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DRUG DELIVERY ASPECTS
DRUG DELIVERY ASPECTS
EXPECTATIONS AND REALITIES OF
MULTIFUNCTIONAL DRUG
DELIVERY SYSTEMS
VOLUME 4

Edited by

RANJITA SHEGOKAR, PHD

Capnomed GmbH, Zimmern, Germany
Elsevier
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 Contributors
Hend Abd-Allah Department of Pharmaceutics
and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt
Sara M. Abdel Samie Department of Pharmaceutics
and Industrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Cairo, Egypt
Mona M.A. Abdel-Mottaleb Department of Phar-
maceutics and Industrial Pharmacy, Faculty of
Pharmacy, Ain Shams University, Cairo, Egypt;
PEPITE EA4267, Univ. Bourgogne Franche-Comt
e,
Besançon, France
Abid Riaz Ahmed Merck Healthcare KGaA,
Darmstadt, Germany
Akash Chavrasiya Department of Pharmacy, Birla
Institute of Technology and Science, Pilani,
Hyderabad, India
Yanping Chen Center of Emphasis in Infectious
Diseases, Department of Molecular and Transla-
tional Medicine, Paul L. Foster School of Medicine,
Texas Tech University Health Sciences Center El
Paso, El Paso, TX, United States
João Dias-Ferreira Department of Pharmaceutical
Technology, Faculty of Pharmacy, University of
Coimbra, Coimbra, Portugal
Diana Diaz-Ar
evalo Immunology Functional
Group, Foundation Institute of Immunology of
Colombia-FIDIC, School of Medicine and Health
Sciences, Universidad del Rosario, Bogota, D.C.,
Colombia
Sachin Dubey Formulation, Analytical and Drug
Product Development, Glenmark Pharmaceuticals,
La Chaux de Fonds, Switzerland
Alessandra Durazzo CREA-Research Centre for
Food and Nutrition, Rome, Italy
Thomas D€ urig R&D and Innovation, Ashland
Pharma and Health & Wellness, Ashland Specialty
Ingredients G.P., Wilmington, DE, United States
vii
Riham I. El-Gogary Department of Pharmaceutics
and Industrial Pharmacy, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt
Muhammad Irfan Department of Pharmaceutics,
Faculty of Pharmaceutical Sciences, GC University
Faisalabad, Faisalabad, Pakistan
Nirmal Jayabalan Department of Pharmacy, Birla
Institute of Technology and Science, Pilani,
Hyderabad, India
Anđelka B. Kova cevi
c Department of Pharmaceuti-
cal Technology, Faculty of Biological Sciences,
Institute of Pharmacy, Friedrich-Schiller University
Jena, Jena, Germany
Dharmesh Mehta Business Development, Gangwal
Chemicals, Mumbai, India
Joana Portugal Mota Lecifarma—Laboratório
Farmac^eutico, Lda, Várzea do Andrade—Cabeço
de Montachique, Lousa; CBIOS-Research Center
for Biosciences and Health Technologies, Lusófona
University, Lisbon, Portugal
Mostafa Nakach Sanofi R&D, Vitry sur Seine,
France
Maha Nasr Department of Pharmaceutics and
Industrial Pharmacy, Faculty of Pharmacy, Ain
Shams University, Cairo, Egypt
Ridahunlang Nongkhlaw Department of Phar-
macy, Birla Institute of Technology and Science,
Pilani, Hyderabad, India
Parameswar Patra Department of Pharmacy, Birla
Institute of Technology and Science, Pilani,
Hyderabad, India
Antonello Santini Department of Pharmacy,
University of Napoli “Federico II”, Napoli, Italy
Ahmad Abdul-Wahhab Shahba Kayyali Chair for
Pharmaceutical Industries, Department of Pharma-
ceutics, College of Pharmacy, King Saud Univer-
sity, Riyadh, Saudi Arabia
viii Contributors
Ranjita Shegokar Capnomed GmbH, Zimmern,
Germany
Vaibhav Sihorkar Formulations, NCE and Innova-
tion, Sai Life Sciences Limited, ICICI Knowledge
Park, Genome Valley, Hyderabad, Telangana,
India
Sarabjit Singh Formulation Research, CIPLA,
Mumbai, India
Eliana B. Souto Department of Pharmaceutical
Technology, Faculty of Pharmacy, University of
Coimbra, Coimbra; CEB—Centre of Biological
Engineering, University of Minho, Gualtar Cam-
pus, Braga, Portugal
Haiyan Wang Key Laboratory of Oral Medicine,
Guangzhou Institute of Oral Disease, Stomatology
Hospital of Guangzhou Medical University,
Guangzhou, Guangdong, People’s Republic of
China; Center of Emphasis in Infectious Diseases,
Department of Molecular and Translational Medi-
cine, Paul L. Foster School of Medicine, Texas Tech
University Health Sciences Center El Paso, El Paso,
TX, United States
Yongyong Yan Key Laboratory of Oral Medicine,
Guangzhou Institute of Oral Disease, Stomatology
Hospital of Guangzhou Medical University,
Guangzhou, Guangdong, People’s Republic of
China; Center of Emphasis in Infectious Diseases,
Department of Molecular and Translational Medi-
cine, Paul L. Foster School of Medicine, Texas Tech
University Health Sciences Center El Paso, El Paso,
TX, United States
Mingtao Zeng Center of Emphasis in Infectious
Diseases, Department of Molecular and Transla-
tional Medicine, Paul L. Foster School of Medicine,
Texas Tech University Health Sciences Center El
Paso, El Paso, TX, United States
 Preface
The book series titled Expectations and Real-
ities of Multifunctional Drug Delivery Systems
covers several important topics on drug-deliv-
ery systems, regulatory requirements, clinical
studies, intellectual properties trends, new
advances, manufacturing challenges, etc., writ-
ten by leading industry and academic experts.
Overall, the chapters published in this series
reflect the broadness of nanopharmaceuticals,
microparticles, other drug carriers, and the
importance of the respective quality, regulatory,
clinical, GMP scale-up, and regulatory registra-
tion aspects.
This series is destined to fill the knowledge
gap through information sharing and with orga-
nized research compilation between diverse
areas of pharma, medicine, clinical, regulatory
practices, and academics.
Expectations and Realities of Multifunctional
Drug Delivery Systems is divided into four
volumes:
Volume 1: Nanopharmaceuticals
Volume 2: Delivery of Drugs
Volume 3: Drug Delivery Trends
Volume 4: Drug Delivery Aspects
The specific objectives of this book series
are to:
1. provide a platform to discuss opportunities
and challenges in development of
nanomedicine and other drug-delivery
systems;
2. discuss current and future market trends;
ix
3. facilitate insight sharing within various
areas of expertise; and
4. establish collaborations between academic
scientists, and industrial and clinical
researchers.
Innovative cutting-edge developments in
micro–nanotechnology offer new ways of pre-
venting and treating diseases like cancer,
malaria, HIV/AIDS, tuberculosis, and many
more. The applications of micro–nanoparticles
in drug delivery, diagnostics, and imaging are
vast. Hence, Volume 4: Drug Delivery Aspects
in the book series mainly reviews advances in
drug delivery areas via targeted therapy with
improved drug efficiency at a lower dose, trans-
portation of the drug across physiological bar-
riers, as well as reduced drug-related toxicity.
The focus of this volume is on GMP scale-up,
regulatory, functional, and preclinical aspects
of drug delivery systems.
One of the contributions by Abdel-Mottaleb
et al. (Chapter 1) discusses new trends in drug
delivery area using hyaluronic acid, as an ingre-
dient as well as an active. The pharma industry is
always in search of new and functional ingredi-
ents. Hyaluronic represents one such example,
and is currently being explored as a drug deliv-
ery agent for a wide range of routes like nasal,
pulmonary, ophthalmic, topical, and parenteral.
The authors review its potential in wound
healing, osteoarthritis, tissue engineering, cancer
targeting, atherosclerosis, diabetes treatment,
theranostic, imaging applications, and so on.
x Preface
Chapter 2, by Shegokar, highlights basic
understandings on preclinical testing. This topic
has recently been seriously picked up by indus-
try and regulatory bodies. However, it is not
easy to get a clear view on what types of preclin-
ical strategy one must select for specific drug
delivery types. Multiple CROs (contract
research organizations) exist to support indus-
try/academics on this, but it is a time-
consuming and costly discussion. Therefore,
the focus of this chapter is to give readers a basic
understanding of preclinical phase and desired
testings, guidelines available, and an overview
of regulatory requirements, and upcoming
trends in the field.
The contribution by Ahmed et al. in
Chapter 3 describes the importance of aqueous
film coatings. The main aim of this chapter is to
provide an overview on the curing (postthermal
treatment) and storage stability of aqueous
coated dosage forms. In this chapter, a novel
approach of aqueous coating—solidification of
Self-NanoEmulsifying Drug Delivery Systems
(SNEDDS)—is discussed, which can signifi-
cantly enhance the solubility and stability of
poorly soluble drugs.
A chapter by Shegokar and Nakach
(Chapter 4) reviews industrial scale-up aspects
of the most employed technique in nanoparticle
production, milling. Very few reports are pub-
lished in literature; however, they describe the
scale-up of one particular drug delivery system.
The focus of this chapter is to give readers an in-
depth understanding of the milling technique
for production of nanoparticles, GMP settings,
regulations associated with it, and an overview
of industrial challenges.
The compilation by Sihorkar and D€ urig in
Chapter 5 aims at discussing amorphous solid
dispersion as a formulation enabling technology
to a commercially viable technology with a
plethora of marketed products across the global
pharmaceutical space. The authors discuss the
industrial perspective of ASD technology,
which has benefited immensely from newer-
generation excipients, especially polymers and
processing techniques like HME. This chapter
provides readers an in-depth understanding
of key parameters in formulating stable solid
amorphous dispersions from an industrial point
of view.
Chapter 6 by Nongkhlaw et al. highlights
opportunities and challenges in formulating bio-
pharmaceuticals. It is another trend spotted in
pharmaceutical drug delivery in addition to
nanotechnology. The authors describe in detail
technological potential, industrial advantages,
technologies available, and limitations of the
same for local (targeting brain, pulmonary, ocu-
lar, oral, etc.) and intravenous drug administra-
tion of biopharmaceuticals. At the end of the
chapter, an overview of formulation strategies
and ingredient choice is provided.
Nanotechnology is good, but to what
extent? It is slowly posing health hazards not
only though automobiles but also though food,
medicine, cosmetics, and detergents. The
topic presented by Souto et al., in Chapter 7
describes the regulatory and ethical issues in
nanoparticles, materials, and particles (NMP)
research. The chapter further outlines the pre-
sent and future of nanotechnology, and its
applications along the axis of social and ethical
concerns.
Chapter 8 by Singh and Mehta reviews steril-
ization opportunities and challenges for drug
delivery systems. The team of authors highlights
key points like the role of formulations, physical
forms, choice of sterilization technique, and reg-
ulatory requirements. An industrial perspective
is given on this topic by discussing common
mistakes and ways to overcome these.
Botulism is a paralytic disease caused by intox-
ication with neurotoxins produced by Clostridium
botulinum. Currently, vaccines and antibodies are
the only two primary means of treating botulism.
The work by Yan et al. (Chapter 9) highlights the
vaccine development strategies for botulism. This
chapter provides an overview of new vaccine and
immunotherapeutic developments like vaccine
 Preface
vector updates, immune sequence optimization,
and recombinant antibodies.
Chapter 10 by Kovacevic discusses challenges
in the delivery of nonsteroidal antiinflamma-
tory drugs (NSAIDs), and highlights the recent
advances on the use of polymeric nanoparticles,
nanoemulsions, and nanosuspensions/nano-
crystals, intended for oral, topical, parenteral,
and ocular administration to overcome the asso-
ciated challenges. These new strategies can
increase drug-associated poor aqueous solubility
and thereby reduce the dose-associated adverse
effects. An overview of various nanotechnological
approaches for delivery of antiinflammatory
drugs is provided in this chapter.
Nowadays, patients and consumers demand
safer and more natural products for regular
infections. This resulted in the evolution of
the nutraceutical market. However, many
xi
nutraceuticals have limitations like high dose,
poor bioavailability, and toxicity and stability
problems. In maximizing their therapeutic
potential to their full extent, nanotechnology
plays a key role. The last contribution by
Abdel Samie and Nasr (Chapter 11) describes
food to medicine transformation of stilbenoid
via vesicular and lipid-based nanocarriers.
In summary, I am sure this book volume
and the complete book series will provide you
great insights in areas of micro-nanomedicines,
drug delivery sciences, new trends, and regu-
latory aspects.
All the efforts of experts, scientists, and authors
are highly acknowledged for sharing their knowl-
edge, ideas, and insights about the topic.
Ranjita Shegokar, PhD
Editor
 C H A P T E R

1
Versatile hyaluronic acid nanoparticles
for improved drug delivery
Mona M.A. Abdel-Mottaleba,b, Hend Abd-Allaha,
Riham I. El-Gogarya, Maha Nasra
a
Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University,
Cairo, Egypt
b
PEPITE EA4267, Univ. Bourgogne Franche-Comt
e, Besançon, France

1 Introduction several thousand repeating disaccharides mole-

Although more than 80 years have passed since
the discovery of hyaluronic acid (HA), it still sur-
prises researchers with its unique physicochemical
properties and physiological roles in the human
body. HA was first discovered by Karl Meyer
and John Palmer in 1954 [1]. They isolated an
unknown material from the vitreous bodyof a
bovine eye, containing two sugar molecules
including “uronic acid.” So, by connecting the sub-
stitute name for the vitreous—“hyaloid”—with
the name of a component of that polysac-
charide—“uronic acid”—the name of HA was
adopted for this material. HA was first used com-
mercially as a substitute for egg white in bakery
products. Later on, its first medical application
for humans was initiated as a vitreous replacement
during eye surgery in the late 1950s [2].
HA belongs to a group of substances called
mucopolysaccharides belonging to the glycos-
aminoglycans (GAGs) family [1, 2]. HA includes
cules in the backbone. The molecular weight of
HA molecules differs owing to the variable
number of these repeating disaccharide units
in each molecule, its molecular weight ranges
from 1 to 10,000 kDa [3]. HA has an unusual
mechanism of biosynthesis and exceptional
physical properties. Sodium hyaluronate is the
predominant form of HA at physiological pH.
Sodium hyaluronate and HA are collectively
referred to as hyaluronan. Due to the fact that
HA exists as a polyanion, it can self-associate
and can also bind water molecules giving it a
stiff, viscous quality with jelly-like consistency
that causes it to behave like a lubricant [2].
HA was found to be abundantly distributed
in cellular surfaces, in the basic extracellular
substances of the connective tissues of verte-
brates, in the synovial fluid of joints, in the vitre-
ous humor of the eye, and in the tissue of the
umbilical cord; all this attracted significant
attention regarding its medical applications

Drug Delivery Aspects 1 # 2020 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-12-821222-6.00001-4
2 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
[4]. Although HA has a very simple structure,
almost everything else concerning the molecule
is unusual. Sometimes its role is mechanical and
structural, such as in synovial fluid, the vitreous
humor, or the umbilical cord. In other cases, it
can interact in low concentrations with cells to
trigger important cellular responses. HA’s char-
acteristics, including its consistency, biocom-
patibility, and hydrophilicity, have made it an
excellent moisturizer in cosmetic dermatology
and skin-care products. Moreover, its unique
viscoelasticity and limited immunogenicity
have led it to be used for viscosupplementation
in osteoarthritis treatment, as a surgical aid in
ophthalmology, and for surgical wound regen-
eration in dermatology. In addition, HA has cur-
rently been explored as a drug delivery agent for
different routes such as nasal, pulmonary, oph-
thalmic, topical, and parenteral [2]. Hence the
use of nanotechnology would combine the out-
standing properties of HA being biocompatible,
biodegradable, nontoxic, and able to bind spe-
cific receptors with the different advantages of
nanoparticles such as enhanced therapeutic
effects and targetability. In this chapter, the
use of HA nanoparticles as a versatile drug
delivery system will be discussed by highlight-
ing the different production techniques based
on the chemical and biological properties of HA.
2 Hyaluronic acid
2.1 Chemistry
The exact chemical structure of HA was
determined by Weissman and Meyer in 1954.
As already noted, HA belongs to a group of sub-
stances called mucopolysaccharides belonging
to the GAGs family. It is an unbranched nonsul-
fated GAG composed of repeating disaccharides
[β-1,4-D-glucuronic acid (known as uronic acid)
and β-1,3-N-acetyl-D-glucosamide], as shown
in Fig. 1.1. Both sugars are spatially related to
glucose in the beta configuration, thus allowing
COO– CH2OH
H H O O
O
O H
OH H H H
H OH H
H OH H N HCOCH3
Glucoronic acid N-acetyl glucosamine
FIG. 1.1 Chemical structure of hyaluronic acid unit.
all its bulky groups (the hydroxyls, the carboxy-
late moiety and the anomeric carbon on the
adjacent sugar) to be in sterically favorable equa-
torial positions, while all the small hydrogen
atoms occupy the less sterically favorable axial
positions. Thus, the structure of the disaccharide
is energetically very stable [3].
The HA backbone is stiffened in physiological
solution via a combination of internal hydrogen
bonds, interactions with solvents, and the chemi-
cal structure of the disaccharide. HA molecular
investigations suggested that the axial hydrogen
atoms form a nonpolar face (relatively hydropho-
bic) and the equatorial side chains form a more
polar face (hydrophilic) which leads to a twisted
ribbon structure for HA called a coiled
structure [4].
Owing to this conformational behavior as
well as its high molecular weight, the solutions
of HA are very viscous and elastic. At very
low concentrations, chains entangle with each
other, leading to a mild viscosity (molecular
weight dependent). However, HA solutions at
higher concentrations have a higher than expec-
ted viscosity due to greater HA chain entangle-
ment that is shear-dependent. For instance, a 1%
solution of high molecular weight HA can
behave like jelly, but when shear stress is
applied, it will easily shear thinly and can be
administered via a fine needle [2]. HA is there-
fore a “pseudo-plastic” material. This rheologi-
cal property (concentration and molecular
weight dependent) of HA solutions has made
 2 Hyaluronic acid
it ideal as a lubricant in biomedical applications.
There is evidence that hyaluronan separates
most tissue surfaces that slide along each other.
The extremely lubricious properties of hyaluro-
nan, meanwhile, have been shown to reduce
postoperative adhesion formation following
abdominal and orthopedic surgery.
HA has several interesting medical, pharma-
ceutical, food, and cosmetic uses in its naturally
occurring linear form. However, chemical mod-
ifications of the HA structure represent a strat-
egy to extend the possible applications of the
polymer, obtaining better performing products
that can satisfy specific demands and can be
characterized by a longer half-life. During the
design of novel synthetic derivatives, particular
attention is paid to avoid the loss of native HA
properties such as biocompatibility, biodegrad-
ability, and mucoadhesivity [5]. HA can be chem-
ically modified by crosslinking or conjugation
reactions. These chemical modifications mainly
involve two functional sites of the biopolymer:
the hydroxyl (probably the primary alcoholic
function of the N-acetyl-D-glucosamine) and the
carboxyl groups [6]. Furthermore, synthetic
modifications can be performed after the deace-
tylation of HAN-acetyl groups [7].
Conjugation reactions usually consist of add-
ing a monofunctional molecule onto one HA
chain by a single covalent bond, while crosslink-
ing employs polyfunctional compounds to link
together different chains of native or conjugated
HA by two or more covalent bonds. Crosslinked
hyaluronan can be prepared from native HA
(direct crosslinking) or from its conjugates.
Crosslinking is normally intended to improve
the mechanical, rheological, and swelling prop-
erties of HA and to reduce its degradation rate,
to develop derivatives with a longer residence
time in the site of application and controlled
release properties [5].
Conjugation of drugs to HA was reported as
early as 1991. This approach aimed to form a pro-
drug by covalently binding a drug to the HA
backbone through a bond that ideally should
3
be stable during the blood circulation and
promptly cleaved at a specific target site [8].
Owing to HA solubility, it is possible to perform
the reaction in water. However, in the aqueous
phase, some reactions are pH-dependent and
need to be performed in acidic or alkaline condi-
tions, which have been shown to induce signifi-
cant HA chain hydrolysis [8, 9].
2.2 Sources
HA is a natural polymer biologically synthe-
sized by cells in the body by an enzymatic pro-
cess. HA production is a unique, highly
controlled, and continuous process. Approxi-
mately half of our body’s HA is distributed in
the cutaneous region. It is produced and
secreted by cells including fibroblasts, keratino-
cytes, or chondrocytes with varying molecular
weights between 50 and 3000 kDa. The Golgi
network is the production site for most GAGs.
In tissues such as skin and cartilage where HA
comprises a large portion of the tissue mass,
HA is synthesized in large amounts. It is natu-
rally synthesized by hyaluronan synthases
(HAS1, HAS2, and HAS3), a class of integral
membrane proteins [10]. The three enzymes
are located on different chromosomes, produc-
ing HA with different molecular weights.
HAS1 and HAS2 proteins are responsible for
the synthesis of high molecular weight HA
(2  106 Da) with the latter more active catalyt-
ically than the former, whereas the enzyme
HAS3 is the most active but can only synthesize
short HA chains from 200,000 to 300,000 Da. The
different molecular weights of HA chains can
lead to different effects on cell behavior. HA per-
forms its biological actions according to two
basic mechanisms: it can act as a passive struc-
tural molecule and as a signaling molecule. Both
mechanisms of action have been shown to be
size-dependent [11].
As mentioned above, HA has an essential
functional component of almost all tissues in
the vertebrate organism. Thus, various animal
4 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
tissues, for example, in rooster combs, shark skin,
and bovine eyes have been used as sources of iso-
lation and production of high molecular weight
HA. Since HA in biological materials is usually
present in a complex linked to other biopolymers,
several separation procedures must be applied to
obtain a pure compound, such as protease diges-
tion [10]. HA was initially isolated from bovine
vitreous humor and later from rooster combs
and human umbilical cords [11]. The mean
molecular weight of the commercially available
“extractive” HA preparations obtained from ani-
mal tissues is mostly in the range from several
hundred thousand Da up to approximately 2.5
MDa [12]. However, it has been observed that
the HA products obtained from rooster combs
caused some allergic responses. Further, the tech-
nology has been developed recently toward bac-
terial fermentation to reduce the production cost
and complex purification processes. Such alter-
native sources include attenuated strains of Strep-
tococcus zooepidemicus and Streptococcusequi for
the production of HA. The bacterium secretes
the HA into fermentation broth and this behavior
is an additional advantage for isolation of the HA
directly from broth without the need for homog-
enizing bacterial cells [13, 14]. However, the risk
of mutation of the bacterial strains, and possible
co-production of various toxins, pyrogens, and
immunogens, decreases the application of fer-
mentative HA in clinical practice. This is also
why HA samples originating from rooster combs
are still currently preferred for human treatment
in cases when the HA material is designated for
injection, in the eyes or synovial joints. However,
these are also not ideal sources of HA, as all HA
products obtained from rooster combs are obli-
gated to carry warnings for those who are allergic
to avian products. Thus, alternative sources for
production of HA are presently a subject of
research [12].
One of the promising potential candidates is
a genetically modified bacterial strain, Bacillus
subtilis, carrying the A gene from Streptococcus
equisimilis encoding the enzyme HA synthase.
Such an engineered strain could produce HA
with the molecular weight in the 1 MDa range.
The advantage of using B. subtilis is that it is
easily cultivated on a large scale and does
not produce exotoxins or endotoxins, and
many products manufactured by this microor-
ganism have received a GRAS (generally rec-
ognized as safe) designation in the early
1960s. At present, microbiologically produced
HA has been approved for treatment of super-
ficial wounds as well as for use in the cosmetic
industry [12].
2.3 Physiological role
HA differs from other synthetic polymers in
that it is biologically active. Together with
HA’s outstanding viscoelastic nature, its bio-
compatibility and nonimmunogenicity have
led to its use in several clinical applications.
HA was described as an ubiquitous carbohy-
drate polymer that is part of the extracellular
matrix [15]. A human body weighing 70 kg con-
tains 15 g of HA. The greatest amount of HA is
present in the skin, followed by the synovial
fluid, the vitreous body, and the umbilical cord.
It can also be found in places where friction
occurs: the joints, tendons, sheaths, pleura, and
pericardium [16].
In the human body, HA occurs in many
diverse forms, circulating freely, decorated with
a variety of HA-binding proteins (hyaladherins),
tissue-associated, intercolated into the extra-
cellular matrix by electrostatic or covalent bind-
ing to other matrix molecules. It comprises a
major portion of the intimate glycocalyx that
surrounds all cells. HA can be tethered to cell
surfaces by any of the membrane-associated
receptors. Recent evidence indicates that HA
also exists within cells, though little is known
of the form or function of such HA [17].
HA is a major component of the synovial
fluid, and was found to increase the viscosity
of the fluid. Along with lubricin, it is one of
the fluid’s main lubricating components. It is
 2 Hyaluronic acid
considered an important component of articular
cartilage, presenting a coat around individual
chondrocytes and providing its resistance to
compression. The molecular weight (size) of
HA in cartilage decreases with age, but the
amount of it increases [18]. HA possesses a num-
ber of protective physiochemical functions that
may provide some additional chondroprotec-
tive effects in vivo, explaining its longer-term
effects on articular cartilage. HA decreases the
nerve impulses and nerve sensitivity associated
with pain. In experimental osteoarthritis, HA
has protective effects on cartilage [19]. Exoge-
nous HA enhances its synthesis together with
proteoglycan in chondrocyte, reduces the pro-
duction and activity of proinflammatory media-
tors and matrix metalloproteinases, and alters
the behavior of immune cells. These functions
are manifested by the scavenging of reactive
oxygen-derived free radicals, the inhibition of
immune complex adherence to polymorphonu-
clear cells, and the inhibition of leukocyte and
macrophage migration and aggregation [20].
A lubricating role of hyaluronan in muscular
connective tissues to enhance sliding between
adjacent tissue layers has also been suggested.
A particular type of fibroblasts, embedded in
dense fascial tissues, has been proposed as being
cells specialized for the biosynthesis of the
hyaluronan-rich matrix. Their related activity
could be involved in regulating the sliding
ability between adjacent muscular connective
tissues [18].
HA is also a major component of skin. More
than half of the total body’s HA is present in
the skin [8], where it plays a structural role that
depends on its unique hydrodynamic properties
and its interactions with other extracellular
matrices molecules (ECM) components. HA
excellent consistency and tissue-friendliness
and being one of the most hydrophilic molecules
in nature has caused it to be described as
nature’s moisturizer [4]. It gives the skin its
properties of resistance and maintenance of
the shape, and supports the preservation of
5
the natural degree of hydration of the skin cells.
Its concentration in the body tends to decrease
with aging, and a lack of it leads to a skin weak-
ness promoting the formation of wrinkles [7].
While it is abundant in extracellular matrices,
HA also contributes to tissue hydrodynamics,
movement and proliferation of cells, and partic-
ipates in a number of cell surface receptor inter-
actions, notably those including its primary
receptors, CD44 and RHAMM. Upregulation
of CD44 itself is widely accepted as a marker
of cell activation in lymphocytes. HA’s contribu-
tion to tumor growth may be due to its interac-
tion with CD44. The receptor CD44 participates
in cell adhesion interactions required by tumor
cells.
On the cellular level, HA is highly hygro-
scopic and this property is believed to be impor-
tant for modulating tissue hydration and
osmotic balance. Because of its hygroscopic
properties, hyaluronan significantly influences
hydration and the physical properties of the
extracellular matrix. Hyaluronan is also capable
of interacting with several receptors, resulting in
the activation of signaling cascades that influ-
ence cell migration, proliferation, and gene
expression [21].
2.4 Turnover and elimination pathways
The concentration of HA in the human body
varies from a high concentration of 4 g/kg in
umbilical cord, 2–4 g/L in synovial fluid, 0.2
g/kg in dermis, about 10 mg/L in thoracic
lymph, and the lowest of 0.1–0.01 mg/L in nor-
mal serum [10]. Depending on the location of
HA in the body, most of it is catabolized within
days. Studies suggested that the normal half-life
of HA varies from 1–3 weeks in inert tissues
such as cartilages, to 1–2 days in the epidermis
of skin, to 2–5 min in blood circulation. The path-
ways involved in HA catabolism include turn-
over (internalization and degradation within
tissue) and release from the tissue matrix,
6 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
drainage into the vasculature, and clearance via
lymph nodes, liver, and kidneys.
In structural tissues like bone or cartilage
with no or little lymphatic drainage, HA degra-
dation occurs in situ with other ECM such as col-
lagens and proteoglycans. On the other hand, in
skin and joints, a minimal fraction (approxi-
mately 20%–30%) of HA degrades in situ. Since
HA is restricted to the small intracellular space
of skin tissue, its half-life is slightly longer for
days and weeks [22].
3 Preparation of hyaluronic acid
nanoparticles
3.1 Conjugate formation
One of the most commonly used techniques
for the preparation of HA nanoparticles is the
preparation of HA-drug nano-conjugates by
establishing a covalent bond between the drug
of interest and HA which could improve solubil-
ity, pharmacokinetic profile, and in vivo plasma
half-life of the conjugated drugs. In most cases,
the conjugation is designed to be cleaved after
reaching the target site, and in cases of cancer,
these conjugates usually have higher accumula-
tion in the tumor sites due to the enhanced per-
meation and retention effect (EPR) [23]. Due to
the presence of multiple functional groups on
the backbone of HA like hydroxyl and carboxylic
acid groups, it was possible to get conjugated to
various compounds and macromolecules such as
paclitaxel [24, 25], sodium butyrate [26, 27], and
ovalbumin [28].
The use of HA drug conjugates is kind of con-
verting the drug into a prodrug derivative in
which the link between the HA and the drug
molecule should achieve certain criteria. The
most important is that the bond should be stable
extracellularly to give the required in vivo half-
life, and should be cleaved easily intracellularly
to achieve the desired effect. It is evident that the
release of intact drug molecule without affecting
the chemical structure is another required prop-
erty [29]. The two most common sites for the
chemical conjugation of drugs to HA are the
hydroxyl groups and the carboxylic acid func-
tionalities while the terminal aldehyde group
can only be used to prepare terminally modified
HA-ligand conjugates as well as to graft HA
oligomers to another polymer carrying amino
groups.
3.2 Self-assemblies formation
Another type of HA nanoparticles are the
ones prepared by HA association with hydro-
phobic polymers with the resulting conjugates
able to self-aggregate in the form of hydrophobic
micelles. Examples include the hydrophobic
association of HA to poly(lactic-co-glycolic acid)
(PLGA) [30], poly(ethylene glycol)-poly
(ε-caprolactone) copolymers (PEG-PCL) [31],
or tetradecylamine [32]. These amphiphilic
self-assembling HA derivatives have been pre-
pared by coupling the carboxylic groups of the
hydrophilic HA through carbodiimide chemis-
try to different hydrophobic moieties such as
PLGA [33], tetradecylamine [32], and PCL [31].
This kind of nanoparticle would combine the
hydrophobic core needed for the encapsulation
of many therapeutic agents, and the hydrophilic
shell provides longer circulation times by reduc-
ing unwanted protein adsorption [23].
Hydrophobically modified HA (HMH) was
prepared by the covalent conjugation to the
hydrophobic tetradecylamine (TDA) using
1-ethyl-3 (3-dimethylaminopropyl) carbodiimide
(EDC) and hydroxysulfosuccinimide (sulfo-
NHS). This reaction was able to produce nano
self-aggregates upon its dissolution and sonica-
tion into aqueous phosphate buffer solution [32].
The self-associated HMH nanoparticles ranged
in size between 197 and 285 nm depending on
the degree of substitution with minor influence
from the polymer concentration used. The higher
the degree of substitution, the smaller the particles
produced. Amphiphilic HA-5b-cholanic acid
 3 Preparation of hyaluronic acid nanoparticles
conjugates (HA-CA conjugates) were synthesized
by chemical conjugation of hydrophobic bile acid
(5b-cholanic acid) to the hydrophilic HA back-
bone through amide formation in the presence
of EDC and NHS. Varying the molar ratio of CA
to the carboxylic acid of HA varied the degree of
substitution from 2 to 10. For the production of
HA nanoparticles, the produced amphiphilic
HA-CA conjugates were dissolved in a phosphate
buffered saline and the solution was sonicated
using a probe-type sonification system followed
by filtration step. The produced particles ranged
in size from 237 to 424 nm [34].
3.3 Ionic nanocomplexes formation
Since HA is a polyanionic polysaccharide, it
can be easily allowed to react with cationic com-
pounds and form successful ionic complexes.
This reaction can be induced by direct interac-
tion of HA with positively charged cargo mole-
cules like in the case of DNA and plasmids, or it
can happen in the presence of another positively
charged polymer like chitosan [35]. Examples
include the ionic nanocomplex between HA
and TRAIL (Tumor necrosis factor Related Apo-
ptosis Inducing Ligand), which was highly sta-
ble and long circulating compared to the
native TRAIL [36]. Similarly, biopolymeric
amphiphiles were prepared from the EDC
mediated coupling reaction between HA and
deoxcycholic acid producing self-assembled
nanocarriers in the size range of 100–600 nm.
The strong interaction between HA and chitosan
(CS) even led to the faster release of the
entrapped negatively charged cargo molecules,
whose release could have been hindered in case
of interaction with chitosan alone. CS-HA plas-
mid nanoparticles were prepared by simple
mixing of both solutions under magnetic stirring
and DNA was then added to form complexes.
The mixture was vortexed for 3–5 s and then left
at room temperature for the complexes to form
completely [37]. It was found that the size of
the nanoparticles increased and the zeta
7
potential decreased upon increasing the ratio
of HA to CS. Similarly, HA could ionically inter-
act with positively charged metallic compounds
such as the anticancer drug cisplatin. Cisplatin-
HA nanoparticles were prepared by simple
mixing of HA and the drug, forming nano-
particles in the size range of 80–160 nm. Being
large in amount, the release of the drug from
the particle matrix was accompanied by dis-
integration of the particles [38].
3.4 Nanogels formation
HA nanogels can be prepared by either phys-
ical or chemical crosslinking of HA to provide
colloidal stable particles in the micro or nano
range. The physical crosslinking would depend
on noncovalent attractive forces between the
polymer chains such as hydrophobic interac-
tions, hydrogen bonding and ionic interactions.
However, chemical crosslinking would provide
particles of higher stability and subsequently
longer half-lives. To prepare chemically cross-
linked nanogel particles, it is preferable to
spatially localize the HA molecules and crosslin-
kers in very small volumes to achieve the
required reaction in the nano range. This could
be successfully achieved using the inverse
w/o microemulsion technique, which is
described in Fig. 1.2 [29]. Hyaluronan micro-
spheres were prepared by the surfactant-aided
homogenization of HA aqueous solutions and
crosslinker in mineral oil. This step is followed
by the initiation of crosslinking by adding
EDC [39]. Other examples of nanogel particles
formation by amidation crosslinking were
described in the literature [40, 41]. However, this
microemulsion chemical crosslinking tech-
niques demands high energy mechanical stir-
ring or ultrasonication and the use of organic
solvents, which are not considered favorable
conditions for the labile molecules such as pro-
teins and nucleic acids. Physical crosslinking is
considered to be much milder in terms of affect-
ing the labile structures of drug molecules [29].
8 1. Versatile hyaluronic acid nanoparticles for improved drug delivery

Crosslinker or
coupling agent

HA and Aqueous
drug phase
Surfactant Organic
phase HA

Nano
gel
= Crosslinks
= Drug

FIG. 1.2 Inverse phase water in oil microemulsion technique for the preparation of chemically crosslinked HA nanogels.
Reprinted with permission from Ossipov DA. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin
Drug Deliv 2010;7:681–703. https://doi.org/10.1517/17425241003730399 and Taylor and Francis Ltd.

4 Applications of HA nanoparticles
4.1 Skin applications
Although HA is present in most of the bio-
logical fluids and tissues and extracellular
matrix of soft connective tissues, skin is consid-
ered to be most HA-abundant tissue in the
human body. Upon aging, especially after the
age of 20, skin content of HA continuously
decreases [42].
The unique properties of HA, including its
biocompatibility, biodegradability, viscoelastic-
ity, and nonimmunogenicity, have made it an
ideal material for cosmetic and biomedical
applications. It exerts a hydrating effect on the
skin, which may help to enhance the penetration
of different drugs through the skin. However, its
own penetration is very limited due to the high
molecular weight as well as the enzymatic
degradation risk. It was reported that cross-
linked HA proved to permeate through the skin
deeper layers making it suitable carrier for trans-
dermal applications [42]. HA itself as macro-
molecule is not able to penetrate the skin
beyond the surface layers due to the strong bar-
rier properties [43]. HA nanoparticles prepared
by the anionic interaction with the cationic poly-
mer protamine were able to penetrate the skin
and deliver the HA to the dermis while free
HA penetrated no further the stratum corneum.
Therefore, these nanoparticles were considered
promising for the effective skin delivery of HA
to contribute to barrier recovery following UV
irradiation [44]. Similarly, nanoparticles of qua-
ternized cyclodextrin-grafted chitosan associ-
ated with HA have been also proven
promising for cosmetics and skin hydration
applications. Their skin hydrating ability as well
as their safety on human skin fibroblasts were
demonstrated in vitro [45].
Besides the use of nanoparticles for the drug
delivery purposes, some cosmetic applications
 4 Applications of HA nanoparticles
have been proposed. Preparations of slightly
crosslinked HA are used as fillers for augmenta-
tion, to fill facial wrinkles and depressed scars.
Such HA gels are more effective in maintaining
cosmetic corrections than collagen-based prod-
ucts. Unlike collagen-based fillers, HA is
extremely elastic, providing the elasticity
required by spaces in which it is injected and
the hyaluronate preparations are more sus-
tained. Examples of the use of HA NPs for cos-
metic applications are described in Table 1.1.
4.2 Osteoarthritis
The use of intra-articular HA injections as a
viscosupplement to restore the normal viscosity
of synovial fluid in osteoarthritis patients is a
well-established therapeutic strategy [11].
Cationic polymeric nanoparticles linked to hya-
luronate proved to be effective in the production
of ionically crosslinked hydrogels in situ to
increase the retention time of a model drug in
the synovial cavity [50].
Gene therapy has been proposed as a treat-
ment modality for targeting specific pathological
TABLE 1.1 Cosmetic applications of HA and HA NPs.
Drug HA nanoparticles
HA Polyion complex formation with the
cationic polymer Protamine
HA Polyion complex formation with
quaternized cyclodextrin-grafted
chitosan
HA HA/lysine NPs by ionic interaction
between HA and lysine
Vitamin E HA-lipid nanoparticles with ethanol
injection technique
Lutin Chitin nanofibril-hyaluronan
nanoparticles by ionic gelation
Vitamin E HA-based nanoemulsion
9
mechanisms and hence helping to treat the
underlying disease origin. HA-CS-plasmid
nanoparticles were prepared as novel nonviral
gene delivery vector for the treatment of osteoar-
thritis. They utilize the ability of HA to bind to the
CD44 to be internalized by the targeted cells by
the endocytosis pathway. The transfection effi-
ciency of these NPs was found to be superior to
the CS-plasmid NPS, suggesting them as a safe
and effective nonviral gene delivery vector to
chondrocytes [37].
4.3 Tissue engineering
HA as one of the main components of body
tissues has frequently been investigated for
tissue engineering applications. HA-based
sheets serve as a matrix for soft tissue, cartilage,
bone, and skin growth, and as a substrate for
tissue regeneration and remodeling. Three-
dimensional scaffolds of HA-based materials
can facilitate restructuring of tissues and assist
in regaining function. These materials are ideal
for tissue reconstruction, as there is no host
immune response, and are particularly useful
Particle
size (nm) Application Reference
100 Dermal HA delivery to restore the skin [44]
barrier properties in damaged skin
235 Enhanced antiwrinkle effects and [45]
moisturizing properties
134 Topical dermal filler [46]
200 Wound healing and localized delivery [47]
for cellular skin layers regeneration
40–200 Antiaging [48]
59 Skin care and enhancement of drug [49]
percutaneous absorption
10 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
for burn and trauma patients. Stem cells require
an HA-rich environment for maintaining the
undifferentiated state. Vascular endothelial cells
can be selected, as well as aortic smooth muscle
cells for the construction of heart valves, by
seeding onto HA sheets and membranes. How-
ever, due to HA’s high solubility and fast elim-
ination, its use for scaffold fabrication and
structural stability has been challenging. To
overcome these limitations, modification and
crosslinking of HA have been proposed. Various
examples on the use of crosslinked HA nanopar-
ticles for drug delivery are presented in
Table 1.2. Water-soluble carbodiimide crosslink-
ing, polyvalent hyadrazide crosslinking, and
other techniques have been introduced for tissue
engineering applications of HA. Chemical cross-
linking is expected to extend the HA degra-
dation process in vivo and provide long-term
stability for the various applications in orthope-
dics, cardiovascular medicine, and dermatology.
Elsewhere, photocrosslinked HA hydrogels
have been also introduced for different applica-
tions such as cartilage tissue engineering, cardiac
repair, molecule delivery, valvular engineering,
control of stem cell behavior, and microdevices
[10]. HA as a natural polymer has been mixed
with poly(lactic-co-glycolic acid) nanoparticles
to develop an in situ crosslinkable system with
drug delivery potential. Although such a system
has shown favorable mechanical properties for
tissue engineering, its biocompatibility and tox-
icity in vivo is still a major concern [64].
4.4 Cancer targeting
Self-assembled polymeric nanoparticles have
been investigated for cancer therapy due to their
ability to encapsulate the chemotherapeutic
agents and release them on a sustained manner.
This is even enhanced by rendering their surface
hydrophilic which would enhance their circula-
tion time leading to higher accumulation into
the tumor tissue with the known EPR effect of
nanoparticles [34, 65]. In addition to this passive
targeting strategy, active targeting can be also
achieved by binding these NPs to targeting
moieties to recognize and bind to the tumor cells
and being internalized by receptor-mediated
endocytosis. Since HA is distinguished by its
ability to bind to various cancer cells that
overexpress CD44, it has been conjugated to
various drug loaded nanoparticles as a targeting
moiety [66]. CD44 is overexpressed in many can-
cers of epithelial origin and therefore the use of
HA nanocarriers could increase the targetability
and the retention into the cancer tissue [66].
However, the use of HA nanoparticles as a tar-
geting moiety and drug carrier is a new trend,
and extensive studies are needed to understand
the factors affecting the affinity to CD44 and the
internalization mechanisms of HA nanoparti-
cles. It was found that the slow CD44 represen-
tation (24–48 h) led to limited availability of HA
internalization receptors. Therefore, a higher
affinity nanoparticle and a higher degree of clus-
tering would lead to a lower number of internal-
ized particles. On the other hand, lower affinity
systems might lead to less clustering with more
efficient HA-mediated delivery of drug pay-
loads [67]. Amphiphilic HA-CA conjugates
nanoparticles, which self-assemble into hydro-
phobic core nanosized particles, surrounded
by a hydrophilic HA shell efficiently accumulate
into the tumor site compared to the pure water-
soluble HA after systemic administration. The in
vivo biodistribution of these NPs in tumor-
bearing mice was investigated using a noninva-
sive near-infrared optical imaging technique,
which revealed a significant accumulation of
the HA NPs in the tumor site. The accumulation
was much stronger than in normal tissues and
was size dependent, which embraces the role
of the EPR passive targeting pathway. However,
when animals were preinjected with high doses
of HA polymer before the injection of HA NPs,
the NPs accumulation in the tumor site was
remarkably attenuated, suggesting that the
interaction between the HA from the NPs to
the CD44 receptors on the cancer cell surface is
 4 Applications of HA nanoparticles 11

TABLE 1.2 Various applications of HA NPs in drug delivery.

Particle
size
Drug HA nanoparticles (nm) Application Reference

Plasmid DNA Polyion complex of HA and chitosan 100–300 Nonviral vector for gene [37]
and plasmid DNA delivery for chondrocytes

siRNA Inverse w/o emulsion (nanogel) 200–500 Selective targeting of HCT-116 cells [51]
Plasmid DNA Dihydrazide mediated crosslinking 5–20 μm Controlled rate DNA delivery [39]
Doxorubicin HA-PEG-PLGA polymeric 93–186 Selective tumor targeting [33]
nanoparticles by nanoprecipitation
HA Self-assemblies of hydrophobic 350–400 Tumor targeting [52]
5β-cholanic acid-HA
HA Self-assemblies of hydrophobic 237–424 Passive and active tumor targeting [34]
5β-cholanic acid-HA
Doxorubicin Bioreducible core-crosslinked 148 Tumor targeting [53]
polymeric micelle based on
hyaluronic acid
Cy5.5 and Self-assemblies of amphiphilic 200 Theranostic system for cancer [54]
doxorubicin iodinated hyaluronic acid
Paclitaxel Hyaluronate-cholanic acid micelles 258 Targeting CD44 overexpression in [55]
cancer cells
Cy5.5 (PEG)-conjugated self-assembled HA 217–269 Cancer therapy and diagnosis [56]
nanoparticles
Cy5.5 Self-assemblies of hydrophobic 237–424 Targeting stabilin-2 and CD44 [57]
5β-cholanic acid-HA receptors overexpressed in
atherosclerosis
89
Cy7 or Zr EDC and sulfo-NHS crosslinked HA 90 Theranostic application in [58]
and cholanic acid atherosclerosis

pEGFP or pβ-gal Hyaluronic acid-chitosan ionotropic 100–235 Gene transfer and targeting to ocular [59]
as model plasmid gelation cells
Dexamethasone HA-chitosan nanoparticles – Enhanced ocular bioavailability [60]

Insulin Reverse-emulsion-freeze-drying 182 Oral insulin delivery [61]

Perfluoropentane Oil-in-water (O/W) emulsification 350 Ultra-long-acting, liver-specific, [62]
Ultrasound contrast agent
CuS and Cy5.5 EDC and sulfo-NHS crosslinked HA 227 Image-guided photothermal [63]
and cholanic acid therapy of cancer
12 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
also responsible for the high accumulation on
the tumor site [34, 52]. The use of core cross-
linked HA micelles prepared with a simple
method of disulfide bond formation loaded with
doxorubicin has shown an enhanced therapeutic
efficiency and tumor accumulation as well as
improved stability in vivo compared to the
uncrosslinked micelles and the free drug. The
superior activity was correlated to the ability
of these carriers to unload the drug inside the
tumor cell only with the micellar structure disso-
ciated in response to the glutathione at the intra-
cellular level. 2,3,5-Triiodobenzoic acid (TIBA)
was conjugated to an HA oligomer as a
computed tomography (CT) imaging modality
and a hydrophobic residue and self-woven
nano-assemblies were produced for the tumor-
targeted delivery of doxorubicinm which
presented a promising theranostic system for
cancer diagnosis and therapy of tumors that
express CD44 receptors [54]. Polymeric nano-
particulate micelles of HA-CA paclitaxel were
found to be specific and efficient chemothera-
peutic treatment for CD44 overexpressing
tumors and cancer cells [55]. However, a major
drawback of HA-based conjugates or nanoparti-
cles for cancer targeting is their preferential
accumulation in the liver after systemic admin-
istration. PEGylated HA-NPs formed self-
assembled nanoparticles in the size range of
217–269 nm of improved cancer accumulation
and targetability compared to HA-NPs when
tested in tumor-bearing mice [56].
4.5 Atherosclerosis
The use of HA-NPs has been proposed as a
potential tool for both diagnostic and thera-
peutic applications in atherosclerosis. A major
observation in the pathogenic process of athero-
sclerosis is the overexpression of receptors of
HA such as stabilin-2 and CD44. Selective stron-
ger accumulation of HA-NPs in atherosclerotic
lesions was observed, which was probably
explained by an active targeting mechanism
after systemic administration [57]. Amine func-
tionalized oligometric hyaluronan conjugated
with cholanic ester and labeled with fluorescent
or radioactive nanoparticles was tested for
targeting atherosclerosis associated inflam-
mation. The 90 nm particles accumulation was
30% higher in atherosclerotic aortas than wild
type controls. The plaques treated with the HA
nanoparticles contained 30% fewer macro-
phages compared to control and free HA treated
groups. Therefore, these nanoparticles were
proposed for PET imaging of atherosclerosis-
associated inflammation due to their favorable
targeting potential [58].
4.6 Ocular drug delivery
Due to the strong defensive mechanisms of the
eye, the transport of drugs via topical instillation
in the eyes is limited, causing restricted bio-
availability. The use of hyaluronan-coated chito-
san nanoparticles was investigated for the
enhanced ocular delivery of dexamethasone.
The HA-coated nanoparticles have shown
2.14-fold higher AUC0-24h compared to dexa-
methasone solution, which was explained by
the prolonged precorneal retention caused by
the highly mucoadhesive HA [60]. HA-chitosan
nanoparticles with sizes between 100 and 235
nm were also tested for ocular gene delivery
and were able to achieve high transfection
efficiency without affecting cell viability [59].
HA is also particularly useful as a space-filling
matrix in the eye to maintain the shape of
the anterior chamber during surgeries. HA solu-
tions also serve as a viscosity-enhancing compo-
nent of eye drops and as an adjuvant to eye tissue
repair.
4.7 Insulin sensitivity and diabetes
Recently, it has been demonstrated that CD44
receptors in pro-inflammatory cells in obese
adipose tissues are involved in the development
of adipose tissue inflammation and insulin
 4 Applications of HA nanoparticles
resistance in type 2 diabetes patients. Therefore,
empty HA nanoparticles were used as a thera-
peutic tool for adipose tissue inflammation and
insulin resistance by selectively accumulating
and clustering the CD44 receptors in inhibiting
the interaction of the low molecular weight HA
with these receptors, leading to improved insulin
sensitivity and glycemic control. The HA nano-
particles are hence proposed as a therapeutic
agent in the treatment of type 2 diabetes patients
[68]. HA nanoparticles have also been proposed
for the oral delivery of insulin as an alternative
for insulin injections for the treatment of diabe-
tes. Insulin-loaded HA nanoparticles were pre-
pared by a reverse emulsion freeze-drying
method in sizes of approximately 180 nm. The
particles produced had very high entrapment
efficiency up to 95%. These pH-sensitive nano-
particles provided the required protection for
insulin from the acidic environment of the stom-
ach and at the same time had no effect on the junc-
tion integrity of epithelial cells, which is an
important parameter to consider in case of
chronic use of medicaments, which is the usual
case of insulin. The presence of HA enhanced
the transcellular efflux of insulin transported
through Caco-2 cell monolayers via the transcel-
lular pathways. The results of permeability
through a rat small intestine showed that insulin
transport through the duodenum and ileum was
enhanced, and the therapeutic efficiency of the
produced nanoparticles was also proved in a dia-
betic rats model [61].
4.8 Theranostic and imaging
For higher diagnostic procedures’ efficiency
for different diseases, there is a great need for
instant real-time imaging techniques that could
favorably target certain organs or tissue types.
One of the most commonly used imaging tech-
niques in medical fields is ultrasound imaging.
New echogenic HA nanoparticles have been
developed and presented as an ultra-long act-
ing, liver-specific ultrasound contrast agent.
13
The particles were prepared with the oil in a
water emulsification method and contained per-
fluoropentane as an ultrasound gas precursor.
The particles were more stable compared to the
conventional microbubbles used for ultrasound
(US) imaging. Their long circulating properties
allowed for several systemic circulations fol-
lowed by intense accumulation at the liver,
which embraced their potential as a targeting
imaging system for diagnosis of liver diseases.
The targetability to the liver was related to both
the small hydrodynamic size of the particles as
well as the high affinity of HA to the liver. More
interestingly, the prepared particles were able to
discriminate between the normal liver tissues
and liver cancer in a liver tumor-bearing mice
model. The cancerous tissue was found to be
more compact than the normal tissue, and hence
lower US signals were observed [62].
Iron oxide-based magnetic nanoparticles
bearing HA on the surface have been developed
to target activated macrophages for imaging and
therapeutic applications in inflammatory dis-
eases. The particles were prepared by
co-precipitation procedures followed by post-
synthetic functionalization with both HA and
fluorescein. Particles expressed a significant bio-
compatibility and stability in serum. Significant
uptake by activated macrophages was observed,
which was HA dependent. In addition, the mag-
netic core of the particles was found to be only
transiently present in the cells, which indicates
lower risks of toxicity. Fluorescein was found
to be successfully delivered to the cellular nuclei,
which showed the potential of using these nano-
carriers also as a targeted drug delivery and
molecular imaging [69]. HA nanoparticles were
synthesized and labeled with the near infrared
dye Cys5.5 for imaging purposes. For using the
same particles but for photothermal purposes,
an additional step of loading the particles with
CuS was performed. The obtained particles had
a size range of around 200 nm. The fluorescent
signal of Cys5.5 was quenched by the presence
of CuS. When the enzyme hyaluronidase was
14 1. Versatile hyaluronic acid nanoparticles for improved drug delivery
added to the particles, the fluorescence signal
was restored in a time-dependent manner, which
suggested that the particles could be used as a
nanoprobe to be activated by the highly overex-
pressed hyaluronidase in the cancerous tumor
areas. Therefore, the CuS-loaded HA nanopar-
ticles were used for photoacoustic imaging,
utilizing the strong absorbance of the CuS. After
intravenous administration, the particles accu-
mulated in the tumor area over time, and when
irradiated with a laser, a good tumor inhibition
rate of 89.74% was observed on day 5, indicating
the great potential of these nanoparticles for
theranostic applications [63].
5 Clinical status
As already discussed, HA seems to be a very
promising molecule for utilization as a vehicle or
as an active ingredient for many drug delivery
purposes. The use of HA as a dermal filler and
for intra-articular delivery is already well estab-
lished, and several products have been proved
safe and approved by the FDA for clinical use
[70]. However, most of the scientific research
conducted on the use of HA and HA NPs for
clinical applications in other areas is still in the
phase of laboratory research and preclinical
evaluation. This may be due to the consideration
of HA as a new chemical entity every time it is
linked or conjugated to a drug or any molecules
for changing their properties. Due to the com-
plex processes involved in such reactions and
the various factors to be studied, HA and its
derivatives are also difficult to industrialize [1].
However, several products have reached phase
II and phase III clinical trials. Alchemica oncol-
ogy in Australia have produced several
HA-based systems for the management of
cancer such as HA-irinotecan, HA-DOX, and
HA-5FU. A phase I clinical trial was conducted
on 12 patients, and HA-Irinotecan has proved
to be safe and well tolerated while preserving
the anticancer activity of irinotecan [23]. Another
phase II trial was done on 41 patients, and
showed the advantages of the HA nano formula-
tions in terms of progression-free survival and
safety [71]. Unfortunately, phase III clinical trials
did not achieve the expected results and the
reason for this is not yet clear [1]. Therefore,
the industrialization and widespread clinical
utilization of HA and HA NPs as drug carriers
are still rich areas that require more extensive
research and have a long way to go.
6 Conclusion
HA is one of the most important natural com-
ponents of human tissues, making it a biocom-
patible, biodegradable, and promising carrier
for various drug delivery applications. The use
of nanotechnology has enhanced to a great
extent the benefits achieved from this biomate-
rial. This could be by using systems that can
actively target certain tissues or achieve
enhanced penetration or permeation through
certain body barriers. Future studies are needed
to enhance the fabrication techniques and
ensure the required efficacy and safety of the
produced nanosystems.
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 C H A P T E R
2
Preclinical testing—Understanding
the basics first
Ranjita Shegokar
Capnomed GmbH, Zimmern, Germany
1 Introduction
Preclinical testing or assessment is the interlink
between drug discovery (i.e., bench—in vitro, in
silico, in vivo—research and development, drug
candidate selection) and clinical testing and ulti-
mate availability of the drug product to the
patient (i.e., bedside). It takes for a drug at least
12–15years of development cycle to go from
lab to clinics. Huge amounts of data about a
product’s performance, stability, effectiveness,
and safety in animals/humans are generated
throughout the complete cycle. It is not only
about years, but also about compilation of
experts’ knowledge from diverse competences
such as chemists, biologists, pharmacists,
regulatory scientists, toxicologists, statisticians,
veterinarians, patent lawyers, clinicians, infor-
matics, etc. The average cost of bringing a drug
from the lab to market is around USD $1–3
billion, depending upon the nature of the drug
(small vs large molecules). The high attrition
rate in drug development cycles also drives up
the costs of medications that reach the market [1].
The typical journey of a chemical entity to
pharma product (Fig. 2.1) is lengthy and spread
Drug Delivery Aspects 19
https://doi.org/10.1016/B978-0-12-821222-6.00002-6
over several years. It starts with synthesis of the
chemical entity for specific biological product
and ends with a “dosage form” (tablet/capsule/
injectable, etc.) for “specific indication” in
patients. In API industry, mostly IND candidate
testing is extended from synthesis screening until
Phase 1 testing, while generic drugs have a
quicker route via bioequivalence studies. The
synthesis step involves standardized SOPs to
GLP R&D practices. However, preclinical and
Phase 1 are strictly done as per GLP and GCP
guidelines, respectively. At the end, commercial
manufacturing is performed as per GMP guide-
lines. On the other hand, pharma companies
may start with either their own IND or IND
supplied from an API manufacturer. Multiple
in vitro, in vivo, and in silico screening assays
and testing are performed during preclinical as
per GLP guidelines. Depending upon product
strategy, the formulation phase may start early
or later during this stage, and sometimes last until
Phase II. Phase III always tests a final formulation
for one specific indication.
The process starts with identification of a
“biological target” using bioinformatic and phe-
notype databases for that particular disease
# 2020 Elsevier Inc. All rights reserved.
20 2. Preclinical testing—Understanding the basics first

FIG. 2.1 Exhaustive journey of drug candidate from lab to clinic (**the numbers are based on literature information, as the
reported numbers are very diverse, hence the highest number is presented). The color intensities of bars indicate potency of
practices from high (dark color) to low (neutral color).

condition. Once the target is identified, chemical
agents (called active pharmaceutical ingredients
or APIs) are synthesized or obtained by other
suitable high-throughput screening of method.
These compounds and their modified versions
are then tested for their effectiveness to the
selected biological target by using various
in vitro, ex vivo, and in vivo models. A further
choice of optimal performing API is made, and
later this new drug candidate is registered as
an investigational new drug (IND) and subse-
quently tested in various clinical trials in its final
dosage form (Phases I–IV, fewer patients to
more patients) to evaluate their safety, perfor-
mance, dose determination, etc.
As the IND is initially tested in humans in
Phase I studies, generally a small population is
selected (10–15 healthy humans). In this phase,
mainly pharmacokinetic data on IND are col-
lected by administrating a single subtherapeutic
dose specifically to evaluate whether the
performance of the IND is comparable to pre-
clinical data. Once the expected data are
obtained, the candidate is tested for safety and
tolerance (20–100 healthy humans). Various
parameters like maximum tolerated dose/esca-
lated dose, relevant and serious adverse effects
(AEs), and pharmacological, pharmacody-
namic, and pharmacokinetic parameters are
evaluated in Phase I. Depending upon the focus
on disease and market need, multiple INDs or a
single one enter Phase I for initial screening.
In Phase II, the therapeutic efficacy of a chem-
ical entity is evaluated in several hundred
patients. The study protocol agreed among
experts and regulatory clearly defines the selec-
tion of population especially inclusion and exclu-
sion criteria, dose selected, and AEs. Beside the
IND’s biological therapeutic efficacy, response
to the IND vs. placebo and before/after treat-
ment data are evaluated in Phase II. It is a deter-
mining clinical phase to assess the safety of the
 1 Introduction
drug in the complete cycle and to obtain prelim-
inary data on the IND’s effectiveness, although it
is not comprehensive enough to provide suffi-
cient evidence across a wide population.
Phase III studies are mainly performed to con-
firm further therapeutic benefits and safety of the
IND in the indication of choice and involves
usually up to 10,000 subjects. The population
size of Phase III varies depending upon the
indication, e.g., in case of platin resistant ovarian
cancer on an average between 100–500 patients
are recruited. Depending upon the scope and
effect of different dosages, combinations with
other therapeutic agents are investigated with
the aim of gathering information on indications
and contraindications over a longer period of
time compared to Phases I and II (Fig. 2.2).
Phase IV clinical studies occur after market-
ing approval, and are generally lengthy. Patient
information across different regions/continents
is collected and analyzed. The cohort of study
focus is broad (10,000 patient population). The
main aim of this phase is to gather additional
data on safety, efficacy, and uses in other indica-
tions. Phase IV studies can bring both positive
and negative outcomes, e.g., finding effective-
ness of a drug in new indications, thereby
opening new market opportunity (positive out-
come) finding special AEs in wide population
FIG. 2.2 Scope and duration of clinical trial phases.
21
may restrict use of IND only for specific indica-
tion (negative outcome) [2–4].
The budgeting on development costs at each
phase is generally based on a bottom-up analysis
of historical clinical trial costs. The cost may vary
depending upon the type of clinical trial, number
of patients, type of archetype, type of study
design, number of clinical centers, CRO (high to
low rating), location of clinical sites, and proce-
dure implemented. R&D and other costs like
basic research through lead optimization, chemis-
try, manufacturing, and controls (CMC), good
manufacturing practice (GMP), manufacturing
build-up and scale-up costs, regulatory or regis-
tration fees (post-Phase III), and all postmarket
commitments (e.g., Phase IV pharmacovigilance
studies) are on the top of this. A research paper
by Terry et al. has impressively compiled info
on clinical cost calculations from various sources
and interviews. Tables 2.1 and 2.2 provide
details on development cost and probability of
success depending on the type of archetype and
clinical phase planned [5].
Based on the data, preclinical phase needs fair
(1–4 years) amount of time to complete compared
to complete cycle (10–18years). Hence the main
aim of this chapter is to illustrate basic and regu-
latory understanding on the fundamentals of
the preclinical phase and challenges involved.
22 2. Preclinical testing—Understanding the basics first

TABLE 2.1 Development cost assumptions per phase per archetype designated at preclinical and clinical phases.
Cost estimates per phase ($ millions)
Preclinical Phase 1 Phase 2 Phase 3
Lower Lower Lower Lower
bound, bound, bound, bound,
upper Point upper Point upper Point upper Point
Archetype bound estimate bound estimate bound estimate bound estimate

Vaccine Simple 3.3, 10.0 6.7 1.8, 2.7 2.2 7.4, 19.0 13.2 56.6, 165.6 111.1
Complex 8.3, 24.9 16.6 1.9, 3.0 2.5 7.8, 20.0 13.9 67.9, 198.7 133.3
New Simple 2.5, 7.5 5.0 1.8, 2.7 2.2 3.7, 7.9 5.8 11.5, 54.1 32.8
chemical
Entity (NCE) Innovative 5.0, 10.0 7.5 4.4, 5.3 4.8 3.9, 8.3 6.1 12.1, 55.4 34.5
Complex 7.5, 12.5 10.0 6.9, 7.9 7.4 4.1, 87 6.4 12.6, 59.6 36.1
Repurposed Simple Ν/A N/A N/A N/A 3.7, 7.9 5.8 10.0, 25.2 17.6
drug
Complex 2.5, 7.5 5.0 1.7, 2.7 2.2 3.7, 7.9 5.8 10.0, 25.2 17.6
Biologic Simple 5.4, 16.2 10.8 1.9, 3.0 2.4 4.5, 10.5 7.5 27.7, 80.5 54.1
Complex 16.2, 27.0 21.6 7.0, 8.3 7.6 5.0, 11.6 8.3 30.5, 88.5 59.5

Selection and
validation of Regulated trials
markers Development beyond EUO/CE

Assay development 1.0, 5.0 3.0 1.0, 3.0 2.0 1.0, 6.0 3.5
Diagnostics Simple technical platform development N/A N/A 50.0, 150.0 100.0 1.0, 6.0 3.5

Obtained from article (5) under open access permission Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding global health product R&D: the portfolio-to-
impact model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res 2018;2:24.

2 Preclinical studies basic preclinical data to IND application espe-

Preclinical studies are performed in in vitro,
in vivo, ex vivo, and in silico models to obtain
basic information about the safety and biologi-
cal efficacy of a drug candidate before testing it
in a final target population, i.e., humans. Pre-
clinical studies or tests are mainly performed
in compliance with GLP/GSP guidelines (good
laboratory practice and good scientific prac-
tices) to ensure reliability and reproducibility
of results. The FDA/EMA require supporting
cially on toxic effects, safety profile, pharma-
cokinetics, and pharmacodynamics. The data
from preclinical trials must be accurate, reli-
able, and based on the best suitable and compa-
rable model available to the target population.
Typically, this means that the IND or drug
product must undergo a series of robust
tests and experiments using in vitro, in vivo,
ex vivo, and in silico models as per the needs
of the focused indication and regulatory
guidelines.
 3 Regulatory aspects of preclinical studies 23

TABLE 2.2 Probability of success (attrition rate) and cycle time (length of phase) per phase and archetype
Length of phase (years) Probability of success (%)
Archetype Preclinical Phase 1 Phase 2 Phase 3 Preclinical Phase 1 Phase 2 Phase 3

Vaccine Simple 3.36 1.57 2.23 2.33 41.0 68.4 45.9 70.8

Complex 3.33 1.97 3.71 3.50 41.0 50.0 21.6 63.6

New Simple 2.49 1.80 3.38 3.18 65.0 59.7 38.8 69.1
chemical
Entity (NCE) Innovative 2.70 1.81 3.35 3.10 60.0 51.9 28.4 57.8
Complex 2.87 1.93 3.51 2.80 55.0 57.2 19.7 40.3
Repurposed Simple 0.00 0.00 2.14 2.14 100.0 100.0 45.7 68.1
drug
Complex 2.33 1.63 2.14 2.14 75.0 58.5 45.7 68.1

Biologic Simple 3.29 1.62 2.47 2.10 75.0 66.2 44.3 70.9
Complex 3.24 1.49 4.16 3.38 77.0 69.6 32.2 62.5
Diagnostics Assay 1.00 1.25 1.33 0.00 50.0 100.0 100.0 100.0
development
Diagnostics Simple technical 0.00 2.50 2.00 0.00 100.0 75.0 100.0 100.0
platform
development

Adapted from (5) under open access permission Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding global health product R&D: the portfolio-to-impact
model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res 2018;2:24.

3 Regulatory aspects of preclinical studies
Preclinical studies mainly follow a combina-
tion of:
• GLP (21CFR, 58) and compliance monitoring;
• OECD principles;
• ICH/M3;
• Common Technical Document/CTD module
4 (article 6 of Regulation (EC) No. 726/2004,
and with respect to the Annex I to Directive
2001/83/EC);
• Pharmacopoeial or codex requirements;
• 3R principles;
• Local animal ethical committee guidelines
and requirements; and
• ISO regulations
The “set of tests” changes as per type of
products, e.g., category (pharma, food, or veter-
inary), type (chemical or device), use (human/
non-human), and other many more factors.
Table 2.3 lists a few tests reported in the litera-
ture for medical devices. As discussed above,
the list is exhaustive. The type of tests to be per-
formed must be decided as per intended use of
product.
For further details, readers are requested to
refer to the above regulatory guidelines and full
articles on this topic. The literature contains vast
information on disease, organ, and bioactivity-
specific assays and preclinical tests. A list of reg-
ulatory guidelines and supporting articles is
provided in Tables 2.3–2.5.
24 2. Preclinical testing—Understanding the basics first

TABLE 2.3 Typical tests for medical device. TABLE 2.4 Typical tests for chemical entity and
pharma drugs—cont’d
Functional tests
Mitochondria membrane potential assay
Functional implantation studies (bone, dental, Apoptosis analysis by flow cytometry
hemostasis, cardiovascular, etc.) other specific tests
Identification and quantification of degradation products
(ISO 10993-15) • Cell angiogenesis assay
Packaging validation* (ISO 11607)
EO residue determination* (ISO 10993-7) Matrigel plug assay
Tube formation assay
Biocompatibility testing Platelet activation assay
Co-culture angiogenesis assay
Sensitization other specific tests
Irritation
Pyrogenicity* • Related assays
Implantation
Cell migration assay
Hemocompatibility
Tumor invasion assay
Microbiological testing of medical devices Chemotaxis assay
Cellular phosphorylation assay
Test for sterility Cell cycle assay
Endotoxins Endotoxin assay
Bioburden Genetic stability testing
Test for specified microorganisms/microbial limit test Endocrine disruption
Test for antimicrobial preservation Drug-drug interaction
Test for antimicrobial efficacy Drug efficacy test
Evaluation of reusables for the intended reprocessing 3D angiogenesis assay
procedure (cleaning, disinfection, sterilization) 3D invasion assay
Inflammation assay
Receptor binding assay
Enzyme inhibition
20 messenger analysis
TABLE 2.4 Typical tests for chemical entity and Plasma stability
pharma drugs. Plasma protein binding assay
• Cell viability assays other specific tests

Thymidine incorporation assays • In vitro permeability and transporter assays

Cell Titer-Glo cell viability assays Caco-2 permeability assay
MTS assay MDCK permeability assay
ATPlite cell viability assay Parallel artificial membrane permeability assay
SRB assay (PAMPA)
other specific tests Transporter assays, e.g., P-glycoprotein (P-gp), BCRP,
• Cell proliferation assays OCT2
other specific tests
MTT cell proliferation assay
cyQuant direct proliferation assay • Type of toxicity assays
BrdU cell proliferation ELISA assay 2D-based hepatotoxicity assay
Label-free and real time proliferation assay using 3D-based hepatotoxicity assay
IncuCyte Cardiotoxicity
other specific tests Neurotoxicity
• Cell apoptosis assays Nephrotoxicity
Genotoxicity
Caspase-Glo 3/7 activation assay Carcinogenicity
Reaction oxygen species (ROS) assay other specific tests
 5 Animal models and type of tests
TABLE 2.4 Typical tests for chemical entity and
pharma drugs—cont’d
• Animal tests
Teratogenicity
Pharmacokinetic
Local tolerance
Pharmacodynamic
Fertility studies
Single dose toxicity in animal
Repeated dose toxicity in animal
other specific tests
TABLE 2.5 Regulatory guidelines and related
publications.
 Sanjay Jain, Michael Edwards, Louise Spencer,
Regulatory Rapporteur—Vol 13, No 6, June 2016,
European Medicines Agency (2007a). Advances and
challenges in the development of drug delivery systems—
A European perspective
 Guideline on strategies to identify and mitigate risks for
first-in human trials with investigational medicinal
products. London: European Medicines Agency.
Retrieved 18 Oct, 2019, from http://www.ema.europa.
eu/docs/en_GB/document_library/Scientific_
guideline/2009/09/WC500002988.pdf
 European Medicines Agency (2007b). Guideline on
requirements for first-in-man clinical trials for potential
high-risk medicinal Retrieved 18 Oct, 2019, from http://
www.ema.europa.eu/docs/en_GB/document_library/
Scientific_guideline/2009/09/WC500002989.pdf
 European Medicines Agency (2009). ICH guideline
M3(R2) on non-clinical safety studies for the conduct of
human clinical trials and marketing authorisation for
pharmaceuticals. London: European Medicines Agency.
Retrieved 18 Oct, 2019, from http://www.ema.europa.
eu/docs/en_GB/document_library/Scientific_
guideline/2009/09/WC500002720.pdf
 Innovation in Development, Regulatory Review, and Use of
Clinical Advances—A Vital Direction for Health and
Health Care Michael Rosenblatt; Christopher P. Austin;
Marc Boutin; William W. Chin et al., https://nam.edu/wp-
content/uploads/2016/09/Innovation-in-Development-
Regulatory-Review-and-Use-of-Clinical-Advances.pdf
 ICH efficacy guidelines https://www.ich.org/page/
efficacy-guidelines
 ICH safety guidelines https://www.ich.org/page/
safety-guidelines
 M3-nonclinical safety studies/ICH https://www.ich.
org/page/multidisciplinary-guidelines
25
4 Preclinical testing and models used
4.1 In vitro assays/cell line assays
In vitro assays to evaluate basic effects are
simple, fast, and cost-efficient. Dedicated cell
lines, tissue, blood cells, organ cultures, or com-
ponents of the same are used. As these assays
are performed under GLP, they allow tight con-
trol over experimental parameters and design of
experiments. Furthermore, depending upon the
intended use of the drug, other tests might be
needed like carcinogenicity, genotoxicity, and
reproductive toxicity. For example, carcinoge-
nicity is performed in two species (rat and
mouse). It involves a number of animals and a
specific study design plan as per the advice of
the regulatory authorities. The costs of such
studies are in excess of $ 2.5 million and they
are typically performed as 2-year bioassays.
Although the basic information provided by
these assays is helpful, one disadvantage is that
isolated cells may not mirror the exact perfor-
mance when they are in vivo, due to missing
the microenvironment of that particular organ.
Depending upon the target disease, indication,
and organ, cell line assays are selected. Mostly
cell line assays are used as in vitro model sys-
tems. Cell lines provide an indefinite source of
biological material if grown under optimum
conditions. The major challenge includes
selection of a proper cell line, strict control of
experimental conditions, and proper design of
the experiment to avoid cross-contamination
and misleading results (Tables 2.6 and 2.7).
5 Animal models and type of tests
5.1 In vivo evaluation
Various animal models are reported in the
literature, including rats, mice, guinea pigs,
hamster, rabbits, cats, monkey apes, and dogs.
One must refer to specific disease physiology,
26 2. Preclinical testing—Understanding the basics first
TABLE 2.6 Key references on in vitro models (as per diseases/target organ).
In vivo and in vitro models for scanning drug substances in
malaria: Prestudy
In vitro and in vivo models of HIV latency
In vitro and ex vivo corneal penetration and absorption models
In vitro skin models in the optimization of skin formulations
In vitro models of Mycobacterium tuberculosis dormancy, and in
vivo models of latent tuberculosis infection
In vitro three-dimensional (3D) models in cancer research: an
update
Improved in vitro human tumor models for cancer gene therapy
Beyond 3D culture models of cancer
In vitro models for evaluation of periodontal wound healing/
regeneration
In vitro cell and tissue models for studying host–microbe
interactions: a review
In vitro and in vivo methods for studying retinal ganglion cell
survival and optic nerve regeneration
In vitro models to study human lung development, disease and
homeostasis
In vitro modeling of the interaction between human epithelial
cells and lymphocytes upon influenza infection
Towards 3D in vitro models for the study of cardiovascular
tissues and disease
In vitro models of the blood-brain barrier
In vitro models of the blood-brain barrier for the study of drug
delivery to the brain
In vitro cardiac tissue models: current status and future
prospects
In vitro models of the blood-brain barrier
Nonanimal models of wound healing in cutaneous repair: in
silico, in vitro, ex vivo, and in vivo models of wounds and scars
in human skin
In vitro psoriasis models with focus on reconstructed skin
models as promising tools in psoriasis research
Further development of an in vitro model for studying the
penetration of chemicals through compromised skin
Turkiye Parazitol Derg. 2017 Sep;41(3):156–163. https://
doi.org/10.5152/tpd.2017.5365
Adv Exp Med Biol. 2018;1075:241–263. https://doi.org/
10.1007/978-981-13-0484-2_10
Drug Deliv Transl Res. 2016 Dec;6(6):634–647
Acta Pharm Hung. 2017;87(1):3–12
Klin Lab Diagn. 2019;64(5):299–307. https://doi.org/10.
18821/0869-2084-2019-64-5-299-307
Mol Carcinog. 2013 Mar;52(3):167–82. https://doi.org/
10.1002/mc.21844. Epub 2011 Dec 7
Hum Gene Ther. 2015 May;26(5):249–56. https://doi.
org/10.1089/hum.2015.028. Epub 2015 Apr 20
Sci Transl Med. 2015 Apr 15;7(283):283 ps9. https://doi.
org/10.1126/scitranslmed.3009367
Periodontol 2000. 2015 Jun;68(1):41–54. https://doi.
org/10.1111/prd.12079
Br J Nutr. 2013 Jan;109 Suppl 2:S27–34. https://doi.
org/10.1017/S0007114512004023
Methods Mol Biol. 2018;1695:187–205. https://doi.
org/10.1007/978-1-4939-7407-8_16
Physiology (Bethesda). 2017 May;32(3):246–260.
https://doi.org/10.1152/physiol.00041.2016
Influenza Other Respir Viruses. 2016 Sep;10(5):438–42.
https://doi.org/10.1111/irv.12394. Epub 2016 May 17
Drug Discov Today. 2016 Sep;21(9):1437–1445.
https://doi.org/10.1016/j.drudis.2016.04.014. Epub 2016
Apr 23
Acta Neurobiol Exp (Wars). 2011;71(1):113–28
Mol Pharm. 2014 Jul 7;11(7):1949–63. https://doi.org/
10.1021/mp500046f. Epub 2014 Apr 18
Adv Drug Deliv Rev. 2016 Jan 15;96:203–13. https://
doi.org/10.1016/j.addr.2015.09.011. Epub 2015 Sep 30
Methods Mol Biol. 2014;1135:415–37. https://doi.org/
10.1007/978-1-4939-0320-7_34
Wound Repair Regen. 2017 Apr;25(2):164–176.
https://doi.org/10.1111/wrr.12513. Epub 2017 Feb 20
Exp Biol Med (Maywood). 2017 Jun;242(11):1158–1169.
https://doi.org/10.1177/1535370217710637
Toxicol In Vitro. 2017 Feb;38:101–107. https://doi.org/
10.1016/j.tiv.2016.10.004. Epub 2016 Oct 14
 5 Animal models and type of tests 27

TABLE 2.6 Key references on in vitro models (as per diseases/target organ)—cont’d
In vitro skin models as a tool in optimization of drug formulation Eur J Pharm Sci. 2015 Jul 30;75:10–24. https://doi.
org/10.1016/j.ejps.2015.02.018. Epub 2015 Mar 5
Improved in vitro models for preclinical drug and formulation Eur J Pharm Biopharm. 2018 May;126:57–66. https://
screening focusing on 2D and 3D skin and cornea constructs doi.org/10.1016/j.ejpb.2017.11.014. Epub 2017 Dec 2

TABLE 2.7 Key references on ex vivo models (as diseases/target organ).

Human lung ex vivo infection models
Ex vivo models of chronic granulomatous
disease
Human ex vivo wound healing model
Cell Tissue Res. 2017 Mar;367(3):511–524. https://doi.org/10.1007/
s00441-016-2546-z. Epub 2016 Dec 20
Methods Mol Biol. 2019;1982:587–622. https://doi.org/10.1007/978-
1-4939-9424-3_35
Methods Mol Biol. 2013;1037:255–64. https://doi.org/10.1007/978-1-
62,703-505-7_14

Modeling tuberculosis pathogenesis through ex Tuberculosis (Edinb). 2017 Dec;107:126–132. https://doi.org/10.1016/

vivo lung tissue infection j.tube.2017.09.002. Epub 2017 Sep 12

Epithelial ovarian cancer experimental models
Ex vivo rabbit and human corneas as models for
bacterial and fungal keratitis
In vivo and in vitro models of type 2 diabetes in
pharmaceutical drug discovery
Human pulmonary 3D models for translational
research
Oncogene. 2014 Jul 10;33(28):3619–33. https://doi.org/10.1038/onc.
2013.321. Epub 2013 Aug 12
Graefes Arch Clin Exp Ophthalmol. 2017 Feb;255(2):333–342. https://
doi.org/10.1007/s00417-016-3546-0. Epub 2016 Nov 14
Diabetes Obes Metab. 1999 Mar;1(2):75–86
Biotechnol J. 2018 Jan;13(1). https://doi.org/10.1002/biot.201700341.
Epub 2017 Sep 20

metabolic pathways, target organ, specific regu-
latory guidelines, and requirements of drug
product and financial aspects before selecting
an animal model. Animal studies are strictly gov-
erned by ethical committees to avoid unneces-
sary harm to animal species. Appropriate study
protocol and related explanations are needed to
obtain official ethical approval to perform animal
studies.
Advances like noninvasive imaging, micro-
sampling, and telemetric monitoring are valu-
able methods to obtain relevant data. Unlike
in vitro cell line assays, in vivo models do not
allow too much control on experimental param-
eters due to variations in animals. The most
popular animal models are mice and rats;
however, these models have several limitations
on the pharmacokinetic and pharmacodynamic
fronts. Despite the limitations, rodent models
are still helpful to get basic information and ideas
on toxicity at the preliminary stages of develop-
ment. Advances in rodent models like the one
below provides new platform to test drug sub-
stances, not to mention this test methodology its
also has its own limitations. Some examples
include:
• Immune deficiency mice;
• Cell line derived xenograft (CDX) models;
• Patient-derived xenograft (PDX) models;
• Orthotopic tumor models; and
• Genetically engineered mice (GEM). etc
28 2. Preclinical testing—Understanding the basics first
5.2 Pharmacodynamics
In a pharmacodynamics study, the effects of a
drug on the body/biological environment (in this
case, animal) are observed based on the concentra-
tion of the drug. This is generally referred to as the
dose response relationship, categorizing the drug
into the category potent, cytotoxic, or safe. Drugs
are evaluated for desired pharmacological effects
and AEs. These studies indicate the therapeutic
index and therapeutic window of a drug.
5.3 Pharmacokinetic
Pharmacokinetic and biodistribution studies
determine how a body handles a drug, meaning
how it distributes and eliminates the drug.
These effects are studied by checking the drug
distribution profile in an organ or particular tis-
sue (target site), and by plasma profiling.
Absorption, distribution, metabolism, and
excretion (ADME) of the drug is key information
when determining safer dose ranges and drug
TABLE 2.8 Key references on animal models (diseases/target organ).
Animal models for cutaneous vaccine delivery
Animal models used to study direct peripheral nerve
repair: a systematic review
Animal model of asthma, various methods and
measured parameters: a methodological review
Animal models of spinal cord injury: a systematic
review.
Animal models in cardiovascular research:
hypertension and atherosclerosis
Animal models for percutaneous absorption
Animal models of osteoarthritis: classification, update,
and measurement of outcomes
Animal models in influenza research
Animal models as a tool in hepatocellular carcinoma
research: a review
administration regime for the next clinical
trial, e.g., oral medication is challenged by first
pass effect whereas intravenous medication is
not, which has an effect on bioavailability. Fur-
thermore, this study can be extended to deter-
mine a drug’s affinity for plasma proteins and
the drug’s molecular interaction with a tissue
or organ’s microenvironment. During metabo-
lism, a drug is altered chemically into pharma-
cologically active or inert metabolites. It is vital
to ensure a therapeutic effect; an adequate and
safe steady-state concentration of the drug
should be maintained in the body. Multiple fac-
tors affect metabolism, such as health condition,
age, race, type of dosage form, etc. Lastly, clear-
ance is mainly achieved via the renal and hepatic
routes, and the metabolites are excreted from the
body at the end. Depending upon the adminis-
tration route, the requirement for preclinical
testing changes, e.g., for a dermal product, bio-
distribution studies are not required, more
imp would be local tissue distribution studies
(Tables 2.8 and 2.9).
Eur J Pharm Sci. 2015 Apr 25;71:112–22. https://doi.org/10.1016/j.
ejps.2015.02.005. Epub 2015 Feb 14
Neural Regen Res. 2020 Mar;15(3):491–502. https://doi.org/10.
4103/1673-5374.266068
Iran J Allergy Asthma Immunol. 2016 Dec;15(6):445–465
Spinal Cord. 2017 Aug;55(8):714–721. https://doi.org/10.1038/
sc.2016.187. Epub 2017 Jan 24
Biomed Res Int. 2015;2015:528757. https://doi.org/10.
1155/2015/528757. Epub 2015 May 3
J Appl Toxicol. 2015 Jan;35(1):1–10. https://doi.org/10.1002/
jat.3004. Epub 2014 Oct 27
J Orthop Surg Res. 2016 Feb 2;11:19. https://doi.org/10.1186/
s13018-016-0346-5
Methods Mol Biol. 2018;1836:401–430. https://doi.org/10.
1007/978-1-4939-8678-1_20
Tumor Biol. 2017 Mar;39(3):1010428317695923. https://doi.
org/10.1177/1010428317695923
 5 Animal models and type of tests 29

TABLE 2.8 Key references on animal models (diseases/target organ)—cont’d

Animal models for evaluation of oral delivery of
biopharmaceuticals
Search for an animal model to investigate selective
pulmonary vasodilation
Animal models in myopia research
Latest animal models for anti-HIV drug discovery
Animal models of tuberculosis: lesson learnt
Animal Models of Tuberculosis: an overview
Animal models in tuberculosis research—where is the
beef?
Malaria in pregnancy: the relevance of animal models
for vaccine development
Animal models of efficacy to accelerate drug discovery
in malaria
J Control Release. 2017 Dec 28;268:57–71. https://doi.org/10.
1016/j.jconrel.2017.09.025. Epub 2017 Sep 19
Lab Anim. 2017 Aug;51(4):376–387. https://doi.org/10.
1177/0023677216675384. Epub 2016 Nov 25
Clin Exp Optom. 2015 Nov;98(6):507–17. https://doi.org/10.
1111/cxo.12312
Expert Opin Drug Discov. 2015 Feb;10(2):111–23. https://doi.
org/10.1517/17460441.2015.975201. Epub 2014 Oct 28
Indian J Med Res. 2018 May;147(5):456–463. https://doi.org/
10.4103/ijmr.IJMR_554_18
Microbiol Spectr. 2016 Aug;4(4). https://doi.org/10.1128/
microbiolspec.TBTB2-0004-2015
Expert Opin Drug Discov. 2015;10(8):871–83. https://doi.org/
10.1517/17460441.2015.1049529. Epub 2015 Jun 13
Lab Anim (NY). 2017 Oct 6;46(10):388–398. https://doi.org/10.
1038/laban.1349
Parasitology. 2014 Jan;141(1):93–103. https://doi.org/10.1017/
S0031182013000991. Epub 2013 Jun 21

The use of animal models in diabetes research Br J Pharmacol. 2012 Jun;166(3):877–94. https://doi.org/10.
1111/j.1476-5381.2012.01911.x

TABLE 2.9 Key references on in silico models (diseases/target organ).

In silico ADME-Tox modeling: progress and prospects
In silico skin model: a multiscale simulation study of drug
transport
In silico models for hepatotoxicity
Expert Opin Drug Metab Toxicol. 2017 Nov;13(11):1147–1158.
https://doi.org/10.1080/17425255.2017.1389897. Epub 2017
Oct 13
J Chem Inf Model. 2017 Aug 28;57(8):2027–2034. https://
doi.org/10.1021/acs.jcim.7b00224. Epub 2017 Jul 31
Methods Mol Biol. 2016;1425:201–36. https://doi.org/10.
1007/978-1-4939-3609-0_11

In silico models of stem cell and developmental systems Theor Biol Med Model. 2014 Jan 8;11:1. https://doi.org/
10.1186/1742-4682-11-1

In vitro and in silico liver models: current trends, challenges
and opportunities
In silico modeling for tumor growth visualization
In silico models for acute systemic toxicity
ALTEX. 2018;35(3):397–412. https://doi.org/10.14573/altex.
1803221. Epub 2018 May 28
BMC Syst Biol. 2016 Aug 8;10(1):59. https://doi.org/10.1186/
s12918-016-0318-8
Methods Mol Biol. 2016;1425:177–200. https://doi.org/
10.1007/978-1-4939-3609-0_10

Continued
30 2. Preclinical testing—Understanding the basics first
TABLE 2.9 Key references on in silico models (diseases/target organ)—cont’d
In silico modeling of the immune system: cellular and
molecular scale approaches
In silico prediction of genotoxicity
In silico cancer research towards 3R
In silico assessment of adverse drug reactions and associated
mechanisms
The use of in silico models within a large pharmaceutical
company
In silico modeling of gastrointestinal drug absorption:
predictive performance of three physiologically based
absorption models.
5.4 Toxicology
In toxicity studies, the safety of a drug and its
dose relation are determined. Additionally, this
study is used to establish biomarkers for moni-
toring AEs in later clinical phases. The toxicity
studies are mainly performed for single and
multiple dose, i.e., single and repeated dose tox-
icity over a period of months, to analyze toxic
effects of the drug and multiple administrations.
Toxicity studies are planned based on the
intended drug administration regime and the
route of administration. Selecting the appropri-
ate species for a program of work is one of the
most critical aspects of drug development to
enable first in man application and/or dose
administration. Various regulatory guidance
including the ICHs and M3R2 gives explicit
information on these approaches, and will indi-
cate other applicable regulations to the test drug
type, route of administration, and the intended
indications. Generally, these follow the use of
two species (a rodent and nonrodent) for small
molecules. For biotherapeutics, on the other
hand, this is mostly akin to pharmacological rel-
evance and/or receptor targeting/occupancy,
and may typically result in the use of single
species (nonhuman primates or genetically
Biomed Res Int. 2014;2014:371809. https://doi.org/10.
1155/2014/371809. Epub 2014 Apr 6
Food Chem Toxicol. 2017 Aug;106(Pt B):595–599.
https://doi.org/10.1016/j.fct.2016.12.013. Epub 2016 Dec 12
BMC Cancer. 2018 Apr 12;18(1):408. https://doi.org/
10.1186/s12885-018-4302-0
Drug Discov Today. 2016 Jan;21(1):58–71. https://doi.
org/10.1016/j.drudis.2015.07.018. Epub 2015 Aug 10
Methods Mol Biol. 2016;1425:475–510. https://doi.org/
10.1007/978-1-4939-3609-0_20
Mol Pharm. 2016 Jun 6;13(6):1763–78. https://doi.org/
10.1021/acs.molpharmaceut.5b00861. Epub 2016 May 5
modified murine species, etc.) programs. Mar-
mosets are also useful nonhuman primate alter-
natives, but are relatively challenging to handle
and are significantly less robust due to their
nature and practicalities such as blood sampling
limitations and breeding.
The dog is arguably the most widely used
nonrodent species for regulatory toxicology, fol-
lowed closely by nonhuman primates and, in
more recent times, minipigs. The rodent of
choice falls mainly into two categories/species,
rats and mice, with the majority of rodent toxic-
ity studies being performed in the rat model/
species. It has also been demonstrated over the
years that using two species that are distinct
phylogenetically provides a far superior cover-
age of a chemical’s risk to humans.
5.5 Advances in the field
Advances in bioinformatics, virtual reality,
and the internet of things over the past decades
have made in silico studies a supporting and
next-generation model. Computer simulations
are used to predict in vivo and in vitro perfor-
mances of chemical entities. For the in silico
model, structure-activity relationship (SAR)
and quantitative structure-activity relationship
 6 Conclusion
(QSAR) are commonly used approaches in the
field of testing. These in silico models are very
complex and are based on compiled knowledge
from statistics, mathematics, bioinformatics, bio-
chemistry, and molecular biology. In addition,
from a preclinical perspective for example, the
no observed adverse effect level (NOAEL) [6]
has typically served as the focal point for select-
ing the most appropriate dose levels for the con-
duct of first in human (FIM) studies. The NOAEL
continues to be used and followed extensively as
prescribed in various regulatory guidance docu-
ments, for selecting safe multiples of animal
doses for extrapolating to humans within the
food, pharmaceutical, and chemical industries.
There have been several publicized limitations
of this approach in recent times, and a resurgence
of a collaborative effort to focus on the advance-
ment of existing/novel alternatives to comple-
ment the use of this well-established method in
regulatory science. A few of these perceived
alternatives are the BenchMark Dose (BMD)
models [7] and/or adverse outcome pathways
(AOPs) [8], both of which are in silico-based
models and are fully implemented by prominent
regulatory agencies to support human safety.
There are still some reservations for virtually
all these concepts, but the coming decades could
see their full use and adoption.
5.6 Databases
In June 2016, the FDA commissioner proposed
building a database that would gather informa-
tion before clinical trials, meaning from preclini-
cal research (lab animals or cells/cell lines). This
proposal of a ClinicalTrials.gov database for pre-
clinical work received mixed reactions from the
scientific community. Currently, if a molecule
becomes successful, all the drug development
data become trade secret; if it does not, the data
are stored in archives, and very rarely published
despite their high scientific value. The idea of this
database supports learning and sharing from un-
and successful trials. Even though the idea is
31
interesting, the scientific community is unsure
about the future of it, due to several reasons, such
as reliability, funding, legal aspects, government
and regulatory scrutiny, etc. https://www.
preclinicaltrials.eu is trying to promote transpar-
ency in preclinical research. The main objective of
this database is to increase transparency, avoid
duplication of animal studies, reduce bias report-
ing, increase data sharing on protocols, study
designs, and outcomes, and foster research
collaborations. The UK’s Catapult database
(https://ct.catapult.org.uk/resources/cell-and-
gene-therapy-catapult-uk-preclinical-research-
database) provides one-stop information for
ongoing preclinical research activity related to
cell, gene, and other advanced therapies. Else-
vier’s PharmaPendium database (https://
www.elsevier.com/solutions/pharmapendium-
clinical-data) collects data on drug safety, the
FDA Adverse Event Reporting System (FAERS),
and ADME and drug-drug interactions. This
database offers risk-benefit analyses and drug
candidate assessments. Researchers can search
FDA and EMA regulatory documents, commit-
tee meeting minutes, FAERS, data on phar-
macokinetics, efficacy, safety, metabolizing
enzymes’ and transporters’ potential drug-drug
interactions, and guidance on validation of the
best animal model. Other databases include
http://fprd.ihes.fr (a French preclinical data-
base) and https://dtp.cancer.gov (the NCI’s
Development Therapeutics Program, DTP).
6 Conclusion
The high costs of drug development (for both
successful and unsuccessful candidates) results
in exorbitant drug prices, leading to high health-
care expenditure. The only solution to reduce
this burden is open communication between
all science streams involved during the com-
plete cycle, better alignment on “regulatory
requirement” and “right experimental design,”
along with transparency in sharing result
32 2. Preclinical testing—Understanding the basics first
and interpretation. This helps to reduce costs
associated with drug development at each
milestone, including the preclinical stage. It is
clear that preclinical is a turning point for any
drug, and involves millions of dollars. Selection
of optimal experimental design, study protocol,
assay, cell lines, and animal model with signifi-
cant translation value have a tremendous impact
on the completion of the preclinical phase with
desired results. A significant amount of research
is needed in generating sophisticated preclinical
models to understand and evaluate the effects of
disease-related factors, cellular microenviron-
ments, and therapy-related (drug + device)
parameters. Although 3D bioprinting, in silico
are showing some promise, but still the question
is whether these techniques are reducing—
long experiments—to get better results or
they are just generating more information
in the pool of existing data. Databases can
certainly help, but questions about data protec-
tion, safety, company policy, and openness
to share all information remain unanswered.
Importantly, outsourcing preclinical studies
to Contract Research Organizations (CROs)
can support companies and speed up pre-
clinical development to a significant extent.
Optimal, cost-effective, and timely collaborative
research is the way forward to reduce health
expenditure.
References
[1] Honek J. Preclinical research in drug development. Med
Writ 2017;26(4).
[2] Mahan VL. Clinical trial phases. Int J Clin Med 2014;
5:1374–83. https://doi.org/10.4236/ijcm.2014.521175.
[3] https://www.hqsc.govt.nz/assets/Consumer-Engage-
ment/Publications/Training-guide/019-clinical-trials-
research-review.pdf.
[4] The International Conference on Harmonisation of Tech-
nical Requirements for Registration of Pharmaceuticals
for Human Use (ICH). www.ich.org.
[5] Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding
global health product R&D: the portfolio-to-impact
model (P2I), a new tool for modelling the impact of
different research portfolios. Gates Open Res 2018;2:24.
[6] Dorato MA, Engelhardt JA. The no-observed-adverse-
effect-level in drug safety evaluations: use, issues, and
definition(s). Regul Toxicol Pharmacol 2005;
42:265–74.
[7] Haber LT, Dourson ML, Allen BC, Hertzberg RC,
Parker A, Vincent MJ, Maier A, Boobis AR. Benchmark
dose (BMD) modeling: current practice, issues, and
challenges. Crit Rev Toxicol 2018;48(5):387–4159.
[8] Edwards, et al. Adverse outcome pathways—organizing
toxicological information to improve decision making.
J Pharmacol Exp Ther 2016;356(1):170–81.
 C H A P T E R

3
Aqueous polymeric coatings: New
opportunities in drug delivery systems
Abid Riaz Ahmeda, Joana Portugal Motab,c, Ahmad Abdul-Wahhab
Shahbad, Muhammad Irfane
a
Merck Healthcare KGaA, Darmstadt, Germany
b
Lecifarma—Laboratório Farmac^eutico, Lda, Várzea do Andrade—Cabeço de Montachique,
Lousa, Portugal
c
CBIOS-Research Center for Biosciences and Health Technologies, Lusófona University,
Lisbon, Portugal
d
Kayyali Chair for Pharmaceutical Industries, Department of Pharmaceutics, College of Pharmacy,
King Saud University, Riyadh, Saudi Arabia
e
Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, GC University Faisalabad,
Faisalabad, Pakistan

1 Introduction polymer dispersions can be prepared by emul-

Aqueous film coatings have been extensively
applied to solid substrates as tablets, pellets,
granules, and capsules, for several purposes.
They include odor and taste masking, improve-
ment of dosage form appearance, protection of
the drug from environmental conditions (light,
oxygen, and water vapor), isolation of incompat-
ible materials, easy identification of drug
products for both patients and healthcare profes-
sionals and to modify drug release (Table 3.1)
[5–7]. Aqueous-based coatings are an attractive
alternative to organic-based coatings, due to
environmental reasons and reduced processing
time (high polymer content) [8–11]. The aqueous
sion polymerization of a monomer (latex) or by
emulsification of a polymer (pseudolatex) [12];
physical properties and method of preparation
of pure aqueous dispersion are summarized in
Table 3.2. The formation of a thin and transparent
film from aqueous latex or pseudolatex disper-
sion takes place in parallel with evaporation of
water. Film formation is illustrated in Fig. 3.1.
During spraying of the aqueous dispersion
onto a substrate, water evaporates at a constant
rate and the particles form a closed and ordered
packing. Deformation and fusion of the colloidal
particles lead to a film with water-filled cavities.
Finally, particle coalescence occurs when the
capillarity forces are strong enough [13–15].

Drug Delivery Aspects 33 # 2020 Elsevier Inc. All rights reserved.

https://doi.org/10.1016/B978-0-12-821222-6.00003-8
34 3. Aqueous polymeric coatings: New opportunities in drug delivery systems

TABLE 3.1 Examples of aqueous based polymers used for immediate and modified release coatings.
Immediate release Polymers (Trade name) Ref.

Taste and odor masking • Hydroxy ethylcellulose (Natrosol), hydroxy proylcellulose (Klucel), hydroxy [1–3]
propylmethylcellulose (Methocel)
Moisture protective
• Polyvinyl alcohol-polyethylene glycol graft copolymer (Kollicoat IR)
Easy identification • Polyvinyl alcohol-polyethylene glycol graft copolymer and polyvinyl alcohol
(Kollicoat Protect)
Improving ingestion • Aminoalkyl methacrylate copolymer (Eudragit E 100)
and swallowing
Isolation of
incompatible materials

Improving appearance
Modified release
Sustained release • Ethylcellulose (Aquacoat ECD and Surelease) [2, 4]
• Ammonio methacrylate (Eudragit RS 30 D, Eudragit RS 30 D and combination of them)
• Ethylacrylate methylmethacrylate (2:1) (Eudragit NE 30 D and Eudragit NM 30 D)
• Polyvinyl acetate (Kollicoat SR 30 D)
Enteric/colonic release • Cellulose acetate phthalate (Aquacoat CPD)
• Hydroxypropyl methylcellulose acetate succinate (AQOAT)
• Methacrylic acid copolymer (Eudragit L 30 D 55)
• Polymethylacrylate-co-methyl methacrylate-co-methacrylic acid (Eudragit FS 30 D)

TABLE 3.2 Physical properties and method of preparation of pure aqueous dispersions.
Aqueous
dispersion Polymer Additives (%) Method of preparation Tg (°C) MFT (°C) Ref

Aquacoat Ethylcellulose Sodium dodecyl Emulsification of 90 81 [30, 31]

ECD sulfate (0.9–1.7) polymer (pseudolatex)
Cetyl alcohol
(1.7–3.3)
Surelease Ethylcellulose Oleic acid (1.9) Inverse emulsification 50 32 [32, 33]
Dibutyl sebacate (pseudolatex)
(3.5)
Aquacoat Cellulose acetate Poloxamer (7) Emulsification of 40 Information [16, 34]
CPD phthalate polymer (pseudolatex) not found
AquaSolve Hydroxypropyl Oleic acid (1.9) Redispersion of a Depends on N/A [35]
methylcellulose Dibutyl sebacate polymeric powder substitution
acetate succinate (3.5) degree
Eudragit Ammonio Sorbic acid (0.25) emulsification of 55 45 [1, 36]
RS 30 D methacrylate type B NaOH (0.1) polymer (pseudolatex)
Eudragit Ammonio — N/A 50 40
RL 30 D methacrylate type A
 1 Introduction 35

TABLE 3.2 Physical properties and method of preparation of pure aqueous dispersions—cont’d
Aqueous
dispersion Polymer Additives (%) Method of preparation Tg (°C) MFT (°C) Ref

Eudragit Ethylacrylate Nonoxynol 100* Emulsification 8 5 [1, 37]

NE 30 D methylmethacrylate (1.5) polymerization (latex)
(2:1)

Eudragit N/A PEG stearyl ether N/A N/A N/A [1, 38]
NM 30 D (0.7)

Eudragit Methacrylic acid Sodium lauryl N/A >100 25 [1, 39]

L 30 D 55 copolymer sulfate (0.7)
Polysorbate
80 (2.3)
Kollicoat Polyvinyl acetate Polyvinyl N/A 46 18 [40, 41]
SR 30 D pyrrolidone (2.9)
Sodium dodecyl
sulfate (0.1)

N/A, not applicable.

polymeric dispersion. The MFT depends on

the elastic modulus (resistance to particle defor-
I mation). Another important characteristic is the
Aqueous dispersion deposited on glass transition temperature (Tg) of the polymer
surface [18] (Fig. 3.2). Below the Tg, the polymer exists in
Water evaporation and
a rigid and glassy state with extremely limited
particles packing
polymer segment mobility. Above the Tg, the
polymer is in a soft and rubbery state, and the
II
polymer chains present a significant movement.
Closely packed spheres with water filled Where the Tg of a polymer is higher than the
voids Water evaporation and desired operating coating temperatures, a plas-
polymer deformation ticizer should be added to obtain good film for-
mation. Generally, plasticizers reduce the
III
cohesive forces along with polymer chains, lead-
Apparently continuous polymer ing to an increase in flexibility, reduction in ten-
film Water diffusion and sile strength, and reduction of Tg and MFT,
polymer interdiffusion facilitating the polymer particle coalescence
[19–22]. A plasticizer should be compatible (mis-
cible) with the polymer and show no (or little)
FIG. 3.1 Film formation of thin films from polymeric tendency to migrate [23]. Plasticizers are high
lattices. boiling point organic compounds, with low
vapor pressures and different water solubility
In order to obtain a good-quality film (free of (Table 3.3) [12].
cracks), the coating temperature is usually The water-soluble plasticizers dissolve in the
10–20°C above the minimum film formation polymer aqueous dispersion, whereas the water
temperature (MFT) [5, 16, 17] of the aqueous insoluble plasticizers are emulsified in the
36 3. Aqueous polymeric coatings: New opportunities in drug delivery systems
aqueous phase of the dispersion. The most widely
used plasticizers are: triethyl citrate (TEC) and tria-
cetin (TA) as water-soluble plasticizers and acetyl-
triethyl citrate (ATEC), acetyltributyl citrate
(ATBC), dibutyl sebacate (DBS), diethyl phthalate
(DEP), and tributyl citrate (TBC) as water-
insoluble plasticizers. The decrease of the MFT
and thus the effectiveness of the plasticizer depend
on the plasticizer amount and its effectiveness for
each aqueous polymeric dispersion. The effects
of type and concentration of plasticizers on MFT
of aqueous polymeric dispersions are shown in
Table 3.4. In addition, the type and amount of
plasticizer can also change the diffusivity of the
coating and thus the drug release rate [42–45].
In the case of polymers with low Tg or where the
plasticizer strongly decrease the Tg and MFT, gli-
dants and antitacking agents are used to avoid
tackiness or agglomeration during coating, dry-
ing, curing, and storage. Talc, magnesium stea-
rate, kaolin, and glycerol monostearate are
frequently used. The content of the antitacking
agent depends on its nature—for example, talc
is used in the range of 25%–100% (w/w, based
on dry polymer), while 5%–20% (w/w, based on
dry polymer) of glycerol monostearate is highly
effective [5, 46]. Pigments are generally added to
polymeric dispersions to enable easy product
identification and to improve the elegance of
pharmaceutical dosage form. In addition, tita-
nium dioxide has been incorporated into film
coating formulations as an opacifying agent
to improve the stability of light-sensitive drugs
[47, 48]. The concentration of pigments should
not exceed critical pigment volume concentration
(CPVP) and the CPVP is characteristic of each
polymer-filler combination [49]. Surfactants such
as sodium lauryl sulfate, poly vinylpyrrolidone,
and polysorbates are used to stabilize the aqueous
polymeric dispersion [1]. Pore formers can also be
incorporated in the coating to modify the drug
release. Polyethylene glycol, polyvinylpyrroli-
done, hydroxypropyl cellulose, and in particular
hydroxypropyl methylcellulose (HPMC) [24, 50]
have been used to increase permeability of the
coatings. However, destabilization of the aqueous
dispersions may occur [51]. Recently, polyvinyl
pyrrolidone, polyvinyl alcohol-polyethylene gly-
col graft copolymer, and carrageenan have been
successfully used to modify drug release from
aqueous-based coatings [52–54] as Aquacoat
ECD-coated dosage forms without destabilizing
the aqueous dispersion.
After completion of the coating process, a
curing step (short thermal treatment) is regularly
performed to improve polymer particle coales-
cence. This curing step is of utmost importance
to promote complete film formation from a poly-
mer aqueous dispersion [55, 56]. The curing can be
performed in the coating equipment or in an oven.
A curing temperature 10–20°C above the MFT of
the aqueous dispersion is recommended to pro-
mote the “further gradual coalescence” (micro-
scopic coalescence). The specific temperature,
time, and presence of humidity on the curing step
is dependent on each formulation as aqueous
polymeric dispersion properties, plasticizer type,
presence/absence of talc, and coating conditions,
to name a few. Tackiness and agglomeration can
occur during the curing process, and to circum-
vent these problems, a thin HPMC overcoat can
be used [46, 57] or talc can be blended with coated
dosage forms before placing them in the oven [16].
During the curing step, drug migration through
the coating can occur, especially if the drug has
a low melting point and affinity for the polymeric
coating [56, 58]. In this case, a subcoating can avoid
drug migration during the curing step [58].
After the curing step, coated dosage forms are
stored under high temperature and humidity
conditions (the International Committee on
Harmonization recommends 25°C/60% RH and
40°C/75% RH) for a defined time. Often, drug
release rate decreases after these storage condi-
tions due to the well-known physical aging.
Physical aging or enthalpy relaxation is observed
with all amorphous polymers. Amorphous mate-
rials are not in thermodynamic equilibrium when
they are cooled below their Tg [59]. Since this is an
energetically unfavorable state, the polymer
chains in a glassy film will reorient to achieve
the equilibrium over time [60].
Another random document with
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 VIII
The word gentility is constantly in the mouths of vulgar people; as
quacks and pretenders are always talking of genius. Those who
possess any real excellence think and say the least about it.
 IX
Taste is often envy in disguise: it turns into the art of reducing
excellence within the smallest possible compass, or of finding out the
minimum of pleasure. Some people admire only what is new and
fashionable—the work of the day, of some popular author—the last
and frothiest bubble that glitters on the surface of fashion. All the
rest is gone by, ‘in the deep bosom of the ocean buried;’ to allude to it
is Gothic, to insist upon it odious. We have only to wait a week to be
relieved of the hot-pressed page, of the vignette-title; and in the
interim can look with sovereign contempt on the wide range of
science, learning, art, and on those musty old writers who lived
before the present age of novels. Peace be with their manes! There
are others, on the contrary, to whom all the modern publications are
anathema, a by-word—they get rid of this idle literature ‘at one fell
swoop’—disqualify the present race from all pretensions whatever,
get into a corner with an obscure writer, and devour the cobwebs and
the page together, and pick out in the quaintest production, the
quaintest passages, the merest choke-pear, which they think nobody
can swallow but themselves.
 X
The source of the love of nature or of the country has never been
explained so well as it might. The truth is this. Natural or inanimate
objects please merely as objects of sense or contemplation, and we
ask no return of the passion or admiration from them, so that we
cannot be disappointed or distracted in our choice. If we are
delighted with a flower or a tree, we are pleased with it for its own
sake; nothing more is required to make our satisfaction complete; we
do not ask the flower or tree whether it likes us again; and, therefore,
wherever we can meet with the same or a similar object, we may
reckon upon a recurrence of the same soothing emotion. Nature is
the only mistress that smiles on us still the same; and does not repay
admiration with scorn, love with hatred. She is faithful to us, as long
as we are faithful to ourselves. Whereas, in regard to the human
species, we have not so much to consider our own dispositions
towards others, as theirs towards us; a thousand caprices, interests,
and opinions, may intervene before the good understanding can be
mutual; we not only cannot infer of one individual from another, but
the same individual may change to-morrow: so that in our
intercourse with the world, there is nothing but littleness,
uncertainty, suspicion, and mortification, instead of the grandeur
and repose of nature.
 XI
It has been objected to the soothing power of Nature, that it cannot
take away the sharp pang of vehement distress, but rather barbs the
dart, and seems to smile in mockery of our anguish. But the same
might be said of music, poetry, and friendship, which only tantalize
and torment us by offering to divert our grief in its keenest
paroxysms; but yet cannot be denied to be enviable resources and
consolations of the human mind, when the bitterness of the moment
has passed over.
 XII
Every one is a hero, the circumstances being given. All that is
necessary is, that the outward impression should be so strong as to
make a man forget himself. A woman rushes into the flames to save
her child, not from duty or reason—but because the distracting terror
for another banishes all recollection of, and fear for, herself. For the
same reason, a person throws himself from a precipice, because the
apprehension of danger gets the better of and confounds the sense of
self-preservation. The doctrine of self-love, as an infallible
metaphysical principle of action, is nonsense.
 XIII
The heroical ages were those in which there was a constant
question between life and death, and men ate their scanty meal with
their swords in their hands.
 XIV
The hero acts from outward impulse; the martyr from internal
faith, and so far is the greater character of the two. And yet it may be
doubted whether the latter is properly a voluntary agent, or whether,
if he could do it unperceived, he would not abstract himself from the
scene, instead of becoming a sacrifice and a witness to the truth.
 XV
What shews that persecution and danger act as incentives rather
than impediments to the will, is that zeal generally goes out with the
fires that kindle it; and we become indifferent to a cause, when life,
property, and limb are no longer endangered. He is the real
philosopher who loves truth for its own sake, not in the spirit of
contradiction: he the genuine friend of freedom and justice, who
hates oppression and wrong after they have ceased, and as long as
the very name of them remains, as well as while it is a bone of
contention between infuriated sects and parties.
 XVI
If reform were to gain the day, reform would become as vulgar as
cant of any other kind. We only shew a spirit of independence and
resistance to power, as long as power is against us. As soon as the
cause of opposition prevails, its essence and character are gone out of
it; and the most flagrant radicalism degenerates into the tamest
servility. We then say as others say; sail with the stream; no longer
sacrifice interest to principle, but are in a pitiful majority. Had events
taken a different turn in 1794, who can predict what the popular cry
would have been? This may point out how little chance there is of
any great improvement in the affairs of the world. Virtue ceases with
difficulty; honesty is militant. The mass of mankind, who are
governed by indolence and habit, fall in with existing events and
interests; the imaginative and reasoning part fall out with facts and
reality; but could they have their way, and model the world at their
pleasure, their occupation would be gone; or if all governments were
wise and good, the character of the patriot would become obsolete,
and a sinecure. At present there is a very convenient division of
labour; and each class fulfils its vocation. It is essential to the
triumph of reform that it should never succeed.
 XVII
We talk about the cant of politics or religion, as if there were no
cant but that which is common to the multitude. But whenever any
two individuals agree about any one thing, they begin to cant about
it, and take the echo of one another’s voices for the verdict of truth.
Half-a-dozen persons will always make a quorum of credulity and
vulgarity.
 XVIII
When people have done quarrelling about one set of questions
they start another. Motion is necessary to mind as much as to
matter; and for ‘an ultimate end,’ Hobbes denies that there is any
such thing. Hence the tendency to all Ultra opinions and measures!
Man is seldom contented to go as far as others, unless he can go
beyond them, and make a caricature and a paradox even of the most
vulgar prejudice. It is necessary to aim at some kind of distinction—
to create some difficulty, were it only for the sake of overcoming it.
Thus we find that O’Connell, having carried his cause, would not let
the ‘agitation’ subside without turning it into a personal quarrel: the
way was opened to him into the House, and he wanted to force his
way there by an ex post facto inference; the banns of marriage were
published between him and parliament, and he would fain, with the
petulance of opposition, seize a seat there.
 XIX
Truth itself becomes but a fashion. When all the world
acknowledge it, it seems trite and stale. It is tinged by the coarse
medium through which it passes.
 XX
Erasmus, in his ‘Remains,’ tells a story of two thieves, who were
recommended by their mother to rob every one they met with; but
warned, on peril of their lives, to avoid one Black-breeches
(Hercules). Meeting him, however, without knowing him, they set
upon him, and were slung across his shoulder,—where Hercules
heard them muttering behind his back, a long way off, ‘This must
surely be he that our mother warned us of.’ In contempt and pity he
let them escape. What modern wit can come up to the grotesque
grandeur of this invention?
 XXI
People addicted to secresy are so without knowing why; they are so
not ‘for cause,’ but for secresy’s sake. It is a mixture of cowardice and
conceit. They think, if they tell you any thing, you may understand it
better than they do, or turn it in some way against them; but that
while they shut up their mouths they are wiser than you, just as liars
think by telling you a falsehood they have an advantage over you.
There are others who deal in significant nods, smiles, and half-
sentences, so that you never can get at their meaning, and indeed
they have none, but leave it to you to put what interpretation you
please on their embryo hints and conceptions. They are glad to find a
proxy for their want of understanding.
 XXII
It is the force and violence of the English mind that has put it into
the safe custody of the law, and it is every man’s disposition to act
upon his own judgment and presumption, without regard to others,
that has made it absolutely necessary to establish equal claims to
curb them. We are too much in a state of nature to submit to what
Burke calls ‘the soft collar of social esteem,’ and require ‘the iron rod,
the torturing hour,’ to tame us. But though the foundations of liberty,
life, and property, are formally secured in this way from the
ebullitions of national character, yet the spirit breaks out upon the
surface of manners, and is often spurted in our face. Lord
Castlereagh was wrong in saying that ‘liberty was merely a custom of
England;’ it is the indigenous growth of our temper and our clime;
and woe to him who deprives us of the only amends for so many
disadvantages and failings! The wild beast roaming his native forests
is respectable though formidable—shut up in Exeter ‘Change, he is
equally odious and wretched.
 XXIII
It was a long time made an argument for not throwing open the
galleries of noblemen and others to the public, that if permission
were given they would be filled with the lowest of the rabble, and
with squalid wretches, who would run up against well-dressed
people, and damage the works of art. Nothing could be more false
than this theory, as experience has shown. It was in vain to quote the
example of foreign countries, as it was said the common people there
were kept more in subjection; but if they are tamer, ours are prouder
for that very reason. The National Gallery in Pall-Mall is now open to
all the world; and, except a shabby artist or two, who ever saw a soul
there who was not, if not well-dressed, yet dressed in his best, and
behaving with decency, instead of trying to turn the place into a bear-
garden, as had been predicted.[42] People will not go out of their way
to see pictures unless they have an interest in them, which gives the
title, and is a security against ill consequences; much less will any
class of people obtrude themselves where they are pointed at as
inferior to the rest of the company, or subject themselves to looks of
scorn and disgust, to see any sights in the world. There is no man so
poor or low but he loves himself better than pictures or statues; and
if he must get snubbed and treated with contempt to indulge his
admiration of celebrated works, he will forego the latter.
Comparisons are odious; and we avoid them. The first object of
every human being (high or low, great or small) is to stand well with
himself, and to appear to the best advantage to others. A man is not
very fond of passing along the streets in a thread-bare coat, and
shoes with holes in them. Will he go in this trim into a group of well-
dressed people to make himself ridiculous? The mind, so far from
being dull or callous on this point, is but too sensitive; our jealousy of
public opinion is the ruling passion, a morbid disease. Does not the
consciousness of any singularity or impropriety of appearance
immediately take off from our pleasure at a play? How seldom we
observe an interloper in the dress circle; and how sure he is to pay
for it! If a man has any defect or inferiority, this is certain, he will
keep it in the back-ground. If a chimney-sweeper or scavenger had a
ticket to a ball, would he go? Oh! no; it is enough to bear the sense of
our own infirmity and disgrace in silence, and unnoticed, without
having it wrought to agony by the glare of contrast and ostentation of
insult! What linendraper or grocer’s son would dine with a prince
every day though he might, to be crushed into insignificance, and
stifled with ironical civility? Do we not observe the difficulty there is
in making servants and mechanics sit down, or keep on their hats in
speaking to their betters, for fear of being thought to encroach, and
made liable to a rebuff in consequence? Assuredly, then, the great
may throw open their palace-doors and galleries of art without
having to dread the inroad or outrages of the mob, or fancying that
any one will go who is not qualified to appear, or will not come away
with his mind and manners improved. The wooden shoes and mob
caps in the Louvre or the Vatican do no harm to the pictures on the
walls: but add a new interest to them, and throw a pleasing light on
human nature. If we are behind other nations in politeness and
civilization, the best way to overtake them is to tread in their steps.
 XXIV
It is at the same time true that familiarity breeds contempt; or
that the vulgar, if admitted to an intimacy and footing of equality, try
to make you feel all your defects, and to pay for the superiority you
have so long usurped over them. The same pride that before kept
them at a distance makes them ready to throw down any barrier of
deference or distinction the moment they can do so with impunity.
No one willingly admits a superiority in another; or does not secretly
prefer himself to the whole universe beside. The slave would kill the
tyrant, whose feet he kisses; and there is no Turk so loyal that he
would not cut off the head of the best of Sultans, if he was sure of
putting the diadem upon his own.
 XXV
The strongest minds are governed more by appearances than by a
regard to consequences. Those who pretend to be the greatest
calculators of their own interest, or the main-chance, are the very
slaves of opinion, and dupes of shallow pretension. They are often so
mad in this respect, that they think neither better nor worse of the
oldest friend they have in the world than the first person they happen
to be in company with does, or the last rumour they heard gives him
out. Their circumspection amounts to looking three ways at once,
and missing the right point of view at last. They would rather speak
to a well-dressed fool in the street than to the wisest man in a thread-
bare suit. I know an author who succeeds with a set of second-hand
thoughts by having a coat of the newest cut; and an editor, who
flourishes about the town in virtue of a pair of green spectacles. Lay
out all you are worth in decking out the person of a vulgar woman,
and she will cut you in the very finery you have given her; lay it out
on your own back, and she will be ambitious of your least notice.
People judge of you not from what they know, but from the
impression you make on others, which depends chiefly on
professions, and on outward bearing and bravery. De non
apparentibus et non existentibus eadem est ratio. If a man has no
opinion of himself, how the deuce should any one else? It is like
electing a person member of parliament who refuses to come
forward as a candidate. On the other hand, let a man have
impudence in lieu of all other qualifications, and he needs not
despair. The part of quack or coxcomb is a favourite one with the
town. The only character that is likely to get on by passing for a poor
creature is the legacy-hunter. Nothing can be too low or insignificant
for that. A man is only grateful to you in the other world for having
been a foil to him in this. A miser (if he could) would leave his
fortune to his dog, that no human being might be the better for it, or
no one that he could envy in the possession of it, or think raised to an
equality with himself.
 XXVI
We complain of old friends who have made their fortunes in the
world and slighted us in their prosperity, without considering those
who have been unsuccessful, and whom we have neglected in our
turn. When our friends betray or desert us, we cling the closer to
those that remain. Our confidence is strengthened by being
circumscribed; we do not wish to give up a forlorn hope. With the
crumbling and decayed fragments of friendship around us, we
maintain our point to the last; like the cobbler, who kept his stall and
cooked his beef-steak in the ruins of Drury Lane. Bonaparte used to
speak of old generals and favourites who would not have abandoned
him in his misfortunes if they had lived; it was perhaps well for them
that they were dead. The list of traitors and the ungrateful is too
much swelled without any probable additions to it.