocrys-
tals, polymer-based nanoformulations, protein-drug conjugates, surfactant-based
nanoformulations, dendrimers, metallic-based NPs, and others (Khan et al. 2011;
Xu et al. 2014). Nanocarriers can act as drug vehicles into or across the skin, as
penetration enhancers by altering the intercellular lipids in skin layer, and serve as
a depot for sustained release and rate limiting membrane barrier for the modulation
of systemic absorption (Marwah et al. 2016). The polymers used for the nanocarri-
ers and their applications are presented in Table 6.2, and Fig. 6.2 presented the dif-
ferent nanocarriers like liposomes, ethosomes, transfersomes, micro- and
nanoemulsions, dendrimers, micelles, nanocapsules, nanospheres, nanostructured
lipid carriers (NLCs), and solid lipid nanoparticles (SLNs) (Khan et al. 2011; Gupta
et al. 2012; Silva et al. 2015).
Table 6.2 Polymers, nanocarriers and their main applications

Materials source Materials Nanocarrier Applications
Natural materials or Chitosan Drug and gene
derivatives Dextran delivery
Gelatine
Alginates Beads
Lipids Liposomes
(phospholipids) Ethosomes
Transferosomes
Transethosomes
Virosomes
Starch
Carbon-based carriers Carbon Fullerenes Photodynamics
Drug delivery
Polymers Branched polymers Dendrimers Drug delivery
Polylactic acid Polymeric
Polyethyleinemine nanoparticles
Block copolymers
Poly(Ɛ-caprolactone)
Magnetic Ferrofluids SPIONS Imaging (MRI)
USPIONS
Metals Silver Ag NPs Photodynamics
Gold Au NPS Drug delivery
Imaging
Others Cd-selenides Quantum dots Imaging
Zn-selenides In vitro diagnostics
Silica NPs Gene delivery
MRI magnetic resonance imaging, NPs nanoparticles, SPIONs superparamagnetic iron oxide par-
ticles, USPIONs ultrasmall superparamagnetic iron oxide particles
Fig. 6.2 Schematic representation of different nanocarriers. NPs nanoparticles, NLCs nanostruc-
tured lipid carriers, SLNs solid lipid nanoparticles, SPION superparamagnetic iron oxide particles,
and CNTs carbon nanotubes
Lipidic-Based Nanocarriers
Lipidic-based nanocarriers include vesicular nanosystems, NLCs, and SLNs as

shown in Fig. 6.3. Vesicular nanosystems are known by their great potential to
incorporate active molecules for administration which improve their therapeutic
efficacy (Agrawal et al. 2015; Mota et al. 2017).
The advantages of these nanosystems are their great feasibility in the incorpora-
tion of lipophilic drugs, enhancement of physical stability and penetration across
the skin, ability to prevent degradation, and low cost (Uchechi et al. 2014; Agrawal
et al. 2015; Mota et al. 2017). The interaction of these systems with SC is mainly
due to their matrix. Natural lipids present advantages, such as the presence of high
dermophilicity and hydration properties (Esposito et al. 2007; Mota et al. 2017).
These nanocarriers have displayed interesting physicochemical features (as
enhancement of drug absorption, decrease of the entrapped drug degradation, and to
Fig. 6.3 Schematic representation of vesicular nanosystems
provide a sustained drug release) which are adequate for drug delivery through the
skin (Menezes et al. 2016).
Vesicles can be used in dermal and transdermal drug delivery, mainly due their
action as drug carriers delivering the entrapped drug molecules into or across the
skin; they can act as penetration enhancers following the penetration of the indi-
vidual lipid components into the SC and by consequence an alternation of the inter-
cellular lipid lamellae within this skin layer or even they can be a depot with
sustained release of dermally active compounds and used as a rate-limiting mem-
brane barrier for the modulation of systemic absorption which provide a control
transdermal delivery system (Alexander et al. 2012).
Liposomes
Liposomes are spherical, vesicular nanosized structures (self-closed bilayer vesicles

of colloidal dimensions from dispersion of membrane-like lipids in aqueous sol-
vents). They can be self-assembling closed colloidal structures, composed by
a phospholipid and cholesterol bilayer membrane surrounding an aqueous
core (unilamellar vesicles), or several of them involved by phospholipid bilay-
ers (multilamellar vesicles) (Barry 2001; Narasimha Murthy and Shivakumar 2010;
Cevc and Vierl 2010; Ascenso et al. 2011; Parboosing et al. 2012; Gupta et al. 2012;
Weissig et al. 2014; Singh et al. 2015; Nunes et al. 2015; Wu et al. 2015; Garg et al.
2016; Gültekin-Özgüven et al. 2016; Mota et al. 2017).
Moreover, liposomes are amphiphilic and biocompatible nanosystems having
ease of modification, and they can encapsulate hydrophilic or lipophilic compounds
(Trotta et al. 2002; Nounou et al. 2008; Cho et al. 2008; Ascenso et al. 2011, 2015;
Akbarzadeh et al. 2013; Ferreira 2014; Uchechi et al. 2014; Ganesan and Choi
Table 6.3 Classification of Size Classification

vesicular systems
500–5000 nm Multilamellar vesicles (MLV)
100–500 nm Unilamellar vesicles (LUV)
20–100 nm Small unilamellar vesicles (SUV)
>1 μm Giant unilamellar vesicles (GUV)
0.1–1 μm Oligolamellar vesicles (OLV)
20–500 nm Medium unilamellar vesicles (MUV)
2016; Mota et al. 2017). They were the first generation of nanosystems and the most
popular nanosized drug carrier aggregates (Jain 2008; Cevc and Vierl 2010;
Akbarzadeh et al. 2013; Ferreira 2014; Ganesan and Choi 2016; Mota et al. 2017).
The conventional liposomes or mixed lipid micelles, which have low elasticity,
present a low ability to penetrate the skin barrier (Cevc and Gebauer 2003; Menezes
et al. 2016). With the aim of passing through the skin, these systems need to have a
high adaptable membrane (Cevc and Gebauer 2003). The lipidic vesicles can be
classified into unilamellar and multilamellar vesicles as presented in Table 6.3
(Akbarzadeh et al. 2013; Ferreira 2014; Rajabi and Mousa 2016; Mota et al. 2017).
Unilamellar vesicles present a single phospholipid bilayer sphere enclosing an
aqueous solution forming on the inside of the other with smaller size, and on other
side the multilamellar vesicles present a concentric phospholipid spheres separated
by water layers (Akbarzadeh et al. 2013; Mota et al. 2017).
These vesicles generally present a size distribution of 25–2500 nm. The most
common lipid used is phosphatidylcholine (Benson 2005; Nounou et al. 2008;
Alexander et al. 2012; Akbarzadeh et al. 2013; Ferreira 2014; Uchechi et al. 2014;
Weissig et al. 2014; Ascenso et al. 2015; Ganesan and Choi 2016; Gültekin-Özgüven
et al. 2016; Mota et al. 2017).
These nanosystems can encapsulate hydrophilic (in aqueous inner space) or
hydrophobic (in phospholipid bilayer membranes) drugs (Weissig et al. 2014; Singh
et al. 2015; Gültekin-Özgüven et al. 2016). The size distribution is again a key fac-
tor for therapeutic applications. In order to better control particle size, it is conve-
nient to pass liposomes several times under high pressure through membranes with
defined pore size (Weissig et al. 2014). In terms of stability, vesicle fusion, aggrega-
tion, and leakage of the encapsulated material may occur (Gültekin-Özgüven
et al. 2016).
The first liposomal drug in skin delivery was reported by Mezei and Gulasekharan
in 1980, with triamcinolone acetonide which facilitates the accumulation of the
drug within the epidermis and dermis with low systemic levels. The first liposomal
topical product commercialized was Pevaryl® with econazole in 1994 by Cilag AG
(Benson 2005; Elsayed et al. 2007; Ascenso et al. 2011; Weissig et al. 2014; Mota
et al. 2017).
In general, it is not expected that the liposomes can penetrate viably the skin,
although occasional transport processes were also reported by Mezei in 1982 (Mezei
and Gulasekharam 1982; Barry 2001). Liposomes can affect the permeability of SC,
which could occur by the association of the molecules that penetrate into the skin
with intact liposomes, or the molecules that cross the skin could be affected by the
interaction with liposomes or with the liposome compounds. However, these vesi-
cles can also cross the skin by the pilosebaceous units (Ascenso et al. 2011).
Liposomes also enhance the skin deposition presenting a reduction or absence of
effect in percutaneous permeation or systemic absorption (Alexander et al. 2012).
Studies are being done with the aim of improving cutaneous delivery and reducing
toxicity (Gupta et al. 2012).
Liposomal formulation may involve an alternation of the pharmacokinetics (PK)
of the free drug and have three different mechanisms of action (or combinations).
The mechanisms of action include the increase of permeability and retention effect
(EPR effect), targeting of the mononuclear phagocytic system (MPS) and slow or
sustain drug release (typically multilamellar liposomes (MLVs)) (Weissig
et al. 2014).
MLVs are multiple bilayer membranes and each membrane is surrounded by
aqueous layers (Weissig et al. 2014; Wu et al. 2015). Those liposomes are ideal to
accommodate low-molecular-weight hydrophilic molecules. The mechanism of
drug delivery probably involves a slow disintegration (e.g., membrane by mem-
brane), releasing the encapsulated drug (Weissig et al. 2014).
These systems also present disadvantages which are in general associated with
their low solubility and in vivo stability which are probably consequences of phos-
pholipid oxidation and hydrolysis-like reaction (Akbarzadeh et al. 2013; Mota et al.
2017). They also present problems that are associated with clinical application such
as difficulties in sterilization and large-scale production with adequate physical and
chemical stability (Azeem et al. 2009).
Liposomes can be prepared by physical dispersion (by handshaking (thin-film
hydration) and non-handshaking methods), solvent dispersion (with ethanol and
ether injection methods), double-emulsion method, reverse-phase evaporation
method, dehydration-rehydration method, and stable plurilamellar vesicle and
freezing–thawing method (Weiner et al. 1989; Fan et al. 2007; Bai et al. 2014; Singh
et al. 2015; Mota et al. 2017). The layer-by-layer electrostatic deposition method
has been considered as an effective method to enhance the stability of these vesicles.
However, other methods may exist, which could be used to increase stability such
as freeze-drying, powder bed grinding, fluidized bed drying, or spray-drying. Spray-
dying method is the preferable method in the production of dry liposomal powders
because the process is low cost and time- and energy-consuming (Gültekin-Özgüven
et al. 2016).
The properties of liposomes can be influenced by the lipidic composition, sur-
face charge, size, and the preparation method (Akbarzadeh et al. 2013; Mota et al.
2017). Liposomes constitute unsaturated phosphatidylcholine species from natural
sources (e.g., soybean phosphayidylcholine or egg). Natural sources led to more
permeable and less stable bilayers when compared with liposomes with saturated
phospholipids bearing long acyl chains (e.g., dipalmitoylphosphatidylcholine)
which are rigid and present a rather impermeable bilayer structure (Akbarzadeh
et al. 2013; Ferreira 2014; Mota et al. 2017). It is also possible to use cholesterol;
this compound contributes to a stabilization of the structure which results in an

increase of the bilayer’s transition temperature and thus the rigidity of liposomes
(Benson 2005).
Recent studies were contributed to the application of liposomes in delivery of
interferon, gene delivery, and cutaneous vaccination (Benson 2005). Some authors
named these vesicles as pharmacosomes. By definition, they are amphiphilic phos-
pholipid complexes of drugs (mainly poorly soluble drugs) which bind to phospho-
lipids by covalent, elestrostatic, or hydrogen bonding (Singh et al. 2015; Zylberberg
and Matosevic 2016). These vesicles have colloidal dispersions where the drug is
covalently bounded to lipids. Pharmacosomes can be presented as ultrafine vesicu-
lar, micellar, or hexagonal aggregates, which is determined by the chemical struc-
ture of the drug–lipid complex (Singh et al. 2015). This system present an advantage
in comparison with liposomes; they improve delivery of poorly soluble drugs (Singh
et al. 2015; Zylberberg and Matosevic 2016). Other advantages of these nanocarri-
ers are the possibility to incorporate hydrophilic and lipophilic drugs; the aggrega-
tion is dependent on the concentration; they have high entrapment efficiency (due to
the covalent linkage); and the loss is probably due to hydrolysis (Singh et al. 2015).
This system was firstly described in the 1980s, which demonstrated a signifi-
cantly better dissolution profile when compared with free drug. The common
method used to prepare pharmacosomes is by solvent-evaporation following the
drug complexation step (Zylberberg and Matosevic 2016).
In 2001, Pierre et al. developed a liposomal formulation containing
5-aminolevulinic acid for skin cancer therapy with the help of photodynamic ther-
apy. Results revealed a reduction of mean size of 500 nm to 400 nm after loading.
Furthermore, different amounts of drug were localized along the skin layers: 10 μg
rested in SC and 60 μg rested in the epidermis (without SC) and dermis. This for-
mulation has been revealed to provide a good performance with a slower perme-
ation and delivery of the drug (Pierre et al. 2001).
Liposome products are also known to provide good skin protection and excellent
qualities (Ganesan and Choi 2016; Mota et al. 2017). In 1965, Bangham et al.
reported the first liposome (Garg et al. 2016; Mota et al. 2017). Liposomes act as
drug reservoir, when applied into the skin surface, and they only remain in the upper
layer of the SC (Ascenso et al. 2015; Mota et al. 2017). It is also known that lipo-
somes can enhance drug penetration into the skin (specially in the epidermis and
dermis), with localized drug delivery and reduced percutaneous absorption (Nounou
et al. 2008; Mota et al. 2017).
Some drugs used in liposomes for drug delivery through the skin are triamcino-
lone, progesterone, tretinoin, and tetracaine (Garg et al. 2016). Recently, they have
been used in hair growth products containing curcumin with particle size ranging
between 213 and 320 nm (Ganesan and Choi 2016). The use of this type of nano-
systems in the cosmetic area is generally related to skin dehydration, skin care, and
enhancement of skin flexibility. By this reason, other class of vesicular systems,
such as transfersomes and ethosomes was developed (Uchechi et al. 2014; Wischke
et al. 2016).
Ethosomes
Ethosomes are composed of phospholipids (20–45%), water, and high concentra-

tions of short-chain alcohol (in general, ethanol between 20 and 45% and isopropyl
alcohol or propylene glycol (up to 15%)) (Touitou et al. 2000). The phospholipids
which could be used in the ethosomal formulations are soybean phosphatidylcho-
line, egg phosphatidylcholine, hydrogenated phosphatidylcholine, dipalmitoylphos-
phatidylcholine, and disterylphosphatidylcholine (Syeda and Krishna 2015; Garg
et al. 2016; Mota et al. 2017). For alcohols, ethanol and isopropyl alcohol (IPA) could
be used. Both can provide softness for the vesicle membrane and act as skin pene-
tration enhancers (Syeda and Krishna 2015; Mota et al. 2017).
The presence of phospholipids and ethanol (in high concentration) can provide a
synergistic effect which is responsible for the deeper distribution and penetration in
the lipid bilayers of the skin (Singh et al. 2015). The ethanol is an efficient perme-
ation enhancer and when combined with phospholipids can be affected by the inter-
cellular region of SC (Touitou et al. 2000; Bendas and Tadros 2007; Jain et al. 2007;
Babaie et al. 2015; Fathi-Azarbayjani et al. 2015; Sharma et al. 2016; Mota
et al. 2017).
Furthermore, ethosomes can also contain polyglycols (e.g., propylene glycol or
Transcutol RTM®) acting as enhancers for skin penetration. Other possible com-
pound of ethosomes is cholesterol, providing stability to the vesicle membrane
(Syeda and Krishna 2015; Mota et al. 2017).
Ethosomes can present sizes from 30 nm to several microns. One factor that
affects the size is their composition, mainly the higher content of lipids which is
associated with larger vesicles. On the other hand, the higher content of ethanol is
associated with a reduction of size vesicles (Ainbinder et al. 2010; Barupal et al.
2010; Verma and Pathak 2010; Chourasia et al. 2011; Maheshwari et al. 2012;
Zhang et al. 2012, 2014a, b; Ghanbarzadeh and Arami 2013; Zhu et al. 2013; Mota
et al. 2017). Increase in ethanol quantity is associated with skin irritation and con-
tact dermatitis (Akhtar and Pathak 2012; Mota et al. 2017).
These vesicles are able to deliver drugs through the skin layers due to their low
penetration through the biological membranes. By this reason, they can be used as
proficient carrier system for dermal and transdermal delivery (Godin and Touitou
2005; Bendas and Tadros 2007; Elsayed et al. 2007; Jain et al. 2007; Nounou et al.
2008; Rao et al. 2008; Ainbinder et al. 2010; Gupta et al. 2012; Alexander et al.
2012; Kumar et al. 2014; Babaie et al. 2015; Fahmy 2015; Singh et al. 2015; Garg
et al. 2016; Mota et al. 2017). Other characteristic which contributes to a better
penetration in the skin is its soft deformable and malleable property (Choi and
Maibach 2005a; Akhtar and Pathak 2012; Bodade et al. 2013; Fathi-Azarbayjani
et al. 2015; Mota et al. 2017).
Ethosomes were discovered by Touitou in 1996 for skin drug delivery (Elsayed
et al. 2006; Bendas and Tadros 2007; Nounou et al. 2008; Rao et al. 2008; Fahmy
2015; Menezes et al. 2016; Mota et al. 2017). In 2000, Touitou et al. prepared etho-
somes with 2% of Phospholipon 90® and 30% of ethanol and water. These etho-
somes have a high percentage of minoxidil (83±6%). The results demonstrated that
the amount of minoxidil that permeated the skin was 673.0±92.0 μg/cm2, and the
amount of minoxidil in the skin was 69.6±11.0 μg/cm2 (n = 8) (Touitou et al. 2000).
In comparison with other lipidic nanovesicles like liposomes and transfersomes,
ethosomes present higher transdermal fluxes through the skin (Jain et al. 2007;
Ainbinder et al. 2010; Chen et al. 2010; Fahmy 2015; Mota et al. 2017).
They are three types of ethosomes: classic ethosomes, binary ethosomes, and
transethosomes. Usually, classic ethosomes are used to incorporate drugs between
130 Da and 24 kDa of molecular weight. Binary ethosomes are very similar with the
classic ethosomes but present in their composition other type of alcohol (propylene
glycol (PG) or IPA) (Abdulbaqi et al. 2016; Mota et al. 2017). The zeta potential of
ethosomes is generally negative (Ascenso et al. 2013; Abdulbaqi et al. 2016; Mota
et al. 2017). These types of vesicular nanosystems are known by their potential of
dermal and transdermal delivery with an efficient intercellular delivery for hydro-
philic and lipophilic drugs (Chen et al. 2010; Abdulbaqi et al. 2016; Mota et al. 2017).
The advantages of ethosomes are related to their thermodynamic stability, high
loading efficiency and EE, and easy process of preparation (Touitou et al. 2001;
Verma and Pathak 2010; Zhu et al. 2013; Mota et al. 2017). Moreover, another
advantage is their possibility to deliver large molecules (peptides and protein mol-
ecules) (Godin and Touitou 2005; Singh et al. 2015). However, a disadvantage is
possible, the skin’s irritation due to excipients and enhancers of drug delivery sys-
tems (Singh et al. 2015).
Ethosomes could be used for the delivery of large and diverse groups of drugs
like fluorescent probes (quantum dots) in the skin by their quantity with a low pro-
file risk. Their toxicological profiles is well-documented in the scientific literature
(Touitou et al. 2001; Verma and Pathak 2010; Zhu et al. 2013; Mota et al. 2017).
Ethosomes can be incorporated in creams, gels, and patches because of their high
stability, easy application, and better skin permeation and therapeutic results, being
mainly used for transdermal drug delivery (Singh et al. 2015; Abdulbaqi et al. 2016).
However, the mechanism responsible for this permeation into deeper layers is still
unclear (Singh et al. 2015).
Ethosomes can be prepared by cold method (conventional thin-film hydration
method) or hot method (addition of aqueous phase in a controlled manner to the
alcoholic solution of phosphatidylcholine) presented in Fig. 6.4 (Chourasia et al.
2011; Singh et al. 2015; Garg et al. 2016; Mota et al. 2017).
Formulations of ethosomes that are available in the market are: Cellutight EF®
(topical cellulite cream increases metabolism and breakdown of fat), Decorin
cream® (antiaging and hyperpigmentation), Nanominox© (with 4% of minoxidil for
hair growth promotion), Noicellex® (topical anticellulite cream), Skin genuity®
(powerful cellulite buster which claims that it reduces orange peel), and Supravir
cream® (acyclovir for treatment of herpes infection) (Garg et al. 2016; Mota
et al. 2017).
Fig. 6.4 Cold and hot methods to obtain ethosomes
Transfersomes
Transfersomes are (ultra)deformable vesicles which penetrate SC by intracellular or

transcellular routes as represented in Fig. 6.3 (Kulkarni et al. 2011; Gupta et al.
2012; Ascenso et al. 2014). The mechanism for penetration through the skin seems
to be the extracellular pathways between cells, followed by an osmotic gradient due
to the water evaporation present in the surface of vesicles, and by consequence they
start to dry out (Cevc and Blume 2003; Simões et al. 2005; Kulkarni et al. 2011).
This mechanism contributes to an easily and very reproducible transport of drugs
associated in the transfersomes into and across the skin (Cevc and Blume 2003).
This system presents another advantage when compared with liposomes, which is,
the improvement of elasticity (Kulkarni et al. 2011; Zylberberg and Matosevic 2016).
In their composition, alcohol (ethanol, more than 10%, or methanol), lipids
(5–10%, phosphatidylcholine – soya phosphatidylcholine, egg phosphatidylcho-
line, dipalmitoylphosphatidylcholine), surfactants (10–25%), and edge activa-
tor (EA, a single chain with an high radius curvature destabilizing the lipid bilayers
and improving the deformability of the bilayers such as, sodium cholate, sodium
deoxycholate, Span 60®, Span 65®, Span 80®, Tween 20®, Tween 60®, Tween 80®,
oleic acid, and dipotassium glycyrrhizinate), and they make together an aqueous
compartment surrounded by a lipid bilayer and buffer (pH 6, 5–7) agent (as a hydrat-
ing medium, e.g., saline phosphate buffer, pH 6.4) (Elsayed et al. 2006; Fireman
et al. 2011; Ghanbarzadeh and Arami 2013; Mota et al. 2017). The ratio of surfac-
tants (individual and total amount) with phospholipids is responsible for the flexibil-
ity of these vesicles (Kulkarni and Yadav 2011; Ascenso et al. 2014). In consequence
of their composition, they can encapsulate hydrophilic, lipophilic, and amphiphilic
drugs. The value of zeta potential of transfersomes is still controversial.
Ghanbarzadeh et al. suggested that zeta potential of transfersomes is negative,
mainly due to the presence of phosphatidylcholine (Ghanbarzadeh and Arami
2013). Nevertheless, other studies have reported a positive value for zeta potential
(Ferderber et al. 2009; Ghanbarzadeh and Arami 2013; Ascenso et al. 2013; Uchechi
et al. 2014; Mota et al. 2017). Transfersomes present size values between 10 and
100 nm (Rajabi and Mousa 2016; Mota et al. 2017). Transfersomes measuring more
than 200–300 nm can squeeze through the skin and penetrate the SC barrier sponta-
neously (Barry 2001; Benson 2005).
The first generation of these nanocarriers was discovered by Cevc et al. in 1992.
Those transfersomes can penetrate the intact skin carrying therapeutic concentra-
tions of drugs under non-occluded conditions (Cevc and Blume 1992; Ascenso et al.
2014, 2015; Mota et al. 2017).
The advantages of transfersomes are the possibility to accommodate drug mol-
ecules with a wide range of solubility and large molecules incapable of diffusing
into the skin (as insulin, interferon) (Kulkarni et al. 2011; Singh et al. 2015).
Furthermore their expensive preparation process, and chemical instability result
in oxidative degradation predisposition (Singh et al. 2015; Prasurjya et al. 2013;
Sachan et al. 2013; Mota et al. 2017).
Cevc et al. also demonstrated that transfersomes can achieve systemic circula-
tion through transdermal osmotic gradient and hydration forces (Cevc and
Blume 1992).
The methods used to prepare transfersomes are thin-film hydration technique
which involves a three-step formation of thin-film by the mixture of phospholipids
and surfactant in volatile organic solvent, evaporation of the solvent, and ultimately,
the hydration of the thin-film with buffer (pH 6.5) by soft stirring for 1 h with ade-
quate temperature or by lipid film hydration technique which involves a dissolution
of lecithin and EA in a mixture of ethanol/chloroform, followed by
solvent-evaporation and hydration with phosphate buffer (pH 7.4) by gentle stirring
for 15 min (Sinico and Fadda 2009; Rattanapak et al. 2012; Prasurjya et al. 2013;
Sachan et al. 2013; Singh et al. 2015; Mota et al. 2017).
Thin-film hydration technique can be optimized by changing the lecitin–surfac-
tant ratio, hydration medium, type of surfactants, and effect of solvent (Prasurjya
et al. 2013; Mota et al. 2017). To prepare small vesicles, sonication can be used
(Sachan et al. 2013).
It has been described that this nanosystem prolonged the release and improved
the biological activity in vivo (Choi and Maibach 2005a; Mota et al. 2017).
The presence of phospholipids in transfersomes led to an improvement of skin
hydration, presenting several advantages in cosmetic and dermatological areas (e.g.,
faster onset of active effect, longer times of action, controlled release of active due
their action as depot). The biological action is not affected by mechanical abrasion,
and they can carry low- and high-molecular-weight actives (Choi and Maibach
2005a; Prasurjya et al. 2013; Sachan et al. 2013; Mota et al. 2017). The structure of
transfersomes contributes to a possible encapsulation of molecules with different
solubilities, and it can protect the drug against metabolic degradation (Prasurjya
et al. 2013; Sachan et al. 2013; Mota et al. 2017). Transfersomes exhibit a high
deformable structure and a good affinity for the skin, presenting inertia and safety.
In fact, they are able to squeeze through the pores in the SC, and they can localize
or rarely have transdermal effects (Elsayed et al. 2007; Ferderber et al. 2009; Zheng
et al. 2012; Fathi-Azarbayjani et al. 2015; Mota et al. 2017).
These vesicles can act by two different mechanisms: as drug carrier systems (the
intact vesicles across the SC carrying vesicle-bound drug molecules into the skin)
and as penetration enhancers (the vesicles cross the SC and change the intercellular
lipid lamellae, which facilitate the penetration of free drug molecules into and
across the SC) (Elsayed et al. 2007; Mota et al. 2017).
The rapid penetration across the intercellular lipid pathway of the transfersomes
through the SC is due to the osmotic gradient (Rajabi and Mousa 2016; Garg et al.
2016; Mota et al. 2017). On the other hand, the penetration of these vesicles through
the pores in SC is due to the deformation of vesicles in the SC, and in the rest of the
epidermis occurs the reformation of vesicles (Choi and Maibach 2005a; Benson
2006; Nounou et al. 2008; Ascenso et al. 2015; Mota et al. 2017). The transfersomes
with encapsulated large molecules can pass through the intact skin via the gap junc-
tion proteins in transdermal immunization. These vesicles are not detrimental to the
intact skin (Choi and Maibach 2005a; Mota et al. 2017). When applied onto the
skin, the dehydration of transfersomes surface by evaporation causes an increase
in osmotic pressure between the highly hydrated regions inside the skin and the
dehydrated transfersomes surface, promoting vesicle penetration through SC (Cevc
et al. 2002; Sinico and Fadda 2009; Mota et al. 2017).
These vesicles and ethosomes are examples of the emerging nanotechnologies
being currently used for the delivery of phytocompounds mainly for skin care (e.g.,
antiaging, moisturizers, sunscreens, skin's cleanser, hair’s care, lip’s care, nail’s
care) and for the prevention of skin-related diseases (Ganesan and Choi 2016; Mota
et al. 2017).
Transfersomes can encapsulate many drugs or antigens and they can be applied
in transcutaneous immunization (Simões et al. 2005; Gupta et al. 2012; Alexander
et al. 2012; Duangjit et al. 2014; Ascenso et al. 2014; Singh et al. 2015; Marwah
et al. 2016). In 2014, Duangjit et al. made transfersomes with 0.77% of phosphati-
dylcholine, 0.04% of cholesterol, and 0.10% of cetylpyridinium chloride in order to
encapsulate meloxicam (0.08%). The size of these vesicles was 83.0±4.4 nm with
an EE of 88.5±1.4%. The amount of drug localized in the skin was around 5 μg/cm2
(Duangjit et al. 2014).
Transethosomes
Transethosomes are lipidic vesicles, similar with transfersomes and ethosomes.

They present ethanol (>30%) and an EA, a penetration enhancer, or a surfactant
(Gupta et al. 2012; Ascenso et al. 2015; Menezes et al. 2016; Chen et al. 2017).
Transethosomes can be named as ultradeformable vesicles (Song et al. 2012;
Ascenso et al. 2015). The first transethosomes were introduced by Song et al. in
2012 (Song et al. 2012). These vesicles have an irregular spherical shape and higher
values of vesicle elasticity which can lead to high skin permeation/penetration.
These vesicles present the same advantages with ethosomes and transfersomes.
The mechanism of skin penetration is probably a fusion of mechanism (Song et al.
2012; Ascenso et al. 2015). In 2012, Song et al. made three different types of trans-
ethosomes. All of them present 30 mg of Lipoid S100®, 300 μL of ethanol, 700 μL
of water, and 3 mg of voriconazole. The first transethosomes present 5.3 mg of
Tween 80®, the second present 5.3 mg of sodium taurocholate®, and the third pres-
ent 5.3 mg of oleic acid. Their sizes were similar, i.e., 191.0±42.0 nm, 229.0±26.0 nm,
and 146.0±5.0 nm for the first, second, and third transethosomes, respectively. The
third have the highest permeation rate (11.6±1.1 μg/cm2/h), cumulative drug amount
permeated for 12 h (96.0±10.2 μg/cm2), drug amount deposited after 12 h in SC
(1.7±0.4 μg/cm2), and in the dermis/epidermis (5.4±1.5 μg/cm2). These results
could be due to the presence of oleic acid in the vesicular system (Song et al. 2012).
Niosomes
Niosomes are analogous to liposomes with the same physical properties but differ-
ent chemical properties in monomer units (Khan et al. 2011; Kasliwal 2012). They
have an intrinsic skin penetration-enhancing properties (Gupta et al. 2011;
Hamishehkar et al. 2013; El-Menshawe and Hussein 2013; Kumar et al. 2014).
Niosomes can control and sustain drug release and can enhance drug permeation
through the skin (improving the SC properties by the reduction of TEWL and by
the increase of the skin smoothness via reloading lost skin lipids) (Hamishehkar
et al. 2013; El-Menshawe and Hussein 2013).
Niosomes are non-ionic surfactant vesicles (NSVs), presenting a microscopic
lamellar structures (Barry 2001; Khan et al. 2011). These vesicles contain hydrated
non-ionic surfactants and cholesterol or its derivatives (Ascenso et al. 2011;
Moghassemi and Hadjizadeh 2014; Hua 2015). Niosomes can have sizes between
10 and 1000 nm (Khan et al. 2011).
These vesicles present many advantages, such as high biocompatibility, low tox-
icity and immune system activation, and capability to be synthesized by suitable
components for targeted drug delivery. Niosomes can improve the horny layer prop-
erties by the reduction of transepidermal water loss (TEWL) and by the increase of
smoothness through replenishment of skin lipids loss followed by corneocyte fusion
(Hua 2015; Moghassemi et al. 2017). Furthermore, they can modify the structure of
SC due their surfactant properties, in order to make the layer looser and more per-
meable (Hua 2015).
Niosomes can act as depots releasing the drug by a controlled manner through its
bilayer providing sustained release of the enclosed drug (hydrophilic and lipo-
philic), or they can provide a targeted drug delivery (Moghassemi and Hadjizadeh
2014; Singh et al. 2015; Moghassemi et al. 2017).
Niosomes can be classified into unilamellar vesicles (UV) and with more bilay-
ers (MLV), depending on the method used in their preparation (Azeem et al. 2009;
Khan et al. 2011; Moghassemi and Hadjizadeh 2014; Singh et al. 2015). Niosomes
can be classified into small unilamellar vesicles (SUV) and large unilamellar
vesicles (LUV) (Moghassemi and Hadjizadeh 2014). Different types of specialized

niosomes like proniosome, surfactant ethosomes, elastic niosomes, polyhedral nio-
somes, discomes (disk-shaped vesicle), aspasome (ascorbyl palmitate vesicle), sur-
factant ethosomes, and others also exist (Moghassemi and Hadjizadeh 2014).
These vesicles have amphiphilic bilayer structure with polar region oriented out-
side and apolar region oriented inside (Khan et al. 2011; Kasliwal 2012; Hamishehkar
et al. 2013).
The formation of niosomes consists in hydration of mixture of a single-alkyl
chain non-ionic surfactant (ethers such as polyglycerol alkyl, dialkylpolyglycerol,
dialkylpolyethylene, glucosyldialkyl, polyoxyethylene, dialkympolyethylene, and
crown), PEG-polyglycerol, ester-linked surfactants, Brij®, polysorbates (Tweens®
(20 and 40)), sorbitan esters (Spans®)), and cholesterol (Azeem et al. 2009; Khan
et al. 2011; Hamishehkar et al. 2013). The non-ionic surfactants could have no
charge (Hamishehkar et al. 2013). The non-ionic surfactants have a hydrophilic
head group and a hydrophobic tail with alkyl, fluoro-alkyl, or steroidal groups
(Azeem et al. 2009). Other compounds like steroidal oxyethylene ethers, laurate
ethers, alkyl galactosides, sorbitan monooleate, and polyoxyethylated hydrogenated
castor oil can be used for the formation of amphiphilic substances (Khan et al. 2011).
The toxicity of compounds used in niosome preparation is still in debate. Hofland
et al. observed an increase of alkyl chain length which is associated with a decrease
of toxicity and that an increase of polyoxyethylene chain length causes an increase
of toxicity. However, another study revealed that neither the polyoxyethylene chain
length nor the alkyl chain length has no influence on skin toxicity of polyoxyethyl-
ene niosomes (during assessment to the cell proliferation of human keratinocytes
in vitro) (Azeem et al. 2009).
The closed bilayers in general do not form spontaneously and usually it involves
some input of energy by physical agitation, heat, or sonication (Azeem et al. 2009).
The properties of these vesicles are described as follows: (a) niosomes are
osmotically active and stable on their own; (b) due the presence of hydrophilic and
hydrophobic domains in their composition, they can accommodate drug molecules
with a wide range of solubilities; (c) they can exhibit flexible structural characteris-
tics (composition, fluidity, size, lamellarity, trapped volume surface charge); (d)
they can improve the bioavailability of poorly soluble drug and enhance skin perme-
ation of those drugs; (e) they can reach the site of action below the administration
route; (f) on their surface, it is possible to attach or anchor positive, neutral, or nega-
tive charges (surface ligands, as carbohydrates, glycoproteins), depending on the
nature and density of charge as well as the stability, permeability, and the interac-
tions with MPS; (g) the surfactants generally used are biodegradable, biocompati-
ble, and nonimmunogenic; (h) niosomal dispersion in the aqueous phase can be
emulsified in nonaqueous phase regulating the delivery of drug; and (i) niosomes
can encapsulate a wide range of molecular weight (Azeem et al. 2009).
The advantages of niosomes are low-cost production during the preparation and
storage, economic for large-scale production, great stability, higher chemical stabil-
ity, easy storage, release of the drug in sustained and controlled manner, stable
structure even in emulsion form, and large number of available vesicle-forming
nonionic surfactants. The use of surfactants allows producing biodegradable and

nonimmunogenic carriers, able to encapsulate large amounts of drug or active com-
pounds in a small volume of vesicles. They can protect the drug from enzyme
metabolism and led to better compliance and effectiveness than conventional oily
formulations. It is easy to control the characteristics of niosomes (shape, fluidity,
and size) by the change in structural composition and production method. Niosomes
can be sterilized (by membrane filtration, autoclaving, and gamma radiation), and
they can be prescribed for different administration routes (oral, parenteral, and topi-
cal), with different dosage forms (semisolids, powders, and suspensions).
The disadvantages associated with nisomes are the aqueous suspensions may
undergo fusion, aggregation, leakage of entrapped drugs, and hydrolysis of encap-
sulated drugs which contribute to a shelf life limitation. The preparation methods of
MLV (extrusion, sonication) are generally time-consuming and need specialized
equipment for the process (Azeem et al. 2009; Khan et al. 2011; Gupta et al. 2011;
Kasliwal 2012; Hamishehkar et al. 2013; El-Menshawe and Hussein 2013;
Moghassemi and Hadjizadeh 2014; Singh et al. 2015). Comparing niosomes and
liposomes, it is possible to conclude that the difference between them is that, nio-
somes are composed by non-ionic surfactant and liposomes by phospholipids; how-
ever, they present the same functionality, with the same physical properties and
acting as amphiphilic vesicles. Niosome’s physical instability during dispersion
may be comparable to that of liposomes; both present risk of aggregation, fusion,
drug leakage, or hydrolysis of entrapped drugs during storage, and in vivo studies
revealed that these two vesicles present similar function (Azeem et al. 2009;
Hamishehkar et al. 2013).
Niosomes have been used mainly in cosmetic industry. First, niosome was made
by Hanjani Vila et al. in 1979, formulated with hydration of mixture of cholesterol
and a single-alkyl chain non-ionic surfactant (Azeem et al. 2009). However, the
patented niosomes for the cosmeceutical purposes were done in 1975 by L’Oreal
(Azeem et al. 2009; Hamishehkar et al. 2013).
Other membrane addictives are charge inducers, such as dicetyl phosphate, stea-
rylamine, and diacylglycerol. Their inclusion contributes to the enhancement of ste-
ric repulsion, leading to increased stability (Azeem et al. 2009).
There are different types of methods to obtain niosomes. Among them thin-film
hydration method (hand shaking method), ether injection method, reverse phase
evaporation method, transmembrane pH gradient uptake process, sonication, heat-
ing method, proniosome technology, dehydration rehydration, freeze and thaw,
microfluidization, dried-reconstituted vesicles and bubble method (Azeem et al.
2009; Khan et al. 2011; Ascenso et al. 2011; El-Menshawe and Hussein 2013;
Moghassemi and Hadjizadeh 2014; Singh et al. 2015). Most techniques require the
use of solvents as chloroform, methanol, diethyl ether, petroleum ether, and others
(Azeem et al. 2009; Khan et al. 2011). Under the ICH Q3, the guideline for residual
solvents, consider that chloroform, dichloromethane, and methanol belong to Class
2 solvents (solvents to be limited); acetic acid, acetone, ethanol, dimethyl sulfoxide
(DMSO), ethyl acetate, diethyl ether, and 1-propanolol belong to Class 3 solvents
solvents which should be limited by GMP or other quality-based requirements); and
petroleum ether belongs to Class 4 solvents (solvents for which no adequate toxico-
logical data was found) (International Conference on Harmonisation for Registration
of Technical Requirements for Pharmaceuticals for Human Use 2011).
Another possible method is by multiple membrane extrusion. Niosomes can also
be prepared from proniosomes by hydration at 80°C followed by agitation for 2
min, resulting in a suspension (Khan et al. 2011). This method also has been used
for the niosome preparation with drugs as valsartan (antagonists of angiotensin),
17β-estradiol (hormones), tenoxicam (AINEs), and others (Moghassemi and
Hadjizadeh 2014).
The preparation of niosomes can be affected by the surfactant structure. A
charged surfactant is intercalated in the bilayers, introducing electrostatic repulsion
between the vesicles which increases the stability. The HLB value has influence on
mean vesicle size and entrapment efficiency. The increase of hydrophobicity leads
to smaller vesicles due to a decrease of free energy. On the other hand, high HLB
and phase transition temperature are associated with high entrapment efficiency.
Surfactants with a small head group are ideal choices for vesicle preparation.
Surfactants with a large head group can be used to form vesicles with gel-state
bilayers, which may aggregate at room temperature. The preparation method influ-
ences the number of bilayers, size, size distribution, entrapment, and permeability
of the vesicles. The surfactant and lipid level affects the entrapment efficiency,
kinetics released, size, and stability of vesicles. The hydration temperature should
be above the phase transition of surfactant – from a gel to liquid phase; a solution
could be the use of surfactant mixtures or by addition of sterols (as cholesterol). The
gel-state niosome contributes to the formation of aggregates. The membrane addi-
tives have an influence on the charge of vesicles, which have influence in stability,
permeability, and interaction with the cells of the reticuloendothelial system. To
overcome this problem, it is possible to use membrane additives such as cholesterol,
diacyl glycerol, steraylamine, and dicetyl phosphate, which can give rigidity to the
bilayers or to impart a negative or a positive charge (Azeem et al. 2009).
Drug or active loading methods into niosomes include direct entrapment (passive
loading) and remote loading (active loading), by using transmembrane pH gradient
(in acidic range), or transmembrane ion gradient (Moghassemi and Hadjizadeh 2014).
With the aim of reducing the niosome’s size, it is possible to use techniques, such
as probe sonication (final size around 100–140 nm), extrusion through filters of
defined pore size, sonication with filtration, microfluidization (sub – 50 nm), and
high-pressure homogenization (yield vesicles <100 nm) (Azeem et al. 2009).
Niosomes can interact with cells through different ways, such as fusion of the
outer layer of the niosome with the plasma membrane, stable adsorption to the cell
surface (nonspecific or by ligand), transfer of lipid molecules between the outer
monolayer of niosome and cell (without association of the entities) or endocytosis
(Azeem et al. 2009).
Furthermore, niosomes can be coated with different types of agents, such as
polyethylene glycol (PEG), hyaluronic acid (HA), and others (Moghassemi and
Hadjizadeh 2014).
Niosomes can be administrated through different routes between them, i.e., sub-
cutaneous, transdermal, and parenteral routes (Azeem et al. 2009; Hamishehkar
et al. 2013). They can reach the dermis layer with slow penetration (Hamishehkar
et al. 2013; Moghassemi and Hadjizadeh 2014). Niosome penetration occurs from
the capillary membrane to extracellular matrix (ECM) (Hamishehkar et al. 2013).
Niosomes are recommended as drug carriers in dermatology for the following
skin disorders: skin cancer, local anesthesia, psoriasis, whitening effect, vitiligo,
hepatitis B immunization (topical immunization), cutaneous inflammation, fungal
and bacterial infections, alopecia, acne and antioxidants (antiaging products)
(Hamishehkar et al. 2013). In 2008, Paolino et al. made niosomes with 5-fluorouracil.
Niosomes with the drug presented a mean size of 479.0±3.2 nm, when compared to
the empty niosomes with 498.0±5.3 nm. The loading capacity of 5-fluorouracil was
45.3±2.2% for conventional method and 40.7±3.1% for sonication method (Paolino
et al. 2008).
Proniosomes are used as stable precursors for the immediate preparation of nio-
somal carrier systems. Proniosomes are prepared by proniosome technology. This
method consists in dispersion of MLV which is converted in SUVs by sonication or
microfluidization or by using French pressure cell (extrusion under high pressure).
In this way, the energy broke down the MLV structure forming SUVs with a high
radius of curvature (Moghassemi and Hadjizadeh 2014).
When proniosomes are applied onto the skin under occlusive conditions, they
originate niosomes. This phenomenon is mainly due to the hydration from the skin
itself, offering a versatile delivery platform technology for drug delivery by trans-
dermal route (Choi and Maibach 2005b; Azeem et al. 2009). Proniosomes are com-
posed of non-ionic surfactants, phospholipids, and alcohols. The presence of
phospholids and non-ionic surfactants can act as penetration enhancers, being
responsible for the increase of the drug permeation. When the alcohol used is of
high degree, it may act as a penetration enhancer (Choi and Maibach 2005b). The
vesicles in contact with SC form aggregates, fusion, and adhesion to the cell sur-
face. These characteristics can be a consequence of a high thermodynamic activity
gradient of the drug at the vesicle–SC surface, improving the penetration of lipo-
philic drugs through the SC. The enhancement of skin penetration is probably a
consequence of their interaction with the SC (Azeem et al. 2009). The mechanism
of proniosomes to modulate drug delivery across the skin is similar with the mecha-
nism of niosomes (Choi and Maibach 2005b).
Examples of drugs incorporated into proniosomes for skin delivery were aceclof-
enac (for arthritis, ankylosing spondylitis), chlorpheniramine maleate (as
anti-allergic), cromolyn sodium (as anti-asthmatic and anti-allergic), estadiol (for
hormonal insufficiencies), flurbiprofen (anti-inflammatory), ethinylestradiol and
levonorgestrel (contraception and hormone replacement therapy), ibuprofen (anti-
inflammatory), ketorolac (anti-inflammatory), levonorgestrel (contraceptive agent),
and piroxicam (anti-inflammatory) (Azeem et al. 2009).
Penetration Enhancer-Containing Vesicles
Penetration enhancer-containing vesicles (PEVs) are an alternative to the phospho-

lipid vesicles presenting optimal delivery performances (Manconi et al. 2017).
These vesicles are similar to transfersomes; however, the EA are replaced by pene-
tration enhancers (PE). In this way, PEVs contain phospholipids (e.g., soya lecithin)
and PE (e.g., 2-(2-ethoxyethoxy) ethanol (Transcutol®), diethylene glycol mono-
ethyl ether (Transcutol P®), capryol-capryol macrogol 8-glyceride (Labrasol®), pro-
pylene glycol (PG), polyethylene glycol 400 (PEG), OramixTM CG110,
LauroglycolTM FCC and cineole). They can also present oleic acid (Mura et al. 2009,
2011, 2013; Chessa et al. 2011; Gupta et al. 2012; Caddeo et al. 2015). PEVs are
able to optimize cutaneous delivery (Mura et al. 2009) and topical drug delivery of
NSAID (Manconi et al. 2012). Mura et al. revealed that the association of soya
phosphatidylcholine (SPC) and Transcutol® does not have a synergic action capable
of improving the drug (minoxidil) diffusion through the skin layers (Mura et al.
2011). However, in 2011, Chessa et al. revealed that a high amount (40%) of hydro-
philic PE facilitate incorporation of drugs (as quercetin) into the phospholipid
bilayer and to avoid drug precipitation in vesicle dispersions (Chessa et al. 2011).
The same study demonstrated that these PE (e.g., PG, PEG, Labrasol®, and
Transcutol®) are able to improve the solubility of drugs (e.g., quercetin and treti-
noin) in vesicle dispersions, as well as to present a synergic effect with phospholip-
ids (e.g., soybean lecithin (Phospholipon® 50, with 45% phosphatidylcholine and
10–18% phosphatidylethanolamine), 1,2-dioleoyl-sn-glycero-3-
phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) ammonium salt
(rhodamine-phosphoethanolamine)) as PE in which PEVs serve as potent nanocar-
riers for skin delivery (Manconi et al. 2011b; Chessa et al. 2011).
Transcutol P® is an efficient PE, non-toxic and biocompatible, and soluble in
water and in oil with a KO/A of 0.7. The presence of Transcutol P® improves the
physicochemical stability (size and zeta pontential). These vesicles cannot be con-
sidered deformable vesicles. Maconi et al. revealed that Transcutol P® had the abil-
ity to improve bilayer fluidity (Manconi et al. 2011a). The study of Castangia et al.
(2014) showed that the PEVs with PEG present an excellent ability as a vesicle
carrier increasing the drug bioavailability in the target tissue (Castangia et al. 2014).
The first report of PEVs was made by Mura et al. in 2009 (Mura et al. 2009).
These PEVs are characterized by a negative zeta pontential and a size range between
100 and 200 nm (Mura et al. 2009; Manca et al. 2013; Caddeo et al. 2014). The
drugs are retained in major amount in SC, without any transdermal delivery (Mura
et al. 2009). New PEVs (nPEVs) which are characterized by positive zeta potential
value and sizes around 35–500 nm also exist (Bseiso et al. 2016).
The techniques to obtain PEVs and nPEVs are thin-film hydration technique,
mechanical shake, and sonication (Mura et al. 2011; Manconi et al. 2011a, b, 2017;
Chessa et al. 2011; Bseiso et al. 2016).
PEVs with quercetin can deliver drugs into the site of inflammation. In the der-
mis, they can inhibit oxidative stress and leukocyte accumulation and stimulate the
repair of the skin damage induced by tetradecanoylphorbol-13-acetate (Caddeo
et al. 2014). In 2009, Mura et al. reported three different PEV formulations contain-
ing minoxidil. All PEVs include 60 mg/mL of soy lecithin, 5.4 mg/mL of dice-
tylphosphate, and 50 mg/mL of minoxidil. The first PEV include 20 mg/mL of
Labrasol®, the second present 20 mg/mL of Transcutol®, and the third present
10 mg/mL of cineole. The sizes ranged from 144±3 nm to 202±3 nm. High EE was
observed in the third PEVs, followed by the first and second. High dose of drug
retained in SC was observed with the same previous tendency; in the epidermis, the
first presented higher value followed by the third and second. In the dermis, the
higher percentage was with the third PEVs, followed by the second and first. The
total amount of minoxidil delivery was higher with the first PEVs followed by the
third and second (Mura et al. 2009).
Solid Lipid Nanoparticles and Nanostructured Lipid Carriers
Recent researches are related to other lipidic NPs that can pass through the skin:
nanostructured lipid carriers (NLCs) and solid lipid nanoparticles (SLNs) (Fig. 6.2)
(Gupta et al. 2012; Duangjit et al. 2014; Mota et al. 2017).
SLNs and NLCs present a great potential for skin delivery of active substances.
They generally present a size range between 10 and 1000 nm (Cevc and Vierl 2010;
Firooz et al. 2015). The advantages of these NPs are low toxicity and the possibility
to be produced in a large scale (Firooz et al. 2015).
Solid Lipid Nanoparticles

SLNs are by definition particles with a solid lipid matrix and a diameter in the nano-
meter range (Parboosing et al. 2012). These systems have sizes between 50 and
1000 nm, composed by biodegradable and biocompatible solid lipids, and many of
them are stabilized by emulsifiers (Vitorino et al. 2011; Nunes et al. 2015).
The advantages of SLNs are higher physical drug stability, better water solubility
and thermal stability, high capacity to incorporate hydrophilic and lipophilic drugs
with high loading capacity, and excellent tolerability (avoidance of organic sol-
vents) (Leyva-Gómez et al. 2015). SLNs have the high ability to protect the drugs
from degradation, control drug release (over various weeks), and they can modulate
drug targeting.
In general, the process to produce SLNs is simple and a low-cost process (Vitorino
et al. 2011; Ascenso et al. 2011; Leyva-Gómez et al. 2015; Nunes et al. 2015;
Madureira et al. 2016).
These systems have been reported to be an alternative to emulsions, liposomes,
and polymeric NPs. However, SLNs also present some disadvantages, such as floc-
culation, aggregation, and compound release during storage time (Nunes et al. 2015).
The first SLN was prepared in 1996 and described by Müller et al. Since then,
SLNs have been studied as carriers for enhanced skin delivery with future applica-
tions in cosmetic and pharmaceutical industries (Müller et al. 1996, 2000, 2002;
Benson 2005; Almeida and Souto 2007; Ascenso et al. 2011; Nunes et al. 2015;
Firooz et al. 2015).
SLNs can be prepared by high-pressure homogenization (HPH) described by
Müller and Lucks, which contribute to obtain a large-scale production with a cost-
effective and relatively simple way; by microemulsion-based technique, described
by Gasco in 1993, which includes the use of organic solvents (HPH/solvent-
evaporation/diffusion); W/O/W double emulsion method; membrane contractor
technique; phase inversion; or high-speed stirring and/or ultrasonication technique
(Müller et al. 2000; Souto and Müller 2005; Almeida and Souto 2007; Vitorino et al.
2011; Ascenso et al. 2011).
The HPH method consists in the use of hot and cold dispersion techniques, which
is also applicable in thermal-sensitive compounds (Almeida and Souto 2007;
Ascenso et al. 2011).
The SLNs obtained by microemulsion-based technique have diameters below 1
μm with a polydispersion index around 0.06–0.9. In this technique, SLNs are
obtained by the contact of a molten lipid (possible containing the drug) with a mix-
ture of water, surfactant, and co-surfactant (optional, preheated to a temperature at
least equal to the melting temperature of the lipid). Afterwards, this microemulsion
is dispersed in water (2–10°C) and washed through diafiltration (Gasco 1993).
Other methods to obtain SLNs can be solvent emulsification–evaporation/solvent
displacement method (described and patented by Fessi et al. in 1992), solvent emul-
sification–diffusion (described and patented by Quintanar-Gerrero et al. in 1999),
lipid particles from supercritical fluid (SCF) technology, high shear homogeniza-
tion, and ultrasound methods (Souto and Müller 2005; Almeida and Souto 2007;
Vitorino et al. 2011), among others.
The solvent emulsification–evaporation method consists in a widespread proce-
dure for the preparation of polymeric microparticles and NPs, but it was firstly used
for SLN preparation by Sjöström and Bergenstahl in 1992 (Sjöström et al. 1993;
Souto and Müller 2005; Almeida and Souto 2007).
SLNs can incorporate lipophilic or hydrophilic drugs but they usually incorpo-
rate lipophilic drugs and only high potent hydrophilic drugs. They seem to fulfill the
requirements for an optimum particulate carrier system, protecting labile com-
pounds against degradation (Almeida and Souto 2007; Ascenso et al. 2011).
SLN can present a polymorphic structure. They change over time toward a more
stable structure that contributes to the expulsion of the active compound or drug.
This problem can be solved by freeze-drying (Leyva-Gómez et al. 2015).
SLNs can be used by dermal and transdermal routes (Vitorino et al. 2011;
Ascenso et al. 2011). They are generally used as a topical application for therapeutic
or cosmetic purposes (as NanoRepair Q10®, Dr. Rimpler). SLNs led to an occlusive
effect which increases skin hydration, wrinkle smoothing, enhancing the penetra-
tion of compounds in specific skin layers, which is a fundamental for the efficacy of
cosmetic treatments (Nunes et al. 2015). In 2005, Souto and Müller made SLNs
with ketoconazole (Souto and Müller 2005). The empty SLNs present 15% of
Compritol®, 2.50% of poloxamer®, and 0.125% of sodium deoxycholate in water.
The loaded SLNs presented 14.25% of Compritol®, 0.75% of ketoconazole, 2.50%
of poloxamer®, and 0.125% of sodium deoxycholate in water. At 4°C of storage

conditions, the free formulation is 211.6±4.3 nm and the loaded formulation is
212.3±7.4 nm, being both monodisperse formulations. By differential scanning
calorimetry (DSC) analysis, they present similar melting points, onsets, and enthal-
pies. However, their integral and crystallinity indices were different for empty and
loaded SLNs.
SLNs are commercialized by Skyepharma (London, UK), nowadays named
Vectura (Cevc and Vierl 2010).
Nanostructured Lipid Carriers

NLCs are colloidal systems composed of liquid and solid lipid matrix with crystal
lattice which creates spaces for the drug (Ascenso et al. 2011; Vitorino et al. 2014;
Firooz et al. 2015). The cores of these NLCs are composed of physiological and
biodegradable lipids which are stabilized by aqueous surfactants or emulsifiers and
can solubilize lipophilic drugs or compounds (Vitorino et al. 2014; Firooz et al.
2015). Surfactants reduce the surface tension between two immiscible liquids fol-
lowed by droplet disruption, and they also prevent the recoalescence of droplets by
adsorbing on their surfaces (Firooz et al. 2015). NLCs maintain their solid state and
they can control the release of drug from the matrix (Ascenso et al. 2011). These
NPs have been referred in the literature to improve the drug loading and to increase
the stability on storage, as well as the release properties (Müller et al. 2002; Ascenso
et al. 2011).
NLCs can be classified in different types concerning the production of highly
concentrated particle dispersions and their application as drug delivery systems
(Müller et al. 2002).
These NPs can be prepared by solvent emulsification–evaporation and
high-pressure homogenization methods or by mixing solid-lipids with spatially
incompatible liquid-lipids leading to special nanostructures (Müller et al. 2002;
Vitorino et al. 2014).
They have been studied for dermal delivery in cosmetics and dermatological
preparations mainly due to their chemical similarity with the lipids present in the
skin (Ascenso et al. 2011; Vitorino et al. 2014). This chemical similarity is due to
their hydrophobic character, presence of a solid matrix, high specific surface area,
and high biocompatibility (Vitorino et al. 2014).
In the study of Souto and Müller (2005), they made NLCs with ketoconazole.
The empty NLCs present 10.50% of Compritol, 4.50% of tocopherol, 2.50% of
poloxamer®, and 0.13% of sodium deoxycholate in water. The loaded NLCs pre-
sented 10.13% of compritol®, 4.13% of tocopherol®, 0.75% of ketoconazole, 2.50%
of poloxamer®, and 0.13% of sodium deoxycholate in water. At room temperature
of storage conditions, the free and loaded formulations present particle size higher
than 3 μm. By DSC analysis, they present similar melting points, integrals, and
enthalpies. However, the onsets and crystallinity index were different for empty and
loaded NLCs (Souto and Müller 2005).
Polymeric-Based Nanoparticles
By definition these NPs are composed of a polymeric matrix with therapeutics

attached or encapsulated into its surface (Parboosing et al. 2012). The polymeric
matrix is a biocompatible and biodegradable polymer (natural, semisynthetic, or
synthetic) and chemically inert, non-toxic, and free of leachable impurities. These
NPs’ sizes range between 10 and 1000 nm (Soppimath et al. 2001; Reis et al. 2008;
Parboosing et al. 2012; Danhier et al. 2012; Rosado et al. 2013; Uchechi et al. 2014;
Mota et al. 2017). Polymeric NPs present an enhanced solubility and penetration
into the deeper skin layers (Nounou et al. 2008; Duangjit et al. 2014; Melero et al.
2014; Uchechi et al. 2014; Huang et al. 2016; Mota et al. 2017).
In these NPs, the drug could be dissolved, entrapped, encapsulated, or attached
in the polymer NP matrix (Uchechi et al. 2014; Mota et al. 2017). The drugs can be
conjugated to the side chain of a linear polymer with a linker (cleavable bond)
(Nounou et al. 2008; Cho et al. 2008; Akbarzadeh et al. 2013; Ferreira 2014;
Uchechi et al. 2014; Ascenso et al. 2015; Ganesan and Choi 2016; Mota et al. 2017).
They can be classified into nanocapsules and nanospheres as seen in previ-
ous Fig. 6.2, where nanocapsules are by definition a nanosized structure with a shell
(Parboosing et al. 2012), while nanospheres are matrix systems where the active can
be homogeneously or heretogeneously dispersed.
NPs can be used for topical drug delivery by their potential of protecting the
unstable active ingredients and providing a sustained release over long periods of
time (Uchechi et al. 2014; Scott and Banga 2015; Katikaneni 2015; Gelfuso et al.
2015; Mota et al. 2017).
Couvreur et al. (1977 and 1979) among others authors were responsible for the
development of the first polymeric NPs and many other formulations are now
described in the literature (Couvreur et al. 1977, 1979; Bala et al. 2004; Müller and
Keck 2004; Mota et al. 2017). In general, those NPs are water-soluble; being pos-
sible to present a surface modification (PEGylation) which achieved a selective
accumulation and retention in target tissue, and they can bear specific surface struc-
tures (antigens or ligands) for cell receptor-mediated targeting (Nounou et al. 2008;
Cho et al. 2008; Akbarzadeh et al. 2013; Ferreira 2014; Uchechi et al. 2014; Ascenso
et al. 2015; Ganesan and Choi 2016; Mota et al. 2017).
The penetration and the transport of these NPs into the skin are influenced by
their chemical composition, size and the viscosity of the formulation (Uchechi et al.
2014; Mota et al. 2017).
They have been developed to provide a good alternative for progressive and
long-term delivery of therapeutic agents into the skin (Bala et al. 2004; Mota et al.
2017), such as glucocorticoids, nonsteroid anti-inflammatory drugs, antimicrobial
agents, and anticancer drugs for melanoma (Rosado et al. 2013; Reis et al. 2014;
Rijo et al. 2014; Silva et al. 2015; Mota et al. 2017).
The most common polymers used are poloxamers, triblock copolymers of poly-
oxyethylene and polyoxypropylene (Pluronic®, PF), PLGA (poly(lactic-co-glycolic
acid)), PCL (poly-Ɛ-caprolactone), PLA (poly(lactic acid), PEG
(poly(ethyleneglycol)), PMMA (poly(methyl methacrylate)), PECA (poly(ethyl
cyanoacrylate)), PACA (poly(alkyl cyanoacrylate)), PBCA (poly n-butyl cyanoac-

rylate), PLGA-PEG (poly(D,L-lactide-co-glycolide)-b-poly(ethylene-glycol)),
PCL-PEG (poly(Ɛ-caprolactone)-b-poly(ethyleneglycol)), and their derivatives
(Soppimath et al. 2001; Reis et al. 2006; Reis 2007; Hasan et al. 2007; Andrade
et al. 2015; Leyva-Gómez et al. 2015; Anitha et al. 2016; Mota et al. 2017). The
polymers are susceptible to degradation, depending on the molecular weight and
copolymer ratio, and it can occur during several hours to several months (Danhier
et al. 2012; Mota et al. 2017).
PLGA is usually used due its biodegradability where hydrolysis leads to metabo-
lite monomers (lactic acid and glycolic acid, which are endogenous and easily
metabolized in the body) and excreted as water and CO2, being the most extensively
studied for drug delivery (Rytting et al. 2008; Danhier et al. 2012; Gomes et al.
2013; Reis et al. 2014; Severini et al. 2014; Pereira et al. 2015; Yukuyama et al.
2016; Abrego et al. 2016; Mota et al. 2017). This polymer is approved by FDA and
EMA for drug delivery systems (Danhier et al. 2012; Hung et al. 2015; Yukuyama
et al. 2016; Abrego et al. 2016; Mota et al. 2017). Some advantages of PLGA NPs
are its ability to solubilize and control the in vivo release of lipophilic molecules
(Fernandez et al. 2012). For example, PLGA NPs with azelaic acid promote an
effective and safe treatment for acne vulgaris, and PLGA NPs containing carprofen
propionic acid were proposed for anti-inflammatory diseases (Gomes et al. 2013;
Reis et al. 2014; Parra et al. 2016; Mota et al. 2017).
The disadvantages of using PLGA as a polymer are related to the use of organic
solvents which can damage some drugs (Bala et al. 2004).
Poly-Ɛ-caprolactone (PCL) is a hydrophobic polymer that can control the release
of drug, reducing the drug percutaneous penetration and protecting the drug from
potential photochemical degradation (Silva et al. 2015). Thus, PCL is also a good
candidate for controlled and long-term drug delivery due to its high stability and
slow degradation (Rosado et al. 2013). PCL NPs with fatty acids were successfully
prepared for skin delivery (Silva et al. 2015; Mota et al. 2017). Here, the presence
of fatty acids improved drug entrapment, reducing the burst release, but they also
can act as skin PE (Rosado et al. 2013; Silva et al. 2015; Mota et al. 2017). In 2015,
Silva et al. made polymeric NPs with betamethasone. Empty NPs presented a size
of 328.2±17.2 nm and the loaded formulation was 307.0±15.3 nm, both monodis-
perse formulations. The EE was of 85.9±10.3% (Silva et al. 2015).
Both NPs can be prepared by emulsification/solvent dispersion or by emulsifica-
tion/solvent diffusion methods (Fig. 6.5) (Soppimath et al. 2001; Reis et al. 2006,
2014; Reis 2007; Fernandez et al. 2012; Yukuyama et al. 2016; Abrego et al. 2016).
The emulsification/solvent diffusion presents advantages, such as high yields,
high batch-to-batch reproducibility, and easy scaling-up (Santander-Ortega
et al. 2010).
On the other hand, dendrimers are a class of highly structured control with a 3D
architecture (Xu et al. 2014; Yang et al. 2015; Srinageshwar et al. 2017). Dendritic
polymers are built up from branched repeat units named branch cell monomers
(Parboosing et al. 2012; Yang et al. 2015; Bohr et al. 2017). Dendrimers can be pre-
sented in three layers as follows: a central core (dendrimer core), repeat units (as
Fig. 6.5 Emulsification/solvent diffusion and emulsification/solvent dispersion methods
poly(amidoamine) – PAMAM, poly(propyleneimine) – PPI, triazine,

poly(etherhydroxylamine) – PEHAM, carbosilane, poly(ether imine), phosphorus,
poly-L-lysine, viologen, among others) and surface functionalities (cationic groups)
(Xu et al. 2014; Yang et al. 2015; Srinageshwar et al. 2017). The first dendrimers
were envisioned by Flory in 1980s (Cevc and Vierl 2010; Yang et al. 2015;
Srinageshwar et al. 2017). Dendrimers are synthesized by convergent or divergent
methods (Yang et al. 2015). The first reported synthesis and characterization of
PAMAM dendrimers with ammonium (NH3) or ethylenediamine (EDA) was made
by Tomalia et al. in 1985 (Tomalia et al. 1985). Different strategies were used to
synthesize dendrimers (Wu et al. 2013; Yang et al. 2015). In 1990, Hawker and
Frechet used a convergent synthetic approach to synthesize dendrimers. The first
dendrimers were commercialized in 1993, by the description of Meijer and co-
workers the kilogram-scale synthesis (Yang et al. 2015).
Dendrimers can maintain drug levels in a therapeutically desirable range; they
can increase half-life, solubility, stability, and permeability of drugs. Dendrimers
are able to deliver different types of drugs, allowing drug targeting and improving
delivery efficiency. The increase of the intracellular uptake is due to the modulation
of tight junction proteins (e.g., occludin, actin); however, they are dependent on the
concentration, generation, and surface charge of dendrimers. The dynamics of cel-
lular entry is influenced by the surface groups and the molecular mass of the den-
drimers. The dendrimer’s internalization generally occurs by two mechanisms:
through a clathrin- and caveolae-mediated energy-dependent endocytosis and
through macropinocytosis. Other special characteristics of dendrimers are that these
systems have a low polydispersity and high functionality. They have been
recognized for their versatility in composition and structurally controlled nanoscale

building blocks for drug delivery (Xu et al. 2014).
PAMAM dendrimers are the main family of dendrimers. They can present differ-
ent core units and terminal groups (carboxylic acids) (Wu et al. 2013; Xu et al.
2014). PAMAM is a polycationic dendrimer (Xu et al. 2014). This family presents
open internal structure and cavities for accommodating guest therapeutic molecules
solubilized or targeted for drug delivery (Sadekar and Ghandehari 2012). Other
advantages of this family of NPs are their biocompatibility, non-immunogenicity,
and water solubility, which are highly suitable for drug delivery. They are quickly
eliminated by renal excretion due their small size and high water solubility
(Srinageshwar et al. 2017).
These NPs present an unique architecture, a nanoscale size, and a high density of
surface functionalities (Yang et al. 2015) and they have been shown to be an effi-
cient siRNA delivery system (Bohr et al. 2017).
Cationic dendrimers present a precisely high number of amine groups on the
surface. These dendrimers can encapsulated nucleic acids (through ionic interac-
tions) and protect them from enzymatic degradation (Yang et al. 2015). Polyanionic
dendrimers present anionic charges and they are useful to the encapsulation of cat-
ionic drugs. Polyanionic dendrimers are usually less toxic than polycationic den-
drimers (Xu et al. 2014).
Dendrimers are mainly used in gene delivery. In this way, cationic dendrimers
with various functional ligands (as lipids, fluorous compounds, amino acids, sac-
charides, proteins or peptides, polymer, nanoparticles (Fe3O4, Au, and others) and
cationic moieties have been successfully developed (Yang et al. 2015).
Dendrimers present two principal approaches to carry and sustain drugs: the first
one is, they exploit the well-defined functionalities for the covalent attachment of
drug molecules to the dendrimer periphery, and the second one is, by noncovalent
interactions with the surface groups or internal cores of dendrimers where the drug
is incorporated. In the first, the drug loading can be tuned by different generation
numbers of dendrimers. The second does not require chemical reaction step but it
suffers from a drawback related to the difficulty to control drug release (Dribek
et al. 2007).
Metallic-Based Nanoformulations
Metallic NPs present extensive applications, such as sensing and nanomaterial-

based sensors (Dias et al. 2015; Valdez et al. 2016; Mota et al. 2017). They are natu-
rally long-lived (Cevc and Vierl 2010). The type of the metal used into metallic NPs
can be cobalt, copper, silver, gold, palladium, and platinum (Adelere and Lateef
2016; Mota et al. 2017). Metallic NPs can be synthesized by chemical or physical
methods, which are expensive and potentially dangerous to the environment.
Chemical methods generally involve the use of reducing agents, organic solvents, or
nonbiodegradable stabilizing agents with some toxicological limitations (Bonnia
et al. 2016; Mota et al. 2017). An alternative to this problem is the use of biological
organisms (microorganisms) and plant extracts or, alternatively, the use of different
methods, such as supercritical fluids (Zhang and Erkey 2006; Bonnia et al. 2016;
Mota et al. 2017).
Gold Nanoparticles
Gold nanoparticles (AuNPs or GNPs) are light-absorbing metallic NPs. They are
promising NPs for therapeutic applications, mainly for systemic administration in
order to reach their target tissue (Santos-Martinez et al. 2014; Silva et al. 2014;
Rajeshkumar 2016). AuNPs can present different colors (red, blue, or others) which
depends on the oscillations of conduction band electrons at adequate wavelengths,
size (between 3 and 120 nm), shape, and amount of aggregation (Haiss et al. 2007;
Rajeshkumar 2016). The size of AuNPs and their surface properties seem to have a
key role in intracellular uptake and in cytotoxicity (Santos-Martinez et al. 2014).
The first use of gold was reported in 1857 by Faraday (Haiss et al. 2007). AuNPs
can be produced by three different methods: the Turkevich method (developed in
1954), the Brust–Schiffrin method (developed in 1994), and the seed-mediated
growth-modified method (developed in 2001). The Turkevich method produces
spherical AuNPs based on mild reduction with sodium citrate. Brust–Schiffrin
method consists of stronger reduction agents, such as sodium borohydride. Seed-
mediated growth-modified method is based on different reduction and capping
agents (e.g., shape modulators). Nowadays, there are other methods like the use of
biocompatible and biodegradable polymers which promote the absorption of those
NPs to longer wavelengths, and the optical therapeutic window is around
650–900 nm, called biogenic synthesis. These new methods present some advan-
tages like cost-effective process, biocompatibility, low toxicity, and ease of method-
ologies (Silva et al. 2014).
AuNPs are inert and not easily oxidized (Santos-Martinez et al. 2014; Silva et al.
2014; Rajeshkumar 2016). However, in some cases, they also present some toxic
chemicals (e.g., capping agents), which can cause some concerns in vivo. They can
cause irreversible localized thermal cellular destruction and imaging (Santos-
Martinez et al. 2014; Silva et al. 2014). By their light-absorbing properties, AuNPs
can be applied into optical, imaging, sensor, and cancer therapy (Rajeshkumar
2016). These NPs have shown promising therapeutic applications in cancer treat-
ment by optimizing their characteristics (e.g., size, shape and plasmonic surface
properties) (Silva et al. 2014).
They can be bio-functionalized in photothermal therapy, for greater transport to
the target tissues. An example is AuNPs with aqueous extracts of Plectranthus sac-
catus (a source for reducing and capping agents) which have been shown to be an
attractive system for photothermal therapy in cancer. Others examples are described
in literature, such as cinnamon or arabic gum as contrast agent for detection of can-
cer cells; saponins, phenolic compounds and phytosterols as antimicrobial agents;
gellan gum, arabic gum, and catechin as drug delivery systems (with cell internal-
ization); gallic acid, protocatechuic acid, and isoflavone as antioxidant agents; or
even with Nyctanthes arbor-tristis as stabilizing agent (Das et al. 2010, 2011; Lee
et al. 2014). Sphaeranthus amaranthoides is other stabilizing agent used in AuNPs
(Nellore et al. 2012). Centella asiatica can be used in AuNPs as an effective reduc-
ing and capping agent (Das et al. 2010). AuNPs with Cerasus serrulata present an
antimicrobial activity (Karthik et al. 2016).
Silver Nanoparticles
Silver nanoparticles (AgNPs) are by definition particulate dispersions or solid par-

ticles, with sizes between 1 and 100 nm (Ahamed et al. 2008; Lara et al. 2011;
Prabhu and Poulose 2012; Mota et al. 2017). AgNPs are commercialized by their
singular features (e.g., catalytic, antimicrobial, optical, and sensor (detection) prop-
erties) (Kora and Sashidhar 2014; Talekar et al. 2016; Mota et al. 2017).
The methods of production influence NPs characteristics (size, shape, charge,
coating/corona effects, and dispersions status (e.g., aggregation)) or media compo-
nents, such as peptides, proteins, carbohydrates, and salts or bacterial strain (strain-
dependent acidification)) (Solomon et al. 2007; Amrita et al. 2014; Rizzello and
Pompa 2014; Kora and Sashidhar 2014; Pawar and Bhangale 2015; Bhawana et al.
2016; Mota et al. 2017).
A possible method to form AgNPs is applying the reduction of silver, usually in
the form of nitrate, by sodium borohydride, and it is characterized by a peak of
absorbance around 400 nm (Solomon et al. 2007; Amrita et al. 2014; Rizzello and
Pompa 2014; Pawar and Bhangale 2015; Bhawana et al. 2016; Mota et al. 2017).
During the synthesis reaction, silver ions are slowly supplied to the growing
particles by the limited solubility of Ag2O (Evanoff and Chumanov 2005).
Usually, the solution of AgNPs present a glass yellow color and they can be used
in drug delivery (Solomon et al. 2007; Amrita et al. 2014; Rizzello and Pompa 2014;
Pawar and Bhangale 2015; Bhawana et al. 2016; Mota et al. 2017). AgNPs have
been referred as good candidates for bacterial infections due their strong and broad-
spectrum antimicrobial characteristics (Rizzello and Pompa 2014; Mota et al. 2017).
The mechanism of their antimicrobial effect is still not clear (Prabhu and Poulose
2012; Mota et al. 2017). The silver ions can be released due their promotion by
acidic and aerobic environment (Rizzello and Pompa, 2014). Silver ions can directly
damage bacteria by blocking the respiratory chain, collapsing the membrane poten-
tial, and stopping ATP production (Rizzello and Pompa 2014; Mota et al. 2017).
These NPs are efficient as microbicides in small doses. They present minimal toxic-
ity, as well as minimal side effects (Lara et al. 2011; Mota et al. 2017), but oxidation
can also occur (Evanoff and Chumanov 2005; Mota et al. 2017).
They have shown wound healing properties due to reduction of their level of
matrix metalloproteinase. They also have antitumor (inducing the apoptosis), anti-
viral, antibacterial, anti-inflammatory, and anti-angiogenic effects by inhibition of
angiogenesis and vascular permeability through vascular endothelial growth factor,
interleukin-1β, and advanced glycation end products in bovine retinal endothelial
cells (Prabhu and Poulose 2012; David et al. 2014; Mota et al. 2017).
Recent studies have demonstrated that AgNPs could have cytotoxic,

pro-inflammatory, and pro-apoptotic effects mediated by reactive oxygen species
(ROS) generated in cells (David et al. 2014; Mota et al. 2017). Silver-based prod-
ucts presenting antimicrobial properties have been used in medical (antibacterial
creams), food (antibacterial agents in packaging) and textile (preparation of silver
wools) industries. In this sense, the incorporation of AgNPs has been seen as an
alternative, which contributes to more environmentally safe industries (Dias et al.
2015). Formulations with silver (like gels, creams, and others) have been widely
used in the health sector (as wound widely).
Magnetic-Based Nanoparticles
This group of NPs includes the superparamagnetic iron oxide particles (SPION),
which present sizes >50 nm, and also the ultra-small SPION with sizes below 50 nm
also exist (Weissig et al. 2014).
Those NPs are in general for the use in magnetic resonance imaging (MRI) con-
trast media, magnetic thermoablation, magnetic biopsy, plasmonic photothermal
therapy, near-infrared (NIR) fluorescence imaging, carbon staining, and radio
enhancement (Weissig et al. 2014; Weissig and Guzman-Villanueva 2015).
In general, magnetic NPs are made of zinc oxide, iron oxide, and magnetite. Zinc
oxide NPs (ZnO NPs) are widely used (Wang et al. 2016). However, their antimi-
crobial activity is under study (Janaki et al. 2015; Shaheen et al. 2016). One of the
possible mechanisms behind their antibacterial activity can be the generation of
ROS and UV-blocking properties (Janaki et al. 2015; Shaheen et al. 2016). In the
1970s, it was demonstrated that the phosphoric acid provides a high stability associ-
ated with its anchoring at the metal oxide surfaces through the ionic covalent bonds
between P and O, preserving the intrinsic properties of NPs (Ghobril et al. 2013).
ZnO NPs can be prepared through traditional high-temperature solid-state methods
or chemical precipitation or sol–gel synthesis and hydrothermal methods (Wang
et al. 2016). The sol–gel method consists in an ineffectiveness of aggregates and
poor control on magnetic properties (Gerber et al. 2017).
The advantages of ZnO NPs are related to their multifunctional physical and
chemical properties and easy synthesis. They have a high antibacterial activity that
can depend on the pH of the precursor solution and reagents, the growth of NPs,
morphology, and particle size, among others. They are also chemically stable under
exposure to high temperatures and UV radiation and soluble. ZnO NPs also present
high biocompatibility with human cells and high selectivity, catalytic efficiency, and
non-toxicity and strong adsorption (Janaki et al. 2015; Shaheen et al. 2016; Wang
et al. 2016). However, they can be easily uptaken by the cells and may be respon-
sible to some oxidative stress (Wang et al. 2016). These NPs are widely used in
cosmetics as sunscreens (Janaki et al. 2015; Wang et al. 2016).
Iron oxide can be present in mainly three forms: magnetite (Fe3O4), maghemite
(γ-Fe2O3), and hematite (α-Fe2O3) (Ali et al. 2016). Iron oxide NPs (Fe3O4 NPs)
present a high specific surface area, superparamagnetic behavior (sizes under
10–20 nm), and high magnetization presenting a great interest in biomedical appli-

cations. Fe3O4 NPs have a very good tolerance profile, allowing their clinical use for
MRI diagnosis (Levy et al. 2011; Ali et al. 2016; Gerber et al. 2017). Depending on
the method used, Fe3O4 NPs can present a size between 5 and 60 nm (Ali et al.
2016). These NPs are approved by FDA. They can be synthesized using templates
or by controlled chemical synthesis, such as sol–gel method, oxidation, chemical
coprecipitation, hydrothermal, flow injection, electrochemical, aerosol/vapor phase,
sonochemical decomposition, supercritical method or even using nanoreactors (Ali
et al. 2016; Gerber et al. 2017). There are also physical techniques, such as deposi-
tion of gas phase and electron beam lithography and biological techniques by
microbial incubation (Ali et al. 2016). Another alternative method that produces
Fe3O4 NPs is the polyol method, which is an easy and extremely reliable process to
synthesize these NPs. It is based on the reduction of Fe(III) by glycol solvents (e.g.,
ethylene, diethylene, or polyethylene) where an activator is necessary (e.g., sodium
acetate) that makes the precipitation of iron and be further activated by thermal
dehydration to obtain an iron oxide phase (Gerber et al. 2017).
The advantages of Fe3O4 NPs are related to their easy process of production and
well-controlled interparticle spacing. The process is easily controlled in terms of
particle size and shape with good reproducibility and scalability. However, some
side effects, which could be harmful, on the lungs, irritation and skin cancer (when
skin absorption occurs) are associated to this type of NPs. For the cosmetic industry,
one disadvantage is the contamination of solid waste from sewage treatment (Ali
et al. 2016).
Other Surfactant-Based Nanoformulations
In this category, micelles, emulsions, microemulsions, nanoemulsions, nanogels,

hydrogels, self-microemulsions, and self-nanoemulsions exist (Fig. 6.2).
Micelles
Micelles are by definition spherical nanosized-shaped structures presenting a shell

and a core, composed of a water-soluble and hydrophobic polymer (Parboosing
et al. 2012; Kumar et al. 2014; Andrade et al. 2015). By definition, they have a fatty
core separated from an aqueous solvent by the polar heads (normal micelle) or an
aqueous core separated by the polar heads from a fatty solvent (inverse micelle)
(Cevc and Vierl 2010). This type of nanocarrier is characterized to improve solubil-
ity and increase oral bioavailability of poorly water-soluble drugs: micelles can
encapsulate drugs with different polarities, presenting a small size and hydrophilic
surface (Cevc and Vierl 2010; Parboosing et al. 2012; Andrade et al. 2016). By their
characteristics, these systems are less recognized by macrophages and can present
some possible mucus-penetrating properties (Andrade et al. 2016). Another
characteristic of these systems mentioned in literature is that they could inhibit drug
efflux mechanisms and, by consequence, multidrug resistance (Andrade et al. 2015).
Usually, these systems are spherical but also can present ellipsoidal, cylindrical,
and bilayer shape (Kumar et al. 2014). They present a size between 3 and 20 nm
(Cevc and Vierl 2010; Andrade et al. 2015). As liposomes, micelles can tailor spe-
cific ligands for targeted delivery.
The first micelles were made by Takada et al. in 1985 (Takada et al. 1985).
Polymeric micelles are formed by amphiphilic block copolymers, revealing to have
high stability (in vitro and in vivo). Immunomicelles have monoclonal antibody
molecules in micelle structure and they were obtained by coupling of antibodies
into p-nitrophenylcarbonyl groups on water-exposed termini of the micelle, present-
ing a high specificity and targetability. They consist in a core of the hydrophobic
blocks stabilized by the corona of hydrophilic polymeric chains. The core of these
micelles has been demonstrated to have a high loading capacity, a controlled release
profile for the drug, and good compatibility between the core-forming block and the
drug (Torchilin 2004).
The most common method used to obtain micelles is micellization and forms
part of the phase behavior of many lipids below their polymorphism, usually applied
in soaps. Nevertheless, they also can be prepared by thin-film hydration technique
(Andrade et al. 2015).
In 2015, Firooz et al. made micelles with different drugs, such as clotrimazole,
econazole, and fluconazole. Their loading efficiencies were 20%, 98%, and 83–91%,
respectively (Firooz et al. 2015).
Emulsions
The emulsion systems are composed by surfactant with an unsaturated acyl chains
in great proportions. The value of hydrophilic–lipophilic balance (HLB) indicates
the surfactant action, as presented in Table 6.4 (Pouton 1997).
The value of HLB is important to know which is a better surfactant to be used to
obtain SEDDS (self-emulsifying drug delivery systems) (Singh et al. 2009). To give
Table 6.4 HLB (hydrophilic-lipophilic balance) values, their characteristics and Behavior/
Solubility by HLB value
Value Characteristics Behavior/solubility
1 < HLB < 3 Anti-foam agents (very lipophilic, destabilizing Hydrophobic/oil-soluble
the liquid film) (0–6 HLB)
3 < HLB < 6 Emulsifying agents W/O
7 < HLB < 9 Wetting agents Dispersible in water
(6–9 HLB)
8 < HLB < 16 Emulsifying agents O/W Hydrophilic/water-soluble
13 < HLB < 16 Detergents (improves the contact with surfaces (9–20 HLB)
and solubilize materials)
15 < HLB < 20 Solubilizing agents – oils dispersible in water
great properties of self-emulsification of SEDDS, the surfactant must have a correct

HLB value, and the HLB value decreases with the temperature until critical tem-
perature (the cloud point) (Pouton 1997).
Water/oil emulsions (W/O) can encapsulate hydrosoluble compounds as pep-
tides (Sha et al. 2005).
Water/oil/water emulsions (W/O/W) are double emulsions, which can be used to
encapsulate hydrophilic drugs (Ho et al. 2008). These emulsions like microemul-
sions (ME) have been studied as candidates to be formulated as adjuvants of drugs
(Leclercq et al. 2011).
The ME W/O/W have demonstrated that the emulsion efficiency is dependent on
the droplet size of emulsion. When the droplet size decreases, the surface-to-volume
ratio also decreases. Then again, the interface between droplets and the intestinal
barrier increases; moreover, it increases the passage of drugs, which contributes for
a better bioavailability (Mota 2014).
Microemulsions
The first report of microemulsions (ME) was made by Gasco et al. in 1989 (Gasco
et al. 1989). ME are homogeneously transparent, optically clear and isotropic, ther-
modynamically stable forming low-viscosity colloidal dispersions (Peltola et al.
2003; Mendonça et al. 2009; El Maghraby 2010; Cevc and Vierl 2010; Leclercq
et al. 2011; Bansal et al. 2014). These emulsions are composed of an aqueous phase,
an oil phase, a surfactant, and a co-surfactant (Peltola et al. 2003; Mendonça et al.
2009; El Maghraby 2010; Alexander et al. 2012). ME can be oil-in-water (O/W) or
water-in-oil (W/O). The O/W emulsions consist in oil droplets that are emulsified in
bulk water phase. The W/O emulsions consist in water droplets that are emulsified
in bulk oil phase (Wu et al. 2015).
These systems present a great potential to be used as formulation adjuvant and
also as drug delivery systems (Leclercq et al. 2011). These emulsions are liquids
and transparent due to their droplet size in dispersible phase to be very low, under
150 or 300 nm (Peltola et al. 2003; Cevc and Vierl 2010; Uchechi et al. 2014; Žilius
et al. 2016; Mota et al. 2017).
These emulsions have thermodynamic stability, low viscosity, and great capacity
to encapsulate drugs/compounds, protecting them from degradation, hydrolysis,
and oxidation. It is possible to get a small droplet size (El Maghraby 2010; Alexander
et al. 2012).
These systems are formed spontaneously with adequate amounts of lipophilic
and hydrophilic ingredients, as well as a surfactant and a co-surfactant (Peltola et al.
2003; Mendonça et al. 2009; El Maghraby 2010; Uchechi et al. 2014). However,
some key factors may influence this process like temperature and system composi-
tion (Anton and Vandamme 2011). These systems are macroscopically homoge-
neous but microscopically heterogeneous (Ferreira 2014). ME are
thermodynamically stable due to the addition of a co-surfactant, so coalescence,
cream formation and phase separation have low probability to happen. The low
viscosity is due to the stability between the oil and the aqueous phase, which is
stabilized by the interface film between surfactant and co-surfactant; however, the
viscosity depends on the proportion between the aqueous and oil phase volumes
(Leclercq et al. 2011). They are used as dermal and transdermal drug delivery sys-
tems mainly due to their thermodynamic stability, easy process to make the formu-
lation, and important capacity to incorporate the drug with a small droplet size (El
Maghraby 2010; Uchechi et al. 2014). They are also used as cutaneous drug deliv-
ery due their high solubilization potential for lipophilic and hydrophilic drugs
(Alexander et al. 2012). Particle size and PdI are responsible for the liquid and
transparent appearance of ME (Peltola et al. 2003). These transdermal drug delivery
carriers have high solubility potential for hydrophilic and hydrophobic drugs
(Uchechi et al. 2014).
The first ME was created in the 1940s by Hoar and Schulman (Malik et al. 2012;
Nikhitha et al. 2015). ME are made by emulsification method by the dispersion of
oil phase in aqueous phase containing a surfactant agent and co-surfactant. In gen-
eral, this last co-surfactant was an alcohol with low molecular weight. According to
their internal structure, ME can be classified as oil-in-water (O/W), water-in-oil
(W/O), or as multiple O/W/O or W/O/W ME. ME are promising systems for cos-
metically active ingredients because they are efficient solubilizers (for hydrophilic
and lipophilic compounds), able to enhance the EE (Žilius et al. 2016). In ME, there
is a sub-subcategory named SMEDDS (self-microemulsifying drug delivery sys-
tems) which is very often used (El Maghraby 2010; Anton and Vandamme 2011).
ME are dispersions with a droplet size under 150 nm (Uchechi et al. 2014; Žilius
et al. 2016). These systems are formed spontaneously with adequate amounts of
lipophilic and hydrophilic ingredients, as well as a surfactant and a co-surfactant
(Peltola et al. 2003; Mendonça et al. 2009; El Maghraby 2010; Uchechi et al. 2014).
However, some key factors may influence this process like temperature and system
composition (Anton and Vandamme 2011). These systems are macroscopically
homogeneous but microscopically heterogeneous (Ferreira 2014). The thermody-
namic stability of ME is due to the addition of a co-surfactant, and coalescence,
cream formation and phase separation have low probability to happen. The low
viscosity is due to the stability between the oil and the aqueous phase, which is
stabilized by the interface film between surfactant and co-surfactant; however, the
viscosity depends on the proportion between the aqueous and oil phase volumes
(Leclercq et al. 2011). They are used as dermal and transdermal drug delivery sys-
tems mainly due to thermodynamic stability, easy process to make the formulation
and an important capacity to incorporate the drug with a small droplet size (El
Maghraby 2010; Uchechi et al. 2014). Particle size and dispersion are responsible
for the liquid and transparent appearance of ME (Peltola et al. 2003). These trans-
dermal drug delivery carriers have high solubility potential for hydrophilic and
hydrophobic drugs, the permeation enhancing effect of the ingredients of micro-
emulsions, easy to prepare, present a great diffusion and absorption and the increase
of thermodynamic activity of drug in the carriers (Uchechi et al. 2014).
In 2003, Peltola et al. made microemulsions with estradiol. These microemul-

sions revealed an increase of the estradiol delivery by transdermal route (Peltola
et al. 2003).
Nanoemulsions
By definition NE, also named submicron emulsions, are dispersion of immiscible

droplets with sizes in the “nano” scale (Cevc and Vierl 2010; Parboosing et al.
2012). They are stabilized by surfactants and present sizes between 20 and 500 nm
(Cevc and Vierl 2010). NE, like ME, can be oil-in-water (O/W) or water-in-oil
(W/O). NE are transparent and isotropic dispersed systems of two immiscible liq-
uids, an oily system dispersed in an aqueous system and an aqueous system dis-
persed in an oily system (Fronza and Campos 2004; Uchechi et al. 2014). These
systems are thermodynamically unstable, because some of them need high energy
to produce them (Ferreira 2014; Uchechi et al. 2014). NE are typically O/W and
stabilized by one or more surfactant agents (Anton and Vandamme 2009).
The droplets present a size between 10 to 300 nm, where the flocculation and
coalescence rarely occur, and there exists a superposition of Brownian motion to
sedimentation phenomena with a positive zeta potential value (Bruxel et al. 2011;
Uchechi et al. 2014; Ganesan and Choi 2016; Hamed et al. 2016). However, they are
susceptible to Ostwald ripening and molecular diffusion which are the principal fac-
tors of NE instability (Uchechi et al. 2014; Mota et al. 2017). The Ostwald ripening
is associated with polydispersed droplet size of the internal phase and the difference
of solubility between larger and smaller droplets. Sonneville-Aubrun et al. described
that a small quantity of oil with low solubility in water or non-ionic or high ethoxyl-
ated surfactant agents prevents Ostwald ripening (Sonneville-Aubrun et al. 2004;
Mota et al. 2017). When these problems are overcome, NE can be stable for a long
time due to their small size and an adequate use of surfactants (Ferreira 2014;
Uchechi et al. 2014; Mota et al. 2017).
The first NE was created in the 1940s by the same method of ME; however, they
can be produced using other methods (e.g., high-pressure homogenization, micro-
fluidization, and phase-inversion temperature methods) (Bhatt and Madhav 2011;
Mishra et al. 2014; Uchechi et al. 2014; Mota et al. 2017).
NE present advantages when compared with emulsions and ME, such as higher
surface area (a consequence of the smaller droplet size in NE), and higher skin per-
meation (Hamed et al. 2016).
NE can encapsulate hydrophobic and hydrophilic drugs (Uchechi et al. 2014;
Mota et al. 2017). The advantage of NE is the size which could enhance the bioac-
tivity of hydrophilic and lipophilic compounds. The instability during storage rep-
resents the disadvantage of NE (Ganesan and Choi 2016; Mota et al. 2017). NE are
less used in transdermal drug delivery by the stability problems inherent to the dos-
age form, but they have been used in cosmetic formulations (Uchechi et al. 2014;
Ganesan and Choi 2016; Mota et al. 2017).
As ME, in NE, exists a subcategory, SNEDDS (self-nanoemulsifying drug deliv-

ery systems).
In 2010, Baspinar et al. prepared nanoemulsions with prednicarbate. Each one
revealed monodisperse sizes between 120 and 240 nm (Baspinar et al. 2010).
Hydrogels
Gels are formed by an elastic network of entangled fibers and a solvent where the
liquid component is typically over 90% of their composition. They can be classified
in different gels by their origin (natural or synthetic), by the type of solvent in their
composition (organic or water), and by the nature of the gelling agent (macromol-
ecule or small molecule) (Limón et al. 2015). Hydrogels are by definition a 3D
macromolecular network, formed by cross-linked polymers. They are able to absorb
large amounts of water within their structure, increasing their volume, and they are
soft and pliable (Carvalho et al. 2013; Calixto et al. 2015; Nitta et al. 2016; Yasasvini
et al. 2017). These characteristics have provide them the ability of absorbing water
(Casolaro and Casolaro 2012; Won et al. 2015; Calixto et al. 2015; Yasasvini et al.
2017). Hydrogels present a higher flexibility due their characteristic features, i.e.,
their rubbery and soft texture (Shukla et al. 2016; Yasasvini et al. 2017). Hydrogels
present low inflammatory responses and they can be used as a reservoir for slow
elution of drugs (Won et al. 2015). Furthermore, they offer convenient drug delivery
matrix composed of natural polymers or cross-linked synthetic macromolecules
(e.g., PEG, polyvinyl alcohol (PVA), poly(acrylic acid), poly(ethylene oxide),
poly(vinyl pyrrolidone), polyglycerol esters of fatty acids, carbomers, sodium algi-
nate, chondroitin sulfate, pectin, dextran, carboxymethylcellulose, other cellulose
derivatives, chitosan, gelatin, and gums, among others) (Jodar et al. 2015; Shukla
et al. 2016).
These gels are generally composed of photolabile groups (Kar et al. 2016). Their
properties can be different depending on the type of the polymer used and the cross-
linking step during their production. Because of this, they can be covalently bonded
throughout the whole system to form a polymer (chemical gels), or they can be
composed of smaller subunits that are attached to each other through weaker chemi-
cal forces (hydrogen bonds, coulombic forces, van der Walls interactions, and oth-
ers (supramolecular gels)) (Limón et al. 2015; Paolicelli et al. 2017). The polymeric
matrix is maintained together by the chemical or physical bonds, and its swelling
behavior can be tuned by the introduction of pH-sensitive functional groups
(Paolicelli et al. 2017).
They have been described for being an excellent vehicle for drug delivery for
skin purposes. Mainly when they have the polyacrylic acid (PAA) polymer in their
composition, presenting ability to adhere on biological tissues, commonly known as
bio(muco)adhesion (Carvalho et al. 2013; Calixto et al. 2015; González-Delgado
et al. 2016). The PAA presents biocompatibility and low toxicity (Carvalho et al.
2013). This phenomenon is due to the interaction of the polymeric chains with the
mucus glycoproteins or superficial epithelial cells. This phenomenon contributes to
an increase of the drug absorption and a decrease of administration frequency

(Calixto et al. 2015).
Between the different hydrogels, carbomer hydrogels and thermosensitive hydro-
gels exist. Carbomer hydrogels have been studied in topical administration, present-
ing excellent bioadhesive, and biocompatible properties for clinical uses
(González-Delgado et al. 2016). They are composed of Carbopol® and synthetic
cross-linked acrylic acid polymers which are widely used in the pharmaceutical and
cosmetic industries (Jiménez et al. 2005).
In general, the hydrogels are prepared by mixing the compounds (Dai et al. 2015;
Abdelkader et al. 2016). Hydrogels are first reported in 1979 (Di Colo et al. 1980).
In 2015, Calixto et al. made hydrogels with metronidazole. This system revealed a
drug release of 60–65% depending on the type of hydrogel used (Calixto et al. 2015).
Nanogels
Nanogels (NGs) are similar to hydrogels; they are nanosized 3D systems, which are
chemically or physically cross-linked having the ability to swell (and entrap drugs)
(Sivaram et al. 2015; Sahle et al. 2017; Sahu et al. 2017a, b). This system presents
efficacy and site-specific delivery with a good patient compliance and safety. Their
sustained-release characteristics can reduce dosing frequency (Sahu et al. 2017b).
NGs are composed of an ionic or non-ionic network of amphiphilic polymer
chains and they swell to a considerable volume when they are dispersed in aqueous
media (Sivaram et al. 2015). The biocompatibility of these systems is associated
with an increase of their compliance and acceptance, as polymeric NGs (Sahle et al.
2017; Sahu et al. 2017a, b). They are good candidates for targeting drug delivery
with effective therapeutic potentials. Their sizes are between 10 and 1000 nm (Sahu
et al. 2017a). They are able to change their chemical properties by external stimuli
and enable to target and control drug transport. When compared with conventional
formulations, NGs can highly increase drug penetration. They are powerful candi-
dates for the treatment of severe skin diseases (Edlich et al. 2017).
High water absorptivity, flexibility in design, increased and prolonged circula-
tion time, bioconjugation of active targeting agents, high biocompatibility, high
loading capacity and controlled release of payload, high stability in aqueous solu-
tion, and rapid response to external stimuli are some characteristics of NGs (Sivaram
et al. 2015). NGs are particularly used for encapsulating poorly soluble and poorly
absorbed drugs (Sahu et al. 2017a). One first report with NGs was made by Shin
et al. in 2001 (Shin et al. 2001).
Polymeric NGs have also thermosensitive and pH-responsive features which
contribute to amplification for the therapeutic efficiency of drugs against a variety
of diseases (Sahu et al. 2017a). Polymers, such as poly(N-isopropylacrylamide)
(pNIPAM), poly(glycidyl methyl ether-co-ethyl glycidyl ether) (p(GME-co-EGE)),
and linear polyglycerol derivate (dPG) can be used to produce NGs (Gerecke et al.
2017; Sahle et al. 2017).
The polymeric NGs can be prepared by different methods, and they can be clas-
sified into physical cross-linked NGs and chemical cross-linked NGs. Cross-linked
NGs involve hydrogen bonds or van der Waals forces of attraction, hydrophobic
interactions, electrostatic interactions, and coordination bonds. Chemical cross-
linked NGs involve the amine-based cross-linking, disulfite-based cross-linking,
and photo-induced cross-linking (Sivaram et al. 2015).
Thermoresponsive nanogels (tNGs) are prepared by the incorporation of thermo-
responsive polymers, which are able to undergo conformational changes from an
extended/hydrophilic coil to a globular/hydrophobic state upon heating above the
lower critical solution temperature (LCTS). The tNGs have great potential as drug
delivery systems. Some examples of polymers used in tNGs are pNIPAM, p(GME-
co-EGE), poly(N-vinylcaprolactam), poly(oligo(ethylene glycol)-methacrylate),
and poly(N-dimethylacrylamide), presenting a reversible volume-phase transitions
near the physiological temperature. Generally, tNGs presenting sizes between 100
and 700 nm and a phase transition temperature around 32–37°C (Sahle et al. 2017).
Furthermore, other compounds besides polymers that could be applied into NGs
and confer thermoresponsive properties, such as oligo(ethylene glycol) methacry-
late are also present (OEGMA) (Rancan et al. 2016).
In 2017, Sahu et al. made nanogels with bleomycin. This system presented an EE
of 54.00±0.95% (Sahu et al. 2017b).
Inorganic and Organic Nanoparticles
These types of NPs composed of carbon nanotubes (CNTs), silica NPs (SNPs), and
graphenes (Fig. 6.2).
Carbon Nanotubes
CNTs have hollow structure which could be open or closed at their ends, belonging
to the family of carbon allotropes, with a size ranging from 2 to 20 nm in diameter
(Gilmore et al. 2008; Ménard-Moyon et al. 2010; Karchemski et al. 2012; Jain
2012). They are present in nanometer range with ultralight weight; however, their
lengths can reach several micrometers. CNTs present a large surface area with high
electrical conductivity and have a considerable strength (Jain 2012). These systems
were discovered in the 1950s/1960s and were described for the first time by Sumio
Iijima in 1991 (Gilmore et al. 2008; Zhang et al. 2010; Ménard-Moyon et al. 2010;
Degim et al. 2010; Taber et al. 2012).
CNTs can be classified into two types, single-walled CNTs (SWCNTs) and mul-
tiwalled CNTs (MWCNTs), as illustrated in Fig. 6.2 (Prato et al. 2008; Zhang et al.
2010; Ménard-Moyon et al. 2010; Degim et al. 2010; Karchemski et al. 2012; Jain
2012). SWCNTs are a single graphite sheet seamlessly wrapped into a cylindrical
tube with 0.4–2.5 nm of diameter, while MWCNTs consist of multiple layers of
graphite sheet with different diameters (>100 nm) (Prato et al. 2008; Zhang et al.
2010; Jain 2012).
CNTs present a large loading capability to carry various bioactive agents as
drugs, which have been presenting low toxicity and immunogenicity (Zhang et al.
2010). They are characterized by high surface area with a high aspect ratio, ultra-
light weight, high mechanical strength, high electrical conductivity, and high ther-
mal conductivity and metallic or semiconducting behavior (Ménard-Moyon et al.
2010). However, CNTs present disadvantages for applications in nanomedicine.
They are chemically inert and insoluble. On other hand, the carbon atoms present in
SWCNTs and MWCNTs have some chemical reactivity. MWCNTs present high
resistance to chemicals than SWCNTs, which is important in functionalization of
CNTs. The functionalization is carried out by grafting of chemical functions at the
surface adding new properties to the CNTs (by breaking C=C double bounds, leav-
ing gaps in SWCNTs) like the use of polymers, metals, or biological molecules.
Functionalized CNTs (f-CNTs) are able to bind to other molecules in order to pen-
etrate the biological membranes. They present a relatively low toxicity, improving
their potential application in tissue engineering, and drug delivery (Jain 2012).
CNTs can transport small drug molecules without impairing their therapeutic
efficacy and in some cases enhancing it (Jain 2012). These systems have been used
for diagnosis and treatment of cancer, infectious diseases, and central system disor-
ders (Zhang et al. 2010).
The CNTs characteristics are relevant to drug development. They have the ability
to penetrate cells with the capacity to cross biological barriers. CNTs are biocom-
patible, non-immunogenic when they are shortened, functionalizes and they become
highly dispersible in water. Which means, they become bio-functionalized nano-
tubes with chemical functionalization of the surfaces of template-synthesized in an
high surface area. They become the strongest man-made fiber, they can be chemi-
cally modified to encourage the growth of the calcium crystals. Ultimately,
they present spectroscopic properties (Raman scattering and photoluminescence)
(Ménard-Moyon et al. 2010; Degim et al. 2010; Jain 2012).
The CNTs can present different routes of intracellular passage depending on the
diameter and length differences (Jain 2012).
In case of SWCNTs, they are uptaken into cells by phagocytosis. They present
no adverse effect on the cells for short-term use and if CNTs are cleared out and do
not accumulate in the body. In general, SWCNTs are commonly used as biological
imaging agents (Jain 2012).
MWCNTs can be functionalized with amino groups attached on the surface;
these can act as “nanoneedles,” being able to passively penetrate the cell membrane
(Jain 2012).
CNTs can be functionalized by the use of bioactive carbohydrates, improving
their solubility and biocompatibility which preserve their desired properties. These
CNTs can detect bacteria by binding to specific lectins, delivering glycomimetic
drugs into cells and probing cellular activities as biosensors. These systems consist
of an adsorptive material with a large surface, allowing use to deliver active drug
molecules through skin. They can provide a high loading and enhanced transdermal
penetration for hydrophobic drugs (Jain 2012).
SWCNTs present an antimicrobial effect through cell membrane damage when
in direct contact (in a size-dependent manner) (Jain 2012).
CNTs have the potential to address the challenges of combating infectious
agents, while minimizing toxicity, by dose reduction of standard therapeutics. This
also allow the load capacity to target the activities, regarding the reduction of
the infectious strains and resistant strains (Jain 2012).
Silica Nanoparticles
SNPs present a size between 30 and 1000 nm (Elsabahy and Wooley 2013). The
amorphous SNPs have been investigated for use in biomedical applications due
their characteristics, like high surface area-to-volume ratio, tunable pore size, con-
nectivity, biodegradability, and ease of modification of the surface (Kim et al. 2015).
SNPs can be used for transdermal penetration as drugs (Lin et al. 2006; Kim
et al. 2015).
The size of these NPs influences the surface area, being one of the most critical
factors which contributes to toxicity. SNPs smaller than 100 nm are critical to
understand their toxicity (Kim et al. 2015).
The amount of silica, at micro-scale size, is an important factor; silica is associ-
ated with the development of several autoimmune diseases (systemic sclerosis,
rheumatoid arthritis, lupus and chronic renal disease, silicosis, and lung cancer)
(Kim et al. 2015). For example, SNPs with econazole showed sizes of 3.25 nm
(Firooz et al. 2015).
Graphenes
Graphenes are similar with CNTs but the walls are formed by one-atom-thick sheets
of carbon, in a two-dimensional material (Jain 2012; Shi et al. 2016; Shim et al.
2016). Graphenes have a flat single layer of graphite with stacked layers of carbon
atoms with a honeycomb lattice (Shim et al. 2016).
At room temperature (RT), they have an unusual linear dispersion band structure
and a ultrahigh electron mobility (Shi et al. 2016). These NPs present a great expec-
tation in the biomedical field, by their mechanical properties, biocompatibility,
transparency, and electrical conductivity. Graphenes have mechanical compatibility,
cell adhesion, and low toxicity properties, being a good solution for applications in
scaffold production, sensing, and drug delivery. They can be used as drug delivery
because of their characteristic properties, such as high surface area, biocompatibil-
ity, and versatile chemistry (Reina et al. 2017).
Graphenes were first reported for drug delivery purposes, in the study by Wang
et al. in 2014 (Wang et al. 2014).
Graphenes can be prepared by chemical vapor deposition, graphene oxidation

(GO), reduction, and aqueous dispersions (Reina et al. 2017). The graphene-based
nanosheets (GNS) include the graphenes, graphene oxides, and reduced graphene
oxides (Shim et al. 2016). However, the graphene oxide and reduced graphene are
easier to handle (Reina et al. 2017).
The surfaces of GNS can be modified with polymers (e.g., polyethylene glycol)
and biopolymers, presenting an enhancement of biocompatibility and increased
drug loading (Shim et al. 2016).
The graphenes produced by GO are different from other graphenes, by the pres-
ence of oxygenated groups on the surface, presenting better solubility in aqueous
media, easier handling and a richer surface chemistry. These ones are composed of
two different domains (one the hydrophobic zone, composed of sp2 carbon, and the
other the hydrophilic zone, presenting oxygenated groups). The functionalization of
GO can increase their dispersibility in water or in the cell culture media, which
contributes to a decrease in cell/tissue toxicity and the induction of accumulation to
target cells and tissues. The functionalization can be made with the use of biocom-
patible polymers (e.g., chitosan, polyethylenimine, polyethylene glycol, and oth-
ers). The targeting also contributes to an accumulation of the nanomaterial in the
desired tissues which enhance their therapeutic results, as well a decrease of the side
effects (Reina et al. 2017). The chemical reduction of exfoliated graphene present
advantages as large-scale synthesis and relatively low cost (Kwon et al. 2017). They
have been studied for cancer treatment (Shim et al. 2016; Reina et al. 2017).
In Table 6.5, some nanopharmaceuticals in the market are briefly presented (Choi
and Maibach 2005b; Azeem et al. 2009; Cevc and Vierl 2010; Khan et al. 2011;
Kulkarni et al. 2011; Ascenso et al. 2011; Kasliwal 2012; Hamishehkar et al. 2013;
El-Menshawe and Hussein 2013; Kumar et al. 2014; Moghassemi and Hadjizadeh
2014; Weissig et al. 2014; Firooz et al. 2015; Marwah et al. 2016; Nassiri Koopaei
and Abdollahi 2016).
6.3.5 Safety Issues of Nanomedicine
When nanotechnology is applied to human health, it is necessary to use nanomateri-

als with high level of biodegradability and biocompatibility properties.
In vitro assays of NPs can revealed if they do not have any toxicity to the cells
(Jain 2008). However, some NP characteristics have influence on biological effects
(Jain 2008; Mota et al. 2017). As NPs remain foreign bodies to the organism, it is
also important to considerer the toxicity of their degratadion compounds after (Reis
et al. 2008).
Another important information to the toxicity is the use of solvents in the pro-
duction of nanocarriers; the residual solvents should be quantified and present a
level according to the regulatory limits (Reis et al. 2007, 2008). As was mentioned
above, in general, all the solvents used in the NP production belong to Classes 2, 3,
or 4 of ICH Q3, being their quantities safe for humans (International Conference on
Table 6.5 Nanopharmaceuticals in the market
Name (drug or active Approved by
compounds) Mechanism of action Indication FDA
®
Liposomes AmBisome (amphotericin B, MPS targeting (i.e., contributed by their negative charge). Systemic fungal infections 1997
Nexstar Company, USA) (IV)
DaunoXome® (daunorubicin) Passive targeting via EPR effect (the drug persists at high HIV-related KS (IV) 1996
levels for several days).
DepoCyt® (cytarabine) MVLs, sustained release (cytotoxic concentrations of drug are Lymphomatous malignant 1999/2007
maintained in the cerebrospinal fluid for more 14 days after a meningitis (IV)
single 50 mg injection).
DepoDur® (morphine sulfate) MVLs, sustained release (the drug is released over an extended Treatment of chronic pain 2004
period of time). (administered into the
epidural space)
Doxil® (doxorubicin Stealth® liposomes, passive targeting via EPR effect AIDS-related KS, multiple 1995a
hydrochloride, Janssen) (extravasation of liposomes by passage of the vesicles through myeloma, ovarian cancer
endothelial cell gaps present in solid tumors, enhanced (IV)
accumulation of drug in lesions of AIDS-associated KS after
administration of PEG-liposomal doxorubicin).
Inflexal® V (influenza virus On surface, mimicking the native virus structure (allowing for Influenza vaccine 1997
antigens (hemagglutinin, cellular entry and membrane fusion), by the presence of
neuraminidase)) antigens on surface, provides high immunogenicity.
Marqibo® (vincristine sulfate) Passive targeting via EPR effect (through fenestra in bone Acute lymphoid leukemia, 2012
marrow endothelium). Philadelphia chromosome
negative, relapsed or
progressed (IV)
Mepact TM (mifamurtide) A synthetic muramyl tripeptide–phosphatidylethanolamine in Non-metastasizing 2009
LML, MPS targeting (the drug is an immune stimulant, that is, resectable osteosarcoma
anchored in negatively charged liposomal bilayer membrane). (IV)
Myocet® (doxorubicin) MPS targeting (by MPS “depot,” with slow release into the Metastatic breast cancer 2000 (in
blood circulation, similar with prolonged infusion). (IV) Europe)
(continued)
Visudyne® (verteporfin) Drug solubilization (by IV administration). Photodynamic therapy of 2000
wet age-related macular
degeneration, pathological
myopia, ocular
histoplasmosis syndrome
(IV)
Niosomes Lancome Niosome Plus® Antiaging formulation
Brij® 30
Oramix NS 10®
RetinA®
P90 liposomes®
Lipid-based Abelcet® Amphotericin B, MPS targeting (selective transfer of drug Systemic fungal infections 1995
(non-liposomal) Amphocil® (outside USA) from lipid complex to fungal cell with minimal uptake into (IV)
formulations human cells has been postulated).
Amphotec® Amphotericin B complex with cholesteryl sulfate (1:1), MPS 1996
targeting.
Polymer-based Copaxone® Polypeptide (with an average molecular weight of 6.4kDa, Multiple sclerosis (SC) 1996/2014
nanoformulations which is composed of four amino acids – glatiramer), without
mechanism associated to nanosize, being based on its
resemblance to myelin basic protein; however, glatiramer is
thought to divert as a “decoy” to an autoimmune response
against myelin.
Eligard® Leuprolide acetate (synthetic GnRH or LH–RH analog) Advanced prostate cancer 2002
incorporated in nanoparticles (with PLGH copolymer (SC)
(DL-lactide/glycolide; 1/1, molar), sustained release.
Genexol® Paclitaxel in micelles (20–50 nm) composed of a copolymer Metastatic breast cancer 2001
(poly(ethylene glycol)-poly(D,L-lactide)), passive targeting via Pancreatic cancer (IV)
EPR effect.
Opaxio® Paclitaxel covalently linked to solid nanoparticles with Glioblastoma 2012
polyglutamate, passive targeting via EPR effect (drug release
inside solid tumor by enyzmatic hydrolysis of polyglutamate).
Renagel® Cross-linked polyallylamine hydrochloride with a variable Hyperphosphatemia (oral) 2000
molecular weight, no mechanism attributable to nanosize,
phosphate binder.
Zinostatin stimalamer® Conjugate protein or copolymer of styrene maleic acid and an Primary unresectable 1994
antitumor protein NCS, synthesized by conjugation of one hepatocellular carcinoma
molecule of NCS and two molecules of poly(styrene co-maleic
acid), passive targeting via EPR effect.
Magnetic Feridex® Superparamagnetic iron oxide nanoparticles coated with Liver/spleen lesion MRI 1996 (in 2008
nanoparticles dextran. The size of iron oxide core is 4.8–5.6 nm and the (IV) was
hydrodynamic diameter is 80–150 nm. MPS targeting (80% discontinued)
taken up by liver and >10% by the spleen within minutes of
administration; tumor tissues do not take up these particles and
thus retain their native signal intensity).
FerahemeTM (Ferumoxytol) Superparamagnetic iron oxide nanoparticles coated with Treatment of iron 2009
dextran with a hydrodynamic diameter of >50 nm. MPS deficiency anemia in
targeting (iron release inside macrophages which enter into adults with chronic kidney
intracellular storage iron pool or is transferred to plasma disease
transferrin.
NanoTherm® Aminosilane-coated superparamagnetic iron oxide, 15 nm Local ablation in 2013
nanoparticles, thermal ablation: injecting iron oxide glioblastoma and prostate
nanoparticles exposed to alternating magnetic field causing the and pancreatic cancer
nanoparticles to oscillate, generating heat directly within the (intratumoral)
tumor tissue.
Dendrimers DEP™ docetaxel (Starpharma Poly-lysine dendrimer; the mechanism of action consist in (1) Breast cancer
Holdings Ltd.) preferential accumulation of drug-loaded DEPTM conjugates in
tumors, (2) drugs are released in tumor from DEPTM backbone
according to linker strategy; (3) drugs enter in tumor cells
inducing cell death and tumor regression.
(continued)
PEGylated Adagen® PEGylated adenosine deaminase (by modification of enzyme Adenosine deaminase 1990
proteins, molecule); the mechanism of action is the increase of deficiency – severe
polypeptides, circulation time and reduction of immunogenicity, increase of combined
aptamersb hydrodynamic radius which prolongs circulation and retention immunodeficiency disease
time, decrease of proteolysis and renal excretion and shielding
of antigenic determinants from immune detection without
obstructing the substrate interaction site.
Cimzia® PEGylated antibody (Fab fragment of a humanized anti-TNF-α Crohn’s disease 2008
antibody). Rheumatoid arthritis
Neulasta® PEGylated filgrastim (granulocyte colony-stimulating factor). Febrile neutropenia 2002
Nonmyeloid malignancies
Prophylaxis (SC)
Oncaspar® PEGylated L-asparaginase. Acute lymphoblastic 1994
leukemia
Pegasys® PEGylated interferon α-2b. Hepatitis B and C 2002
PegIntron® Hepatitis C 2001
Somavert® PEGylated human growth hormone receptor antagonist. Acromegaly (second-line 2003
therapy)
Macugen® PEGylated anti-VEGF aptamer. Intravitreal 2004
Neovascular age-related
macular degeneration
Mircera® PEGylated epoetin β (erythropoietin receptor activator). Anemia-associated with 2007
chronic renal failure in
adults
a
It was the first nanopharmaceutical approved by US FDA (Nassiri Koopaei and Abdollahi 2016).
b
The mechanism of action is the same for all.
AIDS acquired immune deficiency syndrome, DEPTM docetaxel, DMPC dimyristoylphosphatidylcholine, DMPG dimyristoyl phosphatidylglycerol, EPR enhance-
ment of permeability and retention effect, FDA US Food and Drug Administration, IV Intravenous, KS Kaposi’s sarcoma, LML large multilamellar liposomes, MLVs
multilamelar vesicles, MPS mononuclear phagocytic system, MRI magnetic resonance imaging, NCS neuronal calcium sensor, PEG polyethylene glycol, SC subcu-
taneous, TNF-α tumor necrosis factor-α, VEGF Vascular endothelial growth factor
Harmonisation for Registration of Technical Requirements for Pharmaceuticals for

Human Use 2011).
In general, the toxicity of nanocarriers also depends on additional and multiple
parameters associated with physicochemistry as size, shape, concentration, charge,
surface coatings, and composition (Rzigalinski and Strobl 2009). For example, lipo-
somes have been used in cancer treatment without any toxicity of NPs (Jain 2008),
while gold NPs (≈2 nm) showed that cationic particles present a moderate toxicity
and the anionic particles present quite no toxicity (Jain 2008; Mota et al. 2017).
Compliance with the Good Manufacturing Practices, Guiding Principles in
Toxicology Assessment, Good Laboratory Practices and Good Distribution
Practices is crucial for public health assurance (Abrantes et al. 2016).
6.3.6 Toxicity
Detailed evaluation of excipient’s toxicity and safety are required by the regulatory
authorities (Abrantes et al. 2016). The methods used to study that should respect the
Guidelines of Principles on the Use of Animals in Toxicology and the Good
Laboratory Practice (Abrantes et al. 2016).
The environmental impact of nanomaterials is of great interest. Some studies
have been made with aim of studying the ecotoxicity of the produced NPs (Matos
et al. 2016; Lopes et al. 2017). It has been verified a bigger decrease in toxicity
when the drug or active compound was encapsulated in NPs than the drug or
active compound when they were isolated (V. Figueiredo 2014; Grillo et al. 2015;
Matos et al. 2016; Lopes et al. 2017). These studies generally involve the ecotoxic-
ity of springtails (Folsomia candida), zebrafish embryos (Danio rerio), and
Pseudokirchneriella subcapitata and silver catfish species (Rhamdia quelen),
Artemia salina L. brine shrimp (Robbens et al. 2010; Hu et al. 2012; Grillo et al.
2015; Matos et al. 2016; Lopes et al. 2017), and rats (Reis et al. 2008). Other species
that can be used are bacteria, sediment organisms, nematodes, Dapnia magna, and
earthworms (Hu et al. 2012). Springtails are species present in the soil, litter, or
vegetation of most terrestrial ecosystems (Lopes et al. 2017). Moreover, zebrafish
model has been used on studies for nanomedical applications, because they are low
cost (Matos et al. 2016). Silver catfish can be presented in waters, and it is important
to evaluate their toxicity with the aim of verifying the contamination of waters (a
common element to most ecosystems) (Lopes et al. 2017).
The study of Lopes et al. (2017) demonstrated that GML NPs present a toxic
effect on soil, and on silver catfish, the toxic effect decreased, and an increase of the
number of fishes that survive was also observed (Lopes et al. 2017). Another study,
revealed that NH2-ORMOSIL NPs do not present significant effects for mortality,
development delay, hatching, or malformations for zebrafish (Matos et al. 2016).
Grillo et al. evaluated the ecotoxicity using Pseudokirchneriella subcapitata, an
alga, and their NPs have not presented toxicity (Grillo et al. 2015). The Al2O3 NPs
revealed no toxic effect on zebrafish and earthworms, a median lethal concentration
(LC50) on D. magna (162 mg/L) and on nematodes (82 mg/L), and a toxic effect on
sediment organisms but only under extremely high-particle concentrations (which
could not be found in the realistic environment) (Hu et al. 2012).
The nature of the contaminant is a crucial factor. For example, the cadmium
contamination in the environment could enhance the risk of new cancer develop-
ment. In general, the exposition to that is associated with tumors in the lungs, pros-
tate, liver, kidneys, pancreas, urinary bladder, and breast (Rzigalinski and
Strobl 2009).
To study its cytotoxicity, Saccharomyces cerevisiae model is commonly used as
preliminary assessment (Reis et al. 2013, 2014; Rosado et al. 2013; Silva et al.
2014, 2015; Roque et al. 2017).
Excipient’s toxicity is not a minor issue due to the interaction with the drug or
active compounds or with the other excipient’s, is leading to a change of potential
between the efficiency and toxicity (Abrantes et al. 2016). To study the excipient’s
toxicity is hard, due to the large number of them and their chemical profiles diver-
sity, as well as the presence of secondary products and contaminants (Abrantes
et al. 2016).
To study the toxicity for dermal, topical or transdermal routes are required some
toxicological tests as acute oral toxicity, acute dermal toxicity, eye irritation, skin
irritation, injectable acute toxicity, local evaluation of application, phototoxicity/
photoallergy, genotoxicity testing, ADME-PK, toxicity 28 days in two species
(Abrantes et al. 2016).
6.3.7 Regulatory Framework for Nanopharmaceuticals
Nanopharmaceuticals are now widely studied by several regulatory and scientific

bodies as the Food and Drug Administration (FDA), European Medicines Agency
(EMA), Environmental Protection Agency (EPA), National Institute for Occupational
Safety and Health (NIOSH), World Health Organization (WHO), and others to
ensure public health. As example, the FDA and EMA release some technical docu-
ments and recommendations for the industry and regulators concerning the evalua-
tion and the approval of the products and devices to ensure public health and quality
of life. Recently, FDA produced some guidelines (e.g., Drug Products, Including
Biological Products, that Contain Nanomaterials Guidance for Industry, December
2017) for products that contain nanoscale materials or properties due to the dimen-
sions. As example, nanocarriers like liposomes and dendrimers will involve more
elaborate investigations related to their safety and effectiveness (Nassiri Koopaei
and Abdollahi 2016).
The United States was the first country to initiate a formal program for govern-
ment funding of research in nanotechnology. Nanotechnology occupies new and
larger molecule drug space, and as a result, it is certain that new drugs or therapies
will come from this sector. But it is of urgent concern to better characterize these
nanopharmaceuticals and their physicochemical properties (size and size
distribution); these systems should require a sophisticated stability and pharmaco-

kinetics and pharmacology (absorption, distribution, metabolism, and excretion –
ADME) studies; their safety and efficacy (evade during shelf life and cause
morbidities and fatalities) are crucial factors for the implementation and further
commercialization of the process (Nassiri Koopaei and Abdollahi 2016).
6.4 Conclusion
In the present chapter, a range of topics of relevance for nanopharmaceuticals in

skin delivery have been highlighted with the aim of providing an overview of the
past, present, and future status of this exciting field. The combination of nanotech-
nology with drug delivery has become possible due to the exponential growth and
development of new formulations through the skin over the course of the last decade.
Most of the initial developments took place in the early 1990s, with the creation of
functionalized NPs, including a wide range of organic and inorganic materials at
different nanoscale sizes. Since then, the growth of other nanocarriers has been
rapid and intense. Today, creative combinations of nanocarriers have pointed to
novel applications. Furthermore, progress is also being achieved in developing bio-
degradable or recyclable nanomaterials and in ensuring their safe use. Nevertheless,
it is important to consider the type of NPs and the final aim. Other important factors
are the solvent and the method used, environmental impact, and its regulatory
framework. What is less certain is whether the current research supply chain can
adapt to its new role without changing its mode of operation. All research groups
should benefit significantly from developing nanopharmaceuticals that will provide
all stakeholders with new options – including, most importantly, health progress.
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0.1080/10717544.2016.1177136
Chapter 7
Biomimetic and Synthetic Gels
for Nanopharmaceutical Applications
Busra Yildiz, Sezer Ozenler, Muge Yucel, Umit Hakan Yildiz,

and Ahu Arslan Yildiz
Contents
7.2 C lassification of Gels 275
7.3 General Properties of Gels 277
7.3.1 Green Synthesis of Nanogels 277
7.3.2 Swelling Behaviors 277
7.3.3 Responsive Behaviors 279
7.3.4 Mechanical Behaviors 285
7.4 Applications of Gels 287
7.4.1 Therapeutic Agent Carrier 287
7.4.2 Diagnostic and Imaging 295
7.4.3 Biosensors 298
7.5 Challenges in the Applications 300
7.6 Future Perspectives 300
References 301
Abstract This review discusses biocompatible hydrogels and stimuli-responsive

3D networking polymeric materials and building blocks of nanopharmaceuticals.
We aimed to outline the basics of gels and hydrogels, responsive behaviors, and
applications such as therapeutic and drug carriers, bioimaging agents, biosensing
elements. The review also covers significant challenges, limitations, opportunities,
new approaches, and future perspective. We review the general aspects of hydro-
gels to refresh the basic knowledge and theory that is covered in the section
“General Properties of the Gels.” It discusses green synthesis of nanogels, swell-
ing, and responsive and mechanical behavior of the gels. We also reviewed the
B. Yildiz · S. Ozenler · U. H. Yildiz (*)

Department of Chemistry, Izmir Institute of Technology (IZTECH), Izmir, Turkey
e-mail: busrayildiz@iyte.edu.tr; sezerozenler@iyte.edu.tr; hakanyildiz@iyte.edu.tr
M. Yucel · A. Arslan Yildiz (*)
Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey
e-mail: mugeyucel@iyte.edu.tr; ahuarslan@iyte.edu.tr
https://doi.org/10.1007/978-3-030-44925-4_7
274 B. Yildiz et al.
broad applications of gels including therapeutic agent carriers, drug, protein, and
genetic material delivery systems. In a separate section, biosensing and imaging
applications have been reviewed from recent reports. Overall we highlighted
advantages of hydrogels and their evolution that providing broad functionality, sta-
bility, and responsiveness as well as low toxicity. We also discussed the limitations
and challenges as well as unique opportunities for developing alternative synthesis
and processing strategies that revolutionize common understandings. In the last
section, as closing remarks, the future needs in nanopharmaceutical applications
have been elaborately articulated, and the combination of biomimetic-synthetic
hydrogels and biocompatibility has been discussed.
Keywords Hydrogels · Biomimetic gels · Stimuli responsive hydrogels ·

Nanopharmaceuticals · Biosensors · Diagnostics
7.1 Introduction
The future aspect in design and synthesis of bioinspired materials is not simply
mimicking their natural counterparts but providing phenomenal properties and
advancements that even recover deficiency of nature-made matter. The gels are one
of the most studied soft matters utilized in varying biotechnological applications
such as tissue engineering, therapeutic agent carrier and delivery, diagnostic, bioim-
aging, and biosensing. The synthetic gels have been dramatically evolved to provide
broad functionality, stability, and responsiveness as well as low toxicity that syn-
chronizes abilities of polymer science, biology, pharmaceutical sciences, and bioen-
gineering. The standards required for nanopharmaceutical applications limit the
synthetic versatility of gel making materials due to their toxic effects. Therefore the
use of hydrogels is getting a golden standard for pharmaceutical applications since
they provide excellent biocompatibility, mechanical strength, as well as responsive-
ness. This review discusses biocompatible hydrogels and stimuli-responsive 3D net-
working polymeric materials holding great promises for the nanopharmaceutical
applications. The review outlines basics of gels and hydrogels, responsive behav-
iors, and applications such as therapeutic and drug carriers, bioimaging agents, and
biosensing elements.
In the first section of the review, the basics of gels and hydrogels have been dis-
cussed, and the general aspects of the hydrogels highlighted by recent reports, for
example, Demirdirek and Uhrich (2017), Quan et al. (2015), and Koetting et al.
(2016), have been reviewed to give overview for pH, temperature, and affinity
responsiveness of gels. The categorization of the hydrogels has been briefly sum-
marized by the recent reports of Wu et al. (2018) (Janmey et al. 2009; Jin et al. 2016;
Omer et al. 2015). These and the other reports have provided short but elaborate
introduction concerning hydrogel terminology and classification. In the next sec-
tion, we highlighted green synthesis of hydrogels by summarizing (Sepideh Khoee
2016; Neamtu et al. 2017; Wang et al. 2013) and other reports that provide broad
7 Biomimetic and Synthetic Gels for Nanopharmaceutical Applications 275
overview of water-based synthesis procedures and their applications. In the next

section, the swelling and responsive behavior of hydrogels were reviewed where pH
(Manchun et al. 2015), thermal-responsive hydrogels (Ullah et al. 2015), as well as
affinity responsive (Franklin and Guhanathan 2015), electro-responsive (Chen et al.
2016a; Gao et al. 2008; Shang et al. 2008; Rahimi et al. 2012), and light-responsive
hydrogels (Bahram et al. 2016) were discussed to highlight the unique properties of
hydrogels. In the next sections, the potential of hydrogels for nanopharmaceutical
applications has been justified by reviewing several studies, for instance, therapeu-
tic agent carrying hydrogels exemplified by Jin et al. (2016), Kulkarni et al. (2017),
Morimoto et al. (2013), and Nishimura et al. (2017), and bioimaging and biosensing
applications were discussed by revision of Wu and Zhou (2010), Xu et al. (2018),
Wang et al. (2017b), and (Jung et al. (2017a), Deligkaris et al. (2010), Drury and
Mooney (2003), and Peppas and Van Blarcom (2016). In the closing sections, it is
discussed the limitations and challenges as well as unique opportunities for devel-
oping alternative synthesis and processing strategies that revolutionize common
understandings in hydrogels to give broad overview concerning the future needs in
nanopharmaceutical and the importance of combination of biomimetic-synthetic
hydrogels.
7.2 Classification of Gels
The term “gel” refers to a material in which a liquid phase and a solid phase are
dispersed in each other and form a three-dimensional (3D) structure. The gels are
primarily classified as natural and synthetic gels that natural or biomimetic gels
have natural origins. The gels which mimic the extracellular matrix (ECM) are most
frequently used in biomedical applications. The weak mechanical strength of these
gels has directed the scientists to synthesize hybrid gels which is a combination of
biomimetic and synthetic gels. Gels are classified in terms of different aspects such
as their responsive behaviors, nature of monomer, cross-linking, and liquid phase.
The Fig. 7.1 illustrates the classification of gels.
The gels are mainly categorized as pH (Demirdirek and Uhrich 2017; Sarkar
et al. 2017), temperature (Quan et al. 2015; Ruel-Gariépy et al. 2004), affinity
(Koetting et al. 2016; Zou et al. 2015), electrical field (Jackson and Stam 2014;
Rahimi et al. 2012), and light-responsive (Chiang and Chu 2015; Hang et al.
2017). The gels are able to sense the variations in these stimuli or (Bahram et al.
2016) are capable of responding to more than one stimuli giving a dual or mul-
tiresponsivity (Wang et al. 2017c; Wang et al. 2010; Wei et al. 2017; Zhang
et al. 2017). The natural, synthetic, and hybrid gels are classified based on the
nature of building blocks. Natural hydrogels can be composed of chitosan
(Ruel-Gariépy et al. 2004; Wu et al. 2018), alginate (Alemdar 2016; George and
Moon 2015; Li et al. 2017a), collagen (Hou et al. 2017), gelatin (Laha et al.
2016; Lien et al. 2008; Yang et al. 2016), fibrin (Janmey et al. 2009), dextran
(Jin et al. 2016; Omer et al. 2015), and hyaluronic acid (Burdick and Prestwich
GELS
RESPONSIVE NATURE OF LIQUID

CROSSLINKING
BEHAVIORS MONOMER PHASE
pH ORGANOGEL
TEMPERATURE NATURAL ALCOGEL
PHYSICAL HYDROGEL
AFFINITY SYNTHETIC
CHEMICAL POLYMER LIQUID
ELECTRICAL HYBRID
LIGHT OLEOGEL
IONIC LIQUID
Fig. 7.1 The classification of gels based on their responsive behaviors, nature, cross-linking type,
and liquid phase
2011) to mimic the nature of tissues for different applications. Poly(acrylic

acid) (Lim et al. 2017a), p(N-isopropylacrylamide-co-N,N-dimethylaminoethyl
methacrylate) (p(NIPAAm-co-DMAEMA)) gels (Chen et al. 2016b; Wang et al.
2008), poly(hydroxyethyl methacrylate) (Seo et al. 2017), polyvinyl alcohol/
poly (sodium maleate-co-sodium acrylate) (Gao et al. 2008) hydrogels can be
common examples of synthetic gels. Additionally, hybrid hydrogels of gelatin/
poly(lactic-co-glycolic acid) (Chung et al. 2016), poly(ethylene glycol)/dextran
(Artzi et al. 2009), chitosan/poly(vinyl alcohol)/genipin (Garnica-Palafox and
Sanchez-Arevalo 2016), poly(acrylic acid)/fibrin (Rahimi et al. 2012), alginate/
polyacrylamide (Fitzgerald et al. 2015), chitosan/carbon dot (Wang et al.
2017b), and gelatin/carbon nanotube (Spizzirri et al. 2013) gels are applied
providing required mechanical strength and biocompatibility for many applica-
tions. Depending on the origin of the cross-linking, gels are categorized as
physical gels and chemical cross-linked gels. In physical gels, there are non-
covalent interactions like H-bonding, π–π interactions, and van der Waals forces
(Aldilla et al. 2017; Bahram et al. 2016; Chen et al. 2014; Liu et al. 2018;
Pourjavadi et al. 2018; Vilaça et al. 2017). Unlike, chemical gels are formed
with covalent bonding (Fuhrmann et al. 2015; Lim et al. 2017a; Quan et al.
2015; Rahimi et al. 2012).
As shown in Fig. 7.1, based on their liquid phase, gels can also be classified, for
instance, when water is the solvent hydrogels and when organic solvent, polymers,
oil, and ionic liquid are dispersed in the gels’ organogel utilized (Hu et al. 2018;
Nishiyama et al. 2017). An alcogel (Özbakır and Erkey 2015; Zhang et al. 2016b),
hydrogel (Cai et al. 2017; Zhuo et al. 2017), polymer liquid gel (Deng and Yang
2017; Song et al. 2017), oleogel (Dhal et al. 2017; Manzocco et al. 2017), and ionic
liquid gel (Kusuma et al. 2018; Marr and Marr 2016) were the most studied gels in
recent literature.
7.3 General Properties of Gels
7.3.1 Green Synthesis of Nanogels
The motivation in green chemistry is to use non-hazardous chemicals or to mini-

mize the use of hazardous chemicals to maintain the environmental sustainability
(Warner and Anastas 1998). Synthesis of nanogels used in pharmaceutical applica-
tions by green synthesis is therefore important for both application efficiency and
environmental protection. Nanogel synthesis by RAFT polymerization in water is
emerging as an appealing approach in green chemistry perspective. There are three
important features of performing green synthesis of nanogels by RAFT polymeriza-
tion in water: (i) using water as a solvent, (ii) obtaining high solid content, and (iii)
performing surfactant-free synthesis (Sepideh Khoee 2016). The green chemistry
also limits the use of cross-linking agents that cause undesirable toxic effects.
Therefore, photo-induced cross-linking method has been chosen for green synthesis
of gels (Neamtu et al. 2017). Hong et al. was synthesized carboxyl-functionalized
magnetic nanogel by using photochemical polymerization (Hong et al. 2007).
Although enzyme-catalyzed reactions with high enantio- and regioselectivity are
attractive for green synthesis, they have low activity when using organic solvents as
the reaction medium. Wang et al. describe a “substrate-imprinted” lipase nanogel
with high activity in organic solvents. Synthesis of chloramphenicol palmitate in
acetonitrile with the lipase-imprinted nanogel gave a yield of ~99%, while the non-
imprinted lipase gave a yield below 60% (Wang et al. 2013). Li et al. synthesized
nanogel using lysozyme and dextran, two natural biomacromolecules, via a green
method that includes Maillard dry-heat process and heat-gelation process (Li et al.
2008). These examples reveal that green synthesis of hydrogels is getting an attrac-
tive field of research in particular for medical grade material production.
7.3.2 Swelling Behaviors
The swelling of gels occurs by absorbing large amounts of water while preserving
their 3D structure called as volume-phase transition. The swelling is primarily gov-
erned by the diffusion of water molecules into the space between hydrogel networks
(chain) depending on the interactions of polymer chain and water. Ionic nature and
hydrophilicity of the chain are the main parameters affecting the polymer-water
interaction. Nanogels are highly swellable because of secondary forces like electro-
static, van der Waals, hydrophobic and hydrophilic attractions, and covalent bond-
ing on polymer backbones (Soni et al. 2016). As represented in Fig. 7.2,
stimuli-responsive gels swell or collapse in the presence of specific environmental
Fig. 7.2 Swelling and shrinking of a gel in response to different stimuli. (a) Swollen gel contains
solvent molecules diffused between polymeric chains; (b) Swollen gel shrinks to collapsed state by
releasing solvent molecules to surrounding when external stimuli is applied
effects. Solvent molecules were diffused into the gel matrix depending on the mor-
phology and backbone structure of gel. In collapsed state, solvent molecules
released from matrix and gel become dry state. The swelling of anionic and cationic
polymers follows opposite mechanism. As shown in Fig. 7.3, in basic media, anionic
poly(acrylic acid) (PAA) becomes deprotonated and swells, whereas cationic poly
(N, N 9-diethylaminoethyl methacrylate) is protonated and thus shrinks by decreas-
ing water content. Acidic conditions give rise to shrinking of PAA and swelling of
poly (N, N 9-diethylaminoethyl methacrylate) hydrogels. This unique property of
gels has been exploited for various pharmaceutical applications, for instance, Liu
et al. designed a sericin/dextran (SDH) injectable hydrogel cross-linked with hydra-
zone and loaded with doxorubicin to prevent tumor growth. The swelling experi-
ments were carried out in PBS at varying pHs: 6.0, 7.4, and 11.0 at 37 °C. When
DEX-Al content increased, the swelling degree of hydrogels fell. This decline may
result of the lower pore size and porosity of gel in formulations with lower percent
of sericin. In acidic conditions, pH 6.0 which is near to isoelectric point of sericin,
swelling decreased compared to basic conditions (Liu et al. 2016).
Kabiri et al. synthesized poly ammonium persulfate 2-acrylamido-2methyl-
propane sulfonic acid (AMPS) (poly (AMPS)) hydrogel and investigated the
swelling behaviors in different solvents, pH conditions, and ion-induced water
uptake. The interactions of solvent molecules and functional groups on polymer
backbone give rise to varying swelling profiles of hydrogels. Swelling at DMSO-
water mixture was highest among acetone, ethanol, methanol, ethylene glycol
(EG), N-methyl-2-yrrolidone (NMP), and dimethyl sulfoxide (DMSO) only.
The highest swelling ratio of hydrogel in DMSO-water mixture is probably due
Fig. 7.3 Ionization of (a) poly(acrylic acid) and (b) poly (N, N′-diethylaminoethyl methacrylate)
responding to pH of environment
to the ionic repulsions between sulfonate groups both in DMSO and poly
(AMPS) hydrogel. Also, the swelling capacity of the hydrogel decreased in
NaCl and CaCl2 solutions to nearly 30 g/g and 15 g/g, respectively, after 10 min
in salt solutions. The decline was considered to be an effect of inhibited anion-
anion repulsions due to cations of salts (Kabiri et al. 2009).
7.3.3 Responsive Behaviors
The volume-phase transition is considered to be a major mechanism governed by

either “osmotic pressure and charge density alterations” or “affinity of the backbone
to the solvent” providing stimuli-responsive behavior of gels. The stimuli-responsive
gels are found to be that sensitive to changes in environmental effects such as tempera-
ture, pH, electrical field, affinity, and light (Ahmed 2015; Goh et al. 2017; Spizzirri et al.
2013; Tan et al. 2006; Yin et al. 2014). Some of these stimuli are within the living body
such as pH, temperature, chemical species, and biomolecules; thus these systems
have potential to be implemented to the body for varying applications. Figure 7.4
represents these stimuli responsivity of gels to different environmental effects.
Ionizable acidic or basic groups and changes in the lower critical solution tempera-
ture (LCST) are affected by the stimuli and cause conformational and structural
Fig. 7.4 Stimuli responsivity of gels to the different effects: light, pH, temperature, electric, and
affinity (enzyme, glucose, antigen, and antibody)
transitions of the nanogel backbone (Soni et al. 2016). These changes in the back-
bone lead to swelling or deswelling selectively based on applied stimuli and making
the gels “smart” or “intelligent.”
pH-Responsive Gels
pH-responsive gels sense the alterations in pH of the surrounding medium and thus
swell or shrink in volume. pH responsivity is provided by the ionic groups on the
backbone of gels. The polyelectrolytes with varying ionizable functional groups in
the backbone lead to repulsion between polymer chains. This repulsion forms a
space for water to diffuse and swell. The pH-responsive gels were well exploited in
various applications, for instance, Manchun et al. have developed pH-responsive
dextrin nanogels (DNGs) to deliver doxorubicin (DOX) into specific tumor sites in
treatment of colorectal cancer. FDNGs were prepared with addition of formalde-
hyde to DNGs, and it is shown that DOX-loaded FDNGs are powerful systems that
can deliver the drug specifically into the nuclei of the cancer cells and significantly
increase antitumor effectiveness compared to free DOX; nonetheless the amount of
DOX release is greater at pH 6.8 and pH 5. Hence pH-responsive FDNGs are prom-
ising drug delivery systems in the treatment of colorectal cancer (Manchun et al.
2015). Table 7.1 exemplifies the pH-responsive gels based on monomeric units. For
example, acidic gels bear carboxylic functional groups in acrylic acid-based gels
Table 7.1 pH-responsive groups on different gels

pH-responsive
Type Monomer group Reference
Acidic Acrylic acid –COOH Beer et al. (2012), Chen et al. (2016a),
Xu et al. (2018) and Zhang et al.
(2016a)
2-Acrylamido-2- –SO3-Na+ Kabiri et al. (2009) and Willett and
methylpropanesulfonic acid Finkenstadt (2015)
(AMPS)
Basic N,N-dimethylaminoethyl –N(CH3)2 Bossard et al. (2006), Chen et al.
methacrylate (DMAEMA) (2016a), Maksimova et al. (2012),
París and Quijada-Garrido (2010) and
Soleimani et al. (2013)
N,N-diethylaminoethyl –N(CH2CH3)2 Çaykara and Ayçiçek (2005) and
methacrylate (DEAEMA) Thavanesan et al. (2014)
and sulfonic acid groups in AMPS gels. In basic gels, dimethylamino (–N (CH3)2)
and diethylamino (–N (CH2CH3)2) groups give basicity to DMAEMA and DEAEMA
gels, respectively.
Thermoresponsive Gels
Temperature-sensitive hydrogels exhibit a change in volume based on the tempera-

ture of the environment. These gels usually contain hydrophobic or both hydrophilic
and hydrophobic groups on polymer backbone. Figure 7.5 exemplifies thermore-
sponsive gels. Temperature-responsive hydrogels are positive or negative respon-
sive based on upper critical solution temperature (UCST) or a lower critical solution
temperature (LCST), respectively. On the other hand, gels physically cross-linking
can respond to temperature changes by sol-gel phase transitions instead of swelling
and deswelling mechanisms. The transition from sol to gel occurs above at a spe-
cific temperature (LCST) due to the solidification of polymers being hydrophobic at
this condition. The sense action of the positive responsive hydrogels depends on the
UCST. Expansion in volume of hydrogel occurs if the temperature is higher than
UCST. These gels become unswollen by releasing the temperature below (Ullah
et al. 2015). Gels with lower critical solution temperature (LCST) can absorb water
at the temperature below the LCST and release water when the temperature rises
from the LCST (Ullah et al. 2015). Poly(N-isopropylacrylamide) (PNIPAAm) is a
well-known thermoresponsive hydrogel with lower critical solution temperature
(LCST) or transition temperature at ~31–32 °C (Lue et al. 2011; Ulijn 2006). Liu
et al. synthesized the hydrogels of N-isopropylacrylamide (NIPA), sodium acrylate
(SA), and sodium methacrylate (SMA) in aqueous solution with a free radical
polymerization technique. The swelling and deswelling measurements at different
temperatures (beginning from room temperature to maximum 60 °C and reverse for
Fig. 7.5 Examples of thermoresponsive gels. (a) Methylcellulose; (b) chitosan; (c) dextran; (d)
poly(N-isopropylacrylamide); (e) poly(ethylene oxide)-poly(propylene oxide)-poly (ethylene
oxide) (Pluronic®); (f) poly(ethylene glycol)-poly(lactic-co-glycolic acid)-poly(ethylene glycol);
(g) poly(ethylene glycol)poly(propylene fumarate)-poly(ethylene glycol); and (h) general struc-
ture of poly(organophosphazenes)
deswelling experiment) demonstrated that swelling and deswelling of the hydrogels

were reversible; meaning these gels are thermoresponsive (Ulijn 2006).
Affinity Responsive Gels
Affinity responsive gels respond to large biological macromolecules as well as to

small chemical constructs. Smart gels responding to the target biomolecules show
volume changes making the gel system appropriate for varying biomedical applica-
tions. To target the biomolecule, gels are combining with the biomolecular recogni-
tion sites during the synthesis. These gels were developed to be responsive to
glucose, proteins, antigens, and other type of biomolecules. Blood glucose level is
kept at an acceptable level by insulin, a peptide hormone excreted by the β cells of
the pancreatic islets of Langerhans, via rising diffusion of glucose to cells, adjusting
carbohydrate lipid and protein metabolism while besides supporting cell division
and growth thanks to its mitogenic effects (Franklin and Guhanathan 2015). The
hydrogels are sensitive to changes in blood glucose concentrations and release the
necessary amount of insulin to bring glucose level to acceptable concentrations

which are of an interest recently.
There are different approaches to make hydrogels as glucose-responsive material
such as entrapping glucose oxidase (Gox), phenylboronic acid and its derivatives,
and lectins (Wu and Zhou 2010). Entrapping glucose oxidase into the gel system is
one of the most used methodologies. In such a system, glucose is oxidized to glu-
conic acid and catalyzed by GOD, and the pH inside the microenvironment decreases
with the increase in the glucose concentration. This causes an increase in the vol-
ume of the pH-sensitive hydrogel, which results in the release of entrapped insulin.
As another example, Li et al. prepared a pH-sensitive self-assembling peptide
hydrogel, IA-0 as an insulin carrier agent combined with Gox and catalase. The
experimental results show that the peptide hydrogel releases the insulin by disas-
sembling to peptide in vitro regulating the blood glucose levels (Li et al. 2017b).
Lectins are applied to responsive systems thanks to their specific binding abilities to
carbohydrates.
Concanavalin A (Con-A) is one of the lectins used for glucose-responsive gels.
In this system, glycosylated insulin is released to the surrounding medium by com-
peting with the free glucose in the medium. For example, Yin et al. designed a
genipin-cross-linked concanavalin A/GEA-chitosan microgels as a glucose-responsive
insulin delivery system. Experimental results demonstrated that release of bioactive
insulin from the gel structure depends on glucose concentrations and is reversible giv-
ing long-lasting therapy for the patients (Yin et al. 2014). Glucose-sensitive hydrogels
are prepared based on the complex formed between phenylboronic acid and polyols
which have a stronger affinity for phenylboronic acid. Triple (pH, temperature, and
glucose) responsive P (DMAEMA-co-AAPBA) hydrogels are produced by copoly-
merization of (2-dimethylamino) ethyl methacrylate (DMAEMA) and 3-acryl-
amidephenylboronic acid (AAPBA) by Wang and coworkers. Depending on the
glucose concentrations at 37 °C, the hydrogels become swollen with different water
uptake ratios. This result is proof of the suitability of P(DMAEMA-co-AAPBA)
hydrogels for glucose-sensitive carrier systems (Wang et al. 2010).
Chen et al. presented dual-responsive supramolecular nanogels which are pre-
pared by a succinct method for delivery of the drugs into cells in cancer treatment.
Doxorubicin (DOX)-loaded pH and reduction of dual-sensitive nanogels show
greater amount of DOX release under acidic (pH smaller than 6) or greater glutathi-
one (GSH) conditions. Therefore, these pH-sensitive nanogels are promising for the
future cancer treatments (Chen et al. 2014). The enzyme-responsive gels are able to
sense the specific enzyme catalysis and used as biosensor and delivery agents. There
are two types of enzyme-responsive hydrogels. One of them shows sol-to-gel or
gel-to-sol phase transitions and is cross-linked by enzymes. The other one is a
hydrogel with enzyme-sensitive fragments on polymeric backbone (Ulijn 2006).
Gels can also be designed to alter their structure and swelling properties as a
response to specific antigens. The antigens are linked to polymeric chains for selec-
tive biomolecule release applications (Ullah et al. 2015).
Xiong et al. have developed bacteria-responsive multifunctional nanogels in
order to deliver antibiotic to bacterial infection sites. The antibiotic release is
stimulated with bacterial enzymes by degrading the polyphosphoester core of the

nanoparticle and transported to bacterial infection sites. Thus the antibiotic is
released and the bacterial growth inhibition is enhanced. Drug activation in situ and
macrophage-targeting properties make bacteria-responsive nanogels significant for
delivery of antibiotics in the treatment of bacterial infectious diseases (Xiong et al.
2012). Hydrogels sensing the antigen-antibody interactions by swelling and
deswelling mechanisms have been attracted by many biomedical applications like
biosensing and immunotherapies. These systems are designed by physical interac-
tions of antibody or antigen with polymeric backbone, chemical modifications of
the backbone with these species, and reversible cross-linking of hydrogel with
antigen-antibody interactions (Goh et al. 2017). Lu et al. designed an antigen-
sensitive hydrogel based on the antibody Fab’ fragment from monoclonal
anti-fluorescein BDC1 antibody (IgG2a) and copolymer
N-isopropylacrylamide (NIPAAm) in the presence of N,N′-methylenebis
(acrylamide) (MBAAm) as a cross-linking agent. Responsivity of the hydro-
gels to antigen fluorescein (FL) and polyamidoamine dendrimer (PAMAM)-
fluorescein (FD) was determined. The hydrogels with 50% (w/w) Fab’ fragment
showed noticeable swelling or shrinking behaviors at 33.7 and 36.8 °C in acetate
buffer (10 mM, pH 5.0) (Lu et al. 2003).
Miyata et al. modified the antigen rabbit immunoglobulin G (IgG) with
N-succinimidylacrylate (NSA) resulting in the vinyl-rabbit IgG and formed a com-
plex of goat anti-rabbit IgG (GAR IgG). Then, a hydrogel of vinyl-rabbit IgG and
GAR IgG with acrylamide (AAm) was prepared using the cross-linking agent N,N′-
methylenebisacrylamide (MBAA) (Miyata et al. 2009).
Electro-Responsive Gels
Gels with electro-sensitive functional groups demonstrate volume changes in

response to electrical field. There are many examples of electro-responsive gel sys-
tems used in different fields like polyvinyl alcohol/poly (sodium maleate-co-sodium
acrylate), chitosan/carboxymethylcellulose hydrogel, chitosan-g-poly(acrylic acid)
hydrogel elastomers, and polyacrylic acid/fibrin hydrogel (Chen et al. 2016a; Gao
et al. 2008; Shang et al. 2008; Rahimi et al. 2012). Gao et al. synthesized electro-
responsive starch hydrogels by cross-linking with glutaraldehyde. Response of the
gel showed rising with increasing strength of electrical field. Efficient response was
observed around 1.2 kV/mm. It is concluded that electrical field applied and starch
concentration in hydrogel formulation affects the electro-sensitivity of material
(Gao et al. 2015). Electro-responsive hydrogel nanoparticles (ERHNPs) modified
with angiopep-2 (ANG) were developed by Ying et al. to ease the transmission of
the drug phenytoin sodium (PHT) which is used for epilepsy treatment. Since the
hydrogel nanoparticles are electro-responsive, rapid (PHT) dispersion easily arose
from the application of electric field. ANG-PHT-ERHNPs enable fast, long-term,
accurate targeting of the brain and enhance the effectiveness of the (PHT) due to
their long circulating property and availability of angiopep-2 (ANG) itself. Therefore
ANG-ERHNPs were found to be useful systems for epilepsy treatment in the future
(Ying et al. 2014).
Shi et al. designed pH- and electro-sensitive hydrogels based on bacterial cellu-
lose nanofibers (nf-BC) and sodium alginate (SA) gels (nf-BC/SA) to examine the
stimuli-responsive delivery of ibuprofen. When pH increased from 1.5 to 11.8, due
to the protonation of alginate in backbone, swelling ratio raised from less than 8
times to more than 13 times compared to weight of collapsed gel. Also, when 0.5 V
electrical field was applied, hydrogel swells to 14 times of its collapsed state. The
swelling in response to pH and electrical field makes the hydrogel as a novel candi-
date for drug delivery applications (Shi et al. 2014).
Light-Responsive Gels
Hydrogels that carry light-sensitive functional groups on the structure are able to
swell or deswell based on the light, UV or visible, subjected to the material (Bahram
et al. 2016). Kang et al. developed near-infrared light-responsive nanogel system
based on Au-Ag nanorods (Au-Ag NRs) coated with DNA cross-linked polymeric
shells for targeted delivery of drugs. DNA cross-linked polymeric shells are devel-
oped to encapsulate anticancer drugs into the gel scaffold, while Au-Ag NRs are
easily functionalized with targeting moieties for identifying cancer cells. Thus, the
encapsulated drug is released with high controllability after dissolving of the coated
gel shells. The light-responsive gel system was used as remote-controlled targeted
drug delivery agent responding to NIR radiation (ng et al. 2011). Poly(N-
isopropylacrylamide) microgels, poly(N-isopropylacrylamide)-b-poly(4-
acryloylmorpholine)-b-poly(2-((((2-nitrobenzyl)oxy)carbonyl) amino)ethyl
methacrylate) (PNIPAM-b-PNAM-b-PNBOC) hydrogels, semi-IPN hydrogels
based on β -cyclodextrin-grafted alginate (β-CD-Alg), and diazobenzene-modified
poly(ethylene glycol) (Az2-PEG) gel systems are other examples of photorespon-
sive gels (Chiang and Chu 2015; Wang et al. 2017c; Zhang et al. 2016a). Hang et al.
created degradable NIR and UV-responsive nanogels from hyaluronic acid-g-7-N,
N-diethylamino-4-hydroxymethylcoumarin (HA-CM) due to their tendency to tar-
get CD44+ tumor cells and to be able to control the transmission of doxorubicin
(DOX) into these cells. The DOX was easily loaded in nanosized and light-
responsive particles (HA-CM), and then activated drug is released into tumor
cells via NIR and UV irradiation. The light-responsive nanogels provide signifi-
cant improvements in cancer chemotherapy (Hang et al. 2017).
7.3.4 Mechanical Behaviors
The mechanical property of gels is one of the most vital points when these gels are
applied for a specific goal. Elastic recovery and the time-dependent recovery of
hydrogels are crucial and based on viscous behaviors (Koetting et al. 2015). The
rubber can be given as an example for a polymer backbone. Rubbery behavior of

gels indicates high ability to deformation and complete recovery. They are subjected
to large deformations and can go back to original states without fractures. The
deformation due to an external force and the elasticity of polymer are highly depen-
dent to chemical composition and structure of the polymer network. The elastic
modulus of the material gives substantial information about the polymer network
and stiffness. The elastic behavior of polymer is affected by several factors like
network structure, monomer composition, cross-linking degree, swelling, ionic
groups on backbone, and entanglement (Erman and Flory 1978; Kang et al. 2017;
Scherer 1999; Yadollahi et al. 2015). Modified reagents at different ratios in the gel
compounds lead to dramatic changes of mechanical properties of resulting gels.
Coutinho et al. modified gellan gum (GG) with methacrylate groups in order to
increase mechanical properties at physiological conditions used in tissue engineer-
ing. Thus GG hydrogels can be crosslinkable by both physical and chemical mecha-
nisms when methacrylate groups were integrated into the GG chain. Young’s
modulus values of hydrogels with different cross-linking ratios were observed to
between 0.15 and 148 kPa (Coutinho et al. 2010). Hachet et al. modified hyaluronic
acid which is a natural polysaccharide abundant in biological tissues with methac-
rylate in order to allow real-time monitoring of gelation during photopolymeriza-
tion. The effect on the gel properties was adjusted by the complete conversion of the
methacrylate groups, and the conversion of all methacrylate groups was taken
advantage of biomimetic hydrogel substrates for two-dimensional cell culture
(Hachet et al. 2012).
You et al. synthesized quaternized chitosan (QCh) and polyelectrolyte complex
(PEC) hydrogels. The mechanical properties of QCH hydrogel revealed its self-
recovery behaviour depending on the charge density. It was reported that the tensile
fracture nominal stress (σb) and work of extension at fracture (Wb) were found to
be as 16.1 MPa and 15.6 MJ/m3, respectively. The results indicate that considerable
potential arise for application in load-bearing artificial soft tissues (You et al. 2016).
Chang et al. synthesized novel superabsorbent hydrogels in aqueous media using
carboxymethylcellulose sodium (CMC), cellulose, and epichlorohydrin (ECH) as a
cross-linker. The results demonstrate that cellulose provides a strong backbone in
the hydrogel, while CMC improves the size of pore. The hydrogels are good candi-
dates for biomaterials because the hydrogel maintains a stable appearance even at a
water swelling rate of 1000 (Chang et al. 2010).
Yuan et al. synthesized hydrogels by using aldehyde methacrylate sodium algi-
nate and amino gelatin (AMSA/AG) in order to obtain injectable photo-cross-linked
(365 nm) hydrogels. The cross-linked double network hydrogels show high swell-
ing ratio, controllable degradation rate and improved mechanical properties as well
as biocompatibility. Hydrogels are good candidates in regenerative medicine as
therapeutic materials in tissue engineering and biomedical applications because
hydrogels contribute to the appropriate environment for cell growth both on the
surface and the inner (Yuan et al. 2017). De France et al. create in situ gelling nano-
composite hydrogels based on hydrazone cross-linked poly(oligo-ethylene glycol
methacrylate) (POEGMA) and rigid rod-like cellulose nanocrystals (CNCs) in
order to improve injectable hydrogel’s mechanical properties. Within the addition of

CNCs, the storage moduli are increased (up to 35-fold increases in storage modu-
lus), and it is suitable for high-strength biodegradable tissue engineering scaffolds
(De France et al. 2016). Also, chain entanglement is using design to create highly
tunable physical and mechanical properties such as protein hydrogels based on
coiled-coil interactions. Tang et al. creates coupling with the cysteine residues near
the N- and C-termini in order to obtain chain entanglement that has reversible prop-
erties thanks to disulfide bonds. The results suggest that hydrogels with a toughness
of 65,000 J m−3 and 3000% extensibility are achieved to mimick the tendon and
cartilage (Tang et al. 2014).
7.4 Applications of Gels
The specific and excellent responsivity of gels to the different stimuli, swelling
properties, and porous structure make the gels very attractive in many fields such as
biomedicine, pharmaceutical industry, biotechnology, environmental applications,
agriculture, and biosensors (Ullah et al. 2015).
7.4.1 Therapeutic Agent Carrier
Over the years, therapeutic agent delivery with gel-based cargoes is becoming more
effective for treatment of diseases and especially outpatient treatments. These thera-
peutic agents can be drugs (Jin et al. 2016; Kulkarni et al. 2017), proteins (Morimoto
et al. 2013), and genetic materials (Nishimura et al. 2017). In these therapies, the
biocompatibility, biodegradability, and responsive and swelling behaviors of the
gels are crucial to obtain a convenient cure. The material properties of the carrier
agent and the interactions between the gel and the therapeutic agent have to be well
understood before applying to drug formulations (Gong et al. 2013; Hu et al. 2009;
Peppas et al. 2006). Furthermore, nanogels are frequently applied in therapeutic
agent carrying systems thanks to their efficient protection of biomolecules from
disruption by rising the half-life cycle in the body (Soni et al. 2016). These
nanoscale-sized gels provide a time-dependent and site-specific release of therapeu-
tic cargoes thanks to being favorable in terms of solubility of hydrophobic drugs,
tumor targeting, and robustness of bioactive agents and lowered cytotoxicity (Zhang
et al. 2016c). Nanogels can be incorporated with different therapeutics like drug,
protein, peptides, and genetic materials (Dhal et al. 2017; Hang et al. 2017; Jin et al.
2016; Lee et al. 2007; Nishimura et al. 2017; Quan et al. 2015; Wu et al. 2012).
Drug Delivery
As mentioned previously, responsive and swelling behaviors of gels make them

suitable cargoes for drug delivery to specific regions in the body. The drug release
mechanism of the gels can vary with the site of action, drug loaded, and the contact
of drug-loaded gel with stimuli. Since the gels are responsive to different environ-
mental stimuli such as pH, temperature, glucose, and electric field, it is possible to
separate drug delivery from gels in terms of responsiveness to specific stimuli.
There are lots of examples to therapeutic agent carrier systems. Some of the recent
works in this field are summarized in Table 7.2. The table represents examples from
the literature in which gels are used as drug carrier materials. These examples cover
the synthetic and hybrid gels as carrier agents due to the weakness of natural gels in
terms of strength and high degradation ratios.
Peng et al. prepared pH-thermal dual-responsive nanogels for delivery of cisplatin
(CDDP) drug in breast cancer treatment. CDDP loaded into nanogels is formed by
conjugating carboxyl groups with the CDDP, and this bond can be broken by H+
Table 7.2 Examples of gel-based drug delivery systems

Polymer Stimuli Drug loaded Reference
Gelatin/PLGA Temperature Indomethacin (IDM) Chung et al.
(2016)
Chitosan/β-glycerophosphate Temperature Paclitaxel Ruel-Gariépy
(C/(GP)) et al. (2004)
Poly(acrylic acid-g-ethylene pH Theophylline Serra et al.
glycol) (2006)
Alginate/CS/β-GP (hydrogel/ pH Emodin (EMO) Cong et al.
micelle) (2017)
Gelatin nanofibers pH Piperine Laha et al.
(2016)
NIPA-APMA-AAc pH, ionic strength, Ibuprofen, Lago et al.
temperature, drug propranolol (2011)
concentration
Salicylic acid-/acrylic acid- pH Salicylic acid (SA) Demirdirek and
based hydrogels Uhrich (2017)
Gelatin/CNTs hybrid microgels Electric field Diclofenac sodium Spizzirri et al.
salt (2013)
Gelatin-g-PNIPAAm (GN) Temperature Antiglaucoma Luo and Lai
(2017)
N-(2-hydroxyl) propyl-3- Glucose BSA Zou et al.
trimethyl ammonium chitosan (2015)
chloride (HTCC) hydrogels
Furfuryl-gelatin (GF)/ Temperature Chloramphenicol García-Astrain
bismaleimide hydrogels antibiotic (ClPh) et al. (2016)
Hyaluronic acid-Pluronic F-127 Temperature Piroxicam (PX) Jung et al.
(HP) hydrogel (2017b)
Sericin/dextran hydrogel pH Doxorubicin Liu et al.
(2016)
and Cl−. It is shown that drug release is faster at more acidic conditions. Besides,
Cl−-triggered release was reduced by the addition of NIPAM into the nanogel con-
structs at body temperature. It is also found that CDDP-loaded nanogels cause
fewer side effects in the body, compared to free CDDP. In conclusion, pH-thermal
dual-responsive nanogel units are appropriate for CDDP delivery in breast cancer
treatment (Peng et al. 2013). Chung and coworkers synthesized biodegradable
gelatin/poly(lactic-co-glycolic acid), (Gtn/PLGA), and hydrogel films by cross-
linking them with 1, 6-hexamethylene diisocyanate (HMDI) to obtain stronger gels
for transport and delivery of the indomethacin (IDM). IDM was mixed with Gtn/
PLGA-NH2 in DMSO solutions to charge it into the gel system. Compressional
stress measurements showed that the Gtn/PLGA gel (15.5 ± 2.2 KPa) was mechan-
ically stronger than Gtn gel (8.9 ± 0.7 Kpa). The MTS assay was applied to deter-
mine the cytotoxicity of the films by incubating L929 on a tissue culture wells. The
gels were not cytotoxic based on the absorbance values when looking at ISO stan-
dards. The gels were encapsulated successfully with IDM with 84.8% efficiency.
When collagenase was added to the solution at 54 h, the release of IDM was seen
up to 96 h for the Gtn/PLGA and Gtn/PLGA-NH2 films (Chung et al. 2016). The
lower pH in tissues with inflammations, infections, and cankered cells from healthy
tissues makes the pH-responsive nanogels valuable materials for drug release sys-
tems. The pH of the extracellular cancerous cells is around 6.5, while normal tissue
and blood have pH ~ 7.4 due to presence of exceeding lactic acid in tumor environ-
ments (Wu and Wang 2016). Gel-based drug delivery systems are getting more
attention in cancer treatments thanks to their superior properties over conventional
chemotherapies. Chitosan/β-glycerophosphate (C/(GP)) thermosensitive hydrogel
was synthesized to cure cancers by delivering paclitaxel, with high remedial man-
ner for several cancer types, which was synthesized by Ruel-Gariépy and cowork-
ers. The solution of monomers gets gelation under physiological temperature at
37 °C, which is an in situ gelling system. Drug was loaded into the chitosan solu-
tion before mixing with GP and gelling. After gelation, in vitro drug release pro-
files were observed in PBS/SDS 0.3% at 37 °C. After 17 days of incubation, the
hydrogel loaded with 64 mg/mL paclitaxel delivered 32% of the drug to the EMT6
tumors on BALB/c mice and restricted the tumor development (Ruel-Gariépy
et al. 2004).
Hydrophilic gemcitabine (GCT) and hydrophobic doxorubicin (DOX) drugs
were loaded into poly(N-isopropylacrylamide)-b-poly(4-acryloylmorpholine)-b-
poly(2-((((2-nitrobenzyl) oxy)carbonyl)amino)ethyl methacrylate) (PNIPAM-b-
PNAM-b-PNBOC) copolymer hydrogels which are sensitive to UV irradiation and
temperature. Figure 7.6 shows temperature and UV-responsive behaviors of the
hydrogels. When the temperature is below the lower critical solution temperature
(LCST), the hydrogels self-assembled to a micelle structure in which photorespon-
sive PNBOC part in the core, hydrophilic PNAM moiety in the middle, and thermo-
sensitive PNIPAM at the corona. Delivery of GCT and DOX drugs was also achieved
by this responsive manner. Entrapment of the drugs into the micellar cores and extra
micellar aqueous phase were achieved at 37 °C at which copolymer formed a physi-
cal hydrogel. The delivery of two drugs was encouraged via UV irradiation because
Fig. 7.6 The representation of dual responsivity (UV and temperature) PNIPAM-b-PNAM-b-
PNBOC triblock copolymers. (Reprinted by copyright permission from Wang et al. (2017c))
of the transition from gel-to-sol and from hydrophobic-to-hydrophilic transition

within micellar cores. After 10 min. UV irradiation, for 8 h incubation of GCT, 90%
of the drug was released, and DOX after 70 h incubation was released nearly 80%.
Release ratios of the drugs were also examined with temperature. After UV irradia-
tion, at physiological temperature (37 °C), release rates of DOX and GCT were
about 20% and 90%, respectively. Above the physiological temperatures at 45 °C,
these ratios were seen as much lower for both drugs (Wang et al. 2017c). Hyaluronic
acid (HA)-based nanogels, HA-(7-N,N-diethylamino-4-hydroxymethylcoumarin)
(HA-CM), were developed by Hang et al. to deliver DOX in response to NIR and
UV irradiations thanks to cleavage of light-triggered urethane bonds between CM
and HA. The CD44-positive MCF-7 cells were targeted by the gel to release the
drug. 74.9% of DOX loaded into HA-CM4 nanogels was delivered with UV irradia-
tion for 5 min after 48 h incubation. The antitumor efficiency of the nanogel system
was observed where 60 min NIR irradiation caused an inhibition of MCF-7 cell
proliferation at 48 h incubation (Hang et al. 2017).
A glucose-sensitive (p (APBA-b-LAMA)) hydrogel was produced with 3-acryl-
amidophenyl boronic acid and 2-lactobionamidoethyl methacrylate as insulin
release system. The hydrogel containing 15.6% drug exhibited 50% release capac-
ity by 4 h incubation at 37 °C in PBS. The hydrogel with lowest LAMA content
showed highest insulin release upto 84% due to lowest boronic ester content. Also,
mouse fibroblast NIH3T3 cells were incubated for 48 h in the hydrogel solutions.
Exceeding 90% of cell proliferation can prove the non-toxicity of the hydrogels. All
these results show the potential of the (p (APBA-b-LAMA)) hydrogel as a glucose-
sensitive insulin carrier system for the treatment of diabetes (Cai et al. 2017). As a
novel drug carrier system, a pH-responsive hydrogel/micelle complex with sodium
alginate hydrogel and chitosan and glycerol 2-phosphate disodium micelle and algi-
nate/CS/β-GP were designed to oral delivery of emodin (EMO) as a model hydro-
phobic drug. In the system, the drug was encapsulated into the CS/β-GP micelle.
The sodium alginate hydrogel was used as carrier of the drug-encapsulated micelle.
The hydrogel/micelle complex showed the pH-specific swelling and thus EMO
delivery to the medium. Carboxylic groups in the alginate hydrogel backbone show
higher swelling degree in basic medium such as SCF. So, the hydrogel/micelle (3:1
and 4:1) systems had six and eight times higher swelling capacity in SCF (pH 7.4)
than in SGF (pH 1.2). The drug release profiles of hydrogel/micelle complex vary-
ing ratio of hydrogel to micelle to the SGF (pH 1.2), SIF (pH 6.8), and SCF (pH 7.4)
environments were quite different from each other. It is possible to say that hydro-
gel/micelle (3:1) can be suitable for a controlled release system in the colon with
about 10, 35, and 90% release amounts after 24 h in SGF, SIF, and SCF, respec-
tively. However, hydrogel/micelle (1:1) system with 28% released amount of EMO
in SGF provides sustained release of EMO in oral intake of drugs. The specific
delivery of EMO in alginate/CS/β-GP carrier system in response to pH in physio-
logical environment demonstrates its suitability for drug release applications with
varying release profiles (Cong et al. 2017).
Khan et al. synthesized genipin-cross-linked gelatin nanoparticles to control the
release of cytarabine drug, which is commonly used to treat acute myeloid leukemia
(AML). The gelatin nanoparticles have size 50–150 nm but 80 nm in general. The
drug was loaded with different ratios from 48.5% to 88.4%. into the nanoparticle
gels. Increasing the gelatin content lowers the delivered amount of drug since it
limits the water diffusion in gel structure. For example, the released amount of cyta-
rabine is between 8 and 8.5 mg/ml in 1 g of gelatin but nearly 3.5 mg/ml in 7 g of
gelatin. Drug release profile of nanoparticles was observed to change with cross-
linking ratio. Higher cross-linking ratio increased the drug release since genipin
cross-linking gives the structure hydrophilicity; thus higher swelling with water
molecules raised the diffusion of drug from nanoparticles. As another parameter, pH
changes in different regions in the body were also examined. Drug release amounts
were 12.4, 8.3, and 9.7 mg/ml at pH 1.8, 7.4, and 8.5, respectively. At pH 1.8 and
8.5, both cytarabine and gelatin have a net charge, and repulsions give rise to release
of the drug. Lower release at pH 7.4 was because of the isoelectric point of gelatin.
Consequently, genipin-crosslinked gelatin nanoparticles are a good candidate for
pH-responsive release of cytarabine to medicate acute myeloid leukemia (AML)
(Khan et al. 2016).
Protein Delivery
In very recent therapies of diseases, proteins, hormones, and peptides are used as
novel drug materials. Gels loaded with these therapeutics are common carrying
agents with their excellent biocompatibility and controlled manner release profiles
in response to different stimuli. Insulin hormone is used as a drug in the treatment
of diabetes. For example, alginate/κ-carrageenan composite hydrogel beads were
synthesized as a pH-responsive insulin aspart carrier agent. The gel was obtained by
gelation of alginate and κ-carrageenan in the presence of insulin and Ca2+ as gelling
agent. The hydrogel beads with 1% w/v of κ-carrageenan showed swelling degree
8%, − 43%, and around 120% in water, SGF (pH 1.2) and SIF (pH 7.4), respec-
tively, after 6 h incubation. – 43% swelling means that gel beads collapsed due to
hydrogen bonding. Looking at the release of insulin aspart in different pHs, 80% of
it was released from 1% w/v of κ-carrageenan beads at SIF, and almost all insulin
aspart remained in the bead due to the shrinking of gel beads after 4 h incubation.
Also, with composite hydrogel beads incubated in acidic gastric medium, biologi-
cally active insulin aspart was observed as 65% of it. All these may prove the pH-
responsive and controlled delivery of insulin aspart from alginate/κ-carrageenan
composite hydrogel beads (Lim et al. 2017b).
Liu et al. designed a triblock copolymer, poly (d, l-lactic acid-co-glycolic acid)-
b-poly(ethylene glycol)-b-poly (d, l-lactic acid-co-glycolic acid) (PLGA-PEG-
PLGA) in situ gelling hydrogels off-loaded with salmon calcitonin and oxidized
calcium alginate (sCT-OCA) as thermoresponsive cure of antiosteopenia. To see the
efficiency in vivo, the hydrogel system was applied to Sprague-Dawley (SD) female
rats with osteopenia model (the initial stage of osteoporosis). Figure 7.7 represents
the sol-gel transition of the hydrogel system and in vivo release of drug after imple-
mentation. Twenty days after the injection of hydrogel solution to the rat, only a
small number of inflammatory cells were detected showing the biocompatibility of
the system. The gels with different polymeric compositions demonstrated varying
release profiles. Some of them release sCT within 30 days in a controlled way. Both
biocompatibility assays and in vivo release experiments indicated the high effi-
ciency of (PLGA-PEG-PLGA) hydrogels in the cure of osteopenia via sustained
delivery of sCT-OCA complex (Liu et al. 2017). Furthermore, chitosan-polyphosphoric
acid (PPA) beads were loaded with four different proteins which are bovine serum albumin
(BSA), whey protein isolate (WPI), insulin, and a casein hydrolysate to see release profiles in
simulated gastric fluid (SGF, pH 3) and simulated intestinal fluid (SIF, pH 7). The delivery of
1% BSA, insulin, WPI, and casein hydrolysate was observed after 8 h as 6.5%, 15%, and
35% and no release in SIF, respectively. The release patterns were quite different in
SGF. Nearly 35% release of casein hydrolysate was observed whereas there was no release
of BSA, insulin and WPI. The results explain the pH-responsive and controlled release of
different proteins in the body (Yuan et al. 2018).
In a recent study, a mixture of bovine aprotinin, myoglobin from bovine skeletal
muscle, human lactoferrin, serum albumin, and bovine thyroglobulin were entrapped
into poly(ethylene glycol) (PEG)-based hydrogel depots synthesized with norborn-
ene (Nb)-modified PEG, the monomer, and different dithiol (SH) cross-linking
Fig. 7.7 In vivo release of (sCT-OCA) from (PLGA-PEG-PLGA) hydrogels. Poly (D,L-lactic
acid-co-glycolic acid)-poly(ethylene glycol)-poly(D,L-lactic acid-co-glycolic acid), PLGA-PEG-
PLGA; sCT,salmon calcitonin; sCTOCA, complex of salmon calcitonin and oxidized calcium
alginate; MPA, methylprednisolone acetate. Reprinted by copyright permission from (Liu
et al. 2017)
agents. 10% w/w PEG-5 k-Nb, the PEG monomer modified with Nb, was cross-
linked with PEG-1.5 k-SH to examine the release of the proteins. Aprotinin and
myoglobin were delivered over than 90% after 8 h incubation at PBS. 90% of lacto-
ferrin and BSA proteins were released after 50 h incubation. A significant amount
of thyroglobulin with greatest molecular weight and diameter were not detected in
SDS-PAGE after 170 h. It can be seen that the diameter and the molecular weight of
the proteins may affect the release ratio from the hydrogel system (Rehmann
et al. 2017).
Delivery of Genetic Materials
Toward novel treatment and diagnosis techniques, oligonucleotides (ONs) (P. Zhang

et al. 2014), small interfering RNAs (siRNAs) (Chi et al. 2017), microRNAs (miR-
NAs) (Lozada-Delgado et al. 2017), and plasmid DNA (Maksimova et al. 2012) are
becoming more attractive tools. Applying these genetic materials as therapeutic
agent has some hindrances like especially in delivery to targeted cells and immune
system’s response (Castanotto and Rossi 2009). To overcome these challenges,
delivery systems with gel-based materials are frequently used.
Wang et.al designed PEG-b-poly(lactide)-b-dimethacrylate (PEGb-PLA-b-DM)
hydrogel systems combined with nanoparticles as represented in Fig. 7.8.
Nanoparticles formed with a cationic (blue) dimethylaminoethyl methacrylate
(DMAEMA) block (m~139) and endosomolytic (black) blocks of DMAEMA, pro-
pylacrylic acid (PAA), and butyl methacrylate (BMA) (n~91). siRNA with negative
charge and NPs with positive charge can form a complex electrostatically. siRNA/
NP complexes targeted to WW domain-containing E3 ubiquitin protein ligase 1
(Wwp1) to deliver siRNA and silencing of Wwp1 were observed. Fifteen days after
implementation to mid-diaphyseal femur fractures, reduction of Wwp1 gene expres-
sion was reached to 23% which is vital for the silencing of gene (Wang et al. 2017a).
Huynh and coworkers synthesized an environmental friendly, catalyst-free, and
photolytically degradable hydrogel system by cross-linking poly(ethylene glycol)-
diphotolabile-acrylate (PEG-DPA) with eight-arm PEG thiol (PEG(-SH)8) with UV
photopolymerization. The negatively charged siGFP, siRNA silencing GFP expres-
sion, was conjugated with positively charged PEI to accelerate cellular uptake.
Release of siRNA in HeLa cells from system was observed after UV radiation to the
hydrogel. Delivery of all siRNA with and without UV cure occurred in 7 and 8 days,
respectively. Percentage of GFP expression in HeLa cells was nearly 75% and 40%
a d
m n
O OO O O O O
HO
N N
DMAEMA DMAEMA PAA BMA
c O
O
O
O
O O
b a b
O O
PEGa - b-PLAb -b-DM
Fig. 7.8 siRNA delivery from PDMAEMA-b-p (DMAEMA-PAA-BMA) nanoparticle system.

(a) Diblock copolymers of cationic DMAEMA block (blue), endomolytic block of DMAEMA
(black), propylacrylic acid (PAA), and butyl methacrylate (BMA). (b) Positively charged NPs and
negatively charged siRNA molecules formed complexes via electrostatic interactions. (c) Chemical
structure of poly(ethylene glycol)-b-poly (lactide)-b-dimethacrylate (PEGa-b-PLAb-b-DM, a = 91,
b = 2 for 4 kDa PEGa-b-PLAb-b-DM macromers). (d) siRNA/NP complexes were incorporated
into degradable PEG-based hydrogels with controlled release characteristics. Reprinted by copy-
right permission from (Wang et al. 2017a)
Fig. 7.9 Delivery of RNA from hydrogels with and without photolytically degradation. Reprinted
by copyright permission from (Huynh et al. 2017)
for no UV-applied and UV-cured hydrogels after 6 day. At the end of the experi-
ment, percentage of GFP expression decreased to almost 40% and 60% in the treat-
ment of hydrogels without UV and with UV. These results showed that the delivery
of siRNA took place by degradation of biocompatible hydrogel system via hydro-
lytical degradation in aqueous media and also UV radiation as can be seen in Fig. 7.9
(Huynh et al. 2017).
Moreover, supramolecular N-protected phenylalanine (Fmoc-Phe-OH) hydro-
gels were used to carry cationic nioplexes bearing fluorescently labeled oligodeoxy-
nucleotide (FITC-ODN) as a model drug. A natural gelator κ-carrageenan was
selected to physically cross-link Fmoc-Phe-OH. The 100% release of model drug
from hydrogel with 1% κ-carrageenan was observed after 300 min in 37 °C which
was the fastest release among other hydrogel formulations. The preparation of
FITC-ODN-containing hydrogel system with κ-carrageenan cross-linking is illus-
trated in Fig. 7.10. The hydrogels without cross-linking released nearly all FITC-
ODN in 1.5 h, whereas the hydrogel with κ-carrageenan cross-linking showed
sustained-release profile and delivered it in 5 h totally. Biocompatibility and high
release efficiency of the hydrogel system make it a proper material for carrier vehi-
cles of oligonucleotides (Grijalvo et al. 2016).
7.4.2 Diagnostic and Imaging
Simplicity of formation, biocompatibility, and high stability of hybrid micro-

nanogels make them useful for chemical and biochemical sensing and disease
diagnoses. The monitoring of the biochemistry and biophysics of live cells over
time and space are facilitated by hybrid micro-nanogels’ intracellular probing
ability; therefore they make contribution to the explanation of intricate biological
processes and the progress of novel diagnoses (Wu and Zhou 2010). Hui Xu et al.
developed an assay for multiplex detection of Down’s syndrome which is based on
inverse opal structure hydrogel barcodes with poly(ethylene glycol) diacrylate
a Glycerol-based b
OSO3-
structure O OH
O
OH O O O
O O
H 2N O Hydrophobic HN
Fmoc OH OSO3-
O alkyl chains n
Fmoc-Phe-OH k-Carrageenan
Polar Lipid-1
head group
Model drug (FITC-ODN)
HOw(H4C2O) (OC2H4)xOH O O OH
(OC2H4)yOH
O HOOC
w+x+y+z = 20 (OC2H4)zOR
O NH
R=
CH2(CH2)5CH2CH2(CH2)6CH3 O Oligodeoxynucleotide
( 5'-CGGAGGTACATTCGACTTGA-3')
Polysorbate-80 (Tween-80)
Heating
Sonication
Nioplexes
z>0
Lipid bilayer
Fig. 7.10 Preparation of supramolecular hydrogel system carrying FITC-ODN-bearing cationic

nioplexes. (a) Preparation of cationic niosomes containing FITC-ODN. (b) Illustration of the
encapsulation and in vitro release studies of niosomal FITC-ODN from Fmoc-Phe-OH-based
hydrogels (hydrogel-1) and Fmoc-Phe-OH hydrogels physically cross-linked with κ-carrageenan
(hydrogels-(2–4)). Reprinted by copyright permission from (Grijalvo et al. 2016)
(PEG-DA), poly(ethylene glycol) (PEG), and acrylic acid (AA) hybrid components.
The stabilities of the inverse opal structure are provided by the polymerized
(PEG-DA) hydrogel, the interconnected pores of the inverse opal structure induced
by (PEG) supply channels for biomolecules to diffuse into the voids of whole bar-
codes, and the probe immobilization is provided by (AA) (Xu et al. 2018). Chitosan-
carbon dot (CD) non-toxic hybrid nanogels (CCHN) can be incorporated by
pH-sensitive chitosan and fluorescent CDs into a single nanostructure for near-
infrared imaging (NIR) and NIR/pH dual-responsive drug release to develop thera-
peutic efficacy. The schematic of DOX-loaded gels and injection to the mice can be
seen in Fig. 7.11 (Wang et al. 2017b).
Weitai Wu et al. developed a class of core-shell structured hybrid nanogels to
show the integration of functional building blocks into a single nanoparticle system
in order to make optical temperature-sensing, cancer cell targeting, fluorescence
imaging, and combined chemo-photothermal therapeutic effect simultaneously.
The representative structure of core-shell hybrid nanogels is shown in Fig. 7.12
(Wu et al. 2010).
The storage time of the solvent molecules of the hydrogels can be easily adjusted.
Chan et al. has reported Gd-chelating pullulan nanogels (Gd-CHPOA) showing
stronger contrast as well as longer retention time providing high signal-to-noise
ratio for tumor monitoring (Chan et al. 2015). Temple A. Douglas et al. presented
utilization of hydrogel microparticles which are N-isopropylacrylamide (NIPAm)
and acrylic acid (AAc) microparticles and amine-containing dyes to detect Lyme
disease in urine test by discriminating and concentrating bacterial proteins. Lyme
disease proteins are discriminated and concentrated in urine with the existence of
Fig. 7.11 Incorporation of DOX-loaded chitosan-CD nanogels into the mice for NIR imaging and
release of doxorubicin. (Reprinted by copyright permission from Wang et al. (2017b))
Photothermal
Responsive theraphy
hydrogel
Chemotheraphy
Drug
Optical sensing
Ag-Au
bimetallic Cellular imaging
Nanogel
NP
(D < 100 nm)
Hyaluronic Targeting
acid
Fig. 7.12 Schematic illustration of multifunctional core-shell hybrid nanogels. The Ag-Au bime-
tallic NP (10 _ 3 nm) core is NIR resonant and highly fluorescent. The thermoresponsive nonlinear
PEG-based gel shell cannot only manipulate the fluorescence intensity of Ag-Au NP core but also
trigger the release of drug molecules encapsulated in the gel shell under the local temperature
increase of targeted pathological zones or the heat generated upon NIR irradiation. HA, a known
targeting ligand, can be readily semi-interpenetrated into the surface networks of gel shell at a light
penetration depth. Reprinted by copyright permission from (Wu et al. 2010)
hydrogel microparticles 100 times more compared to lack of them. This test can be
extended not only for detection of Lyme disease but also for any infectious diseases
(Douglas et al. 2011). Hah et al. synthesized methylene blue-conjugated hydrogel
nanoparticles followed by monomer combination with methylene blue dye.
Hydrogel nanoparticles were combined with the F3 peptide specifically which

bound to tumor cells specifically and used in the photodynamic therapy (PDT)
method (Hah et al. 2011).
7.4.3 Biosensors
Physical properties of hydrogel make it very responsive to certain stimuli such as

pH, temperature, and light (Jung et al. 2017a). That stimuli-responsive characteris-
tic of hydrogel is proper for biosensing mechanism in the field of biotechnology,
drug delivery, and tissue engineering (Deligkaris et al. 2010; Drury and Mooney
2003; Peppas and Van Blarcom 2016). Biological modified hydrogels are good can-
didates for biosensing application due to its thermodynamically favorable interac-
tions (Sorokin et al. 2006). Srinivas et al. synthesize microgel particles for protein
detection. They prepare a monomer solution by mixing poly(ethylene glycol) diac-
rylate, poly(ethylene glycol), Darocur 1173, and 3X Tris-EDTA buffer with differ-
ent ratios. The microgel particles are then functionalized with specifically modified
DNA aptamers. They claim that the assay is highly sensitive to human α-thrombin,
which is important for diagnosis of cardiovascular disorders, with a limit of detec-
tion of 4 pM (Srinivas et al. 2011). In another aptamer-modified hydrogel research,
Zhu et al. demonstrate a colorimetric aptamer cross-linked hydrogel platform for
cocaine detection as seen in Fig. 7.13 schematically. The platform is designed with
DNA-grafted polyacrylamide and cross-linked with linker aptamer; they further
encapsulate gold nanoparticles (AuNPs) inside the hydrogel. The basic working
principle of the assay is based on sol-gel transition. When the target is introduced to
the system, it binds to the aptamer, and the gel is dissolved, and encapsulated AuNPs
are released (Zhu et al. 2010).
Crulhas et al. fabricated a biosensor platform to detect reactive oxygen species
released from prostate cancer cells. The solution of poly(ethylene glycol) (PEG),
superoxide dismutase (SOD), and ferrocene (Fc) is polymerized onto gold elec-
trode-patterned glass side. In this study, hydrogel matrix is used to entrap superox-
ide dismutase which is the enzyme dismutating O2− to O2 and H2O2, and the
electrocatalytic response of the sensor is measured by electrochemical impedance
spectroscopy and cyclic voltammetry (Crulhas et al. 2017). Ho Roh et al. synthesize
tetragonal microhydrogels, composed of code and probe region, by UV polymeriza-
tion of PEG700 and PEG 200. These code regions of microhydrogels are encoded
by quantum dots with different wavelengths 615 nm for red, 535 nm for green, and
460 nm for blue. The probe region of microhydrogel is modified with ssDNA oligo-
mers as a miRNA detection part. They declare that the microhydrogel biosensor is
a promising sensing platform for detection of miRNAs of Alzheimer’s disease
within the sensitivity of attomole (Roh et al. 2016). A label-free DNA biosensor is
fabricated by Jeong et al. using gold nanowires (AuNW). The working principle of
the biosensor is based on attachment of DNA strand onto AuNWs and then gelation
of DNA-AuNW system by rolling circle amplification in the presence of target
Fig. 7.13 DNA cross-linked hydrogel for signal amplification and visual detection. Reprinted by
copyright permission from (Zhu et al. 2010)
DNA. When the target DNA exists, it forms the circular DNA template and then
makes the template and elongates with rolling circle amplification by polymerase
enzyme; it finally yields an effective gelation of DNA strand-AuNW. Jeong et al.
suggest that the proposed biosensor is developed for detection of single-stranded
pathogen DNA (Jeong et al. 2015). A hepatitis B core antigen (HBcAg) biosensor
based on poly(acrylic acid) (PAAc) is fabricated by Lim et al. Firstly, anti-HBcAg
is immobilized to poly(acrylic acid) hydrogel. The sensing of HBcAg occurs when
it binds to anti-HBcAg and then induces the swelling of PAAc hydrogel. Lim et al.
believe that the biosensor can be evolved to a portable tool to diagnose hepatitis B
virus from human serum in a cost-effective and rapid way (Lim et al. 2017a).
7.5 Challenges in the Applications
Despite of the promising virtues of hydrogels, there are still significant challenges
according to the applications. In biomedical field, low mechanical strength of
hydrogels can appear as a challenge. The combination of nanoparticle and hydrogel
is one of the solutions to overcome the challenges in biomedical field. As an exam-
ple of nanoparticle-hydrogel system (Zhao et al. 2015), Liu et al. synthesize dopa-
mine-modified poly(ethylene glycol) as tissue-adhesive hydrogel (Liu et al. 2014).
Dopamine modification provides strong mechanical property to PEG hydrogel by
binding to Laponite and improves adhesive properties. Another significant draw-
back of hydrogel is hydrophobic drug delivery. As it is well-known that hydrogels
are quite hydrophilic complexes, thus it does not tend to interact with hydrophobic
drugs such as cancer drugs (Sharpe et al. 2014). Hybrid hydrogel networks are
developed to handle the problem of hydrophobic drug delivery (Caldorera-Moore
et al. 2015). Schoener et al. synthesize an amphiphilic interpenetrating networks
(IPN) for delivery of hydrophobic drugs (Schoener et al. 2011). The IPN consists of
two polymeric networks, methacrylic acid grafted with poly(ethylene glycol), P
(MMA-g-EG) as hydrophilic part and poly (n-butyl acrylate) (PBA) as hydrophobic
part. That hydrogel network is suitable for both loading and releasing of hydropho-
bic and small therapeutic agents.
7.6 Future Perspectives
The gels are considered the most studied soft matter that has been dramatically
evolved to provide broad functionality, stability, and responsiveness as well as low
toxicity. The use of gels in nanopharmaceutical applications limits the synthetic
versatility of gel making materials due to their toxic effects. However the limita-
tions and challenges provide unique opportunities for developing alternative syn-
thesis and processing strategies that revolutionize common understandings. The
future needs in nanopharmaceutical applications require a combination of biomi-
metic-synthetic hydrogels that control biocompatibility and responsiveness. The
biomimetic-synthetic hybrid hydrogels are holding a great promise for nanophar-
maceutical applications by their multiplex combination.
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Chapter 8
On-Chip Drug Screening Technologies
for Nanopharmaceutical
and Nanomedicine Applications
Rabia Onbas, Rumeysa Bilginer, and Ahu Arslan Yildiz
Contents
8.2 M icrofabrication of Microfluidics 314
8.2.1 Photolithography and Soft Lithography 314
8.2.2 Microcontact Printing 315
8.2.3 Replica Molding 316
8.2.4 Mold Fabrication 316
8.2.5 Etching 316
8.2.6 Rapid Prototyping 317
8.2.7 Substrate Bonding 317
8.3 Applications of Microfluidics for Drug Screening 318
8.3.1 Cell-Based Drug Screening 318
8.4 Commercialization and Marketing 334
8.5 Challenges of on-Chip Technologies for Nanomedicine and Nanopharmaceutical
Applications 335
8.6 Future Perspective and Conclusion 336
References 337
Abstract Conventional drug discovery and screening processes have important

drawbacks particularly in preclinical phase which include in vitro drug screening
test, namely, 2D cell culture, and in vivo test on animal models. It is known that 2D
cell cultures are insufficient to indicate drug response of real tissues and organs.
Animal models also mislead about drug response with the same reason, and there
are some ethical issues about animal models as well. These drawbacks lead to fail-
ure of drug candidate at later phases or after approved by the Food and Drug
Administration (FDA). Since the drug development phases are very costly pro-
cesses, this failure means time and money consumption to drug companies. More
R. Onbas · R. Bilginer · A. Arslan Yildiz (*)

Department of Bioengineering, Izmir Institute of Technology (IZTECH), Izmir, Turkey
e-mail: rabiaonbas@iyte.edu.tr; rumeysabilginer@iyte.edu.tr; ahuarslan@iyte.edu.tr
https://doi.org/10.1007/978-3-030-44925-4_8
312 R. Onbas et al.
importantly if the drug fails after approval, many patients will be exposed and be
affected by its side effects.
Recently, three-dimensional (3D) cell culture systems have been started to be
used in drug discovery and development in order to develop more realistic and more
predictive models for in vivo tests. Advances in tissue engineering, cell biology,
biomaterials, microfabrication, and microfluidic technologies have enabled to
mimic the functions of varied tissues and organs on a single chip. Recent develop-
ments in microfabrication techniques lead to more precise fabrication of microflu-
idic chips that provides miniaturization of the conventional process. It also leads to
development of cell-based high-throughput screening (HTS) platforms that are
known as “organ-on-a-chip” systems. Currently, organ-on-a-chip technologies are
the most promising experimental platforms to reduce animal tests and to improve
the efficiency of preclinical prediction of drugs. Compared to conventional systems,
these platforms are used to reduce sample volume. Besides, combination of 3D cell
culture with microfluidic platform makes it possible to mimic biochemical and bio-
mechanical microenvironment of real tissues. In other words, more physiologically
relevant and realistic models can be created. This potential use of microfluidic sys-
tems in drug discovery has created their own market which is estimated to reach
$3.6–5.7 billion by 2018, and drug discovery is the second largest market area in
microfluidic applications. In this manuscript on-chip drug screening models were
reviewed which aim to improve drug discovery and development. On-chip drug
screening studies from the nanopharmaceutical point of view were reviewed under
the following sections: (1) microfabrication of microfluidics; (2) utilization of
microfluidics for drug screening, in particular organ-on-a-chip drug screening tech-
nologies; and (3) commercialization, marketing, and challenges of microfluidic
drug screening platforms.
Keywords Cell-based drug screening · Drug discovery or screening · Heart-on-a-

chip · High-throughput screening · Kidney-on-a-chip · Liver-on-a-chip ·
Microfabrication · Microfluidics · Nanomedicine · Nanopharmaceuticals ·
Organ-on-a-chip
8.1 Introduction
In the preclinical and clinical phase of drug development and screening, using ani-
mal models and human models is limited because of ethical concerns, availability of
test subjects, and the applicability of test procedures. Moreover, animal models may
be insufficient to predict clinical efficacy of drugs for particular human tissue types,
as well as labor-intensive processes lead to increased cost. It is known that after
preclinical and clinical phases as shown in Fig. 8.1, very few drug candidates reach
the market which generally takes around 12–15 years. Furthermore, over one billion
US$ is spent through all these processes, and “post-marketing” phase follows these
8 On-Chip Drug Screening Technologies for Nanopharmaceutical and Nanomedicine… 313
Fig. 8.1 Schematic outline of new drug development pathway
steps after the drug reaches the market (Haque and Ratemi 2017; Zongjie Wang
et al. 2015). If the drug fails at the late process stages or after approval of the drug,
it would be very devastating for pharmaceutical companies because they have
already spent enormous budget on drug discovery and development. For that reason,
“fail early, fail cheap” attitude is adopted by numerous pharmaceutical companies
(Eder et al. 2016). As said above, drug research and development (R&D) is a long,
complex, and costly process, and also only 10% of all molecules entering phase 1
clinical trials are being approved by FDA at the end. Up to now, available preclinical
experimental models, such as 2D cell cultures or animal models, have been inade-
quate to evaluate the efficacy of drug candidates, leading to most of the drug failures
at clinical phase (Blomme 2016; Esch et al. 2015).
Based on abovementioned obstacles in drug discovery field, it is a requirement to
develop new, effective, and more realistic drug screening models to be able to pre-
dict complex responses of a real human tissue. For that reason, lately, researchers
have been working on in vitro 3D cell culture models for the drug discovery and
development. In vitro models are superior to animal models in terms of systematic,
repetitive, and quantitative analysis of cell or tissue physiology (Fan et al. 2016;
Gupta et al. 2016; Phan et al. 2017; Sabhachandani et al. 2016; Tung et al. 2011;
Zanoni et al. 2016). In addition, these models are cost-effective, easy to control and
manipulate, and time efficient compared to animal models. Tissue engineering, 3D
cell culture, and microfluidics are novel technologies that can speed up the drug
discovery and development processes since these technologies are capable of evalu-
ating numerous different combinations of experimental parameters with 3D cell
culture-based microfluidic high-throughput screening models. Microfluidic systems
have been drawing attention as a potential platform for HTS technology because
they use low sample volume and allow easy manipulation of nanoliter volumes at
low cost, as well as it is applicable for cell-based assays (Du et al. 2016; Elliott and
Yuan 2011; Selimovic et al. 2013; Xu et al. 2011). Currently, various publications
(An et al. 2016; Au et al. 2014; Fan et al. 2016; Toh et al. 2009) have utilized micro-
fluidic approach for drug development and screening studies.
The main aim of this paper is to provide summary of latest developments about
on-chip drug screening platforms which are alternative to classical 2D in vitro
assays and animal models. This paper mainly comprise (i) microfabrication tech-
niques of microfluidics; (ii) screening techniques for cell-based assays; (iii) organ-
on-a-chip technologies for drug screening; (iv) commercialization and marketing
efforts; and (v) challenges in the field.
314 R. Onbas et al.
8.2 Microfabrication of Microfluidics
Microfluidic platforms enable miniaturization, automation, and parallelization of

biochemical assays that control and manipulate small volume of fluids in microliter
to picoliter range (Stone et al. 2004; Whitesides 2006). Microfluidic platforms are
capable of carrying out reactions with a very small volume which means increased
surface-to-volume ratio; hence reactions occur in a very short time. Moreover, it
provides high-throughput analysis via parallel processes which result in the faster
evaluation process. As a result of miniaturization, microfluidic platforms are cost-
effective which are very significant for the pharmaceutical industry especially in the
area of HTS of drug candidates (Bhupinder Singh Sekhon 2010; Mitchell 2001;
Qasaimeh et al. 2013; Pihl et al. 2005).
Preferred microfabrication method and strategy provide certain properties which
influence the function and output of the developed microfluidic chip. In this section
commonly used microfabrication methodologies for on-chip nanopharmaceutical
applications will be discussed in detail.
8.2.1 Photolithography and Soft Lithography
Lithography is one of the earliest and commonly used microfabrication technique

used in the field of microfluidics. Lithography is classified under two main classes,
depending on application procedure, either called as photolithography or soft lithog-
raphy. In photolithography, micropatterns are produced using light source, photore-
sist, and mask. Ultraviolet (UV) light is applied to the photoresist to obtain the
desired pattern through a mask. A developer solution is used to solubilize the unex-
posed parts of the photoresist in order to obtain photoresist pattern. Afterwards, the
main material is applied on the photoresist pattern, and then photoresist is removed
(Tai Hyun Park 2003).
In lithography generally silicon wafers, glass, and organic polymers are pre-
ferred as the matrix material of microfluidic chips. Quartz glass and Pyrex 7740
glass are mostly preferred due to their superior optical and chemical characteristics.
Nevertheless, these are high-priced materials, they have complicated process, and
etching rate is not fast enough to obtain adequate depth (Guanglong Wang et al.
2012). However, fast, cheap, and reliable process can be obtained with soda–lime
glass substrate which has opaque structure (Che-Hsin Lin et al. 2001; Sun et al.
2012). In addition to these materials, polymer materials draw attention lately
because of the low production cost and easy processing steps. Various polymers,
poly(methyl methacrylate) (PMMA) (Schnauber et al. 2016; Yan et al. 2016),
polydimethylsiloxane (PDMS) (Challa et al. 2017; Voicu et al. 2017), polycarbon-
ate (PC) (Retolaza et al. 2018; Zheng et al. 2018), polyester (Krauss et al. 2017;
Tsuchiya et al. 2018), polystyrene (PS) (Aydinoglu et al. 2017; Chen et al. 2017),
and poly(ethyleneterephthalate) (PET) (Li et al. 2017; Zhao et al. 2018), have been
utilized for lithographic techniques.
During photolithography process, conventional silicon etching is commonly
used in microfabrication. However, conventional silicon etching is comparatively
expensive because fabrication steps are complex, and it requires cleanroom facili-
ties. For that reason, an alternative method has been developed which is named as
“soft lithography.” In soft lithography a soft elastomeric material is used for pattern
transfer or surface modification. Due to easy handling, rapid process, and cost-
effective features, soft lithography method is more convenient for the fabrication of
an integrated microfluidic system (Whitesides et al. 2001; Tai Hyun Park 2003;
Xiao-Mei Zhao and Whitesides 1997). Generally a polydimethylsiloxane (PDMS)
stamp is used in soft lithography as an elastomeric material because PDMS is cheap
and it is very easy to use. Moreover, PDMS offers some advantages for biological
applications such as biocompatibility and gas permeability which are important for
cell culture and transparency for optical assays (Randall and Doyle 2005). Also,
porous structure of PDMS leads small molecules to diffuse into the bulk; this prop-
erty has let PDMS be used in microextraction applications to eliminate trace organic
compounds from aqueous samples (Erik Baltussen et al. 1999). On the other hand,
PDMS has some material-related limitations such as non-specific binding of mole-
cules because of its high hydrophobicity, and intake of small molecules. These limi-
tations may affect results of microfluidic studies in drug screening, proteomic
analysis, and cell culture applications (Toepke and Beebe 2006). Alternatively,
poly(methyl methacrylate) (PMMA) offers many advantages. PMMA is an indus-
trial material that is inexpensive, widely available, non-toxic, and easily processed.
PMMA has moderate resistance to wet and dry etching environments. Moreover, it
has high optical transparency which is more transparent than even ordinary soda–
lime glass. More importantly, this material is highly transmissive from mid-infrared
to near-ultraviolet wavelengths that makes it very useful for lithographic patterns
(Carbaugh et al. 2016).
8.2.2 Microcontact Printing
Microcontact printing is a plain method, which is mostly classified under soft

lithography, to form microstructures in an additive process. Microcontact printing
transfers an “ink” onto a surface in a defined pattern by the microfeatures of an
elastomeric stamp which is coated by ink. Process of chemisorption or physisorp-
tion is used for ink transfers from the stamp to the surface. Releasing the PDMS
stamp from the surface fabricates the printed pattern of ink on the surface (Amit
Kumar 1993). Microcontact printing is generally used for 2D fabrication; it is also
possible to combine with other process (e.g., metal plating) and create quasi-three-
dimensional structures (Hidber et al. 1996). It is possible to use many materials as
an ink such as organic small organic molecules, polymers, and nanoparticles on a
solid substrate via appropriate modification of stamp and substrate (Tian et al. 2013).
316 R. Onbas et al.
8.2.3 Replica Molding
In this method it is possible to replicate 3D microstructures by using a variety of

materials. Replica molding process has three steps: (i) development of the desired
patterned master, (ii) transfer of pattern to PDMS and then release of the PDMS
after contacting with the master, and (iii) solidification of a liquid precursor against
the PDMS mold and release of the solidified structure to isolate a replica of the
master (Gates 2005). Replica molding is a reliable, simple, and inexpensive meth-
odology which provides possibility to duplicate highly complex structures in a sin-
gle step. However, the most important limitation of this methodology is the
production of complex and precise structures under certain sizes (Whitesides 1998;
Zhong et al. 2014).
8.2.4 Mold Fabrication
Utilizing molds, namely, injection molding and hot embossing, is one of the mostly
preferred approaches to fabricate microfluidic devices in order to create the patterns
and features in polymers. Injection molding is a reliable, reproducible, and inexpen-
sive process in high volume production cases. However, process parameters have
critical effect to create the pattern which are mold temperature, melt temperature,
holding pressure, injection velocity, holding time, and part thickness. Injection
molding is used for micromolding in the industry as well (Maghsoudi et al. 2017).
On the other hand, hot embossing is preferred by academic labs due to accessibility
and flexibility for low to medium production, and it is highly cost-effective com-
pared to injection molding in terms of set up besides being simple and easy to oper-
ate and providing higher accuracy in the replication of small features. Generally,
computer numerical control known as CNC or precision laser systems are used to
produce molds for hot embossing and injection molding. Yet, surface roughness is
one of the most important limiting factors for these techniques which affects subse-
quent bonding or interferes with optical microscopy and imaging of cells. Polishing
can overcome those obstacles; however polishing also leads to increase of costs and
labor (Berthier et al. 2012; Heckele and Schomburg 2004).
8.2.5 Etching
Etching is an easy to use microfabrication technique that builds topographical fea-

tures on a surface via material removal through physical or chemical processes. This
technique is generally classified as wet or dry etching according to the applied pro-
cesses; either liquid chemicals or gaseous physicochemical processes were utilized,
respectively. Wet etching always results in isotropic profile which means etching
occurs at a same rate in all directions; for that reason it is not possible to obtain
vertical sidewall on amorphous substrates. On the other hand, dry etching process is
capable of forming better vertical sidewalls which have higher aspect ratio.
Moreover, etching limitation is very low for dry etching process, it has higher aniso-
tropicity, and more importantly it is possible to fabricate microchannels on varied
substrates (Betancourt and Brannon-Peppas 2006; Ning et al. 2016).
8.2.6 Rapid Prototyping
Rapid prototyping is a new-generation microfabrication technique that enables fab-

rication of 3D microstructures by using computer-aided models. Rapid prototyping
is a highly recognized and preferred methodology since it is rapid, easy to use, and
compatible with varied parameters. In general, fabrication of micro systems starts
with stereolithography and is completed by additive manufacturing technology or
3D printers (Li et al. 2018; Pradel et al. 2018; Vanderburgh et al. 2017). Varied
materials such as polymers or metals can be processed in a very short time. In this
technique mostly PDMS is used because of its properties such as ease of use and
mechanical elastomeric properties. However, surface chemistry of PDMS and
mechanic strength are not convenient for commercialization. Moreover, it is not
easy to scale up PDMS. Also, thermoplastics are preferred such as PMMA, polysty-
rene, and cyclic olefin copolymers (Carlborg et al. 2011; Matellan and Del Rio
Hernandez 2018; Tan et al. 2018).
Rapid prototyping is preferred not only for microfluidic chip fabrication but also
for creating masters. Creating masters with rapid prototyping provides some advan-
tages over the conventional photolithography and micromachining of silicon. First
of all, production stage of the transparencies lasts hours instead of days or weeks;
hence rapid prototyping saves time (hours compared to days or weeks) and is
cheaper than chrome masks ($20 compared to $500–$1500). The other advantage is
simplicity and flexibility. However, fabrication of channels in microfluidic devices
with a random orientation or to create circular reservoirs because of anisotropic
etching is quite difficult by using this approach (Duffy et al. 1998; Ghaderinezhad
et al. 2017).
8.2.7 Substrate Bonding
Bonding step is a very significant step in microfabrication processes which should

maintain a tight seal in order to prevent leakage without giving any harm to the
channels. There are two types of bonding methods: direct and indirect bonding.
Direct bonding methods are thermal fusion bonding, ultrasonic welding, surface
modification, and solvent bonding. If the bonding needs additional material or
chemical reagent such as epoxy, adhesive tape, or chemical reagent in order to
318 R. Onbas et al.
maintain bonding of substrate, it is named as indirect bonding. As an example,

adhesive bonding and microwave bonding techniques are known as indirect bond-
ing methods (Judy 2001; Silverio and Susana 2018; Tsao 2016).
8.3 Applications of Microfluidics for Drug Screening
Drug discovery is one of the most difficult applied sciences due to complicated
cell–drug interaction and difficulty in optimizing drug pharmacokinetics, physical
properties, selectivity, and so on. Conventional drug screening is carried out by
using well plates (e.g., 96, 384, and 1536 well plates) which are known as static
cultures. These static cultures require replacement of nutrients and elimination of
waste which likely result in contamination and experimental errors (Dittrich 2010;
Tsui et al. 2013). In order to minimize challenges in drug discovery, new-generation
screening technologies, such as microfluidics and organ-on-a-chip technologies,
hold promise to overcome abovementioned challenges (Balakrishnan et al. 2015;
Hung et al. 2005; Jr-Lung Lin et al. 2011). Microfluidic platforms, known as “lab-
on-a-chip” systems, can combine many analytical components in a single device
which leads to less cell and drug sample requirement compared to conventional
systems (from 10- to 1000-fold) (Chun-Wei Chi et al. 2016). In this section (i) cell-
based nanopharmaceuticals, (ii) screening technologies, and (iii) organ-on-a-chip
approaches will be reviewed.
8.3.1 Cell-Based Drug Screening
The lead identification is the first process step of early drug discovery that includes
the screening of millions of compounds present in compound libraries and aims to
recognize the possible therapeutic effects of lead compounds. Most of the time,
conventional HTS techniques are incapable of providing physiologically relevant
results which ends up with a failure of a drug candidate at the end. However, cell-
based HTS techniques give a more realistic biological response and help to acceler-
ate drug validation process. Moreover, utilizing cell-based HTS enables
multiparametric investigation to clarify the structure–function relation, investiga-
tion of binding affinity and pharmacological characteristics of lead compounds, and
recognition of important side effects. However, currently used technology for cell-
based HTS is microwell plates that are high-priced and time-consuming (Kiran
Bhadriraju 2002; Klaus Giese et al. 2002; Upadhyaya and Selvaganapathy 2010).
For that reason, assay miniaturization is required to overcome aforementioned prob-
lems. Miniaturization provides economic advantageous that decreases the develop-
ment and operating costs. Currently, continuous flow assays are carried out by using
microfluidic platforms which are the commonly used approach for assay miniatur-
ization (Sundberg 2000). Cell-based HTS can be performed by using three different
modes of microfluidic platforms to control fluid flow which are perfusion flow mode
(Kimura et al. 2008; Selimovic et al. 2011; Sugiura et al. 2010; Zhu et al. 2004),
droplet-based mode (Abate et al. 2013; Baret et al. 2010; Clausell-Tormos et al.
2008; Kimura et al. 2008; Miller et al. 2012; Trivedi et al. 2010), and microarray
mode (Kwon et al. 2011; Lee 2008; Wei et al. 2005; Wlodkowic et al. 2009) (Du
et al. 2016). Those microfluidic platforms lead to the advancements in in vitro cell-
based drug screening studies, especially organ-on-a-chip approaches. Compared to
routinely used systems, microfluidic platforms give possibility to design overall
structure based on application and allow to analyze the interactions at molecular,
cellular, and tissue level (Sackmann et al. 2014).
Screening Techniques for Cell-Based Interactions
There is an increasing tendency to investigate drug–receptor interaction at the cel-

lular level for nanopharmaceutical applications, that’s why suitable probes and
screening techniques are required. In order to observe drug-induced cell response,
varied sensing platforms are developed which are capable of measuring specific
properties of cells such as size, morphology, membrane capacitance, mechanical
and hydrodynamic properties, and the organization of biomolecules within cells or
adhesion patterns of cells on a surface (Buckmaster et al. 2009; Chen and Zou 2016;
Gossett et al. 2012; Liang et al. 2017; Liu et al. 2011; Shen et al. 2017; Sobiepanek
et al. 2016; Zhao et al. 2017). Besides measuring cellular characteristics, there are
also varied biosensors which were developed to measure specific biomarkers or
metabolic markers that are secreted from cells as a response of drug induction. One
of the most commonly used screening techniques is measuring metabolic activity
for drug response of the cells. The “live–dead assay” which indicates the metabolic
activity of live cells is a significantly used technique for HTS (Clausell-Tormos
et al. 2008; Lee 2005, 2008; Nierode et al. 2016; Takaki et al. 2012; Tung et al.
2011; Worthington et al. 2017). Currently, most of the live–dead assays are com-
mercial kits that utilize fluorescence staining of live and dead cells separately. Live–
dead principle is used by Brouzesa et al. (2009) to develop a microfluidic droplet
screening chip for high-throughput cytotoxicity screening of single mammalian
cells. The cells were encapsulated in a single aqueous microdroplet and combined
with another optically coded droplet from a library that encodes an identified drug
composition and drug concentration in each droplet (Fig. 8.2a). Later on combined
droplets were incubated off-chip for 24 h, and then the droplets were reinjected into
the chip. During this reinjection step, live-dead staining of cells were accomplished
via combination with a fluorescence dye containing droplets (Eric Brouzesa
et al. 2009).
Similar to fluorescence-based live–dead assay, characterization of cell morphol-
ogy is also a preferred method to determine the response of the cells against drug
induction. Detection of cell death is possible by observing cell morphology which
is the very significant viewpoint of various HTS platforms (Carpentier et al. 2016;
Paran et al. 2007; Rana et al. 2017; Yoshii et al. 2015; Zhu et al. 2017; Zhu et al.
320 R. Onbas et al.
Fig. 8.2 Cell-based screening approaches. (a) The droplet-based screening. (Reprinted by copy-
right permission from Eric Brouzesa and Jonathan M. Rothberg 2009). (b) Schematic representa-
tion of the microfluidic device to investigate morphological changes of cells in microfluidic
channel. (Reprinted by copyright permission from Kim et al. 2010)
2012). Based on fluorescence-based live–dead assay approach, Kim et al. (2010)

designed a microfluidic device for label-free in situ monitoring of Cd2+-induced cell
apoptosis, which is a morphology-based image cytometric analysis (Fig. 8.2b). Cell
rounding is a primary characteristic for quantifying early apoptotic cell death which
is quite related with biochemical properties (i.e., reactive oxygen species (ROS)
generation). Also, the new microfluidic platform is a simple, effective, and label-
free in situ monitoring method for cell death identification. In this study, on-chip
image analysis was conducted through cytometry-based cytotoxicity assay that
comprises four steps: (1) loading of the cells into microfluidic chip, (2) toxin (e.g.,
Cd2+) induction of cells through gradient formation, (3) analysis of morphologic
changes of adherent cells via optical techniques, and (4) application of ROS assay
followed by fluorescence image analysis. According to obtained EC50 value and
dose–response curve, system contains 2000 times smaller volume cell numbers
compared to 96 well plates. That means this on-chip screening platform is capable
of lowering sample volume that can contribute to carry out HTS for drug discovery
and development studies (Kim et al. 2010).
Another commonly used, label-free, and non-destructive technique is cell-based
impedance spectroscopy which detects the dielectric characteristics of biological
samples. Unlike the optical detection, electric signals are suitable properties for
recording and processing because there is no light source and optical detector
requirement. In order to measure impedance changes during cell apoptosis, a
microelectronics-based microfluidic chip is designed by Meissner et al. (2011).
HepG2/C3A cells were exposed to acetaminophen, and it was shown that continu-
ous drug perfusion leads to a time- and concentration-dependent impedance
decrease. Moreover, the platform is capable of detecting cellular changes before the
cellular death, and compared to standard expensive and time-consuming viability

assays, the platform has very high sensitivity (Meissner et al. 2011).
As mentioned above, measuring biomarkers or metabolites is another approach
to investigate drug-induced response of the cells. Mass spectrometry is an effective
method for recognition and characterization of cell metabolites and biomarkers
(Bassani-Sternberg and Coukos 2016; Diamandis 2004; Indovina et al. 2013; Kolch
et al. 2005; Kosaka et al. 2017; Lazova and Seeley 2017; Tolson et al. 2004;
Wulfkuhle et al. 2003). Chen et al. designed a microfluidic platform that combines
cell culture chambers, on-chip sample preparation (i.e., a solid phase extraction)
module, and electrospray ionization mass spectrometry (Fig. 8.3a). A microfluidic
chip with stable isotope labeling assisted microfluidic chip electrospray ionization
mass spectrometry is developed for qualitative and quantitative analysis of the cell
metabolites after drug treatment. Developed on-chip system is a promising example
that can be carried out as an on-line multiparameter cell metabolism analysis plat-
form for HTS (Chen et al. 2012).
Another microfluidic platform, which is a combined chemo- and biosensor to
investigate four different parameters such as pH, oxygen, lactate, and glucose for
cellular activity, was developed by Weltin et al. (2014) to monitor human cancer cell
metabolism (Fig. 8.3b). In this study, different from the silicon-based microphysi-
ometer, an optically transparent multiparametric microphysiometer is developed to
detect parameters of living cells on a chip through extracellular acidification, cel-
lular adhesion, oxygen consumption due to cellular respiration, or energy metabo-
lism such as glucose uptake and lactate production. Cell culture metabolism of
T98G human brain cancer cells was detected on-chip. Drug screening application
was carried out by using cytochalasin B; alteration and recovery effects of cellular
metabolism were measured (Weltin et al. 2014).
Besides all abovementioned techniques and approaches, surface-sensitive tech-
niques such as surface plasmon resonance (SPR) spectroscopy and resonant wave-
guide grating (RWG) were also preferred. SPR-based techniques, SPR, surface
Fig. 8.3 Cell-based screening approaches. (a) Schematic diagram of the microfluidic chip that is
combined with cell culture chambers and ESI-MS in order to detect metabolic activity of drug-
exposed cells. Reprinted by copyright permission from (Chen et al. 2012). (b) The microfluidic
platform combined chemo- and biosensor that are capable of the detection of cell metabolism.
(Reprinted by copyright permission from Weltin et al. 2014)
322 R. Onbas et al.
plasmon-enhanced fluorescence spectroscopy (SPFS), localized surface plasmon

resonance (LSPR) spectroscopy, and imaging SPR (i-SPR), can provide very sensi-
tive and specific information about drug–target interaction and binding kinetics in
label-free format (Arslan Yildiz et al. 2013; Baird et al. 2002; Danelian et al. 2000;
Fabini and Danielson 2017; Fang 2015; Liu et al. 2006; Mowla et al. 2017; Pillet
et al. 2011; Walter Huber 2006; Wang et al. 2015; Yasmina and Day 2002).
Organ-on-a-Chip Drug Screening Technologies
In vitro cell culture models are cheaper and faster compared to in vivo models;
however in vitro cell culture mostly remains incapable of mimicking complex cell–
cell and cell–matrix interactions, modeling the drug diffusion kinetics, and deter-
mining the effective drug dose which is important for efficacious and accurate
testing of drugs. Commonly used in vitro cell culture models are two-dimensional
(2D) cultures in which cells are cultured on a plastic substrate and grow as a mono-
layer. 2D cell cultures are incapable of translating the exact response of real tissues
and organs. For that reason, more realistic 3D cell culture models which mimic the
real physiology of human organ and tissues need to be designed to accelerate the
drug discovery and development. Organ-on-a-chip platforms meet the requirement
of HTS by rapid improvement in tissue engineering, stem cell research, develop-
ment of new-generation biomaterials, microfluidics, and microfabrication tech-
niques (Conant et al. 2017; Lanz et al. 2017; Skardal et al. 2017; Yang et al. 2017).
Organ-on-a-chip comprises the structure and function of organs while providing the
biochemical, bioelectrical, and biomechanical properties of cellular microenviron-
ment and extracellular matrix (ECM). Moreover, it is also possible to scale up and
apply the HTS and control over physical factors, such as fluid shear stress and
mechanical distortions. Therefore, organ-on-a-chip platforms are the promising and
featuring experimental platforms for drug screening studies (Shen et al. 2017;
Skardal et al. 2016; Tsui et al. 2013; Zongjie Wang et al. 2015). To date, many stud-
ies have been performed utilizing organ-on-a-chip platforms as it is illustrated in
Table 8.1 and discussed in detail as kidney-on-a-chip, heart-on-a-chip, and liver-on-
a-chip in the following sections.
Kidney-on-a-Chip
The kidneys are one of the most important organs in the body that provides meta-
bolic component balance. Kidneys are exposed to many toxic materials that come
from blood vessels. However, during time, those toxic materials cause a dysfunction
of kidney abruptly, which is named as acute kidney injury, and 19–33% of acute
kidney injury are caused by drug-induced nephrotoxicity. Moreover, new drugs in
preclinical and clinical stages may also cause acute kidney injury suddenly.
According to AstraZeneca drug discovery report, 82% of drug trial failed at pre-
clinical stage, and 8% of them is due to nephrotoxicity problems. Likewise, 52% of
Table 8.1 Example of studies covering drug screening of organ-on-chip with different therapeutic
groups, microfluidic platforms
Therapeutic
group Cell type Microfluidic platform Conclusion References
Kidney-on-Chip
Vasopressin IMCD cells Photolithography; Osmolarity of apical Jang and
and aldosterone PDMS fluidic sample and Suh (2010a)
Na uptake was
changed after
vasopressin and
aldosterone
exposure
Ifosfamide MDCK cells Replica molding; The biochip Choucha
PDMS stimulated early Snouber
inflammatory et al. (2012)
response. While
ifosfamide has no
effect on Petri dish
MDCK culture,
biochip platform
affects the
inflammatory
response through
modulation of the
Nrf-2, ATM, p53,
and NFKB
pathways
Ifosfamide For liver HepG2/ Replica molding; The nephrotoxicity Choucha-
C3a and HepaRG PDMS of ifosfamide has Snouber
cell lines, for not been observed et al. (2013)
kidney MDCK on biochip
cells monoculture of
MDCK cells;
reduction of MDCK
cell number and
disturbance of the
intracellular calcium
release have been
detected in liver–
kidney biochip
compared to
untreated
co-cultures and
treated monoculture
of MDCK cells
Cisplatin Primary human Soft lithography and Cisplatin response Jang et al.
proximal tubular micromolding; PDMS and Pgp efflux (2013)
epithelial cells transporter activity
observed similar
with in vivo
response
(continued)
324 R. Onbas et al.
Therapeutic
– Human proximal All tissues Maschmeyer
tubule cell line, performed high et al. (2015)
human HepaRG viability and
cell line, human discrete
primary hepatic physiological tissue
stellate cells, architecture. This
human juvenile study is the first
prepuce approach for ADME
profiling
Heart-on-a-chip
Haloperidol Cardiomyocyte Agar microculture The results Kaneko
cells chip with infrared demonstrate et al. (2007)
laser photothermal significance of cell
etching size for reliable and
stable outcome
Epinephrine Cardiac cells Spin coating a layer Real-time data Grosberg
of poly(N- collection and et al. (2011)
isopropylacrylamide) analysis were done
and PDMS on the during
surface, microcontact pharmacological
printing of fibronectin exposure
Isoproterenol Cardiomyocytes Spin coating a layer Accurate functional Agarwal
of poly(N- contractile response et al. (2013)
isopropylacrylamide) has been observed
and PDMS on the from the
surface, microcontact microfluidic chip
printing of fibronectin that offers a suitable
in vitro drug testing
platform
Terfenadine Human induced Soft lithography; Fexofenadine did Kujala et al.
and pluripotent stem PDMS not have any effect (2016)
fexofenadine cells on rate-corrected
field potential
duration
prolongation in
human cardiac
tissues; however,
terfenadine led to
significant
rate-corrected field
potential duration
prolongation at
100 nM
concentrations
(continued)
Therapeutic
Doxorubicin Cardiospheres Photolithography; Secretion of creatine Shin et al.
PDMS kinase was sensed (2016)
when the
doxorubicin was
applied with a
dose-dependent
manner (from 1 to
10 μM). The
viability decreased
over a period of
5 days particularly
in the high
concentration; the
beating rates were
changed. As a result,
cardiotoxicity of
doxorubicin was
clearly obtained via
on-chip in vitro
cardiac model
Doxorubicin Human umbilical Bioprinting of GelMa End of the day 6, Kou et al.
vein endothelial and alginate on the beating rate of (2016)
cells PMMA chip the cardiomyocytes
decreased up to
70.5%. Also, the
secreted amount of
von Willebrand
factor, which is an
adhesive and
multimeric
glycoprotein and
takes the role of
hemostasis,
decreased to 76.0%.
The results
indicated that this
platform is a
promising
experimental
platform for
cardiovascular HTS
(continued)
326 R. Onbas et al.
Therapeutic
Isoproterenol Human induced Thermoplastic In vitro contractile Lind et al.
and verapamil pluripotent stem polyurethane and development of (2017)
cell-derived dextran laminar tissues was
investigated for
28 days. Promising
results were
obtained that have
positive and
negative inotropic
responses with
apparent EC50
values compared to
literature
Liver-on-a-chip
– Primary rat Multilayer Results confirmed Liu Tsang
hepatocytes photopatterning of that hepatocytes et al. (2007)
PEG hydrogel adhere to RGDS
motif more likely
compared to other
peptides indicating
functional liver
model can be
produced via
encapsulated
hepatocytes on
RGD peptides
Acetaminophen HepG2 and Photolithography and The results Au et al.
NIH-3 T3 cells etching; Teflon-AF indicated that the (2014)
cells respond to
fibroblast-dependent
contractile behavior,
and their albumin
secretion profiles,
and also cytochrome
P450 3A4 activities.
Acetaminophen
treatment at 5.0 mM
did not result in
apoptotic or necrotic
responses compared
to control cultures
Acetaminophen HepG2/ C3A Bioprinting of Compared to in vivo Bhise et al.
hepatic cells spheroid-laden results, the response (2016)
hydrogel of the microfluidic
platform was similar
to animal and
in vitro models
(continued)
Therapeutic
Acetaminophen Hepatic Bioprinting of Decreasing Knowlton
spheroids GelMA, natural metabolic activity, and Tasoglu
extracellular cell density and (2016)
matrix-derived biomarkers over
hydrogel scaffold 6 days were
observed which
shows the similarity
with animal and
in vitro models
means that
developed
microfluidic
platform may take a
step further in drug
development and
preclinical drug
assays
IMCD inner medullary collecting duct, PDMS polydimethylsiloxane, MDCK Madin-Darby canine
kidney,PEG polyethyleneglycol, RGDS Arg-Gly-Asp-Ser, derived from fibronectin, GelMA gela-
tin methacryloyl, PMMA, poly(methyl methacrylate); HepG2/C3a: liver hepatocellular carcinoma,
HepaRG differentiated hepatic cells; Nrf-2 nuclear factor (erythroid-derived 2)-like 2, ATM ataxia
telangiectasia mutated, NFKB nuclear factor kappa beta, NIH-3T3 fibroblast cells; HTS: high-
throughput screening, pgp p-glycoprotein
the drug trials failed in clinical stages, and 9% of them stem from renal issues
(Huang et al. 2014).
Proximal tubule is a part of kidney that is composed of epithelial cells. Proximal
tubule has vital role to release waste compounds or retake some vital filtered solutes
(Wilmer et al. 2016). Small molecules, drugs, metabolites, and toxins by multispe-
cific transporter appeared in proximal tubule (Jang et al. 2013). Toxic materials
accumulate in the kidney and jeopardize different parts of the kidney, especially in
proximal tube. Therefore, nephrotoxicity-related drug studies usually maintain on
proximal tube due to that drug-induced toxic materials accumulate at this region
(Fig. 8.4a).
In order to predict nephrotoxicity, an organ-on-a-chip platform that can miniatur-
ize organ on microfluidic chip via primary human cell line might be a candidate to
cope and confer a realistic approach to preclinical studies. Organ-on-a-chip also
provides 3D and dynamic cell culture environment in order to analyze pharmacoki-
netics (Kim and Takayama 2015; Jang et al. 2013). Recently, a kidney-on-a-chip by
Jang and Suh was constructed by utilizing PDMS chip (Fig. 8.4b). Primary inner
medullary collecting duct (IMCD) cells were cultured. Cells were treated with argi-
nine vasopressin to induce the water uptake and with aldosterone to induce the Na
uptake. As a result osmolarity of apical fluidic sample and Na uptake was changed
after vasopressin and aldosterone exposure which means the platform can be stimu-
lated with hormonal exposure. Formation of functional renal tubule on chip system
328 R. Onbas et al.
Fig. 8.4 Kidney-on-a-chip approaches. (a) Protein-bound metabolites (red) or drugs are taken
into glomerulus through transporter protein. Filtered vital solutes by glomerulus can retake by
proximal tube. Solely, drugs that are filtered cannot be reabsorbed (green), or drugs that are taken
by transporter protein (red) are eliminated via urine. (Reprinted by copyright permission from
Wilmer et al. 2016). (b) Fabrication of multilayer microfluidic device. The sandwich model con-
sists of microfluidic device and PDMS channel and PDMS reservoir. (Reprinted by copyright
permission from Jang and Suh 2010b). (c) Four-organ-on-a-chip platform. Numbers stand for dif-
ferent tissues: intestine (1), liver (2), skin (3), and kidney (4). (Reprinted by copyright permission
from Maschmeyer et al. 2015). PDMS Polidimetilsiloksan
proved that developed platform can be used for further nephrotoxicity screening
(Jang and Suh 2010b).
Later, Choucha-Snouber et al. designed an organ-on-a-chip platform that mimics
synergic interaction of kidney and liver to screen neurotoxic effect of ifosfamide.
The liver model was created with HepG2/C3a and HepaRG cell lines, while the
kidney model was formed with Madin-Darby canine kidney (MDCK) cell line. In
the microfluidic chip that contains only MDCK cells, no toxicity was observed dur-
ing 72 h ifosfamide exposure. However, in liver–kidney combined microfluidic
chip, nephrotoxicity was observed during 72 h ifosfamide exposure. The results
demonstrated development of systemic organ–organ interaction and the potential of
the system for in vitro drug toxicity assay (Choucha-Snouber et al. 2013). Similarly,
Maschmeyer et al. (2015) conceived an organ-on-a-chip platform that mimics four
organs, liver, skin, kidney, and intestine, nearly in 28 days (Fig. 8.4c). This is the
first in vitro design to detect absorption, distribution, metabolism, and excretion
(ADME) profiling and dose toxicity testing of drug candidates over 28 days
(Maschmeyer et al. 2015).
In order to make standard framework of drug screening on kidney-on-a-chip
platforms, nephrotoxic assays must be matching with in vivo test results. Therefore,
prediction of the system should be assessed with sensitivity (true positives) and
specificity (true negatives), and this is possible with a library of compounds with
already known nephrotoxic effects (Wilmer et al. 2016).
Heart-on-a-Chip
Cardiovascular diseases are the most common causes of death in the United States
according to a report from the American Heart Association (Mozaffarian et al.
2014). Although various drugs have been developed to treat heart and vasculature
diseases, many patients were affected by them that leads to deceleration of drug
discovery and development in approved cardiovascular drugs (Ribas et al. 2016).
Because of the treatment-generated effects, 81 drugs had to be withdrawn from the
market in the United States, Europe, and Asia; also 16 of those caused cardiac
arrhythmias between 1990 and 2013. Furthermore, rate of cardiotoxicity-related
withdrawal is strikingly increased from 5.1 to 33.3% since 1997 (Li et al. 2016).
Current drug discovery models have many important drawbacks such as long
analysis periods and expensive procedures (Ribas et al. 2016). Most importantly,
animal models are not good enough to estimate human responses in cardiovascular
diseases. In other words, positive/negative results in animals generally do not match
to positive and negative results in human counterparts (Bracken 2009; Kaufman
1997; Pandora Pound et al. 2004). Therefore more realistic experimental models
that are relevant to human physiology are required to evaluate the drug candidates.
There are two aspects of drug development for investigation of cardiac muscle
physiology: contractility and electrical characteristics which are significant param-
eters to analyze the cardiac pharmaceuticals and to evaluate potential cardiotoxic
effects of non-cardiac drug candidates. Currently, developing quasi in vivo cardiac
tissue models on microfluidic devices utilizing cardiomyocytes is a very remarkable
study that aims to imitate the elastic modulus of the heart (Joanna Skommer 2015).
Kaneko et al. (2007) examined the change of beating intervals of a four-cell and
nine-cell cardiomyocyte network, which is the cell-based model to assess drug
screening, by stimulation of haloperidol. In this study, on-chip single cell-based
screening system was used in order to monitor states of cells cultured in the micro-
chamber array continuously. The system is comprised of a cultivation microcham-
ber array and a microscopy system. The cardiomyocytes were cultured in each
microchamber. Haloperidol was applied as an antipsychotic drug to measure the
response of cardiomyocyte network. A nine-cell network was found as an appropri-
ate model because beating intervals are similar to a healthy heart. The results show
that size of the cell network is very important to obtain stable and reliable results for
drug screening (Kaneko et al. 2007).
330 R. Onbas et al.
In another study, a “heart-on-a-chip” platform that works based on muscular

thin-film technology was designed by Grosberg et al. (2011). Developed on-chip
platform was capable of measuring contractility, combined with a quantification of
action potential propagation and cytoskeletal architecture in multiple tissues.
Moreover, real-time data collection and analysis during pharmacological interven-
tion are studied. The results showed that quantification of stress, electrophysiology,
and cellular structure are possible with developed on-chip platform (Grosberg et al.
2011). In the following work, a reusable one-channel microfluidic device was
designed and combined with muscular thin film. Muscular thin-film chip has alumi-
num and polycarbonate parts, an aluminum bottom to hold the chip, a transparent
polycarbonate top held in place by three screws, and barbed fittings for fluidic input
and output. Aluminum-based well-shaped chamber allows optical readout of con-
tractility, and polycarbonate is used for creating fluidic top (Fig. 8.5a). In order to
test cardiac contractility, b-adrenergic agonist (isoproterenol) was used in a range
between 1 nM and 100 mM. The obtained responses and pD2 value are alike to the
varied literature data which supports that the developed on-chip technology is com-
patible for generating large amounts of high-quality functional data and HTS
(Agarwal et al. 2013).
Afterwards multi electrode arrays that implement the previously developed on-
chip technology to engineer laminar human cardiac tissues which is derived from
hiPSC-CMs were developed (Fig. 8.5b, c). In Fig. 8.5b, microfluidic connections
were housed in a removable manifold which allowed open well culture of the human
cardiac tissue in custom polyether ether ketone well.
Newly developed on-chip platform provides collagen-based extracellular matrix
with physiologically relevant substrate stiffness. For validation isoproterenol was
applied to record cardiac field potentials. Furthermore, a cardiotoxic pro-drug, terf-
enadine, and its non-cardiotoxic metabolite, fexofenadine, were applied to predict
the response of laminar human cardiac tissues. As a result a heart-on-a-chip micro-
fluidic platform was found as a suitable model for HTS (Kujala et al. 2016).
Fig. 8.5 Heart-on-a-chip models. (a) Exploded view of the fabrication and assembly of a micro-
fluidic device. (Reprinted by copyright permission from Agarwal et al. 2013). (b) Schematic of the
microfluidic multi electrode arrays. (c) Phase contrast image of human cardiac cells in microfluidic
micro-electrode arrays. Scale bar 100 mm. (Reprinted by copyright permission from Kujala
et al. 2016)
Not only physiological properties of cardiac muscle but also cardiovascular bio-
markers are generally analyzed through on-chip platforms for drug screening pur-
poses. Shin et al. (2016) designed an integrated label-free electrochemical biosensing
platform by merging an Au microelectrode and a microfluidic chip, to measure low
levels of biomarkers secreted from a human cardiac-organoid on-a-chip model.
Aptamers which are specific to a cardiac injury biomarker were functionalized to
the sensor chip. Secretion of creatine kinase was sensed when the doxorubicin
which is an anticancer drug was applied with a dose-dependent manner (from 1 to
10 μM). When the viability decreased over a period of 5 days particularly in the high
concentration, the beating rates were changed. As a result, cardiotoxicity of doxoru-
bicin was clearly obtained via on-chip in vitro cardiac model (Shin et al. 2016).
Recently, Zhang et al. (2016) used a different approach in order to construct an
endothelialized myocardial tissue model by bioprinting method. The endothelial
cells were encapsulated in the bioink and slowly migrated along the microfibers to
form a layer of confluent endothelium (Fig. 8.6a). Afterwards, it was combined with
a microfluidic bioreactor perfusion chip for HTS (Fig. 8.6b). Doxorubicin was
applied with the range of 10 mM and 100 mM, and dose-dependent responses were
taken from endothelialized myocardium on-a-chip model. End of the day 6, the
beating rate of the cardiomyocytes decreased to 70.5% and 1.62% for respective
drug doses. Also, the secreted amount of von Willebrand factor, which is an adhe-
sive and multimeric glycoprotein and takes the role of hemostasis, decreased to
76.0% and 35.3%. The results indicated that this platform is a promising experi-
mental platform for cardiovascular HTS (Zhang et al. 2016). 3D printing method
was also applied to design a microphysiological microfluidic platform which was
able to read out the contractile stress of multiple laminar cardiac microtissues.
Figure 8.6c shows that the microfluidic chip has three critical properties: (i) multi-
layer cantilevers including a base layer, an embedded strain sensor layer, and a
tissue-guiding layer; (ii) electrical interconnectors for readout; and (iii) eight inde-
pendent wells. Insert 1: Confocal microscopy image of immunostained laminar
neonatal rat ventricular myocytes, cardiac tissue on the cantilever surface. Blue,
DAPI nuclei stain. White, α-actinin. Scale bar, 10 μm. Insert 2: Still images of a
cantilever deflecting upon tissue contraction. Insert 3: Example resistance signal. In
order to initiate self-assembly of engineered physio-mimetic laminar tissues, neona-
tal rat ventricular myocytes and human induced pluripotent stem cell-derived car-
diomyocytes were used. Drug screening was performed by using isoproterenol and
verapamil to demonstrate response of the neonatal rat ventricular myocyte-based
tissues. In vitro contractile development of laminar tissues was investigated for
28 days. Promising results were obtained that have positive and negative inotropic
responses with apparent EC50 values compared to literature (Lind et al. 2016).
332 R. Onbas et al.
Fig. 8.6 Heart-on-a-chip models. (a) Bioprinting of a microfibrous scaffold using a composite
bioink encapsulating endothelial cells and (b) photograph of the microfluidic bioreactor with an
embedded bioprinted scaffold. (Reprinted by copyright permission from Zhang et al. 2016). (c)
The fully printed final device. (Reprinted by copyright permission from Lind et al. 2016)
Liver-on-a-Chip
Liver is an important organ that is involved in drug metabolism and toxicity; there-
fore it is commonly used as a target model for HTS. As in the kidneys, drug-based
toxicity leads to acute liver failure that ends up with withdrawal of drugs from the
market. According to recent reports, almost 50% of drugs on the market kept respon-
sible for liver failure during preclinical process. Some of those drugs do not show
any effect as liver failure in animal models, which means there is weak correlation
between animal and human models (Bjornsson 2015; Guguen-Guillouzo 2008;
Guillouzo et al. 2007). Therefore, preclinical animal models are required to be
updated with different models based on species-specific variation of hepatocellular
functions between human and animal (Khetani and Bhatia 2008). This requirement
of realistic models for drug screening studies has resulted as a liver-on-a-chip
attempt.
Recently, a continuous flow microfluidic liver-on-a-chip platform is constructed
by Tsang et al. Primary rat hepatocytes were encapsulated inside 3D polyethylene-
glycol (PEG) hydrogel scaffold that is doped with Arg-Gly-Asp (RGD) peptide. In
addition, multilayer photopatterning was performed to incorporate cells into hydro-
gel for organ printing. The adhesion of hepatocytes on peptides was observed by
utilizing varieties of peptides such as RGDS (Arg-Gly-Asp-Ser, derived from fibro-
nectin), YIGSR (Tyr-Ile-Gly-Ser-Arg, derived from laminin), GF-(Hyp)GER (Gly-
Phe-HydroxyPro-Gly-Glu-Arg, derived from collagen), KQAGDV
(Lys-Gln-Ala-Gly-Asp-Val, derived from fibrinogen), and the negative control
RGES (Arg-Gly-Glu-Ser). Results confirmed that hepatocytes adhere to RGDS
motif more likely compared to other peptides indicating functional liver model can
be produced via encapsulated hepatocytes on RGD peptides (Liu Tsang et al. 2007).
With the development of on-chip platforms, drug screening and toxicity studies
started to get more attention; especially liver-on-a-chip platforms were heavily used
for HTS. An on-chip liver organoid is developed by Bhise et al. (2016) for drug
toxicity assay. HepG2/C3A hepatic cells were bioprinted in spheroid-laden hydro-
gel and utilized in continuous flow microfluidic chip. The cells were cultured under
dynamic conditions over a period of 4 weeks and exposed to acute acetaminophen
in order to set up drug toxicity assay. Compared to in vivo results, the response of
the microfluidic platform was similar to animal and in vitro models (Bhise et al.
2016). In another model, similarly hepatic spheroids were encapsulated in a hydro-
gel scaffold via bioprinting technique for purposes of drug screening (Fig. 8.7), and
designed microfluidic platform was used for acetaminophen induction. Decreasing
metabolic activity, cell density, and biomarkers over 6 days were observed which
shows the similarity with animal and in vitro models means that developed micro-
fluidic platform may take a step further in drug development and preclinical drug
assays (Knowlton and Tasoglu 2016).
Fig. 8.7 Microtissue bioprinting system; assembly of the bioreactor chip and perfusion of media.
(Reprinted by copyright permission from Knowlton and Tasoglu 2016)
334 R. Onbas et al.
8.4 Commercialization and Marketing
In 2013 the microfluidic market reached $1.6 billion, and the market is estimated to
reach $3.6–5.7 billion by 2018 (Volpatti and Yetisen 2014). Microfluidic applica-
tions are classified considering their market size: in vitro diagnostics, drug discov-
ery, biotechnology, and ecology. Drug discovery is the second largest market
fragment in microfluidic applications which needs high sample throughput and low
costs per test (Mark et al. 2010). Although there are plenty of academic studies that
show the proof-of-concept devices in microfluidic technology, dispersion of this
technology is still restricted due to limitations. The deficiency of customer develop-
ment and validation of market need are the main limitations. Moreover, investors
have preferred other novel technologies due to absence of a “killer application”
which means revenues greater than $100 million in microfluidics (Blow 2007).
Currently, many companies commercialized microfluidic technologies for geno-
typing, microarray analysis, diagnostics, and drug discovery (Volpatti and Yetisen
2014). As an example, Caliper Technologies (Mountain View, CA) recently showed
that by utilizing on-chip platform only 750 ng of the protein is required which could
screen 750,000 compounds in a drug library against a protein target.
However, in this section of review, mostly “organ-on-a-chip” approaches are
summarized which focus on generating small segments of human tissues and organs
in microfluidic chips for the in vitro nanopharmaceutical applications. Table 8.2
summarizes the companies and corresponding commercialized products for drug
discovery and development.
Hepregen is a spin-off company established in 2008 out of MIT. In 2013, they
launched their commercial product which is known as HepatoPac. HepatoPac is a
“liver-on-a-chip” platform which is designed for both short- and long-term ADME,
toxicology, and efficacy studies along with preclinical drug discovery and
Table 8.2 Commercialized organ-on-a chip products

Founded
Company year Country Product Product property
Hepregen 2008 USA HepatoPac Long-term ADME, toxicology, and
efficacy studies along with preclinical
drug discovery and development
TissUse 2010 Germany 2-organ and Preclinical insight on a systemic level
4-organ chips using human tissue
Nortis 2012 USA Nortis launched Alternative HTS platform to in vivo tests
kidney-on-a- on animals in drug discovery and
chip development process in order to increase
clinical trial success
Tara 2014 USA Heart-on-a-chip Improve predictive cardiac tissue models
Biosystems which are capable of faster, safer, and
more reliable HTS
ADME absorption, distribution, metabolism, and excretion, HTS high-throughput screening
development. Moreover, this platform not only supports human but also rats, mon-
keys, and dogs.
Nortis is one of the most known companies in this field that is founded in
Woodinville, WA, in 2012. Nortis launched its first commercial product in 2015.
Aim of this company is to provide an alternative HTS platform to in vivo tests on
animals in drug discovery and development process in order to increase clinical trial
success. Recently, the new generation kidney-on-a-chip system has been developed
by Nortis that use human kidney cells on kidney-on-a-chip platforms to test the
efficacy of drugs. Developed platform suggests more accurate and realistic, less
invasive drug discovery and development studies.
TissUse is founded in 2010 as a spin-off company from the Technische Universität
Berlin, Germany. A unique and patent-protected “human-on-a-chip” platform has
been developed in order to speed up the development of nanopharmaceutical and
nanomedicine products. This company launched their two-organ and four-organ
chips which have been successfully used for academic and industrial applications.
Importantly, for the first time, their products carried out preclinical insight on a
systemic level using human tissue and enabled the direct prediction of effects of
drugs on near real-life models. Currently, TissUse is working on the development of
a “human-on-a-chip” platform which is comprised of interconnected organs to form
acceptable organismal complexity.
Another important company in drug discovery-related microfluidic market is
Tara Biosystems. It is a New York-based Columbia University spin-off company
which is founded in 2014 and developing “heart-on-a-chip” products. Tara
Biosystems is focused on three crucial areas: cardiac toxicology, heart failure drug
discovery, and precision cardiology. The company’s goal is to improve predictive
cardiac tissue models which are capable of faster, safer, and more reliable HTS.
8.5 C
hallenges of on-Chip Technologies for Nanomedicine
and Nanopharmaceutical Applications
Microfluidic systems occupy significant role in drug screening applications.

However, microfluidic systems bring challenges with itself so there are still engi-
neering and biological problems that need to be solved. Typical challenges of
microfluidic systems are non-standard culture protocols, complex operational con-
trol, and chip design (Halldorsson et al. 2015). Microfluidic systems are very small
platforms that lead to many crucial problems during biological studies with tradi-
tional hydraulic system. Small volumes have low tolerance to errors in flow; other-
wise large errors mostly cause high shear stress which damages cells (Darby et al.
2010). Moreover, some materials that are used in microfluidic systems cause obsta-
cles and limit their usage for varied applications. PDMS is a commonly used mate-
rial in microfluidic systems. However, PDMS hinders chip size, fabrication process,
and scale-up, as well as hydrophobicity of PDMS prevents cell attachment, causing
336 R. Onbas et al.
to hamper cell–matrix relation (Ning et al. 2016). Furthermore, establishing proper

size of each organ for functional organ activity, controlling the stability of the sys-
tem, and accomplishing expected cell density are another limitations of microfluidic
systems in terms of cellular studies (Wikswo et al. 2013).
8.6 Future Perspective and Conclusion
Drug discovery is going through very rapid developments which leads to transfer of
research knowledge to a commercially available product. Especially nanopharma-
ceuticals have been started to be used in varied fields such as pharmaceutical dis-
covery, precision medicine, and diagnostics. Lately, pharmaceutical industry mostly
preferred cell-based assays to predict the toxicity of drug candidates. First of all, it
is possible to recognize allosteric modulators and get clear information on com-
pounds about cell permeability and stability in the cell and cytotoxicity-related
studies. Moreover, cell-based assays represent more realistic physiological environ-
ment for HTS. However, application of molecular or biochemical-based assays is
quite limited due to their specificity to their target. It is not possible or easy to pre-
pare or purify all drug targets for biochemical assays. In addition to this, activity of
compounds in molecular-based assays is not the same with the activity in the cel-
lular environment which means that it is not easy to indicate tissue-related responses
(An and Tolliday 2010; Yang et al. 2012).
Miniaturization is an important issue in cell-based HTS. Microfluidic technology
is the most efficient way to decrease reaction volume. Moreover, combination of
two important research areas tissue engineering and microfabrication generates a
new-generation drug screening platforms, organ-on-a-chip model. The main pur-
pose of organ-on-a-chip model is to develop human-on-a-chip systems using human
cells and tissues and utilize it for drug screening instead of animal testing. These
models have been proven as promising platforms for drug discovery. Additionally
on-chip platforms provide decreased drug development time and costs and better
prediction of drug responses compared to animal models. In order to reach this
purpose, some features of organ-on-a-chip models should be upgraded for toxico-
logical and metabolic tests such as reliability, controllability, and practicality
(Alessandro Polini et al. 2014; Williamson et al. 2013; Zongjie Wang et al. 2015).
Although there is a fast development of microfluidic platforms, still there are no
commercially available drugs that were discovered utilizing microfluidic technolo-
gies. Organ-on-a-chip platforms have been used effectively in the laboratories, but
skillful operators are required most of the time. In addition still current systems
meet the demand such as multi-well plate platforms. The most important limitation
that needs to be overcome is they are not cost-effective enough to handle the com-
pounds for HTS compared to currently used models (Chandrasekaran et al. 2016).
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Chapter 9
Synthesis of Some Bioactive Nanomaterials
and Applications of Various
Nanoconjugates for Targeted Therapeutic
Applications
Sabyasachi Chakrabortty, Sunil Kumar Vimal, and Sanjib Bhattacharya
Contents
9.2 S ynthesis of Biologically Relevant Nanoparticles 349
9.2.1 Carbon Materials 349
9.2.2 Noble Metals and Metal Oxides 352
9.2.3 Semiconductor Nanocrystals 353
9.3 Surface Functionalization Strategy 355
9.3.1 Non-covalent Surfactant Replacement 355
9.3.2 Covalent Attachment 356
9.4 Targeting Capabilities 357
9.4.1 Cellular Target 360
9.4.2 Subcellular Target 361
9.5 Therapeutic Evaluation 363
9.5.1 Nanoparticles as an Imaging Tool 369
9.6 Conclusion 370
References 371
S. Chakrabortty
Max-Planck-Institute for Polymer Research, Mainz, Germany
Department of Chemistry, SRM University, AP – Amaravati, Guntur, Andhra Pradesh, India
e-mail: sabyasachi.c@srmap.edu.in
S. K. Vimal
International Institutes for Integrative Sleep Medicine (WPI-IIIS), University of Tsukuba,
Tsukuba, Ibaraki, Japan
Department of Pharmaceutical Science, Southwest University, Chongqing Shi, China
S. Bhattacharya (*)
Department of Pharmaceutical Science, Southwest University, Chongqing Shi, China
e-mail: sanjib2017@swu.edu.cn
https://doi.org/10.1007/978-3-030-44925-4_9
348 S. Chakrabortty et al.
Abstract In the last two decades, the boom in nanoscience has heightened the
material science research to a different dimension. Synthesis of different tunable
sizes, propriety of new types of individual, and composite nanomaterials have
inspired researcher to use various application domains such as semiconductor opto-
electronics, photonics, catalysis, industrial tool, biomedical options, and too many
to list them. In this chapter, we mainly focus on the synthesis of uncommon or
newly emerging nanoparticle such as carbon dots, nanodiamonds whereas briefly
mention other nanoparticles. Since these carbon dot-based nanomaterials are rela-
tively new and their biological applications are limited, we have listed the biological
applications of various other types of well-known nanocomposites including quan-
tum dot nanocrystals. Therefore, this book chapter should give a comprehensive
overview of chemistry of newly emerging nanomaterials and biological application
of biocompatible nanomaterials that is well studied and information available.
Keywords Carbon Dots and Quantum dots · Nanoparticle Synthesis · Surface

functionalization · Targeting · Therapeutics · Nano-bio interfaces
9.1 Introduction
Delivery of bioactive compounds to its desired tissues remains an old challenge

faced by the pharmaceutical research. However, more success was achieved in the
last decade for the enhancement of targeting efficacy and reduction of nonspecific
effects. In that respect nanoscience has revolutionized the area of targeted drug
delivery and pharmaceutical research due to immense application potential. The
tunable chemical, physical, and biological behavior of nanoscale materials can be
resourceful to utilize them in biomedical purposes (Petros and DeSimone 2010;
Zhang et al. 2008). Among many available inorganic nanomaterials, quantum dots,
noble metal, and metal oxides offer rich optical window and bio analytical proper-
ties that can easily be applied to address various biological studies. Of late, the
world have witnessed the use of nanoparticle system for the applications such as
bioimaging, targeted drug delivery, drug particle nanoconjugate, therapeutic uses,
theranostics, clinical application, and many more (Zhang et al. 2008; Wang and
Wang 2014). Coupling the advances in bioscience and nanoscience work, drug
development by both academia and pharma industry has made a new dimension of
formulating therapeutics for combating various deadly disease which was difficult
to manage in the past. We hope these new-generation nanoparticles have great
potential for the advancement in the study of many important intracellular pro-
cesses, high-resolution cellular imaging, tumor targeting, long-term in vivo investi-
gation of cell trafficking, and diagnostics. In this book chapter, we particularly focus
on synthesis, functionalization, and targeting application of carbon dots and nano-
crystal with few metal oxide nanomaterials. Similarly, quantum dot nanocrystal
possess bioimaging applications as it could not qualify enough to be used as thera-
peutic carrier as the most established combinations content toxic metals. Carbon dot
9 Synthesis of Some Bioactive Nanomaterials and Applications of Various… 349
and nanodiamonds are relatively younger compare to other nanomaterials; thus they
warrant further investigation for their biomedical use. These issues of using carbon
dot, nanodiamond, and other nanocrystals remain realistic for biological application
with optimism that over time more studies will clarify whether nanodiamond or
carbon dot particles could serve even better biomedical purpose. For this reason, we
have elaborated and presented the biological application of those nanomaterials and
nanoconjugates in this book chapter which have been extensively studied biological
applications and some of them reached to the clinic. Therefore this book chapter
overlays a generalized and simplified approach chemistry of nanoparticle synthesis
and ligand functionalization to make nanoconjugates with a blend of putative bio-
logical applications of biocompatible nanocomposites only.
9.2 Synthesis of Biologically Relevant Nanoparticles
Colloidal nanocrystals (NCs) are nanoscaled materials that could be nicely dis-
persed in solvents. Depending on their material composition and shapes, NCs
exhibit different physicochemical properties such as high electron density (e.g.,
metal nanoparticles), efficient and stable fluorescence (e.g., semiconductor NCs or
commonly known as quantum dots (QDs)), phosphorescence (e.g., doped oxide
materials), or ferromagnetism (e.g., iron oxide or cobalt nanoparticles). However,
synthesis of these nanoparticles with defined size, shape, and compositions is
always challenging. We will mainly focus on the synthesis of broadly three compo-
sitionally different nanomaterials, namely, (i) carbon, (ii) noble metal and metal
oxides, and (iii) semiconductor nanocrystals. In the following section, the synthetic
schemes of these colloidal nanoparticles will be addressed.
9.2.1 Carbon Materials
Among all the vital elements present in periodic table, the main building block for
all organic compounds, carbon is the most essential element for all living organisms
(Lide 2004).
Although there are many allotropes present for carbon materials, the two most
popular crystalline forms are graphite and diamond. In bulk form, both consist of
infinite carbon network and do not show any interesting physical or chemical prop-
erties. However, nanosized carbon materials exhibit various unique physicochemi-
cal properties such as photoluminescence (PL) behavior, photothermal properties,
magnetic readout, facile surface functionalization, etc. Moreover, being biologi-
cally inert, these materials can easily be employed in addressing biologically rele-
vant questions due to their biocompatibility. In this part, we will mainly discuss
about the synthesis of two main classes of nano-carbon materials, namely, carbon
nanodots (C-dots) and florescent nanodiamond (fND).
Fig. 9.1 Schematic synthesis of C-dots, prepared via “top-down” and “bottom-up” approaches.
Also, their modifications including functionalization, doping, and nanohybrids were shown.
(Adapted from Ref. (6), with permission Wang and Hu 2014)
C-dots constitute an interesting class of lately discovered nano-carbon materials

which possesses excellent PL properties. In addition, C-dots could be attractive in
terms of applications where size, cost, and biocompatibility issues are critical. Since
the inception in 2006(Sun et al. 2006), carbon dots are known as clusters of carbon
atoms with diameter ranging typically below 10 nm, which contain some fraction of
impurities in their structures such as oxygen, hydrogen, or nitrogen. They possessed
excellent properties such as good biocompatibility, low toxicity, photo chemical
stability (less photo-bleaching and photo-blinking), good up-conversion properties,
strong photoluminescence, environmental friendly, and low cost.
C-dots can be synthesized using different synthetic approaches (Zhu et al. 2013)
which include “top down” and “bottom up” (Fig. 9.1) and are feasible to surface
functionalization (Wang and Hu 2014; Roy et al. 2015). C-dots were first discovered
by Xu et al. (2004) during the purification of single-walled carbon nanotubes.
However, effort to prepare C-dots appeared few years later. The methods for top-
down synthesis are arc discharge, laser ablation, chemical ablation, and electro-
chemical oxidation, where large carbon structures are broken down to C-dots (Lim
et al. 2015). Conversely bottom-up methods include microwave irradiation and
hydrothermal/solvothermal preparation. C-dots synthesized from small precursors
such as carbohydrates, citrate, and polymer/silica nanocomposites through afore-
mentioned synthetic methods. Zhu et al. (2009) prepared C-dots by irradiating a
solution of poly(ethylene glycol) (PEG) and saccharide in 500 W microwave oven
for 2–10 min (Zhu et al. 2009). In most of the cases researcher used water as solvent
with the functional molecules to be incorporated on C-dots backbone as the co-

material during synthesis. This strategy in turn helped to get desired material
depending on their applications as water is used as solvent. In addition, chemical
modification and surface passivation using various organic, polymeric, and inor-
ganic or biomaterials enhances fluorescence and physical properties of C-dots.
NDs are another class of unique nano-carbon materials having highly stable sp3
carbon in the core with amorphous and reactive sp2 carbons on their surfaces
(Shenderova et al. 2006). They are mostly prepared by two methods, (i) detonation
method and (ii) high-temperature/high-pressure (HTHP) synthesis. In detonation
method, large quantities of NDs were prepared with low cost but normally suffered
from aggregation in solution (Shenderova et al. 2006). In HTHP pathway, NDs can
be produced with various sizes ranging from a few nanometer to micrometer with
better crystalline structures (Boudou et al. 2009; Boudou et al. 2013).
In addition, several methods such as arc discharge, laser ablation, chemical abla-
tion, and electrochemical oxidation were available in the literature. However, the
quality of NDs produced was not optimal (very low throughput and poor quality). It
required several other treatments to use them in biological applications. Under vis-
ible light, a perfect diamond crystal is transparent. However Nanodiamond (ND)
could display intense color (fluorescent nanodiamond (fND)) due to defects in their
crystal lattices. Those are generally substituted with nitrogen, silicon, etc. and
vacancy defects which are capable of absorbing as well as emitting visible light
(Doherty et al. 2013). Since fNDs are made of nontoxic carbon materials and pro-
cesses desired optical properties, they are envisioned as promising materials for
bio-applications. Recently, Wu et al. in 2015 revealed that even anticancer drug
doxorubicin can easily be surface functionalized with nanodiamond and thus NDs
can be used as drug delivery vehicles (Fig. 9.2).
Fig. 9.2 Schematic diagram of functionalized fluorescent nanodiamond (Jee and Lee 2009)
9.2.2 Noble Metals and Metal Oxides
Next, various synthetic schemes of noble metal (Au, Ag, Pt, Co, etc.) have received
more attention due to their escalation in usage in different applications in the last
two decades. Depending on their size and shape, they possess unique physicochemi-
cal properties. Thus, developing new and promising methods to synthesize noble
metal are of great interest to the researcher. As similar with any other nanoparticles
synthesis, noble metals were also synthesized by both “top-down” and “bottom-up”
approaches (Pareek et al. 2017). Figure 9.3 clearly showed various approaches
which was implemented for the synthesis of noble metals. For example, gold
nanoparticles can be synthesized in different shape and size, such as hollow, spheres,
rods, prisms, cages, etc (Grzelczak et al. 2008).
Similarly, metal oxides, especially magnetic iron oxides (Fe3O4), have become
interesting due to its wide application in biology. They can be used in both magnetic
separation when attached with bio-entity and act as magnetic contrasting agent
(MRI) for bioimaging. Generally, Fe3O4 was synthesized via aqueous route (Qu
et al. 2011). However, highly crystalline, monodisperse Fe3O4 nanoparticles were
synthesized via hot injection method in organic solvent (Sun and Zeng 2002).
Typically, hot injection method referred to as the preparation of monodisperse
Syntheis of
Noble metal
Nanoparticle
Top-down Bottom-up
Micro-
Milling Pyrolysis
patterning
Chemical Micro- Electro- Laser Micro- Green Biological

reduction emulsion chemical ablation wave synthesis methods
Prokaryotes Eukaryotes
(Bacteria) (Fungi, Algae,
Plants)
Fig. 9.3 Various synthetic schemes for the preparation of noble metal nanoparticles (Pareek
et al. 2017)
nanoparticles at elevated temperature (more than 200 °C) with sudden introduction

of precursors to a pre-heated organic solvent (high boiling point). This produces
tiny nucleation of the target nanoparticles, which will grow homogeneously across
all particles to obtain better monodispersity. Often, the existing solvent and/or sol-
vent mixtures act as passivating agent to impart colloidal stability. Thereafter,
researcher used the surface functionalization techniques to bring the nanoparticles
from organic medium to water for biological activity. This will be explained in
detail in later section.
9.2.3 Semiconductor Nanocrystals
Colloidal semiconductor nanocrystals (NCs), especially known as quantum dots

(QDs), are often mentioned as “artificial atoms (Yin and Alivisatos 2005), which
represent a class of materials which have been intensively investigated over the past
three decades. In general, QDs are crystalline, nanometer-sized, clusters of semi-
conductor material which can be manipulated in solution for favorable QD/solvent
interaction. Typically, they are in the size range of 1–10 nm, composed of few hun-
dred to a few thousand atoms depending on their material choices. Interestingly,
when the physical dimension of these materials become comparable to the bulk
exciton Bohr radius (as, e.g., ~5.6 nm for CdSe NC case), quantum confinement
effects become predominant. As a result, they possess discrete electronic energy
levels (Gur et al. 2005), which manifested in their size-dependent absorption and
emission behavior. Qualitatively it can be understood by considering a “particle-in-
a-box” model, where smaller dimension particles emit at higher energy (bluer
region) and larger particles show red region emission (Fig. 9.4). Thus, depending on
their size, the same QDs exhibit very narrow fluorescence at different wavelength as
depicted in Fig. 9.4. Mainly, this emission behavior is largely explored in biological
applications. The wet-chemical synthesis of QDs was pioneered by Murray et al. in
1993, where the organometallic precursors pertaining to the respective semiconduc-
tors were injected into a high boiling organic solvent at elevated temperatures
(Fig. 9.4). This yields highly monodispersed spherical-shaped nanoparticles with
better crystallinity. In general, these bare QDs exhibit lower fluorescence quantum
yield (QY) because the presence of surface related trap states essentially unlocked
fast non-radiative relaxation pathways for photoexcited carriers. To improve their
fluorescence efficiency, Hines and and Guyot-Sionnest in 1996 demonstrated a
growth of another semiconductor material of higher bandgap to effectively passiv-
ate the core surface. This pioneering method along with many other scientists essen-
tially opened the field where we observe near-explosive growth in their applications.
Since the deeper understanding in preparing better nanomaterials such as choice of
precursors, ligand used, solvent effects, etc. has evolved significantly, it is relatively
easy to create various NCs with different shape, size, and composition for tailoring
both the physical and chemical properties. To date, several semiconductor NCs have
been reported with size ranging from rods (Chakrabortty et al. 2010), tetrapods
a b
c d
Particle in box
n2h2
En =
Absorbance (arbitrary units)

8m*L2 115Å
83Å
65Å
51Å
43Å
37Å
decreasing size
32Å
23Å
~19Å
~16Å
~12Å
300 350 400 450 500 550 600 650 700 750
Wavelength (nm)
Fig. 9.4 (a) A digital image of the experimental setup for producing high-quality colloidal quan-
tum dots. (b) The size-dependent optical properties of CdSe nanocrystals. Photograph of CdSe
NCs in hexane excited with a single light source showing a wide range of colors by tuning the size
of NCs. (c) This shows the increase in spacing between the energy levels as the size of the NC
become thinner. (d) The increasing size of the CdSe nanocrystals shows red shift in their first
absorption peak, indicating the involvement of smaller bandgap. (Adapted with permission Murray
et al. 1993)
(Mishra et al. 2012), to cubes (Liu et al. 2009) (Fig. 9.5) where some of them pos-
sesses increasing QY approaching to unity. Despite preparing better quality semi-
conductor NCs in hot injection approach where organic solvent played the major
role, it always imparts challenges to utilize them with biological environment, i.e.,
aqueous medium. In contrast, a comprehensive review was recently published (Jing
et al. 2016) about how to synthesis aqueous-based better quality QDs which are dif-
ferent from conventional hot injection method. Here, the principle of solubility
products, hard/soft-acid/base (HSAB), and the effect of pH were considered as
directed influence upon the growth and kinetics of the NCs. However, it remains a
great challenge to synthesize water-based QD with narrow size distribution, batch
to batch variants, and the presence of different ions (such as hydroxyl and others)
also made it more complex. Thus, it imposes difficulties to use them for further
applications. To avoid aqueous route, there are plenty approaches in the literature
where surface functionalization strategy was adopted to make them water soluble.
In the next part of this chapter, we will discuss those in detail.
Fig. 9.5 Representative low-resolution TEM image of synthesized monodisperse (a) CdSe spheri-
cal nanocrystals, (b) CdSe seeded CdSe nanorods, and (c) CdSe cubes. (Adapted from (Mishra
et al. 2012; Liu et al. 2009), with permission from (Mishra et al. 2012; Liu et al. 2009))
9.3 Surface Functionalization Strategy
Surface modification of nanoparticles remains an elusive task as it can facilitate the

wide application of nanotechnology with the help of versatile chemistries. One must
address the issue of biocompatibility and water solubility before using them into
biological environment. Different nanomaterials require different strategies to mod-
ify their surface for making them soluble in aqueous environment and compatible
with tissues and cells. Broadly, they are divided into two methods, (i) non-covalent
approach and (ii) covalent attachment.
9.3.1 Non-covalent Surfactant Replacement
This approach has been employed when the nanoparticles is produced in nonpolar
solvent (i.e., organic) especially in the case QDs and metal oxides. To solubilized
them in aqueous buffer, the hydrophobic surface ligands need to be replaced by
either hydrophilic or amphiphilic ones. Different solubilization strategies have been
envisioned over the past two decades, including (i) encapsulation by another layer
of amphiphilic co-polymers (Gao et al. 2004), silica shells (Gerion et al. 2001), or
micelles made from phospholipids (Dubertret et al. 2002), polymer shells (Pellegrino
et al. 2004), etc. and/or (ii) complete ligand exchange with thiol-containing mole-
cules (Pathak et al. 2001; Chan and Nie 1998), peptides (Pinaud et al. 2004), den-
drons (Guo et al. 2003), oligomeric phosphine (Kim and Bawendi 2003), etc. The
pictorial representation of QD stabilization and subsequent functionalization was
depicted in Fig. 9.6. In addition, the long-term, aggregation-free usage of QDs in an
aqueous environment via suitable surface coating also represents a key challenge.
Recently, Wu et al. in 2010 have demonstrated a novel biopolymer hybrid of defined
chain length which can be used to impart colloidal stability to nanoparticles. This is
a classic example of complete ligand exchange of as-synthesized QDs, which are
Fig. 9.6 QDs solubilization and functionalization strategies; As synthesized TOPO (trioctylphos-
phine oxide)–passivated QDs can be solubilized in aqueous buffer by addition of a layer of amphi-
philic molecules containing hydrophilic and hydrophobic moieties or by exchange of TOPO with
molecules that have a coordinating end (usually a thiol group, SH) and a hydrophilic end. (Adapted
from Medintz et al. 2005 with permission)
initially prepared in organic solvent. To make the conjugate water soluble, this pep-
tide backbone has been modified in such a way that it contained poly(ethylene
oxide) side chains for better water solubility and reduction of non-specific interac-
tion and many thiol and amine anchoring moiety for better surface passivation of the
QDs. Figure 9.7 clearly depicts the schematic diagram on how the biopolymer was
transformed to an effective surface anchoring group. Furthermore, the stability of
such nanoparticle–biopolymer conjugate under various physiological conditions
such as high pH, low pH, presence of salt, high glucose, and under trypsin enzyme
was performed. In all cases, those functionalized biopolymer provided excellent
stability of the conjugate (Chakrabortty et al. 2017).
9.3.2 Covalent Attachment
Concurrently, numerous nanoparticle–bioconjugates are prepared based on stan-

dard covalent conjugation techniques where different types of cross-linking strate-
gies are employed. Depending on their combination of nanoparticles and biological
applications, either the surface of the nanoparticles or its surface-bound passivating
Fig. 9.7 Synthesis of polypeptide hybrids where the Human Serum Albumin (HSA) protein was
further functionalized with poly ethylene oxides (PEO) and thioctic acid (TA). This biopolymer
further anchored to CdSe/CdZnS QD surface through its sulfur coordination. (Adapted from Wu
et al. 2010), with permission)
ligand can directly be conjugated to the biological entity. Nanoparticles possessing

correct functional group, which can be further reacted with some other functionally
through covalent conjugation without affecting the colloidal stability of the nanopar-
ticle, will be suitable, and these strategies can be applied to the nanoparticles that
are discussed earlier in this chapter. Some comprehensive examples presented in
Fig. 9.8 (Sapsford et al. 2013) clearly demonstrated the possible chemistries which
can be very helpful. Additionally, these covalent bonds are quite stable in harsh
biological environment such as low pH, oxidation, presence of enzyme, possibilities
of displacement by other molecules, etc. In the next section, usage of these func-
tional strategies will be described for targeting diseased cell or tissues.
9.4 Targeting Capabilities
The prospect of engineering functional nanoparticles that selectively aimed to

destroy the region of interest in our body would certainly remain as challenging
concept (Peer et al. 2007; Rao et al. 2015; Min et al. 2015). This have driven
researchers to engineer plethora of different nanoparticle conjugates which exhib-
ited unique physicochemical properties with programmable biological and medical
functions (Nel et al. 2009; Albanese et al. 2012).
Ligand Substrate Ligand Attached to Substrate Reaction

O O
NP S Michael
NP SH N Addition
N
O O
O H
NP NH2 NP N
Epoxide
Opening
HO
O O
O Amidation
NP NH2 N O
NP NH
O
NP HOOC NP
NH2 NH
Amide Bond
Formation
O
H2N O
NP COOH NP Amide Bond
HN Formation
H
NP
H2NHN
NP CHO N NH Imine
O Formation
O
NP O
NP CHO H2NO N Imine
H Formation
N
N
NP N Click
NP N3 Chemistry
X H
N Addition of
NP XCN
NH2 NP NH Amine to
x = O, S Cyanates
NP Cross
NP Methathesis
Diels-Alder
NP N Reaction
NP N
Fig. 9.8 Wide examples of nanoparticle (NP) functionalization chemistries. (Adapted from
Sapsford et al. 2013 with permission)
However, the clinical translations of those conjugates are limited because those
particles face both biological and physical barriers once injected into the body. The
nature of the barriers includes protein absorption, renal clearance, phagocytic
sequestration in reticular endothelial system, flow and shear forces, diffusion, etc.
These impart a major drawback in case of administrated nanoparticles in reaching
target tissues or cells (Florence 2012; Lazarovits et al. 2015; Nichols and Bae 2012).
However, nanoparticle conjugates composed of mainly inorganic materials are
advantageous in comparison to other materials (Wilhelm et al. 2016). Firstly, they
provide higher delivery efficacy while comparing to tradition organic materials. In
addition, nanoparticles that possess hydrodynamic diameter smaller than 100 nm,
are pharmacokinetically optimum in size with better delivery efficiency. Next and
more importantly, the active targeting strategies largely outperform the passive tar-
geting approaches. In general, passive targeting is controlled by the properties of
targeted tissue such as local concentration of enzymes, pH, tissue permeability fac-
tor of drug particulate system, etc. For example, when nanoparticles are accumu-
lated in tumor tissues due to leakier vasculature and defective lymphatic drainage,
we commonly referred that as enhanced permeation and retention (EPR) effect. In
contrast, a target tissue-specific modification can easily improve better delivery effi-
ciency (Wang and Kohane 2017). As we discussed in the previous section, one can
easily incorporate the targeting moieties to the nanoparticle backbone efficiently by
covalent conjugation, and the chance of better administration of drug can easily be
manipulated with nanoparticles. To illustrate this phenomenon, Fig. 9.9 clearly
depicts the active and passive targeting strategies. Finally, the shape of the particles
Fig. 9.9 Illustration of active and passive targeting

also plays a critical role as rod-shaped nanoparticles have a tendency towards better
delivery efficiency than spherical, platelike, or other shapes. The nanorods would
penetrate through the cell membrane more efficiently than the nanospheres of the
same effective hydrodynamic size (Chauhan et al. 2011), which render them supe-
rior in both uptake and clearance mechanism from the body. This finding can also
be correlated with the findings that small enhancement in nano therapeutic penetra-
tion that may result significant improvement therapeutic effectiveness (Chauhan
et al. 2011).
9.4.1 Cellular Target
Based on current understanding, it is believed that all nano carrier systems are
focused on either non-specific endocytosis or receptor mediated endocytosis (RME).
Mostly conventional targeting ligands can be characterized into a few different cat-
egories, including small molecules, peptides, aptamers, and antibodies. The small
molecule drugs have also attracted considerable attention as potential single-multiple
targeting ligands due to their low molecular weight low production costs and adjust-
able chemistry to allow the functionalization on single nanoparticles. These ligands
are generally pre-conjugation onto an anchoring molecule followed by post-inser-
tion via physical interactions onto nanoparticle surfaces (Kamaly et al. 2012; Mishra
et al. 2010; Fang et al. 2013). A variety of ligands such as monoclonal antibodies
(mAbs), polyunsaturated fatty acids (PUFAs), folic acid, riboflavin, nicotinic acid,
transferrin, oligo peptides, hyaluronic acid, and aptamers have been exploited as the
tumor-targeting modules (TTMs) and various diseases to construct targeting drug
conjugates (Bareford and Swaan 2007; Rajendran et al. 2010). The employment of
targets which are distinctively expressed in certain organs offers the possibility to
further enhance the specificity of a treatment. Prostate-specific membrane antigen
(PSMA) is a tissue-specific receptor that has been efficiently used to target antican-
cer drug-loaded nanoparticles. The first generation of prostate-specific nanoparti-
cles incorporated PSMA-binding aptamers on their surface to promote internalization
by cancer cells. Additionally, numbers of specific targets have been investigated to
optimize the interactions of therapeutic and diagnostic nanomaterials with diseased
cells (Wang and Wang 2014). Likewise, in a mouse xenograft model, one single
intra-tumoral injection of aptamer-functionalized nanoparticles loaded with
docetaxel was able to show a considerably higher proportion of complete tumor
regression and significantly prolonged survival rates (Dhar et al. 2011). Acute
myeloid leukemia (AML) affected cells expressed CD33 surface antigen which
could be used as a cellular target to identify and destroy leukemic cells using anti-
CD33 monoclonal antibody. Folic acid, which is essential in many metabolic pro-
cesses for cell survival, has shown high specificity in recognizing folate receptors
that are overexpressed in many types of tumor cells. There are several examples of
folate-conjugated nanoparticles in drug delivery, including liposomes, polymeric
nanoparticles, and dendrimers (Pan et al. 2003). These nanoparticles have
demonstrated to be effective in treating ovarian, breast, lung, renal, and colon can-
cers, (Pan et al. 2003; Low et al. 2008) and a number of those drug conjugates are
currently in clinical and preclinical development (Xia and Low 2010; Dehaini et al.
2016). Another cancer cell-specific anti-nucleosome monoclonal antibody 2C5
(mAb 2C5), which recognizes the surface of various tumor cells via tumor cell
surface-bound nucleosomes, was also attached to polymeric micelles, making the
resulting micelles capable of specifically targeting a broad range of tumors (Torchilin
et al. 2003). However, the presence of these large biological macromolecules (Ab or
Ab fragments) can seriously compromise their circulation times in the bloodstream
and their ability to traffic to their intended destination in vivo (Simard and Leroux
2010). Therefore, large efforts have been put in the development of less immuno-
genic targeting moieties (e.g., peptides, small molecules) which might possibly
have progressive futures for in vivo applications (Veiseh et al. 2009).
9.4.2 Subcellular Target
Subcellular-specific bioimaging molecular design which can be used as either diag-

nostics or therapeutic tool have emerged as vital strategies in recent biomedical field
(Rajendran et al. 2010). In particular, for drug delivery, subcellular targeting is envi-
sioned as great prospects to not only enhance the effect of drug molecules by trans-
porting it to the targeted organelles rather potentially minimizing the side effects as
compared to nondirectional targeting (Rajendran et al. 2010). Targeted NPs can
bind to targets localized on the cell surface and enter into the cell through endocy-
tosis either receptor mediated or non-specific endocytosis. However, if the target is
located intracellular, NPs and their cargo may not be able to reach the target of inter-
est due to intracellular sequestration of the NP or due to the lack of subcellular tar-
geting abilities. For example, NPs targeted to the nucleus must enter the cell
membrane, escape endosomal/lysosomal pathways, possess a way to interact with
the nuclear pore complex, and be small enough to cross the nuclear membrane.
Targeting ligands such as the nuclear localization signal (NLS) and NPs conjugated
with receptor-mediated endocytosis (RME) peptides improved and effective deliv-
ery to the nucleus (Kang et al. 2010). Delivery to mitochondria remains most excit-
ing, and challenging subcellular target due to its biological barriers includes outer
and inner mitochondrial membrane, negative membrane potential compared to
other cellular membranes, and electrostatic interactions between the NP and mito-
chondria. Initially, most of the studies are based on liposomal NPs or metal oxide
for delivery to the mitochondria as depicted (Breunig et al. 2008; Chakrabortty et al.
2017). Advanced nanomaterial based drug delivery overcome physiochemical
obstacle (negative membrane potential and double membrane) of cell. This efficacy
is achieved by using stearyl triphenyl phosphonium (STPP) which exhibits both
cationic and lipophilic properties. STPP-targeted liposomes were fabricated with
anticancer agents such as ceramide and scarleol (Patel et al. 2010) to target the mito-
chondria. Recently, NPs synthesized with a lipophilic triphenyl phosphonium
Fig. 9.10 Confocal microscopy image of HeLa cells incubated with protein-coated fluorescent Au
clusters. Here, the protein moiety was functionalized with lipophilic cation (triphenyl phosphine).
(a) shows the emission from small Au clusters. (b) HeLa cell under white light. (c) Fluorescence
originated from commercially available mito-tracker and (d) overlay of all three images. Inset in
(d) shows the line scanning profile of fluorescent intensity (along the white arrow; green colour
from fluorescent Au and red colour from mito-tracker). (Adapted from Chakrabortty et al. 2017,
with permission)
(TPP) cation (Fig. 9.10), which is known to cross into the mitochondrial matrix
space and subregions to deliver acting therapeutics (Marrache and Dhar 2012). To
date, the number of effective subcellular targeting successfully achieved and experi-
mental and theoretical efforts has been made using nanomaterial-based targeted
delivery to the nucleus (Pouton et al. 2007), cytosol (Vasir and Labhasetwar 2007),
mitochondria (Yamada and Harashima 2008), endosomes (Rajendran et al. 2010),
and lysosomes (Lloyd 2000). Overall, the effect of varying charge, size, and target-
ing molecules of NPs have been optimized and identified through in vitro studied
which showed that the targeted NP improved the efficacy and decreased the toxicity
and obesity compared with nontargeted NPs or the individual therapeutics alone
(Dehaini et al. 2016; McNamara and Tofail 2017; Pang et al. 2017).
9.5 Therapeutic Evaluation
Over the last two decades, there is increase in the development of number of
nanoparticle-based therapeutic products by more than 150 companies for nanoscale
therapeutics. Additionally, more than 20 nanoparticles therapeutics are being cur-
rently in clinical use (presented in Table 9.1) (Bobo et al. 2016; Anselmo and
Mitragotri 2016; Weissig et al. 2014). Many nanoparticle systems have been used in
the clinic to either treat or diagnose disease. The brighter example of this progress
in the clinic is Doxil’s, a polyethylene glycol (PEG) functionalized liposomal doxo-
rubicin approval which has been made it possible by the extensive efforts of using
nanoconjugates in preclinical, commercial, and clinical studies. Furthermore, the
overall outlook of nanoparticle drug delivery systems is promising, as they are also
being developed for treating and curing not only cancer but a large number of other
diseases. Abraxane, is the pioneering example of a commercially drug-loaded
nanoparticle and consists of human serum albumin nanoparticle conjugates encap-
sulating an anticancer drug paclitaxel. It appeared on the market at the beginning of
2005, and it is now approved for different tumor types, including breast, lung, and
pancreatic cancer. More recently approved products such as Marqibo, MEPACT,
and Onivyde also have a strong presence in clinical trials. Moreover, oral delivery
of particles has been approved clinically for imaging applications (e.g., Gastromark),
local delivery of particles has been widely used in the clinic as depot delivery sys-
tems for the extended delivery of a variety of biologics including peptides and other
small molecules (e.g., DepoCyt), and topical application of particles has been
approved clinically to increase penetration of biologics across the skin barrier (e.g.,
Estrasorb). Alongside a number of nanoparticles are clinically approved as contrast
agents and widely used in imaging likewise, SonoVue are fluorocarbons or sulfur
hexafluoride encased in lipid shells, and Optison is formulated as human serum
albumin encased perflutren (Dehaini et al. 2016; McNamara and Tofail 2017; Bobo
et al. 2016; Anselmo and Mitragotri 2016; Weissig et al. 2014). Moreover nanopar-
ticles functionalized with targeting ligands are expected to improve the therapeutic
index of nanoparticle. Targeted therapeutics has better selectivity treating diseases
without affecting normal tissue. There has been increasing interest in applying
molecular targeting to nanoparticle therapeutics and formulate biofunctionalized
targeted pharmaceutics. Targeted nanoparticles have several potential advantages
such as increased uptake into target cells, more partition within target tissue, higher
therapeutic efficacy, and lower toxicity. The first targeted nanoparticle entering clin-
ical trials was MCC-465, a immunoliposomal formulation of doxorubicin conju-
gated to the F(ab)2 fragment of the human monoclonal antibody GAH (Matsumura
et al. 2004). Another immunoliposome, HER2-targeted liposomal formulation of
doxorubicin, is awaiting clinical trials (Noble et al. 2004). Nanoparticles, function-
alized with folate, had more than 6.7-fold cell uptake compared to non-targeted
nanoparticles (Kim et al. 2005), and aptamer-targeted nanoparticles had 77-fold
increase in intracellular uptake in in vitro model of prostate cancer (Farokhzad et al.
Table 9.1 list of FDA-approved nanoparticle therapies and diagnostics

Name of product/
manufacturer and Approved application/
approved year Nanopharmaceutical specification indication
Polymer nanoparticles – Synthetic polymer particles combined with drugs or biologics
Adagen® PEGylated adenosine deaminase enzyme, Adenosine deaminase
pegademase bovine improve circulation time and decreased deficiency-SCID
(Sigma-Tau immunogenicity
Pharma.) (1990)
Cimzia® PEGylated antibody fragment Fab’ Crohn’s disease, Rheumatoid
certolizumab pegol fragment of a humanized anti-TNF-alpha arthritis, Psoriatic arthritis,
(UCB) (2008, 2009, antibody, improved circulation time, ankylosing spondylitis
2013) decrease proteolysis, and greater stability
in vivo
Copaxone® Glatopa Random copolymer of L-glutamate, Multiple sclerosis (MS)
(Teva) (1996, 2014) L-alanine, L-lysine and L-tyrosine, large
amino acid-based polymer with controlled
molecular weight and clearance
characteristics
Eligard® (Tolmar) Leuprolide acetate; synthetic Advanced prostate Cancer
(2002) gonadotropin-releasing hormone (GnRH)
or luteinizing hormone-releasing hormone
(LHRH) incorporated in nanoparticles
composed of PLGH copolymer
Macugen® (Bausch PEGylated anti-VEGF aptamer (vascular Intravitreal neovascular
& Lomb) (2004) endothelial growth factor) aptamer, age-related macular
improved stability of aptamer as a result of degeneration
PEGylation
Mircera® PEGylated chemically synthesized ESA Anemia associated with
(Hoffman-La (erythropoiesis-stimulating agent), chronic kidney disease in
Roche) (2007) improved stability of aptamer as a result of adults
PEGylation
Neulasta® PEGylated filgrastim (granulocyte colony Febrile neutropenia, in
pegfilgrastim stimulating factor), improved stability of patient with nonmyeloid
(Amgen) (2002) protein through PEGylation malignancies; prophylaxis
Pegasys® PEGylated interferon alpha-2a protein, Hepatitis B and C
(Genentech) (2002) improved stability of protein through
PEGylation
PegIntron® (Merck) PEGylated IFN alpha-2A protein, Hepatitis C
(2001) improved stability of protein through
PEGylation
Renagel® Cross-linked poly(allylamine Chronic kidney disease,
[sevelamer hydrochloride); phosphate binder increase hyperphosphatemia
hydrochloride] circulation and therapeutic delivery
(Sanofi) (2000)
Somavert® (Pfizer) PEGylated human growth hormone Acromegaly
(2003) receptor antagonist, improved stability of
protein through PEGylation
(continued)
Name of product/
Oncaspar® (Enzon Polymer-protein conjugate (PEGylated Acute lymphoblastic
Pharma.) (1994) L-asparaginase), improved stability of leukemia
Krystexxa® Polymer-protein conjugate (PEGylated Chronic gout
(Horizon) (2010) porcine-like uricase), improved stability of
Plegridy® (Biogen) Polymer-protein conjugate (PEGylated Multiple sclerosis
(2014) interferon beta-1a), improved stability of
ADYNOVATE Polymer-protein conjugate (PEGylated Hemophilia
(Baxalta) (2015) factor VIII), improved stability of protein
through PEGylation
Opaxio® phase III Paclitaxel covalently linked to Glioblastoma
(2012) polyglutamate nanoparticles, passive
targeting via EPR effect
Liposome formulations combined with drugs or biologics
DaunoXome® Daunorubicin citrate encapsulated HIV-associated KS
(Galen) (1996) (non-PEGylated), passive targeting via
EPR effect increased delivery to tumor site
DepoCyt® Cytarabine encapsulated in multivesicular Lymphomatous malignant
(Sigma-Tau) (1999, liposomes; sustained release form meningitis
2007) maintains the cytotoxic concentration
of drug in the cerebrospinal fluid
and tumor site
Marqibo® (Onco Vincristine sulfate encapsulated in Philadelphia chromosome-
TCS) (2012) sphingomyelin and cholesterol liposomes, negative acute lymphoid
passive targeting via EPR effect, increased leukemia (tertiary)
delivery to tumor site
Onivyde® Irinotecan encapsulated in liposomes, Metastatic pancreatic cancer
(Merrimack) (2015) increased delivery to tumor site; lower (secondary)
systemic toxicity arising from side-effects
AmBisome® Amphotericin B encapsulated in Systemic fungal and
(Gilead Sciences) liposomes, reduced nephrotoxicity protozoal infections, visceral
(1997) leishmaniasis parasite in
SCID patient
DepoDur® (Pacira Morphine sulfate encapsulated in Opioid analgesic (post-
Pharmaceuticals) multivesicular liposomes, sustained release operative), administered into
(2004) of drug epidural space
Visudyne® (Bausch Verteporfin in liposome, increased delivery Macular degeneration, wet
and Lomb) (2000) to site of diseased vessels; photosensitive age-related; pathological
release myopia, ocular
histoplasmosis syndrome
Doxil®/Caelyx™ Doxorubicin hydrochloride encapsulated AIDS-related KS, ovarian
(Janssen) (1995, in Stealth® liposomes, passive targeting cancer, multiple myeloma
2005, 2008) via EPR effect, improved delivery to site (secondary)
of disease; decrease in systemic toxicity of
free drug
(continued)
Name of product/
Abelcet® (Sigma- Amphotericin B lipid complex, MPS Systemic fungal infections
tau) (1995, 1996) targeting selective transfer of drug from
lipid complex to fungal cell, reduced
toxicity
Curosurf® (Chiesi Liposome-proteins SP-B and SP-C, Pulmonary surfactant for
farmaceutici) (1999) increased delivery for smaller volume, respiratory distress
reduced toxicity syndrome
Surfactant-based nanoformulations
Estrasorb™ Emulsion of estradiol in soybean oil Hormone replacement
(Novavax) (2003) (micellar estradiol) controlled delivery of therapy during menopause
therapeutic
Diprivan® (1989) Oil-in-water emulsion of propofol in Sedative–hypnotic agent for
soybean oil/glycerol/egg lecithin, drug induction and maintenance
solubilization rendering drug of anesthesia
biocompatible and enhancing ease of
administration
Fungizone® (AMB) Lyophilized powder of amphotericin B Systemic fungal infections
(1966) with added sodium deoxycholate, drug
solubilization rendering drug
biocompatible and enhancing ease of
administration
Protein nanoparticles combined with drugs or biologics
Abraxane® ABI-007 Albumin-bound paclitaxel nanoparticles, Metastatic breast cancer,
(Celgene) (2005, passive targeting via EPR effect, improved non-small cell lung cancer,
2012, 2013) solubility and delivery to tumor pancreatic cancer
Ontak® (Eisai Inc) Fusion protein binds to interleukin-2 Cutaneous T-cell lymphoma,
(1999) receptor, followed by receptor-mediated CD25 positive persistent or
endocytosis; fragment A of diphtheria recurrent disease
toxin then released into cytosol where it
inhibits protein synthesis, targeted T-cell
specificity; lysosomal escape
Kadcyla® (2013) Immunoconjugate. Monoclonal antibody Metastatic breast cancer
(against human epidermal growth factor
receptor-2)–drug (DM1, a cytotoxin acting
on microtubule) conjugate, linked via
thioether
Nanocrystals
Emend® (Merck), Aprepitant as nanocrystals, surface area Antiemetic, emesis
(2003) allows faster absorption and increases
bioavailability
Tricor® & Fenofibrate as nanocrystals, fenofibrate as Hypercholesterolemia,
Triglide® (Lupin insoluble drug-delivery microparticles, hypertriglyceridemia
Atlantis), (2004) passive diffusion increases bioavailability
simplifies administration
(continued)
Name of product/
Rapamune® (Wyeth Sirolimus, increased bioavailability Immunosuppressant
Pharma.) (2000)
Megace ES® (Par Megestrol acetate as nanocrystals, Anorexic, cachexia
Pharma.) (2005) increased saturation solubility reduced
dosing
Avinza® (Pfizer) Morphine sulfate, increased drug loading Psychostimulant
(2002, 2015) and bioavailability
Focalin XR® Dexamethylphenidate HCl, increased drug Psychostimulant
(Novartis) (2005) loading and bioavailability
Ritalin LA® Metyhlphenidate HCl, increased drug Psychostimulant
(Novartis) (2002) loading and bioavailability
Zanaflex® (Acorda) Tizanidine HCl, increased drug loading Muscle relaxant
(2002) and bioavailability
Vitoss® (Stryker) Calcium phosphate, mimics bone structure Bone substitute
(2003) allowing cell adhesion and growth
Ostim® (Heraseus Hydroxyapatite, mimics bone structure Bone substitute
Kulzer) (2004) allowing cell adhesion and growth
OsSatura® (IsoTis
Orthobiologics)
(2003)
NanOss® (Rti
Surgical) (2005)
EquivaBone®
(Zimmer Biomet)
(2009)
Invega® Sustenna® Paliperidone palmitate, allows slow release Schizophrenia,
(Janssen Pharms) of injectable low solubility drugs schizoaffective disorder
(2009 & 2014)
Ryanodex® (Eagle Dantrolene sodium, faster administration Malignant hypothermia
Pharma.) (2014) at higher doses
Inorganic and metallic nanoparticles
Nanotherm® Aminosilane-coated SPION, thermal Local ablation in
(MagForce) (2010) ablation: Injecting iron oxide nanoparticles glioblastoma, prostate, and
exposed to alternating magnetic field pancreatic cancer
causing the nanoparticles to oscillate, (intratumoral)
generating heat directly within the tumor
tissue
(continued)
Name of product/
Feraheme™ Ferumoxytol SPION with polyglucose Treatment of iron deficiency
ferumoxytol sorbitol carboxymethylether, magnetite anemia in adults with CKD
(AMAG pharma) suspension allows for prolonged steady
(2009) release, MPS targeting: iron released
inside macrophages, subsequently enters
into intracellular storage iron pool, or is
transferred to plasma transferrin
Venofer® (Luitpold Iron sucrose Iron deficiency in CKD
Pharma.) (2000)
Ferrlecit® (Sanofi Sodium ferric gluconate
Avertis) (1999)
CosmoFer/INFeD®/ Iron dextran colloid
Ferrisat (Sanofi
Aventis) (1992)
DexIron®/ Iron dextran
Dexferrum® (Sanofi
Aventis), (1996)
Injectafer/Ferinject Iron carboxymaltose colloid Iron-deficient anemia
® (Vifor) (2013)
GastroMARK™; SPION coated with silicone, MPS Imaging agent (Discontinued
umirem® (AMAG targeting superparamagnetic character in 2009)
pharma.) 2001
Feridex®/Endorem SPION coated with dextran, MPS targeting Imaging agent of liver
® (AMAG pharma.) with superparamagnetic character lesions (Discontinued in
1996 2008)
Definity® (Lantheus Perflutern lipid microspheres, ultrasound Ultrasound contrast agent
med. imaging) contrast agent for liver, breast, intraocular,
(2001) pancreatic tumors and
pulmonary diseases
Optison® (GE Human serum albumin stabilizing Ultrasound contrast agent
healthcare) (1997) perflutern microsphere, ultrasound contrast for lymph node, renal cell
agent carcinoma, myocardial
infarction
Nanoparticle vaccines
Epaxal® (Crucell) Liposomes with hepatitis A virus Hepatitis A vaccine
(Discontinued)
Inflexal V ® Liposomes with trivalent-influenza Influenza vaccine
(Crucell) (Discontinued)
Source: website of FDA and pharmaceuticals companies. Partly information used with permission
from Ref. (78)
Abbreviations: EPR enhanced permeability and retention, MPS mononuclear phagocyte system,
SPION superparamagnetic iron oxide, CKD chronic kidney disease, SCID severe combined immu-
nodeficiency disease, KS Kaposi’s sarcoma, PEG polyethylene glycol, PLGH poly- (D, L-lactide
-co-glycolide)
2004). Kirpotin et al. formulated anti-HER2 MAb-liposome conjugates which had

6 times more intracellular uptake in tumor targeting (Kirpotin et al. 2006).
Biofunctionalized targeted nanoparticles also preferentially accumulate in the
tumors when compared to non-targeted nanoparticles. Kukowska-Latallo et al.
demonstrated that folate-targeted PAMAM dendritic polymers concentrated in KB
tumor xenograft in SCID mice shown to achieve greater efficacy (Kukowska-Latallo
et al. 2005). Additionally, anti-HER2 immunoliposomes encapsulating doxorubicin
in tumor xenograft models and transferrin-targeted siRNA nanoparticle is more
effective (Park et al. 2002; Bartlett et al. 2007). To date, the number of nanoparticle
therapeutics is also used as an imaging agent, and technologies that have been
approved for clinical use by the FDA in the United States, or the European Medicines
Agency (EMA) in the European Union (Table 9.1). In bioimaging NPs such as
quantum dots (QDs), gold nanoparticles, magnetic nanoparticles, and their advanced
formulations have been widely used in cellular imaging, tracking of protein in cell,
targeting of integrin αvβ3 (Cai et al. 2006), cell migration-trafficking (Chien et al.
2011), enzyme activities (Tung et al. 2000), and other biological interactions at the
molecular and cellular level (Weissleder et al. 2000; Arvizo et al. 2012; Bhattacharyya
et al. 2010). Super paramagnetic iron oxide nanoparticle (SPION), made from iron
oxide coated with dextran, is extensively used as MRI contrast agent for cancer
imaging and targeting of tumors (Ghosh et al. 2012). Additionally, QDs have been
applied for multiplex molecular imaging of lymph nodes, embryonic stem cells, as
well as tumor cells and vasculature (Kang et al. 2009). Recently, nanodiamond
coated gutta-percha, a novel thermoplastic biomaterial, has been tested for its clini-
cal potential which addresses reinfection and bone loss properties following root
canal therapy (Lee et al. 2017). In the coming years, many more biofunctionalized
targeted nanoparticles including targeted polymeric nanoparticles will be entering
clinical trials.
9.5.1 Nanoparticles as an Imaging Tool
Most frequently bioimaging modalities include magnetic resonance imaging (MRI),

computed tomography (CT), positron emission tomography (PET), and ultrasound
that are used for the diagnosis of diseases. Interestingly, these techniques are less-
invasive, higher efficacy, improved safety, and relatively lower toxicological pro-
files. Nanoparticle-based scaffold have been explored as a platform for the
development of contrast agents as well as drug delivery because of their sensitivity,
biocompatibility, tunable physiochemical properties and improved imaging time
(Sakamoto et al. 2010; Na et al. 2009). Recently, “theranostic” term refers to the
combination of therapy and disease diagnosis. Nowaday’s theranostic nanoparticle
are widely used for molecular imaging to monitor the biodistribution, accumulation
of drug at target site, and quantify the drug release after therapeutic delivery to
achieve a cellular responses associated with diseases (Zhang et al. 2012) (Table 9.1).
Additionally, it could provide key insights into the rational design of targeted
nanocarriers for personalized treatment. Likewise in a prostate cancer, a smart core–

shell quantum dots aptamer (doxorubicin QD–aptamer) was engineered to sense
drug release (Bagalkot et al. 2007). The extracellular domain of PSMA was identi-
fying by A10 RNA aptamer. The intercalation of doxorubicin within the double-
stranded “GC” dinucleotide segment of the A10 aptamer on the surface of QDs
resulted in quenching of both QD and doxorubicin fluorescence. Further, targeted
QD conjugates into PSMA-expressing prostate cancer cells after receptor-mediated
endocytosis and released doxorubicin induced the recovery of fluorescence from
both the QDs and doxorubicin. This system allowed sensing of the intracellular
release of doxorubicin and enabled the synchronous fluorescent localization and
killing of cancer cells. Additionally, drug-containing paramagnetic nanoparticles
also design to target various atherosclerotic plaque lesion components including the
αvβ3 integrin (Winter et al. 2006), fibrin (Flacke et al. 2001), and collagen type III
(Cyrus et al. 2006), allowing both targeted MR imaging and drug delivery. Dextran-
coated superparamagnetic iron oxide nanoparticles (SPIONs) are an extensively
used MRI contrast agent for cancer imaging and targeting of tumors (Ghosh et al.
2012). On the other hand, surface-enhanced Raman spectroscopy (SERS) gold
nanoparticles are used in in vivo imaging in mouse model (Maiti et al. 2012).
Magnetic NP-based imaging systems have shown potential for real-time visualiza-
tion of biological events, such as cell migration and trafficking (Chien et al. 2011),
enzyme activities (Tung et al. 2000), and other biological interactions at the molecu-
lar and cellular level (Weissleder et al. 2000; Arvizo et al. 2012; Bhattacharyya et al.
2010). Furher improvement in this field will concrete the way for detecting the
disease, targeted therapies, improve image guided surgery and overall assessment
for the response to a nanoparticle agent given as theranostic to patient.
9.6 Conclusion
This book chapter certainly provides the information about how the versatile phys-
iochemical property of nanoparticles offers ample opportunities. The tunable opti-
cal, chemical properties of QDs nanoparticles provide immense potential to
construct various nanoconjugates of different size, shape, surface charge, and func-
tional properties. The rich chemistry of the functional nanomaterials is the underly-
ing key force to synthesize different nanoconjugates with a potential of multimodal
applications. These in turn generate wide varieties of nanomaterials with differen-
tial biological properties. These broad range of physical, chemical, and biological
behavior of versatile nanocomposite have been explored as a catalyst for reaction,
semiconducting technology, electrochemical, bio analytical, imaging, carrier of
pharmaceutics, and theranostic application. However, the toxicity and pharmaco-
logical effects of these foreign nanomaterials are a concern before achieving a suc-
cessful translation in clinic. Engineering the nanoparticles with careful design can
often restructure the bio-unfriendly nanoconjugates towards bio-compatible and
bio-orthogonal construct. Given a series of nanocomposites under clinical trial and
some of them are already approved in clinic, this shows quite a bit promise for the
biocompatibility and tolerable toxicity of the nanoformulated biologics for future
application at bedside. Nonetheless, nanopharmaceuticals have mostly been used
for the cancer research application, diagnosis, and therapy, while there applications
for other disease remain elusive. Rich inherent application properties of the nano-
functionalized materials leaves hope for us to come up with more biological, chemi-
cal, physical, and optical exploration.
Acknowledgments This was partly supported by Southwest University to SB and SRM

University AP, Amaravati to SC.
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Index
A Bilginer, R., 311–337

Abdellatif, A.A.H., 73–103 Bioavailability, 2, 21, 24, 27, 31, 34–38,
Agarwal, A., 324 55–57, 74, 77, 78, 81, 88, 89, 96, 97,
Agarwal, N.B., 52 144, 191, 194, 198, 209, 225, 229, 230,
Agrawal, S., 1–58 242, 346, 367
Agulla, J., 49 Biomedical application, 75, 76, 86, 87, 94, 96,
Ahmad, H., 1–58 116, 125, 127, 128, 160, 191, 240, 249,
Akhtar, K., 115–144 275, 282, 284, 286
Akhtar, M.I., 115–144 Biomimetic gels, 275
Ali, A., 53 Biosensors, 193, 248, 283, 287, 298–300,
Almeida, A.J., 205–257 319, 321
Alzheimer’s disease, 38, 47, 54, 298 Blood–brain barrier, 18, 39, 40, 54, 57, 133
Ananda, S., 46 Bossard, F., 281
Andersen, E.S., 168 Brouzesa, E., 319
Aptamer, 168, 169, 172, 174, 213, 254, 298,
331, 360, 363, 364, 370
Araújo, R.S., 15 C
Arbab, A.S., 126 caDNAnano, 160, 170, 174, 175
Arslan Yildiz, A., 311–337 Cai, X., 55
Arya, A., 1–58 Cancer detection, 122, 129–138, 140, 144, 174
Au, S.H., 326 Caparica, R., 181–198
Ayçiçek, I., 281 Carbon dots and quantum dots ·, 348, 350
Castangia, I., 229
Cationic, 4, 15–17, 20–22, 26, 38, 44, 45, 57,
B 77, 83, 124, 163, 235, 236, 255, 278,
Babazadeh, A., 31 294–296, 361
Bana, L., 47 Çaykara, T., 281
Bangham, A., 3 Cell-based drug screening, 318–319
Beer, M.V., 281 Chakrabortty, S., 347–371
Beier, C.P., 46 Chang, M., 168
Benesch, M., 46 Chastellain, M., 126
Berry, C.C., 126 Chen, F., 126
Bhattacharya, S., 347–371 Chen, J., 281, 283
Bhise, N.S., 326, 333 Chen, P.-Y., 51
https://doi.org/10.1007/978-3-030-44925-4
378 Index
Chen, Z.-L., 47 F
Chono, S., 23 Fukuta, T., 50
Choucha-Snouber, L., 328
Chua, S.L., 46
Chung, T.-W., 288 G
Cong, Z., 288 Gaihre, B., 126
Contrast agents, 51, 94, 116–119, 121, 122, García-Astrain, C., 288
129–132, 135–142, 144, 368–370 Gómez-Lopera, S.A., 126
Conventional therapeutics, 210–211 Grosberg, A., 324, 330
Costa, P.M., 51 Grossi, G., 162
Couvreur, P., 233 Guin, D., 126
Crulhas, B.P., 298 Guyot-Sionnest, P., 353
D H
de Almeida, T.S., 181–198 Hau, P., 46
Deddens, L.H., 50 Heart-on-a chip, 322, 324, 329–332, 3385
Deligkaris, K., 275 High-throughput screening, 313, 327, 334
Demirdirek, B., 274, 288 Hines, M.A., 353
Di Legge, A., 46 Holiday junction, 161
Di Stefano, A., 48 Hou, Z., 36
Diagnostics, 57, 213, 334, 336, 348, 361, 364 Hwang, H., 49
Dilna, V., 159–175 Hydrogels, 34, 78, 84, 85, 97,
DNA, 15, 16, 19, 22, 39, 87, 125, 159–175, 245, 246, 257, 274–279,
285, 294, 298, 299 281, 284–300
bricks, 160
Douglas, S.M., 162
Drug delivery, 2, 3, 17, 19–21, 23–27, 38–46, I
48, 57, 58, 75–78, 81, 84–99, 101–103, Ibrahim, M.A., 73–103
126, 142, 161–165, 167–169, 172, 174, Improved performance and stability, 183
175, 183, 190–192, 194, 196–198, Inorganic and organic nanoparticles, 247
208–213, 215, 218–220, 224, 228, Ionic liquid and nanoparticle combined
232–234, 236–238, 241, 243–249, 257, systems, 181–198, 276
280, 285, 288, 289, 298, 300, 348, 351, Iron oxide nanoparticles, 115–144, 253, 367,
360, 361, 363, 366, 369, 371 369, 370
systems, 2, 21, 44, 57, 77, 84–86, 90, 92,
94, 95, 98, 101, 102, 162, 182, 183,
190–191, 196, 197, 211, 220, 232, J
234, 237, 241–243, 245, 247, 280, Jang, K.-J., 323
288, 289, 363 Javed, Y., 115–144
Drug discovery or screening, 313, 318, 320 Jiang, D.W., 168
Drury, J.L., 275 Jin, B., 275
Dwivedi, A. K., 1–58 Jøraholmen, M.W., 26
Ju, L., 15
Ju, R.-J., 51
E Júlio, A., 181–198
El-Toni, A.M., 93 Jung, Y.S., 288
Enhanced drug solubility and loading, 194
Environment, 10, 12, 27, 92, 98, 124, 126,
128, 163, 174, 182, 188–191, 197, 198, K
206, 236, 238, 256, 279, 281, 286, 291, Kabiri, K., 279, 281
327, 336, 354, 355, 357 Kaneda, S., 49
Ethemoglu, M., 52 Kaneko, T., 324, 329
Index 379
Katsnelson, A., 162 Manca, M.L., 24

Kaur, C.D., 21 Manorama, S.V., 126
Kawaguchi, A.T., 49 Marchi, A.N., 168
Khurana, R.K., 34 Marina, N.M., 46
Kidney-on-a-chip, 322, 326, 332–334 Maschmeyer, I., 324, 328
Kim, D.K., 126 Medical imaging, 120
Kim, M.J., 320 Medicinal application, 95
Kirpotin, D.B., 369 Meissner, R., 320
Knowlton, S., 327 Metallic-based nanoparticles,
Ko, S., 168 210, 213, 236–237
Koetting, M.C., 274 Microfabrication, 311–337
Kono, K., 19 Microfluidics, 313–318, 334
Kou, B.Q., 325 Migliore, M., 48
Kucherianu, V., 48 Missirlis, D., 85
Kujala, V.J., 324 Modi, S., 168
Kulkarni, A.D., 275 Mondini, S., 126
Kunisawa, J., 19 Mooney, D.J., 275
Morales, M., 126
Morimoto, N., 275
L Mota, A.H., 205–257
Lago, M.A., 288 Mourtas, S., 47
Laha, A., 288 Müller, R.H., 232
Le, J.V., 167 Murata, M., 23
Lee, H., 168 Mutlu, N.B., 47
Li, H., 16
Li, J., 126, 168
Li, T., 15 N
Li, W., 47 Nahar, M., 24
Lin, C., 168 Nahata, M., 46
Lind, J.U., 326 Nano-bio interfaces, 349, 351, 353
Lipidic-based nanoparticles, 210, 213 Nanocarriers, 3, 39, 44, 77, 84, 211–214,
Liposome, 1–58 218, 222, 229, 250,
Lippens, R., 46 255–257, 370
Liu, J., 288 Nanomedicine, 39, 42, 75, 89, 101, 248,
Liu, M., 162 250, 311–336
Liu, Y., 51 Nanoparticle Synthesis ·, 194, 349
Liver-on-a-chip, 322, 326, 332–334 Nanopharmaceuticals, 73–103, 159–171,
Lopes, L.Q.S, 255 181–197, 205–257, 273–300, 311–336,
Lopes, L.Q.S., 255 364, 366–368, 371
Luo, L.J., 288 Nanotechnology, 3, 26, 39, 74, 75, 78, 94–96,
160–165, 197, 209–210, 212,
250, 256, 257, 355
M Nguyen, H.T., 18
Ma, H., 34 Nishimura, T., 275
Ma, H.L., 126
Ma, X., 168
Macário, I., 52 O
Magnetic properties, 122, 127–129, 239 Olsen, D., 126
Magnetic resonance imaging, 54, 115–144, Onbas, R., 311–337
213, 239, 254, 369 Organ-on-a-chip, 313, 318, 322, 327,
Magnetic-based nanoparticles, 239–240 328, 334, 336
Maiti, K., 30 Origami, 160–162, 165–175
Maksimova, E.D., 281 Ozenler, S., 273–300
380 Index
P Single-stranded, 160, 166, 167, 169,

Pacheco-Torres, J., 51 170, 172, 174, 299
Pardoe, H., 126 Size and morphology control, 183
París, R., 281 Skin delivery, 195, 205–257
Parkinson’s disease, 38, 48, 54 Snouber, C., 323
Paul, K.G., 126 Soleimani, A., 281
Peppas, N.A., 275 Souto, E.B., 232
Perrault, S.D., 162 Spizzirri, U.G., 288
Phachonpai, W., 48 Sriraman, S.K., 20
Pharmaceutical applications, 30, 57, 75, 84, Staple strand, 160, 165, 166, 168, 170, 172
86, 101–103, 273–301, 314, Stimuli responsive hydrogels, 274, 277,
319, 334–336 279, 280
Phospholipid complexes, 27, 28, 30, 35, Stroke, 38, 39, 41, 46, 49, 50, 54–57
36, 56, 218 Sun, W., 162
Phytosome, 1–58, 191 Surface functionalization, 3, 125, 127, 349,
Pierre, M.B.R., 218 350, 353–357
Polymeric-based nanoparticles, 233–236 Surfactant-based nanoformulations,
Portnoy, E., 26 213, 240, 366
Pramod, K., 159–175
Priprem, A., 54
T
Takada, K., 241
Q Tanifum, E.A., 47
Qin, J., 53 Targeted, 3, 17–21, 38, 43–45, 47, 49–51, 57,
Qin, L., 51 76–78, 87–89, 92, 94, 98, 103, 117,
Qu, C., 50 120, 129, 134, 137, 141, 144, 162, 163,
Quan, S., 274 166, 168, 172, 175, 208, 224, 241, 285,
Quijada-Garrido, I., 281 290, 294, 297, 347–371
Targeting, 7, 8, 16, 17, 20, 22, 45, 46, 51,
73–103, 116, 118, 119, 121, 132,
R 135–137, 141, 143, 162, 168–170–174,
Regulatory considerations, 197 209, 217, 230, 235, 246, 250, 253, 284,
Reis, C.P., 181–198, 205–257 348, 357, 359–363, 366, 368–370
Relaxivity values, 117, 119, 132, 137 Tasoglu, S., 327
Robinson, R., 46 Thavanesan, T., 281
Rothemund, P., 160 Therapeutics, 1–58, 77, 82, 87, 88, 91, 95,
Rotman, M., 47 117, 118, 137, 162, 163, 168, 175, 190,
Ruel-Gariépy, E., 288 191, 205–257, 274, 275, 287–296,
300, 323–327, 347–371
Tong-un, T., 48, 53
S Touitou, E., 219
Sabu, C., 159–175 Toxicity, 2, 3, 15–17, 19, 20, 22, 25, 35, 36,
Santos-Rebelo, A., 205–257 38, 44, 45, 53, 57, 77, 86, 87, 91, 94,
Scaffold, 86, 160, 165, 166, 168, 170, 172, 97, 103, 116–118, 122, 129, 141, 144,
174, 196, 249, 285, 287, 327, 163, 166, 182, 184, 188–190, 208, 211,
332, 333, 369 212, 225, 230, 237, 239, 248, 249, 255,
Schiffelers, R., 21 256, 333, 336, 366, 370, 371
Self-assembly, 86, 160, 161, 163–166, 168, Tsang, L., 326
172, 174, 331 Tumor targeting, 14, 20, 26, 91, 121–129, 163,
Serra, L., 288 168, 287, 348, 360, 369
Setyawati, M.I., 168
Shad, N.A., 115–144
Shi, K., 51 U
Shimbo, D., 49 Uhrich, K.E., 274, 288
Shin, S.R., 325, 331 Urban, C., 46
Index 381
V Y
Van Blarcom, D.S., 275 Yan, J., 50
van Rooy, I., 45 Yang, M.M., 15
Veeti, A.T., 162 Yang, Y., 50
Vimal, S.K., 347–371 Yang, Z.Z., 47
Vindigni, G., 168, 171 Yanyu, X., 30
Yildiz, A. A., 273–300
Yildiz, B., 273–300
W Yildiz, U. H., 273–300
Wagner, S., 46 Ying, X., 52
Walsh, S., 168 Yucel, M., 273–300
Wang, H., 275, 290 Yun, X., 50
Wang, S., 249 Yurasov, V., 48
Wang, Z., 49
Wang, Z.J., 16, 49
Weltin, A., 321 Z
Willett, J.L., 281 Zappi, D., 193
Wu, T., 274 Zhang, H., 162
Wu, W., 275, 297 Zhang, J., 126
Wu, Y., 351, 355 Zhang, P., 162
Zhang, Y.-W., 281
Zhang, Y.S., 331, 332
X Zhao, Y., 49, 50
Xia, C.F., 48 Zheng, X., 47
Xiang, Y., 48 Zhou, S., 275
Xiong, M.H., 283 Zhu, H., 350
Xu, C., 15 Zhua, G., 162
Xu, H., 275, 281 Zong, T., 50
Xu, X., 350 Zou, X., 288

Nano-Pharmaceuticals: Principles and Applications Vol.1

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Environmental Chemistry for a Sustainable World 46

Vinod Kumar Yata

Organic Contaminants in Riverine and Groundwater Systems

Sustainable Agriculture Reviews

More information about this series at http://www.springer.com/series/11480

ISSN 2213-7114 ISSN 2213-7122 (electronic)

Karnal, India Vinod Kumar Yata

1 Liposomes vs Phytosomes: Principles, Methodologies,

8 On-Chip Drug Screening Technologies

Dr. Vinod Kumar Yata is an Interdisciplinary Researcher working in the National

Dr. Shivendu Ranjan has completed his BTech and PhD in Biotechnology from

Dr. Eric Lichtfouse, PhD, born in 1960, is an Environmental Chemist working at

the University of Aix-Marseille, France. He has invented carbon-13 dating, a method

Ahmed A. H. Abdellatif Department of Pharmaceutics and Industrial pharmacy,

Sabyasachi Chakrabortty Max-Planck-Institute for Polymer Research,

Sunil Kumar Vimal International Institutes for Integrative Sleep Medicine (WPI-

Hafsa Ahmad, Abhishek Arya, Satish Agrawal, and Anil Kumar Dwivedi

1.10 C NS-Based Therapies: Challenges and Interventions 38

Keywords Liposome · Cationic · Targeted · Phytosome · Phospholipid complexes

1.1 Introduction: General Concepts

Nanotechnology has emerged as a revolutionary tool capable of controlling and

1.2 Liposome: Background

1.2.1 Liposome Discovery and Description

1.2.2 Classification of Liposomes

Table 1.1 Classification of liposomes based on size and lamellarity

Fig. 1.1 Classification of liposomes based on composition and applications

1.2.3 Composition of Liposomes

Largely phospholipids can be of the following five categories:

Phospholipids from Natural Sources

Most commonly employed lipids known as phosphatidylcholine (PC) along with

Table 1.3 Ingredients used for liposome preparation

Modified Natural Phospholipids

Fully Synthetic Phospholipids

Phospholipids with Non-natural Head Groups

Description of Specific Phospholipids

Some of the important phospholipids include phosphatidylcholine (PC), phosphati-

Cholesterol is another important cell membrane component besides phospholipid,

1.3 Preparation Methods for Liposomes

Liposomes are formed spontaneously upon hydration of phospholipids. Few more

1.3.1 Hydration (by Passive Loading)

Fig. 1.2 Schematic representation of liposome preparation methodologies

Methods Based on Replacement of Organic Solvent by Aqueous Media

Ether Injection Method

Methods Based on Detergent Removal

Lipids can be solubilized by using detergents at their critical micelle concentrations

Methods Based on Size Transformation and Fusion

1.3.2 Sizing Stage

1.3.3 Removal of Non-encapsulated Material

1.4 Liposomes: Strategies and Applications

1.4.1 Formulation Strategies

suitable vectors for vaccination-based therapies for cancer. An investigation demon-

dopamine receptors on the blood–brain barrier exhibited strong anti-glioma effi-

NTF2-AlexaFluor488) were delivered with high efficiency into mammalian cell

1.4.2 Therapeutic and Clinical Applications

architecturing liposomal formulations for delivering chemotherapeutic agents in

bovine herpesvirus type 1 (BoHV-1) gD (Rodriguez et al. 2013). Ma et al. reported

Pulmonary Drug Delivery

Ocular Drug Delivery

Liposomes were investigated as carriers for Distamycin A delivery in ocular

Topical Drug Delivery

Diagnostic and Imaging

1.5 Phytosome: Background

1.5.1 Phytosome: Background, Discovery, and Description

Karnal, India Vinod Kumar Yata

1 Liposomes vs Phytosomes: Principles, Methodologies,

8 On-Chip Drug Screening Technologies

1.10 C NS-Based Therapies: Challenges and Interventions 38

1.1 Introduction: General Concepts

1.2 Liposome: Background

1.2.1 Liposome Discovery and Description

1.2.2 Classification of Liposomes

1.2.3 Composition of Liposomes

Description of Specific Phospholipids

1.3 Preparation Methods for Liposomes

1.3.1 Hydration (by Passive Loading)

Methods Based on Replacement of Organic Solvent by Aqueous Media

Methods Based on Detergent Removal

Methods Based on Size Transformation and Fusion

1.3.2 Sizing Stage

1.3.3 Removal of Non-encapsulated Material

1.4 Liposomes: Strategies and Applications

1.4.1 Formulation Strategies

1.4.2 Therapeutic and Clinical Applications

1.5 Phytosome: Background

1.5.1 Phytosome: Background, Discovery, and Description

1.5.2 Properties and Characterization of Phytosomes

1.6 Preparation Methods for Phytosomes

1.7 Therapeutic Applications of Phytosomes

1.7.2 Clinical Applications

1.8 Differences Between Liposomes and Phytosomes

1.10 CNS-Based Therapies: Challenges and Interventions

1.10.1 Drug Delivery to the Brain

Physical Barriers in Brain Targeting

Alteration of BBB in Brain Diseases

Strategies Useful for Drug Delivery to the Brain

Areas of Concern in Brain-Targeted Delivery Systems

1.10.2 Liposomal Interventions Implied in CNS Therapies

2.2 Goals of Nanoparticle-Based Drug Delivery Systems

2.3 Classification of Nanoparticles

2.3.1 Classification of Pharmaceutical Nanoparticles

Nanosized API Particles Instead

Nanoparticles/Nanosuspensions: Stability Issues