10.1007@978 981 13 8954 2 PDF

Nanoparticles
in Medicine
Ashutosh Kumar Shukla

Editor
123
Nanoparticles in Medicine
Editor
Nanoparticles in Medicine
Editor
Physics Department, Ewing Christian College
University of Allahabad
Allahabad, Uttar Pradesh
India
ISBN 978-981-13-8953-5 ISBN 978-981-13-8954-2 (eBook)

https://doi.org/10.1007/978-981-13-8954-2
© Springer Nature Singapore Pte Ltd. 2020

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To my parents.
Preface
This collection intends to highlight the potential applications of nanoparticles in

medicine. Gold and silver nanoparticles, rare earth-doped fluoride nanoparticles,
protein-based nanoparticles, and fungal-mediated nanoparticles have been covered
in different chapters. The applications in different fields of medicine covered in this
volume include cancer therapy, photodynamic therapy, antioxidant therapy, den-
tistry, and caries prevention. Related toxicity issues have been addressed in indi-
vidual chapters and in a separate chapter as well.
I am thankful to the expert contributors for contributing to this volume. My spe-
cial thanks to Prof. Alex I. Smirnov, Department of Chemistry, North Carolina State
University, USA; Prof. Anjana Pandey, Department of Biotechnology, MNNIT,
Allahabad; Dr. Chitta Ranjan Patra, Department of Applied Biology, CSIR-IICT,
Hyderabad; Dr. Monalisa Mishra, Department of Life Science, NIT, Rourkela; and
Dr. S Rajeshkumar, Department of Pharmacology, Saveetha Dental College,
Chennai, who kindly reviewed the manuscripts for this volume.
I sincerely thank Dr. Naren Aggarwal, Executive Editor, Clinical Medicine,
Springer (India) Private Limited, for giving me the opportunity to present this book
to the readers. I also thank Teena Bedi, Associate Editor, Clinical Medicine, Springer
(India) Private Limited, and Mr. Ejaz Ahmad, Project Coordinator (Books), for their
support during the publication process.
It was a good learning experience for me to go through individual chapters and
hope that it will be a good experience for the audience too.
Allahabad, India Ashutosh Kumar Shukla

May 2019
vii
Contents
1 Theranostic Applications of Lysozyme-Based Nanoparticles�� 1

Sourav Das, Manideep Pabba, M. E. Dhushyandhun, and Chitta
Ranjan Patra
2 Emerging Nanomaterials for Cancer Therapy�� 25
Sanjay Kumar, Pratibha Kumari, and Rajeev Singh
3 Application of Nanoparticles in Dentistry: Current Trends�� 55
Subhashree Priyadarsini, Sumit Mukherjee, Janmejaya Bag, Nibedita
Nayak, and Monalisa Mishra
4 Microbial Synthesis of Silver Nanoparticles
and Their Biological Potential�� 99
Annuja Anandaradje, Vadivel Meyappan, Indramani Kumar,
and Natarajan Sakthivel
5 Fluoride Nanoparticles for Biomedical Applications�� 135
M. S. Pudovkin and R. M. Rakhmatullin
6 Gold Nanostructures in Medicine and Biology �� 175
Siavash Iravani and Ghazaleh Jamalipour Soufi
7 Fungus-Mediated Nanoparticles: Characterization
and Biomedical Advances�� 185
S. Rajeshkumar and D. Sivapriya
8 Precautions to Avoid Consequences Leading to Nanotoxification�� 201
Sharda Sundaram Sanjay
ix
About the Editor
Ashutosh Kumar Shukla holds B.Sc., M.Sc., and D. Phil. degrees from the
University of Allahabad. His doctoral research involved using electron spin reso-
nance spectroscopy and optical absorption spectroscopy to investigate transition
ion-doped single crystals. He has been a university teacher and researcher for
17 years and is currently an Associate Professor of Physics at Ewing Christian
College, Allahabad, a University of Allahabad institution. He also served as an
Associate Professor (Pure and Applied Physics) at Guru Ghasidas Vishwavidyalaya,
Bilaspur, C.G. (a central university).
Dr. Shukla has successfully completed research projects in the area of electron
spin resonance, funded by the University Grants Commission, India’s main higher
education body. He has presented his research at various international events in
countries including the USA, UK, Germany, Spain, and Russia. He has published a
number of research papers in peer-reviewed journals and has edited numerous vol-
umes. He has delivered several invited lectures on characterization techniques at
national and international conferences and workshops. He also reviews manuscripts
for a number of international journals.
He has received many scholarships and fellowships, including a National
Scholarship; Government of U. P. Ministry of Higher Education Scholarship; and
research fellowships from the Council of Science & Technology, Lucknow; Council
of Scientific & Industrial Research, New Delhi; and the Indian National Science
Academy-Bilateral Exchange Fellowship.
xi
Theranostic Applications of Lysozyme-
Based Nanoparticles 1
Sourav Das, Manideep Pabba, M. E. Dhushyandhun,
and Chitta Ranjan Patra
Abbreviations
A549 Adenocarcinomic human alveolar basal epithelial cells

ABTS 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
AgNPs Silver nanoparticles
AuNCs Lysozyme-gold nanocluster
CD Circular dichroism
CF Cystic fibrosis
CMC Critical micelle concentrations
COPD Chronic obstructive pulmonary diseases
CuNCs Copper nanocluster
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal growth factor
FRET Forster non-radiative energy transfer
FT-IR Fourier-transform infrared spectroscopy
HeLa Cervical cancer cells (human)
HGT Horizontal gene transfer
hLYS Human lysozyme
HPLC High-performance liquid chromatography
LOD Lowest detection limit
LYMB Lysozyme microbubble
S. Das · C. R. Patra (*)

Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
Department of Applied Biology, CSIR-Indian Institute of Chemical Technology,
Hyderabad, Telangana, India
e-mail: crpatra@iict.res.in
M. Pabba · M. E. Dhushyandhun
Department of Applied Biology, CSIR-Indian Institute of Chemical Technology,
Hyderabad, Telangana, India
© Springer Nature Singapore Pte Ltd. 2020 1

A. K. Shukla (ed.), Nanoparticles in Medicine,
https://doi.org/10.1007/978-981-13-8954-2_1
2 S. Das et al.
MALDI Matrix-assisted laser desorption/ionization

MCF-7 Michigan cancer foundation-7
MD Molecular dynamics
MIC Minimum inhibitory concentration
NAG-3 N-acetyl-d-glucosamine
NC Nanoclusters
ND Nanodiamonds
NHF Normal human foreskin fibroblast
NP Nanoparticles
PBS Phosphate buffered saline
PDRAB Pan-drug resistant Acinetobacter baumannii
PEI Polyethyleneimine
rHlys Recombinant human lysozyme
ROS Reactive oxygen species
SAP Serum amyloid p component
SDS Sodium dodecyl sulfate
snLYZ Self-assembled nanostructured lysozyme
TDD Transdermal drug delivery
THB Theobromine
THP Theophylline
TMB 3,3′,5,5′-Tetramethylbenzidine
US Ultrasound
VRE Vancomycin-resistant Enterococcus faecalis
WT Wild type
XRD X-ray crystallography
XTT 2,3-Bis-(2-methoxy-4-nitro5-sulfophenyl)-2H-tetrazolium-5-
carboxanilide salt
1.1 Introduction
Lysozyme is an enzyme (anti-bacterial) generally distributed in divergent biological

fluids, tissues as well as in animal secretions (Callewaert and Michiels 2010). It has
various important applications such as anti-bacterial, anti-inflammatory, and anti-
proliferative activities (Salton 1957; Ye et al. 2008). Recent reports demonstrate that
the importance of this small globular protein relies on its substantial use as a model
protein in order to know the structure, dynamics, function as well as folding of the
proteins (mainly the underlying principle) (Sonu et al. 2016). The high natural
abundance of this protein leads to the development of various nanoparticles reflect-
ing its broad spectrum of usage. Lysozyme was first discovered in the year of 1909.
But, this protein was first recognized by Alexander Fleming in the year of 1922 who
coined it as “Lysozyme” (Fleming and Wright Almroth 1922). Now-a-days, nano-
technology has been extensively used in the various areas of science and technol-
ogy. The emergence of the nanotechnology in the field of biotechnology leads to the
development of new kind of nanoparticles. Meanwhile, due to several advantages
1 Theranostic Applications of Lysozyme-Based Nanoparticles 3
and lesser toxicity profile, the protein-based nanoparticles have got the remarkable
importance (Tarhini et al. 2017). Recently, scientists are relentlessly making various
kinds of protein-based nanoparticles especially lysozyme proteins useful for numer-
ous applications such as drug delivery, imaging, and sensing. In addition, the
uniquely faceted tunable structure of the protein lysozyme made it useful in making
various nanoparticles including inorganic metals like gold, silver, platinum, silica,
copper, and iron (Ghosh et al. 2014; Li et al. 2017). Furthermore, the most recent
advances move toward the development of new treatment strategies using these
protein-based nanoparticles. Herein, this chapter will mainstay on the theranostic
applications of lysozyme-based nanoparticles and the interactions between lyso-
zyme and the metal nanoparticles. Finally, this chapter will provide a brief overview
on the properties of lysozyme as well as its applications in a concise manner.
1.1.1 Lysozyme and Its Background
Lysozyme is a ubiquitous anti-microbial enzyme generally found in fluids, secre-

tions such as human saliva, milk, tears, even all mucosal surfaces, respiratory tract,
and digestive tract. It is also available in both animals and plants (e.g., papaya latex)
(Murakami et al. 1998; Peeters and Vantrappen 1975). Lysozyme has high thermal
stability having melting point 72 °C (at pH 5.0) and quite stable at higher pH range.
The iso-electric point of this protein is 11.35. According to the reports, in egg white
large amount of lysozyme can be found (Venkataramani et al. 2013). Lysozyme
(14.3 KDa) has 129 amino acids with four cysteine residues. It sustains the con-
densed globular structure with an active site at the protein surface in physiological
condition. Deep down the complicated secondary structure, it has five helical
regions along with the alpha helices, beta sheet, random coil, and beta turns.
According to reports, the crystal form of this protein can be obtained by binding the
trisaccharide (NAG-3: N-acetyl-d-glucosamine) in its substrate groove (Russell
et al. 2017). It has been observed from the crystallographic analysis that the lyso-
zyme (obtained from chicken egg) has high amount of arginine and has no sulfhy-
dryl group. Generally, it demonstrates two conformations, active site (open) and
inactive site (closed) which change accordingly during the moment of action (Choi
et al. 2012). The basic properties of the lysozyme helps in forming the complexes
with negatively charged substances (Salton 1957).
1.1.2 Biological Activity
Lysozyme shows catalyzing activity by hydrolyzing the peptidoglycan residues

such as 1,4-beta-linkages between N-acetyl-d-glucosamine and N-acetylmuramic
acid found in bacterial cell wall (Scanlon et al. 2010). Peptidoglycan which is one
of the primary components of the bacterial cell wall delivers resistance against tur-
gor pressure. Therefore, lysozyme builds a kind of first line defense mechanism
against bacterial infections and colonization by participating in innate immune sys-
tem. Scanlon et al. used the concept of protein engineering to design the human
4 S. Das et al.
Glu35 Glu35
HO O HO O
OH O OH O
H2O
NH NH
O HO O O HO O
RO O O RO OH HO O
NH NH
O OH O OH
n
O O- O O-
Asp53 Asp53
Fig. 1.1 Lysozyme (hLYS) catalyzes hydrolysis of peptidoglycan. A line drawing representing
the two repeating carbohydrate units of the bacterial cell wall: (1,4)-linked N-acetyl muramic acid
and N-acetyl glucosamine. The catalytic residues of hLYS are shown in gray. The “R” group of
N-acetyl muramic acid represents a C2-ether-linked lactic acid moiety that is in turn coupled to
adjacent polysaccharide chains via short, cross-linking polypeptides. The resulting macromolecu-
lar net surrounds individual bacteria and forms the protective sacculus that opposes osmotic stress.
Sacculus integrity and cell viability are compromised by hLYS-catalyzed hydrolysis of the poly-
saccharide chains. Reprinted with permission from Scanlon et al. (2010). Enhanced anti-microbial
activity of engineered human lysozyme. ACS Chem Biol 5:809–818 (Scanlon et al. 2010).
Copyright © 2010 American Chemical Society
lysozyme and showed their mechanisms for enhanced anti-microbial activity

(Fig. 1.1) (Scanlon et al. 2010). Additionally, the mechanism of action can be enzy-
matic (muramidase-dependent) or non-enzymatic (membrane perturbing)
(Callewaert and Michiels 2010; Callewaert et al. 2012; During et al. 1999; Scanlon
et al. 2010). Lysozyme increases the phagocytic activity of some leukocytes (poly-
nuclear) and macrophages non-specifically which supports the immunoregulatory
activity of the same (Ye et al. 2008). According to Bruzzesi et al., the monomer of
lysozyme undergoes dimeric association which is a pH-dependent reversible pro-
cess (Bruzzesi et al. 1965).
1.1.3 Application in Different Fields
Lysozyme has various biological applications like anti-microbial, anti-proliferative,

and amyloid-forming ability.
1.1.3.1 Anti-Microbial Activity

Lysozyme exhibits enhanced anti-microbial activity. Generally, in human airway
fluids lysozyme is the most effective cationic anti-pseudomonal agent (Cole et al.
2002). However, the wild-type human lysozyme is not able to perform well in spe-
cific lung environment during chronic infections. Recombinant human lysozyme
can be used as a therapeutic protein if properly delivered by inhalation techniques
(Shire 1996). Alleviating the functional limitations of the wild-type human lyso-
zyme may help in the development of new anti-microbial therapies. Because of the
cationic (positive) nature of the human lysozyme, it is thought that it can be used to
attract the negatively charged bacterial cell wall. In some cases, due to the dense
cationic charge, hLYS (human lysozyme) can bind with alginate and turns it into its
inactivated form (Scanlon et al. 2010). In order to decrease the immunogenic effect
of the enzyme, Gill et al. used human scaffold protein as the starting template. The
authors demonstrated that charge-engineered human lysozymes could be created
with the help of bio-informatics and structural analysis techniques (Gill et al. 2011).
The authors reported that the re-shaped electrostatic potential of bio-therapeutic
candidates delivered enhanced inhibitory effect against gram-positive (M. luteus)
and gram-negative (P. aeruginosa) bacteria. Additionally, the group demonstrated
that even after modifying the amino acid residues in the variant form, the stability
and global structure were similar to the wild-type form. Finally, the authors shed
lights on the future applicability of the modified human lysozyme for the treatment
of the CF (cystic fibrosis) as well as COPD (chronic obstructive pulmonary dis-
eases). Sometimes, in case of acute and chronic diseases, huge amount of anionic
biopolymer accumulated which concomitantly decreases lysozyme activity. In order
to overcome the pitfalls, human lysozyme was modified with the help of the protein
engineering useful for airway infections. Scanlon et al. functionally interrogated the
human lysozyme (charge-engineered) using a novel high-throughput screen method
(Scanlon et al. 2010). The group used a mouse model having acute pulmonary
infections (Pseudomonas aeruginosa bacterial strain) which showed a large gather-
ing of biopolymers (anionic) that passively deteriorate the lysozyme activity. The
authors illustrated the idea that in the presence of biopolymer (disease-associated),
reduction in the net cationic character improved the anti-microbial activity. Finally,
the authors concluded that the hLYS acted as an adaptable scaffold on which variant
antibiotics (biocatalytic) can be engineered. Hughey et al. demonstrated that lyso-
zyme can act as a food preservative by preventing the spoilage of food from bacte-
rial contamination (Hughey and Johnson 1987). The authors mentioned that four
strains of L. monocytogenes (infected in human through vegetables, milk products
like whole milk or soft cheese) as well as few strains of C. botulinum could be lysed
by the lysozyme activity. The group demonstrated that lysozyme in association with
EDTA exhibited enhanced activity in a consistent manner. The authors explained
that by associating lysozyme with the EDTA increased the activity by removing cell
wall components and exposing the peptidoglycan layer toward lysozyme’s active
site. Finally, the authors mentioned that the rate of bacterial degradation was high
enough in low temperature compared to the higher temperature.
1.1.3.2 Anti-Proliferative Activity of Lysozyme

Apart from the anti-microbial activity, the lysozyme can also act as an anti-
proliferative agent. It is deeply associated with monocyte–macrophage system for
non-specific defense mechanism (Osserman 1976). It has been reported that it is
generally expressed in gastric Paneth cells as well as having an efficient effect on
malignant tumors. Guo et al. demonstrated the anti-proliferative activity toward
gastric cancer cells (MGC803, MKN28, and MKN45) as well as normal fibroblast
(human lung) of recombinant human lysozyme (rHlys) (Guo et al. 2007). The
authors showed that rHlys displayed inhibiting activity of gastric cells at higher
6 S. Das et al.
concentrations, whereas at concentration lower than that it triggered the prolifera-

tion surprisingly. The authors claimed that 100 μg/l was the optimum growth inhib-
iting concentrations. Finally, the group provided a ray of hope for the future
application of recombinant human lysozyme as an anti-proliferative agent in future.
Similarly, Ye et al. reported the anti-proliferative activity of marine-derived lyso-
zyme both in vitro and in vivo conditions (Ye et al. 2008). The authors reported that
the marine-derived lysozyme hindered the proliferation of the endothelial cells as
well as retarded neovasculature in chicken embryos in a dose-dependent manner.
Furthermore, the group mentioned that the lysozyme significantly inhibited the
growth of tumor in tumor-bearing mice (hepatoma 22 or sarcoma 180). The marine-
derived lysozyme exhibited dose-dependent reduction of the branch points when
treated on the hen egg, whereas the hen-derived lysozyme did not show any decrease
in branches (Fig. 1.2). The PBS control group also exerted the similar effects like
hen-derived lysozyme. Finally, the authors claimed that due to several advantages
(abundance, cold-adaption property, optimum temperature stability, easier extrac-
tion property, etc.) of the marine-derived lysozyme, it could be useful for anti-
proliferative activity. Similarly, Mahanta et al. designed self-assembled
nanostructured lysozyme (snLYZ) using simple de-solvation technique (Mahanta
et al. 2015). The self-assembled nanostructure was further characterized using the
different physico-chemical techniques. The as-synthesized snLYZ nanostructured
lysozyme displayed excellent structural and functional stability in wide ranges of
pH. The authors mentioned that the snLYZ exhibited excellent anti-proliferative
activity toward human breast cancer cell line MCF-7 compared to the native lyso-
zyme through ROS generation pathway. Additionally, the authors revealed that the
hemocompatible snLYZ displayed more cell-killing ability when tagged with folic
acid. Finally, the authors shed lights on the future application of the nanostructure
in tissue engineering and regenerative medicine.
1.1.3.3 Amyloid-Forming Ability

Whenever there are mutations in the genes coding for lysozyme, it gives rise to
lysozyme amyloidosis. In order to study the effect, Helmfros et al. expressed the
disease-associated variant F57I with normal wild-type variant (human) lysozyme in
the central nervous system of Drosophila melanogaster model in association with
Fig. 1.2 Effect of marine lysozyme on CAM. Fertilized eggs were incubated continuously for
6 days, and then a window was opened to expose the CAM, and lysozyme at different concentra-
tions was added on sterilized filter paper discs on day 7. The eggs were incubated for another 72 h,
and then the treated CAMs were harvested and photographed. (a) 0 nM/egg (PBS negative con-
trol), (b) 1.2 nM/egg (marine lysozyme), (c) 1.2 nM/egg (hen egg lysozyme), (d) 100 nM/egg
(suramin). (e) Macroscopic assessment of vascular density conducted by counting the number of
branch points within a 100-mm2 area surrounding filter paper in each photograph. The number of
branch points was markedly decreased with marine lysozyme in a dose-dependent manner com-
pared to PBS control; there are no obvious differences between hen egg lysozyme-treated groups
and PBS control (n = 8; mean ± SEM); double asterisk p < 0.001 compared to PBS control.
Reprinted with permission from Ye et al. (2008) Marine lysozyme from a marine bacterium that
inhibits angiogenesis and tumor growth. Appl Microbiol Biot 77:1261–1267 (Ye et al. 2008).
Copyright © Springer-Verlag 2007
a b
c d
e 70
60
number of vessel branch point
50
40
**
30
** **
20
**
10 **
0
PBS
0.6
1.2
1.8
2.4
0.6
1.2
1.8
2.4
sudamin
100nM
marine lysozyme hen egg lysozyme

8 S. Das et al.
or without SAP (serum amyloid p component), member of pentraxins (Helmfros

et al. 2016). The authors indicated that SAP protein helped in innate immune system
through complement activation and assisted in the deposition of amyloid as well as
couples with fibrils of lysozyme. The authors declared that the lifespan of flies
reduced when they expressed amyloidogenic variant F57I compared to WT lyso-
zyme. Additionally, the group observed that the neurological function was vandal-
ized in flies when nerve cells expressed the F57I, which was recovered in the
presence of SAP. Finally, the authors reported that SAP could bind with through
ER-associated degradation pathway in order to impede the aggregation and accu-
mulation of disease-associated lysozymes. The tertiary structure of the proteins as
well as the resultant aggregation (fibrillary) of the human lysozyme resulted in lyso-
zyme systemic amyloidosis owing to their systemic changes in different conditions.
Jafari et al. observed the effect on human lysozyme’s amyloid-forming ability when
stimulated in sodium dodecyl sulfate (SDS) at 500000 ps MD (molecular dynamics)
at two temperatures (ambient) (Jafari and Mehrnejad 2016). Lysozyme, α + β class
of proteins, consists of disulfide bonds (four) along with 19 cationic as well as 11
anionic residues. This stable protein has hydrophobic core having polar surface.
SDS, an anionic detergent, acts as stabilizer to maintain helical structure of the pro-
teins on above critical micelle concentrations (CMC). The structure of lysozyme
and SDS is shown in Fig. 1.3. The authors found out that at temperature more than
the CMC, the helical structure of the protein was disrupted which ultimately ended
in β-sheets formation during stimulation. Finally, the authors mentioned that further
research should be performed on understanding the amyloid-forming ability of
human lysozyme. Sharma et al. mentioned that the globular protein lysozyme trans-
formed into building block of β-amyloids and lower ordered associated species
when heated (Sharma et al. 2016). The author reported that aggregation or spheru-
lites was observed along with dropping of crystallization point when applied higher
temperature. The formation of β-fibril-rich core as well as thin crystalline needles
with outward processions was observed; they were apparently the dimers and tri-
mers of lysozyme having minimal β-sheet content. The authors declared that broom-
like fibril at the edge was observed when they tried to use the needles as seed
materials. Additionally, the group highlighted that benzyl alcohol and arginine com-
pletely eradicated the spherulites formation through crystallization.
1.2 Interaction of Lysozyme with Nanoparticles
The broad repertoire of lysozyme drags its application in different fields. Lysozyme
has been used to make various nanoparticles including gold, silver, platinum, iron,
titanium, and copper. The protein lysozyme not only acts as a stabilizing agent but
also acts as a template or capping agent in order to prepare these nanoparticles.
a Lysozyme
C6-C128
C30-C116 H1
H2
H4
H3
C77-C95
C65-C81
b SDS
O3S O1S
C12 C10 C8 C6 C4 C2
O4 O2S
S
C1 C11 C9 C7 C5 C3
Fig. 1.3 The structures of human lysozyme and sodium dodecyl sulfate. (a) The native structure
of human lysozyme represented as a new cartoon model. The α-helix structures are shown with the
letter H and C6-C128, C30-C116, C65-C81, and C77-C95 represent disulfide bounds in the human
lysozyme structure. (b) The structure of an SDS surfactant molecule with its polar head group (in
red and green) and hydrophobic tail (in cyan and yellow) is shown as a ball and stick model.
Reprinted with permission from Jafari and Mehrnejad (2016) Molecular insight into human lyso-
zyme and its ability to form amyloid fibrils in high concentrations of sodium dodecyl sulfate: a
view from molecular dynamics simulations. PLoS One 11:e0165213 (Jafari and Mehrnejad 2016).
Copyright © 2016 Jafari, Mehrnejad
In view of this rising concern, it is an urgent need to discuss the interaction of
nanoparticles with the lysozyme. According to Wang et al., the interaction of the
lysozyme with the silver nanoparticles (AgNPs) was hydrophobic in nature (Wang
et al. 2017). The conformational changes in the lysozyme structure led to the sur-
face absorption of the protein on the nanoparticles which resulted in a pseudo-
second-order kinetics having hysteresis effect. The group showed that the interaction
10 S. Das et al.
of lysozyme with the nanoparticles was entropy-driven, and there was a reduction in
the alpha-helix content observed from the CD spectra. Similarly, Roy et al. demon-
strated that the interaction of lysozyme with the gold nanoparticles also followed
the same static quenching mechanism as observed by the fluorescence (Roy et al.
2017). The authors mentioned that the binding of lysozyme with the gold nanopar-
ticles was strong, and a single binding site was observed in the lysozyme for gold
nanoparticles. The binding process was thermodynamically stable, impetuous
where hydrophobic interaction played a pivotal role. The authors demonstrated that
there was a Forster non-radiative energy transfer (FRET) between lysozyme and
gold nanoparticles and found out that there was no significant difference in the
alpha-helix structure after interaction with the lysozyme. On the other hand, Yadav
et al. studied the interaction of lysozyme with silica nanoparticles and found that the
interaction depended on their size and surface area of the nanoparticles (Yadav et al.
2016). The authors demonstrated that the electrostatic interaction of lysozyme with
the nanoparticles led to the absorption of the protein on its surface ultimately
resulted in phase change. The authors found out that the phase transformation relied
on the attraction forces (short range) of lysozyme over the repulsive forces (long
range) between the nanoparticles which modeled by the two-Yukawa potential. At
lower concentration of lysozyme, the phase transformation was believed to happen
owing to the elevation of the size of the nanoparticles. Another study led by the
Aghili et al. where the group showed the interaction of protein lysozyme with iron
nanoparticles was because of the effect of both hydrogen bonding and van der Waals
forces (Aghili et al. 2016). The authors revealed that the quenching mechanism fol-
lowed the static and dynamic quenching process. The interaction not only changed
suspension potential but also red shifted the emission maxima corresponding to
tryptophan residue in the synchronous fluorescence spectroscopy. The group
declared that there was no such change in the secondary structure, but considerable
alteration in tertiary structure was observed. Altogether, the authors reported that
the interaction of lysozyme with the nanoparticles resulted from the negative
entropy effect. The decrease in the entropy of the protein (absorbed) was higher than
the increase in the entropy of the surface-attached water molecules, which enabled
this interaction effect. Similarly, Ghosh et al. synthesized the copper nanoclusters
using the lysozyme acted as template (Ghosh et al. 2014). The authors revealed that
the existence of various functional groups such as –NH2, –SH, –COOH, and –OH
delivered stability to the nanoclusters. The group declared that at higher pH the
protein was partially unfolded through unveiling abundant number of such groups
which gave enough space for binding of the nanoclusters on protein surface. The
different applications (therapeutic and diagnostic) of lysozyme-based nanoparticles
are represented in Scheme 1.1.
Scheme 1.1 Schematic
representations of
lysozyme-based Anti-
nanoparticles Microbial
Activity
Bio- Wound
Sensing Healing
Lysozyme-
Based
Nanoparticles
Cell Drug
Imaging Delivery
Catalytic
Activity
1.3 herapeutic Application of Lysozyme-Based

T
Nanoparticles
Lysozyme-based nanoparticles have several applications including anti-microbial,

drug delivery, wound healing, catalytic activity which are outlined below.
1.3.1 Anti-Microbial Activity
Owing to the rampant growth of drug-resistant microbes, alternative approaches

have been developed to provide microbe-free environment (Huh and Kwon 2011).
In view of this rising concern, various nanoformulations are introduced to resist the
infections. Recently, various nanoparticles have been synthesized using metals like
silver, copper, and gold possessing anti-bacterial properties (Zhou et al. 2012). It is
observed that silver ions from silver nanoparticles inhibit DNA replication, inacti-
vate cellular proteins in bacteria and introduce structural changes in the bacterial
cell membrane. These characteristics enable the application of silver nanoparticles
for anti-bacterial purposes such as bandages, food containers, pesticides, and dis-
infection of water. Coating of nanosilver with an anti-microbial protein such as
lysozyme produces a combined effect. Hence, lysozyme is being used as a catalyst
12 S. Das et al.
for the synthesis of lysozyme-coated silver nanoparticles through various methods

such as microwave-assisted method, borohydride reduction, reflux method, and
synthesis in an alkaline medium. Of the described methods, Ashraf et al. identified
that heat-denatured lysozyme-coated silver nanoparticle synthesized by reflux
method (RL Ag NPs) was most effective followed by microwave-assisted (ML Ag
NPs), alkaline medium-based preparation (AL Ag NPs) and borohydride reduction
(BL Ag NPs) (Ashraf et al. 2014). The authors explained that the enhanced effect
of heat-denatured lysozyme-coated silver nanoparticle was because of more expo-
sure of hydrophobic residues on the surface. The authors showed that the nanopar-
ticles were tested on different bacterial species and different strains of the same
species, and the bactericidal activity was measured by the minimum inhibitory
concentration (MIC). The group reported that the RL–Ag NPs exhibited excellent
anti-microbial property against Pseudomonas aeruginosa 3 compared to other
lysozyme-coated silver nanoparticles at various concentrations. The zone of inhi-
bition was highest in RL–Ag NPs followed by ML–Ag NPs, AL–Ag NPs, and the
least was observed in case of BL–Ag NPs as shown in Fig. 1.4. Additionally, the
18 100 ppm
80 ppm
60 ppm
16
40 ppm
20 ppm
14
12
Zone of inhibition (mm)
10
0
AL Ag NPs BL Ag NPs RL Ag NPs ML Ag NPs
Types of Iysozyme stabilized Ag NPs
Fig. 1.4 Bactericidal effect of lysozyme-stabilized Ag NPs against P. aeruginosa 3. Zones of

inhibition were measured in millimeters, whereas concentration of particles applied is given in
parts per million. No zone formation was observed at any dose of positive control (lysozyme solu-
tion). However, clear zone formation was observed in the presence of lysozyme-stabilized Ag NPs
at 10-ppm concentration. Reprinted with permission from Ashraf et al. (2014) Lysozyme-coated
silver nanoparticles for differentiating bacterial strains on the basis of anti-bacterial activity.
Nanoscale Res Lett 9:565 (Ashraf et al. 2014). Copyright © Ashraf et al.; license Springer. 2014
group showed that some strains were resistant to the bactericidal effect, while some
strains were quite sensitive to the nanoparticles. The phenomenon was attributed to
the silver-resistant genes acquired by bacteria through horizontal gene transfer
(HGT). Finally, the group concluded that lysozyme-based silver nanoparticles
could differentiate various strains of bacteria from the same species depending on
their resistance power toward the nanoparticles. Similarly, the combinatory effect
of lysozyme as well as silver was evaluated in nanoformulation against both gram-
negative and gram-positive bacteria. Eby et al. mechanically deposited the hybrid
materials of silver and lysozyme as a coating onto medical instruments (Eby et al.
2009a). The authors observed that this lysozyme–silver nanoparticles exhibited
significant anti-microbial activity which was attributed to the efficient release of
the enzyme for prolonged time. Amidst, the authors observed variable effects in the
different bacterial strains used with some degree of anti-bacterial activity, while no
such effect was observed in fungal or yeast cultures in this experiment. The authors
used a method called lytic assay (in vitro) in order to prove the anti-microbial
effect of the blades and needles by mitigating the normal action of the instruments.
Furthermore, the group concluded that the presence of lysozyme in the coating
improved biocompatibility in implanted devices and enabled uniform deposition of
the coating which improved the adsorption and release of nanoparticles to and
from the coated materials. Likewise, Eby et al. synthesized the lysozyme (from
chicken egg) stabilized silver nanoparticles where it acted as catalyzing agent as
well as reducing agent (Eby et al. 2009b). The authors nicely demonstrated the
formation of the lysozyme-stabilized silver nanoparticles in Fig. 1.5. The authors
Ag + + +
Iysozyme in water + Iyso
+ + + +
Ag
Iysozyme in methanol Ag Iyso
3k
Iyso Ag +
reduced Ag
+ Ag + Ag
+
+
+
Iyso + Ag
+
+ + +
+
Ag Ag
+
Iysozyme in methanol stable dialysis 25k + + +
+ colliod Iyso
Ag+ into water + +
in water Ag + Iyso
Iyso + +
+ +
Ag
Ag +
+
Ag
Ag
Ag
SDS Ag SDS
no colloidal Ag Ag
stability Iyso Ag
Ag
Ag Ag Ag
Iyso
Fig. 1.5 Proposed mechanism of Aglyso nanoparticle formation. Reprinted with permission from
Eby et al. (2009a, b) Lysozyme catalyzes the formation of anti-microbial silver nanoparticles. ACS
Nano 3:984–994 (Eby et al. 2009b). Copyright © 2009 American Chemical Society
14 S. Das et al.
mentioned that in the presence of methanol lysozyme took an amphiphatic form

which assisted in forming stable silver colloids and the surfactant properties of the
protein lysozyme increased. In addition, the colloidal stability of the lysozyme–sil-
ver was maintained after transfer into water. The addition of SDS precipitation and
agglomeration of the nanoparticles was observed. The authors reported that the
non-toxic nanoparticles demonstrated enhanced anti-microbial effect toward
Proteus mirabilis strains (silver resistant) and E. coli strain-consisting pMG101
plasmid. Finally, the authors shed lights on the future usage of this potential com-
posite as novel anti-microbial agents. Likewise, Chen et al. prepared stable lyso-
zyme–gold nanocluster AuNCs using microwave method (Chen et al. 2010). The
authors mentioned that the as-prepared AuNCs acted as a labeling agent on pan-
drug-resistant Acinetobacter baumannii (PDRAB), Vancomycin-resistant
Enterococcus faecalis (VRE) and compared with Au–ovalbumin NCs prepared by
the same method. Sample vials containing bacteria and NCs were seen under UV
illumination, and it was observed that only in the case of Au–lysozyme NCs red-
dish fluorescent bacterial spots were observed while these spots were observed as
white resulting from autofluorescence of bacteria indicating the inability of oval-
bumin NCs to bind to the bacteria. The authors demonstrated that the nanoclusters
exerted excellent anti-microbial activity against Acinetobacter baumannii
(PDRAB) (pan-drug-resistant), Enterococcus faecalis (VRE) (vancomycin-resis-
tant) bacteria compared to free lysozyme resulted from their multivalent interac-
tion of the NCs with the bacteria. Additionally, the authors demonstrated that the
AuNCs did not show toxicity in vitro culture (A549) evincing its biocompatibility.
Altogether, the author shed lights on the future applicability of the clusters as an
anti-microbial agent. On the other hand, Tripathy et al. prepared lysozyme tailored
ZnO nanoparticles using the low temperature solution route (Tripathy et al. 2014).
The as-synthesized nanoparticles exhibited profound anti-bacterial effects against
both the Staphylococcus aureus and Escherichia coli. The authors showed that in
comparison to lysozyme (native), pristine zinc oxide nanoparticles (ZNPs), the
lysozyme-conjugated zinc oxide nanoparticles (L-ZNPs) exerted higher bacteri-
cidal effects in both the strains as evident by the confocal images in Fig. 1.6. The
viable cells emitted green fluorescence, whereas the red fluorescence was seen
from dead cells. The authors revealed that owing to more negative surface proper-
ties of E. coli rather than S. aureus, the difference in inhibitory effects was obtained.
The group explained the difference in the zeta potentials between the surface of the
tailored ZnO nanoparticles and the bacteria ultimately led to cell killing. Altogether,
the authors envisioned the future applicability of these protein-conjugated metal
nanoparticles as nanobiotics for biological applications.
1.3.2 Drug Delivery
Drug delivery is one of the important aspects in the modern healthcare system in
order to cure multiple diseases (De Jong and Borm 2008). Conventional therapies
are poorly available, non-specific, have fast clearance, have various side effects,
a Control Lysozyme ZNPs Lysozyme-ZNPs

S. aureus
E. coli
b Lysozyme-ZNPs conjugates
(positively charged)
Bacteria Cell Death
(negatively charged cell wall)
Fig. 1.6 (a) CLSM images of S. aureus and E. coli followed by treatments and stained with FITC
and PI dyes. The green or red fluorescence is defined as live or dead cells, respectively. Scale
bar = 5 μm. (b) Anti-bacterial action of 5 L-ZNPs conjugates. Reprinted with permission from
Tripathy et al. (2014). Tailored lysozyme–ZnO nanoparticle conjugates as nanoantibiotics. Chem
Commun 50:9298–9301 (Tripathy et al. 2014). Copyright © Royal Society of Chemistry 2014
etc., which retard them from providing efficient therapeutic effects (Cho et al.
2008). The usage of protein-based nanoparticles helps in overcoming these pitfalls.
The following section will describe the lysozyme-based nanoparticles in drug
delivery applications which advances the treatment strategies. Generally, nanodia-
monds (ND) are biocompatible and known to have drug carrier properties. Kim
et al. reported the production of contact lenses fabricated with drug-loaded ND
nanogels (nanodiamond nanogels) in which the drug (timolol maleate) release was
triggered by the lysozyme present in the lacrimal fluid for ocular drug delivery
systems in diseased conditions such as glaucoma (Kim et al. 2014). The authors
showed that polyethyleneimine (PEI) was coated on individual ND and concomi-
tantly cross-linked with chitosan (enzyme-cleavable). These ND nanogels were
loaded with timolol maleate and entrapped into polyHEMa matrix; the resultant
was casted into contact lenses as shown in Fig. 1.7. The authors showed that upon
exposure to lacrimal fluids, the lysozyme cleaved the N-acetylated chitosan which
resulted in degradation of ND nanogels and ultimately released the timolol maleate
from the lens. Additionally, the authors reported that this delivery system overcame
all the drawbacks like wet transportation and drug loss and enabled the release of
16 S. Das et al.
Chitosan
a HO O AcO2 HO O HO O
H O H O
OH O OH
HO n HO HO 0.5
0.5 NH
NH2 NH2
Ac
Timolol
Chitosan (Mw = 57k) N-Acetylated chitosan (DA = 50%)
Oxidation PEI
Pristine ND clusters ND-COOH ND-PEI Timolol-embedded ND-nanogel
Lysozyme
Fig. 1.7 Schematic illustration of our lysozyme-activated drug eluting contact lens. (a) Drug-
loaded ND nanogels are synthesized by cross-linking PEI-coated NDs and partially N-acetylated
chitosan (MW = 57 kDa; degree of N-acetylation = 50%) in the presence of timolol maleate. The
ND nanogels are then embedded in a hydrogel and cast into enzyme-responsive contact lenses. (b)
Exposure to lacrimal fluid lysozyme cleaves the N-acetylated chitosan, degrading the ND nanogels
and releasing the entrapped timolol maleate while leaving the lens intact. Reprinted with permis-
sion from Kim et al. (2014) Diamond nanogel-embedded contact lenses mediate lysozyme-
dependent therapeutic release. ACS Nano 8:2998–3005 (Kim et al. 2014). Copyright © 2014
American Chemical Society
the drug from the lenses. The group checked the cell viability using XTT on using
human trabecular meshwork cells (primary) and found out the biocompatible
nature of the nanogels. Furthermore, ND enhanced the mechanical properties of
lens, helped in maintaining the water content, optical clarity, and oxygen permea-
bility at practical level. The authors declared that the nanogels-embedded system
could be used as an enzyme activator as well as a drug sequester which could be
utilized in other biological systems with few modifications. Meanwhile, the group
reported that byproduct (nanodiamonds) of mining and various refining processes
could be scalable into favorable materials for various therapeutic uses. Likewise,
Sonu et al. observed the behavior of the psychoactive drugs obromine (THB) and
theophylline (THP) in the presence of protein lysozyme as well as their binding
capability with that protein (Sonu et al. 2016). The authors demonstrated that in the
presence of the drugs, there was significant quenching in the fluorescence of the
lysozyme. The group mentioned that stronger binding of the THP drug was
observed compared to THB drug evincing the powerful hydrogen bonding com-
pared to hydrophobic binding. Additionally, the authors mentioned that binding
capacity of the lysozyme with the drugs changed drastically in the presence of the
nanoparticles of Au and Ag which presumably gave the idea of the drug-carrying
capacity of the lysozyme. Finally, the authors envisioned that it gave a superior
perspective of the transportation process of the drugs inside the human plasma. Cai
et al. developed a facile approach for the synthesis of nanogels used for drug deliv-
ery as well as imaging applications (Cai and Yao 2013). The authors revealed that
lysozyme and dextran in the nanogels formation acted like reducing and stabilizing
agents. The authors demonstrated the in-situ production of Au nanoparticles at pH
4 under UV irradiation in the presence of solvated amino acid residues. The group
mentioned that the well-dispersed Au@Lys–Dex nanogels were loaded with doxo-
rubicin which delivered excellent antitumor activity. Additionally, the group
revealed that the as-synthesized nanogels exhibited good optical imaging property
with high contrasting images which included the sub-cellular distribution of the
lysozyme and doxorubicin.
1.3.3 Wound Healing
Lysozyme is a hydrolase enzyme generally found in infected area (skin) and is also
applicable in healing of wounds. The generation of the polyelectrolyte environment
across the wound arena triggers the release of growth factors when lysozyme stays
in the release medium (Picheth et al. 2014). Another study showed that lysozyme
enhanced the healing rate in guinea pigs and the anti-bacterial activity of the enzyme
ameliorated the healing process (Charernsriwilaiwat et al. 2012; Gasior-Chrzan
1988). Charernsriwilaiwat et al. designed CS-EDTA/PVA nanofibers using the lyso-
zyme with the help of electrospun techniques used for wound-healing application
(Charernsriwilaiwat et al. 2012). The authors measured the loaded lysozyme using
the HPLC technique. The author demonstrated cell lysis activity using Micrococcus
lysodeikticus cells acted as a substrate. Additionally, the nanofibers exhibited excel-
lent wound-healing ability in male Wistar rats compared to control (gauze) and
commercially available gauze used as negative as well as positive control in a total
span of treatment. Furthermore, the group observed that the nanofiber mats exerted
biocompatibility toward NHF (normal human foreskin fibroblast) cell line and
released lysozyme quickly to the wound area. Altogether, the group shed ray of
hope on the future applicability of the biodegradable nanofibers as wound dressing
materials. On the other hand, Liao et al. demonstrated the ultrasound (US) captiva-
tion (controlled) technique for the transdermal drug delivery (TDD) using the EGF
(epidermal growth factor)-coated lysozyme microbubble (LYMB) which was
US-mediated (Liao et al. 2018). The group identified that the loading efficacy of the
EGF was higher in LYMB. The authors claimed that upon treatment with the
LYMBs (3 W/cm2 US), significant decrease in the growth of Staphylococcus aureus
was observed. The authors showed the differential wound-healing efficiency among
various treatment groups (control, dressing, LYMB, EGF, and EGF + LYMB) in the
absence of US (upper wounds) and in the presence of US (lower wounds) at various
time periods along with its quantification in Fig. 1.8. They declared that EGF-
LYMBs with US treatment displayed higher wound-healing efficacy and complete
epithelialization in comparison to other control groups in total experimental days.
18 S. Das et al.
Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21

a
Control
Control+US
Dressing
Dressing+US
LYMBs
LYMBs+US
EGF
EGF+US
EGF-LYMBs
EGF-LYMBs+US
b 120
Control
***
Wound healing (%)
Dressing * *** ***

100 ***
LYMBs
EGF ** ***
80 ***
EGF-LYMBs ***
** ***
60 ***
40 ***
20
0
3 6 9 12 15 18 21
US US US US US US US
Time (days)
Fig. 1.8 (a) Photographs of the wounds on mouse skin in a completely untreated animal (day 0)
and in groups C, D, M, E, and EM without US (upper wounds) and with US (lower wounds) at
various treatment time points. (b) Quantification of wound healing in the groups (∗p < 0.05,
∗∗p < 0.01, ∗∗∗p < 0.001). Reprinted with permission from Liao et al. (2018) Ultrasound-mediated
EGF-coated-microbubble cavitation in dressings for wound-healing applications. Sci Rep 8:8327
(Liao et al. 2018). Copyright © Liao et al; 2018 Springer Nature Publishing AG
1.3.4 Catalytic Activity
Lysozyme can act as a good catalyzing as well as a stabilizing agent in the formation
of different nanoparticles which included gold, silver, platinum, titanium, and
copper-based nanoparticles. For example, Yu et al. developed platinum nanoclusters
(ultrafine) using the lysozyme at basic medium (Yu et al. 2014). The as-prepared
nanoclusters (NCs) were characterized by XRD and FT-IR techniques. The authors
identified that the NCs synthesized at certain lysozyme concentration led to opti-
mum fluorescence, whereas concentrations higher or lower than that reduced the
fluorescence. The group demonstrated that the PtNCs exerted good catalyzing abil-
ity toward O2 oxidation of different organic substrates (TMB, ABTS, and dopa-
mine). Additionally, the authors showed the practical application of NCs by using it
in the degradation of methylene blue in the absence of hydrogen peroxide (H2O2)
which gave future direction of the as-prepared NCs.
1.4 iagnostic Application of Lysozyme-Based

D
Nanoparticles
Lysozyme-based nanoparticles have been developed for diagnostic purposes such as

cell imaging and bio-sensing. Cell imaging is the labeling of cells based on the fluo-
rescence properties of the nanoparticles. Similarly, sensing of the molecules depends
on the interaction between the nanoparticles and the molecules present in the
system.
1.4.1 Cell Imaging
Cell imaging is one of the foremost important subsets in biological applications.

Cellular imaging is required in order to visualize the cellular compartments as well
as to explore the cellular mechanism (Parveen et al. 2012). Various nanoparticles
generally used for cell imaging. In this chapter, we will discuss only the lysozyme-
based nanoparticles. For example, Ghosh et al. synthesized copper nanoclusters
(CuNCs) in the presence of lysozyme by single-step reduction procedure (Ghosh
et al. 2014). The authors mentioned that the as-prepared CuNCs displayed higher
quantum yield having tunable emission properties depending on the excitation. The
as-synthesized nanocluster was characterized using MALDI which revealed the
presence of Cu2 and Cu9 in the nanocluster. The authors showed that the agglomer-
ated particles sized up to 2.3 nm, whereas the diameter was observed 0.96 nm for
smaller particles. Moreover, the group claimed that this chemically, photostable
nanoclusters exhibited excellent cell imaging properties toward cervical cancer
cells (HeLa) upon treatment with nanocluster in cell culture conditions. The authors
demonstrated that the compound exerted biocompatible nature when treated the
HeLa cells in a dose-dependent manner. The epifluorescence microscopic images of
cells along with XTT assay result are shown in Fig. 1.9. Furthermore, the in vitro
hemolysis assay using human blood gave future direction of this nanocluster for its
use in vivo system. Another study led by Cai et al. where they prepared the nanogels
for cell imaging applications (Cai and Yao 2013). The as-prepared doxorubicin-
loaded Au@Lys–Dex exhibited excellent optical imaging properties when treated
with KB cells for certain time. Altogether, the authors manifested that the nanogels
system could be used not only as a cell imaging agent but also as a drug delivery
vehicle.
1.4.2 Sensing Applications
In the area of nanotechnology, the sensing of molecules has got immense attention.
With the advent of nanoparticles, the performances along with the sensitivity simply
increased (Yguerabide and Yguerabide 1998). Recently, there are several nanopar-
ticles that have been used for the sensing applications of various molecules, ions,
etc. But, the incorporation of proteins in forming the nanoparticles made it
20 S. Das et al.
125
a b
100
Viable cell (%)

75
50
25
0
l 3 7 .5 .3 .9 .5
ro 2. 5.
nt 11 17 23 34
co
[Cu NCs - composite] (µg/mL)
Fig. 1.9 (a) Epifluorescence microscopic images of HeLa cells under UV light. Cells were incu-
bated with Cu NCs for 2 h (scale bar 50 μm). (b) Cell viability for measuring the cytotoxicity of
the Cu NCs on HeLa cells as measured by XTT assay after 24 h of incubation. Reprinted with
permission from Ghosh et al. (2014) Blue-emitting copper nanoclusters synthesized in the pres-
ence of lysozyme as candidates for cell labeling. ACS Appl Mater Inter 6:3822–3828 (Ghosh et al.
2014). Copyright © 2014 American Chemical Society
beneficial. For example, Wang et al. developed copper nanocluster using lysozyme
for the detection of glucose in blood (Wang et al. 2015). The authors mentioned that
the as-prepared Lys–CuNCs exerted bright orangey-red fluorescence having high
quantum yield. The group showed that the fluorescence of as-prepared copper nano-
cluster was reduced in the presence of glucose and revealed that the reduction of Cu
(I) on the surface of NCs might be the plausible mechanism. Additionally, the group
observed that biosensor demonstrated predominant selectivity toward divergent
interferences such as metal ions, amino acids, and carbohydrates. Finally, the
authors mentioned that the as-prepared Lys–CuNCs could permit the detection of
glucose level rapidly and in a simple way on the variation of its fluorescence.
Similarly, Sun et al. synthesized lysozyme-stabilized silver nanoclusters (Lys-
AgNCs) used for the identification of the sulfide ions (S2−) by their fluorescence
quenching properties (Sun et al. 2016). The authors mentioned that under optimum
conditions this sensor displayed a high detection limit. Furthermore, the group
claimed that the practicability of the probe was proved by using it on real samples
which demonstrated convenient recoveries. Finally, the authors observed that this
economical and facile approach could be used in the detection of sulfide ions from
real water samples which showed magnificent advantages of this method. Altogether,
the authors explained that this fruitful work would give future direction for further
application of the prepared AgNCs. Lin et al. synthesized gold nanoclusters using
the lysozyme type VI used for the detection of Hg2+ and CH3Hg+ (ultrafine) (Lin and
Tseng 2010). The authors claimed that with increment in the concentration of the
lysozyme, a significant blue shift in the peak maxima with concomitant decrease in
the particle size was observed. Additionally, the authors identified that lysozyme
acted as a sole reducing agent and its activity resembled that of citrate in AuNP
production. The fluorescence band of Lys VI-stabilized AuNCs (Au-631) was cen-
tered at 631 nm which found to be highly stable in a solution of glutathione.
Moreover, the authors demonstrated that the nanocluster exhibited lowest detection
limit (LOD) toward Hg2+ compared to other AuNC-based Hg2+ sensors. At last, the
authors claimed that Au-631 could differentiate CH3Hg+ and Hg2+ in the presence of
ethylenediaminetetraacetic acid which gave future direction to this work.
1.5 Future Prospects of Lysozyme-Based Nanoparticles
Certainly, there is a drift in combining the therapeutic as well as diagnostic applications

of nanoparticles for increasing their efficacy in disease management. From past
decades, various scientists have developed such kinds of nanoparticles useful for dual
applications. Among these nanoparticles, lysozyme-based nanoparticles recently
attracted significant attention for their low toxicity, biodegradability, and biocompati-
ble nature. Generally, this feature book chapter showed that lysozyme is a valuable
protein, a favorable choice, used to make numerous nanoparticles having miscella-
neous applications both therapeutic as well as diagnostic. The foregoing discussion
tried to abridge the applications of lysozyme, its properties to develop nanoparticles,
and the elucidation of its interaction with metals. But, there is growing concern for their
future uses. In order for a clinical translocation, various factors have to be taken care:
the stability of protein (lysozyme), loading efficacy, the bioactivity during folding, the
surface modification techniques, and most importantly the toxicity issue. Although it
has been observed that lysozyme-based nanoparticles are less toxic, further studies are
required for long-term fate. In addition, greater efforts should be pointed on the surface
medications, fabrication techniques, etc. which will lead to efficient nanoparticles.
Furthermore, the interaction mechanism of model protein lysozyme with metals should
be explored more accurately. Efforts are seeking more efficient synthesis of lysozyme-
based nanoparticles. However, no such lysozyme-based nanoparticles have got FDA
approval for their biological applications until now. So, with comprehensive knowl-
edge, in-depth studies along with strong ambitions toward development of such
nanoparticles will push the envelope toward their clinical approval in the upcoming
future. This integrated part has attempted to give a future perspective which will pro-
vide useful guidance in developing new strategies fruitful for theranostic applications.
Acknowledgement A generous financial support from DST, New Delhi, for “Nanomission
Project” (SR/NM/NS-1252/2013; GAP 570) to CRP is duly acknowledged. SD is thankful to
UGC, New Delhi, for Senior Research Fellowships. The authors are thankful to the Director,
CSIR-IICT for his support and encouragement and for his keen interest in this work. IICT Manuscript
No. is IICT/Pubs./2019/021 dated January 28, 2019 for this manuscript is duly acknowledged.
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Emerging Nanomaterials
for Cancer Therapy 2
Sanjay Kumar, Pratibha Kumari, and Rajeev Singh
PHOTOTHERMAL/ BID-IMAGING/
PHOTODYNAMIC THERAPY DRUG TRACING
RADIOFREQUENCY
MAGNETIC THERMAL ABLATION
ABLATION
GENE THERAPY/ CHEMOTHERAPY/

IMMUNOTHERAPY CONTROLLED DRUG RELEASE
2.1 Introduction
Nanotechnology includes the orchestrating development of small-sized devices that

can be measured on molecular scale. In few decades, it has gained an upsurge of
interest in practically every field of research areas including information technol-
ogy, optics, electronics, magnetics, biomedicine, and materials development.
S. Kumar · P. Kumari
Department of Chemistry, Deshbandhu College, University of Delhi, New Delhi, India
R. Singh (*)
Material/Organometallics Laboratory, Department of Chemistry, Atma Ram Sanatan Dharma
College, University of Delhi, New Delhi, India
e-mail: rajeev@arsd.du.ac.in

https://doi.org/10.1007/978-981-13-8954-2_2
26 S. Kumar et al.
It has come out with incomparable precision and sensitivity to any other technology
and thus, attracts scientists from many different disciplines such as information
technologists, engineers, physicists, biologists, chemists, and material scientists.
The excursion of nanotechnology in biomedicine includes nano-devices that are
very small in size (less than 100 nm) and can be measured at molecular level. With
such a tiny size, the changed electronic properties of nanoparticles, this in turn
brings a change in their basic properties such as conductivity, melting points, fluo-
rescence, chemical reactivity, and color. This feature enables them to suitably enter
the human cells (>50 nm) and circulate within the blood flow (>20 nm), detect the
very small changes associated with tumor cells, deliver the targeted drug molecules
without perturbing the neighboring healthy cells, and signal back for confirmation
of delivery.
Nanomaterials effectively help in recognition and treatment of cancer even at
very early stage conferring the success of technology that was far beyond the imagi-
nation before. Cancer is one among fatal diseases for which scientists have been
fighting since decades. It includes the diseased state of genes that fails to regulate
the controlled growth of cells. The cells grow in an abnormal manner leading to
formation of tumor that has a potential to spread through the other parts of body in
some cases. The so far used cancer therapies include chemotherapy, radiation, and
surgery. Recently, radiotherapy and gene therapy are also securing increasing atten-
tion to provide an effective cure to cancer problem (Ware et al. 2017).
In chemotherapy, the treatment is given in the form of drugs (for example, cispla-
tin, paclitaxel, and doxorubicin) to destroy the tumorous cells or to stop the process
of division of these cells or to stop them to get the nutrition required to grow. The
major drawback of this therapy includes the killing of healthy cells too, that can be
even more fatal. Apart from that, it is not much successful if cancer is in much
advanced stage. Drug resistance to tumor, in some cases, also accounts for the treat-
ment failure. Bioimaging and tracing of drug toward the tumor site is essential for
target specific action where nanomaterials have pronounced role. Drug cargo is
loaded on nanoparticles surface through binding sites conjugated with tumor recog-
nition receptors like folate (Van Dam et al. 2011). The main objectives of a nanocar-
rier include drug loading capacity, drug internalization, enhanced circulation time,
and bioexcretion. The receptor molecules play an effective role in target specificity
and hence the preparation and surface modification of nanomaterials become more
important in terms of their action.
The process of localized heating through radiofrequency or photothermal proce-
dure is revolutionized by nanomaterials. The localized heating through non-radiative
radiations can kill cancer cells more effectively so stimulus responsive target heating
through nanomaterials has greater impact on tumor cells. Nanomaterials are internal-
ized to tumor site and then stimulated through frequency application leading to local-
ized increase in temperature. The nanomaterials are also used in generating reactive
oxygen species under laser irradiation leading to photodynamic therapy. Beside these
destructive approaches nanomaterials are also used for accelerating the host’s own
immune response through nano injections and by suppressing the over-expression
of genes through siRNA introduction to affected cells (Kershaw et al. 2006).
2 Emerging Nanomaterials for Cancer Therapy 27
Among many advantages of nanomaterials in cancer treatment, the fate of adminis-

trated nanoparticle and their biological persistence in body cannot be ruled out.
However, the pathways for biological excretion are studied but still it needs more
attention in terms of their toxicity (Wang et al. 2017a).
2.2 Nanotechnology and Cancer Therapy
The cancer incidence, prevalence, and mortality rates are at exceedingly high levels,
which indicates the need for an advanced technology to play an important role for
cancer treatment (Ferlay et al. 2015). Cancer is one of the main causes of death
worldwide. One of the causes may be the delay in detection. There are many advan-
tages for developing technologies that can detect cancer at its earliest stages. Due to
the availability of curative treatment, in most cases, the survival rate becomes higher
than 90% for about 5 years (Etzioni et al. 2003). Conventional anatomical imaging
techniques typically detect cancers when they are few millimeters (such as MRI) or
centimeters (such as PET) in diameter but at this stage, they already consist of more
than a million cells and prove to be inappropriate methods due to its low specificity.
Due to the recent progress in nanotechnology, molecular and cell biology, and imag-
ing technologies, the development of new imaging modality became possible which
aims at rectifying the above disadvantage (Fakruddin et al. 2012; Wang et al. 2016a).
Nanotechnology serves as a technology for diagnosis and treatment of cancer. It
provides early diagnosis through improved imaging (Niu and Chen 2018).
Nanotechnology has the ability to increase the selectivity and potency of chemical,
physical, and biological approaches for obtaining cancer cell death while minimiz-
ing toxicity to non-malignant cells (Gmeiner and Ghosh 2015). Materials at the
nanoscale are increasingly being targeted to cancer cells with great specificity
through both active and passive targeting (Rodzinski et al. 2016). There are many
properties of nanomaterials which led them to be used in cancer therapy. The most
important properties of nanomaterials which enables easy diagnostic and therapy
approaches (Jacob and Deigner 2018; Senapati et al. 2018), comprise charge, core
and surface properties, shape, flexibility as well as multivalency. The small particles
comparable to the largest biological molecules such as enzymes, receptors, and
antibodies can better determine the in vivo distribution, targeting ability, and drug
loading capacity (Fang et al. 2009).
Nanotechnology platforms are emerging frontiers in cancer treatment techniques
which include detection of cancer sites and recognition of stages. The treatment
procedures include introduction of drug at target site and further characteristics like
biodegradability, bioavailability, and increased drug retention time at tumor site.
Drug loss during transit process toward site can be prevented with proper loading of
drug on nanomaterial and also their deloading can be triggered with nano-
technological parameters. Intracellular trafficking also plays an important role in
drug carriers and effective enhancement is observed at nanoscale. Nanoscale param-
eters can be adjusted and tuned properties of the materials are helpful in active and
passive targeting of cancer cells. The active targeting of cancer cells is carried out
28 S. Kumar et al.
by different ligands encapsulated drug molecules which are transferred to target

region where they are recognized by various receptors. The receptor molecules
include specific peptide sequences or antibodies which are located on that regions
and helps in delivering the drug at site. The problem of internalization of drug is the
main barrier for therapeutic action as biological system’s internal defense mecha-
nisms standstill many internalization processes. Nanocarrier plays vital role here
and many of them used as internalization agent to overcome biological barriers. CD
44 receptor mediates the internalization through lysosomes carrying graphitic hol-
low carbon nitride nanosphere (GHCN). The surface of GHCN is modified with
hyaluronic acid for biocompatibility. The fluorescent nature of nanomaterial leading
to membrane breakdown through ROS generated species and locally delivering of
material gives theranostic action (Liu et al. 2016a). Folate receptors are widely tar-
geted by scientists to deliver drugs and many nanomaterials into cancer sites
(Ledermann et al. 2015; Alberti et al. 2017). Cell penetrating peptides are also used
for transduction of protein into cells for cancer therapy which uses stimulus respon-
sive nanomaterials for protein therapeutics (Lu et al. 2014). The nano-based drugs
are catching much attention these days as they are more permeable and bio-friendly,
still the elimination of nanodrugs remains a challenge. The local concentration of
nanodrug and its sustainability in that area for longer time is termed as enhanced
permeability and retention (EPR) which serves as basic requirement for therapeutic
action (Quader and Kataoka 2017).
2.3 Nanomaterials as Cancer Therapeutic Agents
There are different types of nanomaterials which are being used for biomedical
applications such as detection techniques, targeted drug delivery, bio-imaging, real-
time imaging of drugs, radiofrequency techniques, and thermal treatment.
Nanomaterials found their place in almost every field of cancer therapy. The follow-
ing categories of nanomaterials depending upon their origin are described in brief of
their role in cancer abolition techniques:
2.3.1 Metal Nanoparticles
Transition metal compounds such as arsenic compounds have historical background

in cancer therapy and have been used for medical treatment for more than twenty-
five centuries. Arsenic trioxide (As2O3) has been used for 10 years in patients with
acute promyelocytic leukemia (APL). As2O3 is safe and effective not only in patients
with leukemia, but also in patients with many other malignancies (Xu et al. 2004).
Similarly, the anti-tumor activity of potassium antimony tartrate in lymphoid malig-
nancies is promising (Lecureur et al. 2002), but it is compromised by cross-resistance
with cisplatin in human ovarian carcinoma (Naredi et al. 1995). Moreover, exposure
to potassium antimony tartrate has been associated with over-expression of the mul-
tidrug resistance-associated protein (MRP1), a drug export pump (Payen et al. 2001).
The first platinum-containing complex to be used in cancer therapy is cisplatin.

It was synthesized in 1844 and was known as Peyrone’s chloride. Rosenberg et al.
reported the inhibitory activity of cisplatin on E. coli division 120 years later
(Rosenberg et al. 1965). Around 30,000 platinum derivatives have been synthesized
and tested against cancer cells but only 30 compounds have reached clinical trials
and more than half of those have already been rejected. Presently, four are used
clinically: cisplatin, carboplatin, oxaliplatin, and nedaplatin (Desoize and Madoulet
2002). Along with these metal ions iron which is considered to be most useful bio-
logical element has also significant contribution. Dietary iron restriction markedly
decreases tumor growth in rodents (Wang et al. 1999). The antibodies which block
transferrin-binding to cellular receptors inhibit cancer cell growth in vitro and
in vivo (Kemp et al. 1995). The anti-tumor effect of bleomycin, an anticancer drug,
is mediated by chelation of iron or copper, to form a complex which degrades DNA
(Burger et al. 1981). By considering the historical background in case of metals
nanoparticles of many of these metals found interestingly applicatory contribution.
Transition metal nanoparticles had vast medicinal history in reference to cancer
treatment and a large number of these metal nanoparticles are being used in various
field of treatment. On the basis of their use in cancer therapy they are categorized
into the following categories:
2.3.1.1 Plasmonic Metal Nanoparticles

The studies of the anti-tumor activity of gold compounds were stimulated by vari-
ous observations. Firstly, patients treated with gold for rheumatoid arthritis (chryso-
therapy) had lower rates of malignancy than other patients (Fries et al. 1985).
Secondly, the efficacy of cisplatin against cancer generated great interest as gold in
the +3 oxidation state is isoelectronic with platinum (II) and forms similar square-
planar complexes. The metal nanomaterials possess entirely different chemical and
physical properties as compared with other bulk materials. In case of noble metals
such as gold and silver the surface properties of these nanomaterials become more
interested in terms of their absorption. They show surface plasmon resonance effect
which leads to strong electromagnetic radiation on nanoparticle surface or in other
words these nanoparticles show strong absorption band and also strong scattering of
light which increases in magnitude than compared with organic dyes (Jiang and Pu
2017). Strong absorption and scattering makes them noble contrast agent in cancer
therapy.
The absorption and scattering is more desirable when it falls near infrared region
(NIR) since most of the biological fluids do not absorb in this region which results
in clearer and more accurate imaging of tumor. Since Au NPs absorb in NIR (650–
900 nm), they are suitable plasmonic metals for cancer imaging (Panikkanvalappil
et al. 2017). Along with imaging this strongly absorbed light can also be converted
into heat and at tumor site for effective thermal treatment. Hence they are also better
photothermal contrast agents. Au NPs in form of gold nanorods, gold nanoshells,
and gold nanospheres have strong plasmonic effect and lead to localized surface
plasmon resonance (LSPR). Surface tenability during Au NPs formation is easier
over other NPs which gives promising impact. Different functionalities can be
30 S. Kumar et al.
installed on these plasmonic nanoparticles (Jiang et al. 2015). In vitro studies of
these plasmonic nanoparticles show they impact cytotoxicity by imposing oxidative
stress. The desired concentration of nanoparticle induces lipids and protein oxida-
tion in cancer cells by producing reactive oxidative spices (ROS) ultimately leading
to cell death (Wei et al. 2015). The compatibility of these nanoparticles with in
biological system remains an issue and various polymer and protein coatings are
applied over surface of these nanomaterials.
Recently, Rohit Srivastava and coworkers (Chauhan et al. 2018) reported glycol-
chitosan stabilized protein from corn which is hydrophobic inherently and when it
is deposited with gold nanoshells, it exhibits effective photo thermal therapy as well
as it acts as biocompatible contrast agent for radiography. Since both Ag and Au are
desirable plasmonic agents in cancer therapy, still AuNPs are preferred over AgNPs
because of structural damage of Ag NPs in vivo and hence plasmonic effect appar-
ently decreased. Taking the advantage of gold as lesser biodegradable and silver as
better plasmonic counterpart into account, Claire Wilhelm and coworkers (Espinosa
et al. 2018) recently prepared a plasmonic hybrid of silver nanoplates covered with
gold nano shell (Ag@AuNPs). They compared the plasmonic effect in NIR of the
prepared hybrid with their individual counterparts. The photothermal efficiencies of
hybrid Ag@AuNPs are found to be enhanced and also the deposition of degraded
Ag in iron storage proteins ferritin was much lesser as compared with AgNPs alone,
hence making the hybrid more bio stable and efficient photo thermal agent.
2.3.1.2 Magnetic Metal Nanoparticles

Metal nanoparticles of cobalt, nickel, and iron are magnetic in nature and can be
used to carry out therapeutic action in cancer treatment by functionalizing the sur-
face of these nanomaterials with therapeutic drug, like DOX, radionuclides for trac-
ing and analyzing the pathways of drugs, genetic materials like siRNA for targeted
gene therapy and with antibodies for immunotherapies of tumor (Gobbo et al.
2015). The magnetic nanoparticle can also induce hyperthermia in alternating mag-
netic field hence contributes toward one more therapeutic technique in cancer aboli-
tion (Espinosa et al. 2016). Magnetic NPs are best contrast agents in magnetic
resonance imaging (MRI). The magnetic NPs which can possibly be used in imag-
ing and hyperthermia treatment, are considered under theranostic platform leading
to multimodal treatment of cancer (Bonvin et al. 2017).
Nanoparticles of iron are superparamagnetic in nature and largely used in ther-
mal ablation techniques as well as guided delivery of targeted drug. The para-
magnetism vanished immediately as external field is removed and also shows higher
contrast per unit particles, hence lesser nanoparticles required for application (Singh
and Sahoo 2014). Moreover, iron oxide nanoparticles are considered to be the best
biodegradable nanomaterials and can be eliminated through normal iron metabo-
lism pathways in body (Nosrati et al. 2018a). Superparamagnetic iron nanoparticles
(SPION) are prepared through facile hydrothermal precipitation methods. Surface
of SPIONs is tuned with various inert organic moieties such as PEG, PAMAM
(Nosrati et al. 2018b) and polysaccharides (Mai et al. 2018) for increasing the bio-
availability. Also the anticancer activity of curcumin (CUM) which is needed in
micro molar concentrations in tumor site is maintained by combining it with mag-

netic NPs (MNP-CUM) and they have been tested in human pancreatic cancer cell
line in vitro as well as in vivo (Yallapu et al. 2013). With the emergence of dual
modalities and synergistic approaches in cancer abolition, magnetic nanoparticles
play important role. Gang Liu and coworkers (Yang et al. 2018a) reported metalla-
aromatic agents functionalized magnetic nanoparticles loaded into micellar carrier
(MA-SPIOs@Alkyl-PEI2k-PEG) for MRI/Photoacoustic imaging which gives
deeper tissue penetration and more accurate tumor location along with enhanced
photodynamic and photothermal therapeutics as metalla-aromatic agents are poten-
tial contrast agents for ROS generation.
2.3.1.3 Semiconductor Metal Nanoparticles

Another important category of metal nanoparticles comprises of semiconductor
materials. The metal oxides are generally semiconductor in nature and by taking the
advantages of their conduction and valence band gap into account reactive oxidative
species (ROS) can be generated on photo irradiation. Due to oxidative stress gener-
ated by these nanomaterials, tumor cells are targeted and abolished, hence these are
used in photodynamic therapy (PDT) (Fan et al. 2016).
The ovarian cancer is considered to be the most lethal form of cancer because of
recurrence and drug resistance. The most common type is epithelial ovarian cancer
(EOC) and recurrence rate is pretty much high in this case which is untreatable
because of cancer stem cell (CSC) expressions. The CSC alters the damaged DNA
expressions and leads to chemo resistance and metastasis. However, in this cancer
type epithelial mesenchymal transition markers such as TWIST regulate these
expressions. Glackin and coworkers (Shahin et al. 2018) reported the knock down
of TWIST by specifically introducing siRNA into EOC overexpressing cells through
nano-vehicle delivery. They reported hyaluronic acid functionalized mesoporous
nanoparticles conjugated siRNA (MSN-HA-siRNA) for effective and enhanced
delivery of siRNA against TWIST in primary cells of epithelial ovarian cancer.
Quantum dots (QDs) are semi-conducting, light-emitting nanocrystals (Gao
et al. 2004). The advantages of QDs include improved signal brightness, size- and
composition-tunable light emission, resistance to photobleaching, and simultaneous
excitation of multiple fluorescence colors (Rhyner et al. 2006; Smith et al. 2006).
Furthermore, different colors of QDs can be simultaneously excited by a single light
source, with minimal spectral overlapping (Guo et al. 2017a). QDs can be used for
cancer diagnosis and therapy with high specificity (Li et al. 2017a). Bagalkot et al.
(2007) worked on a multifunctional QD-aptamer (Apt)-doxorubicin (Dox) conju-
gate [QD-Apt(Dox)] for cancer-targeted imaging and therapy where RNA aptamers
covalently attached to the surface of QD, that act as both targeting molecules and as
drug carrying vehicles. Dox present in the multifunctional conjugate is the thera-
peutic agent for tumor cells, however, it also acts as a fluorescent agent. QDs have
also application in diagnosis where detection of Her2 (hairy-related 2) on breast
cancer cells by linking them with immunoglobulin G (IgG) and streptavidin gives
stronger image signal than organic dyes (Wu et al. 2002). QD-based fluorescent in situ
hybridization was also helpful in cellular labeling and imaging (Valizadeh et al. 2012).
32 S. Kumar et al.
Antibody-conjugated QDs have been used to detect prostate cancer cell marker PSA,
the QD conjugates detected the tumor site in mice transplanted with human prostate
cancer cells (Harma et al. 2001). Multimodal functionalities of such nanomaterials
intensify their use in therapeutic practices as along with imaging and detection pro-
cedure with QDs they simultaneously used in photodynamic therapy (Kuo et al.
2018; Zhang et al. 2018).QDs can also be used as photosensitizers or to activate
other photosensitizers in photodynamic therapy (Dahan et al. 2003).
2.3.2 Carbon Dots
Fluorescent carbon dots (CDs) of size smaller than 10 nm rapidly excrete from the
body (Wang et al. 2017b; Huang et al. 2013). Good biocompatibility (Yang et al.
2009a), low cytotoxicity (Wang et al. 2013), high quantum yield, non-blinking char-
acter, and low cost are considered as an emerging candidate in nano medicine (Sun
et al. 2006). CDs can also be used as photoacoustic contrast agents (Wu et al. 2013),
as photosensitizers for photodynamic therapy (Markovic et al. 2012) and for photo-
thermal therapy (Wang et al. 2014) although CDs have limited reports as a drug
delivery system (Zheng et al. 2014). Zheng et al. (Zheng et al. 2015) synthesized
green fluorescence emitting CDs by a simple one-step microwave synthesis into a
drug delivery system that discriminates cancer cells from normal cells. Fluorescent
CDs have also been used as traceable drug delivery vehicles as doxorubicin (DOX),
a broad-spectrum anticancer agent, can be attached to it through non-covalent bond-
ing between the –COOH group on CDs and the –NH2 group on DOX. This bonding
enables them to be used for cancer specific localized drug release (Zeng et al. 2016).
They carried out a series of in vitro and in vivo studies and reported that carboxyl-
rich green-emitting CDs are nontoxic bioimaging agents for drug biodistribution
research, and demonstrated in vivo study showing that the CDs can be used as a
stable cancer drug delivery system that can selectively kill cancer cells (Zeng et al.
2016). Along with targeted drug delivery, CDs can also be used for imaging purpose
(Matea et al. 2017). CDs are enhanced in target specificity fluorescence quantum
yield by combining with folic acid residue which in turn target cancer through folate
receptor sites (Liu et al. 2018). Surface functionalization is poor in case of CDs,
hence drug carrying capabilities are very less, although charge convertible CDs
were developed and taking advantages of their fluorescent nature into account image
guided drug delivery was carried out in vitro by combining them with an anionic
polymer and dimethylmaleic acid (PEG-(PAH/DMMA) (Feng et al. 2016a).
2.3.3 Carbon Nanotubes (CNTs)
Carbon nanotubes (CNTs) are made up of single or multilayer graphene sheets.

They have unique features such as large surface area, stable nature, and unique opti-
cal properties (Eatemadi et al. 2014). CNTs act as carriers for drug transport and for
tumor imaging and physical ablation (Elhissi et al. 2012). CNTs can be divided on
the basis of number of wall layers present into single-walled CNTs (SWCNTs) and
multiwalled CNTs (MWCNTs) (LIU et al. 2012).Simple CNTs structure cannot be
used in drug delivery directly, it can be used as drug carrier through the peptide,
protein, nucleic acid, or drug molecules. Most of model drugs like DOX, paclitaxel,
loaded with CNTs were reported as drug delivery systems (Li et al. 2017b; Darrat
et al. 2018). Dong et al. discovered the potential of MWCNTs trans activator of
transcription-chitosan (TC) as carriers of DOX against BEL-7402 cells in vitro,
which proved that this drug delivery system had good treatment efficacy on cancer
and revealed its application potential for cancer therapy (Dong et al. 2017).
2.3.4 Organic-Based Nanomaterials
Organic-based nanomaterials are generally naturally or are synthetically engineered

for biomedical purposes. Organic nanomaterials are available in a wide range for
various applications but the more common ones are solid-lipid, liposomes, micelles,
dendrimers, and polymeric nanomaterials (Wicki et al. 2015). From the advanta-
geous point of view these organic-based nanomaterials are more biocompatible and
the triggering as well as monitoring in biological system is convenient than other
nanomaterials. Moreover, biological rejection and longer retention time can also be
ensured by these nanovectors. These are discussed in brief as follows:
2.3.4.1 Solid-Lipid Nanomaterials

Solid-lipid nanocarriers (SLNs) are made up of tolerant lipid component, combin-
ing the benefits of polymeric nanoparticles, liposomes, and fat emulsions (Kang
et al. 2010). SLNs are a potential approach for drug delivery due to their non-toxicity
(Almeida and Souto 2007). Its advantages include good biocompatibility, high ver-
satility, high drug loading efficacy, and better drug release kinetics with long-term
stability (Naseri et al. 2015; Peng et al. 2015).According to a study, curcumin-
conjugated SLN was used on a MCF-7 breast cancer cell line to determine its effi-
cacy against the cancer cells and gradual drug release for 12 h was observed. The
cytotoxic effects were comparatively more than curcumin alone (Mulik et al. 2010).
Truzzi et al. studied the antitumor effects through lymphatic circulation, using
solid-lipid nanoparticles (SLNs) to encapsulate iron oxide nanoparticles and hepa-
rin, to simulate the intestinal lymphatic absorption of oral administration in CaCo-2
cells (Truzzi et al. 2017), which depicted the use of SLNs as an important route of
delivery for oral administration.
2.3.4.2 Liposomes
Liposomes are spherical structures which consist of a hydrophilic core and a hydro-
phobic shell, that enables them to carry both hydrophilic and lipophilic drugs
(Bozzuto and Molinari 2015; Lombardo et al. 2016). They have many advantages in
chemotherapy such as high drug encapsulation efficiency and drug loading capacity,
good stability, specific targeting and lymphatic orientation, sustained release effects,
good biocompatibility, low immunogenicity, and less side effects (Zhang et al. 2017).
34 S. Kumar et al.
Liposomes as nanocarriers of chemotherapy drugs have been used in breast cancer

(Park 2002), ovarian cancer (Allen and Cullis 2013), and Kaposi’s sarcoma treatment
and had a good outcome (Yang et al. 2011).Recently, the liposomes have been used
to target CD45 and/or CD90 of T-cells in vitro and in vivo to employ adoptive immu-
notherapy (Zheng et al. 2017). Wang et al. formulated doxorubicin-liposome (DOX-
liposome) with polymethacrylate derivatives (DOX-ERLP). The in vitro study on
MCF7/Adr cells and liver cancer H22 cells showed DOX-ERLP can cause cancer
cell death efficiently (Wang et al. 2017c). Zhang et al. modified lipid-coated zinc
phosphate hybrid nanoparticles coupled with vaccine based on the specific immune
response in vivo to achieve tumor immunotherapy (Zhuang et al. 2016).
2.3.4.3 Dendrimers
Dendrimers are polymer molecules made from a large number of branched mono-
mers. It has many functional groups attached to its surface which can be used as
chemical reaction sites (Furer et al. 2015).Dendrimers bind to the drugs mainly by
connecting the drugs to the functional groups on its surface (Liu and Frechet 1999).
The dendrimers as a nanocarrier show enhanced permeability and retention effect
(Palmerston Mendes et al. 2017). Nguyen et al. depicted letrozole-loaded
poly(amidoamine) (PAMAM) dendrimer G3.5 coated Hep by an in vitro release test
that showed a potential drug release ability of pH- and redox-responsive PAMAM
dendrimers (Nguyen et al. 2017). Zarebkohan et al. developed a PAMAM-PEG-
serine-arginine-leucine (SRL) nanocarrier to target C6 glioma. In in vitro tests,
green fluorescence protein (GFP)-loaded dendrimer depicted specific target ability,
which indicated the potential of PAMAM-PEG-SRL nanocarrier to be used for gene
delivery (Najafi et al. 2016). Moreover, dendrimers have also been used for psoriasis
skin treatment in an in vitro study (Gungor and Rezigue 2017). Soibermon et al.
worked on G4-PAMAM dendrimer to deliver dexamethasone for corneal inflamma-
tion (Soiberman et al. 2017).
2.3.4.4 Polymeric Micelles

Polymeric micelles are a type of micelles consisted of block copolymers having
hydrophilic and hydrophobic monomer units (Parveen et al. 2012).The core of poly-
meric micelles consists of densely packed polymer matrix which enables the poly-
mer micelles to be utilized as suitable and effective nanocarriers (Gaucher et al.
2010). Liu et al. developed poly(lactic-co-glycolic acid) nanoparticles modified
with transferrin for using it as a carrier for DOX for leukemia K562 cells showing
high expression of transferrin receptor which in turn increased tumor cells killing
ability (Liu et al. 2016b). Gao et al. worked on a micellar system called 7-pep HD
micelles, and the in vitro study on MCF/Adr cells and in vivo study on MDR
tumors-loaded female nude mice depicted that these micelles can effectively target
the MDR (Gao et al. 2017).Wu et al. designed a nano delivery system called anti-
miRNA oligonucleotide-21-human epidermal growth factor receptor-poly(ethylene
glycol)-poly(ε-caprolactone) nanoparticles and then encapsulated trastuzumab with
it to target gastric cancer cells (Wu et al. 2017a). Several series of analyses and
in vitro tests in SCC7 cells proved that the polymeric micelles have good stability,
high cellular uptake, low toxicity and may be used in the delivery of many hydro-
phobic antineoplastic drugs in the future (Nam et al. 2017).
2.3.4.5 Polymeric Nanoparticles

To reduce the side effects of chemotherapy drug specificity and targeted action is
essential step for which delivery agents play vital role. Polymeric nanoparticles
have been proved to be good candidates as drug delivery systems or molecules for
combined treatment and diagnosis of cancer (Parveen and Sahoo 2008; Cegnar
et al. 2005). They have remarkable benefits such as stability, biocompatibility, bio-
degradability, tailorability, and low cost. Polymeric nanoparticles are designed to
accumulate in tumor sites by controlling their hydrodynamic properties or func-
tionalizing their surface with targeting molecules (Thierry 2009). Polymers
responding to particular tumor microenvironment conditions like reduced pH, high
levels of reactive oxygen species, or overexpressed enzymes can be used to trigger
a controlled drug delivery (Huang et al. 2012). Biodegradable polymers are suit-
able for drug delivery since they break down in physiological conditions, which
reduces the toxicity of the carrier and also facilitates drug release (Tiwari et al.
2012; Mahapatro and Singh 2011).
Biodegradable polymeric nanocarriers can undergo anticancer targeting through
various mechanisms which includes passive delivery to the tumor cells by pro-
longed circulation in the blood stream, transport across the endothelium to the
tumor, and ligand-mediated cancer targeting (Mitra et al. 2001; Kreuter et al. 2007).
Moreover, polymeric nanocarriers can also target non-cancer cells that make up the
tumor microenvironment (Din et al. 2017). Poly(lactic-co-glycolic acid) (PLGA) is
one of the most important polymers which is widely used for the encapsulation of
anticancer agents due to their intelligent behavior in response to physicochemical
variations, polymers have generated great attention in the field of controlled drug
release (Tabatabaei Mirakabad et al. 2014). When a drug is encapsulated in poly-
meric nanoparticles, its intracellular uptake by all types of cells is substantially
reduced. The unwanted drug interactions with normal cells can be prevented by this
way (Li et al. 2018a).
2.4 Role of Nanomaterials in Cancer Ablation Techniques
The above-listed nanomaterials are used as vectors for cancer ablation techniques in
various capacities such as binding entities with drug molecules or with genetic
material. Nanomaterials can act itself as drug to inhibit the outgrowth of cells and
their role extends for tracing the real-time pathways of applied drug during in vivo
studies. Organic-based nanomaterials provide various surface functionalities for
drug interaction and providing physiological condition for its specific release.
Cancer ablation starts with early detection of cancer cells and its target analysis fol-
lowed by chemical treatment and then thermal heating the tumor site. Different
ablation techniques are discussed in detail here with their reference with role played
by different nanoparticles.
36 S. Kumar et al.
2.4.1 Nanomaterials as Imaging Probes
Nanoparticles are of a great use in imaging applications due to their high surface
area-to-volume ratio as well as having the ability for numerous sites for chemical
modification which increase imaging sensitivity (Cui and Rao 2017). Bio imaging
is one of those essential requirements in cancer therapy without which imagination
of cancer eradication is not possible. The imaging of tumor site for determining the
extent of cancer in localized area and deciding the therapeutic dose are basic
requirements for therapeutic action. The traditional techniques for cancer detection
such as magnetic resonance imaging, positron emission tomography, X-ray, and
ultrasound pose greater risk of radiation poisoning, hence optical imaging replaces
them with time.
Various fluorescent imaging probes including dyes which absorb in 566–600 nm
range, have limitations as they cannot deeply picturize the tissues because hemoglo-
bin itself absorb around 520 nm. Hence optical imaging techniques generally use
near infrared (NIR) fluorescence imaging which uses wavelengths between 700 and
1000 nm giving rise to real-time imaging with high resolution. These techniques are
non-invasive, auto fluorescent for tissues but they require NIR probes which must
be active in given range of wavelength and must be biocompatible. The probes must
have high quantum yield and better stability in terms of photochemical and biologi-
cal barriers. The main problems in bio imaging are finding out the suitable contrast
agent and then insertion of these agents into cancer site. Many nanomaterials are
inserted to biological system as contrast agents which are generally fluorescent in
nature with the help of biocompatible moieties to prevent their rejection. The image
guided interventions can be done easily such as drug tracing, surgery, and RNA
transfection at more accuracy and more effectiveness (Li 2014).
The nanomaterials used as contrast agents show high degree of fluorescence phe-
nomenon and generally get excited easily in low energy irradiations such as NIR
exposure. The localized enhanced concentration of these materials expresses the
images of affected areas and also traces the drug pathways. The combined effects by
nanomaterials are also observed along with imaging which includes localized heat
ablation and drug release. The NIR active probes are basically categorized into two
parts: the organic NIR probes and inorganic NIR probes. These are discussed in
detail as follows:
The organic dyes are considered to be the best among imaging probes. They are
bio compatible and can easily interact with receptor organic molecules and easily
recognize them such as folate receptor, antibodies, and peptides. Hence they can be
suitable candidate for target specificity and imaging. The dye molecules are recog-
nized to have better photo physical properties and greater biostability than other bio
imaging agents. The low quantum yield and lesser hydrophilicity remain drawbacks
in their application which leads them toward lesser fluorescent time and aggrega-
tion, respectively. The near infrared fluorescent dye Cy5.5 was conjugated with gly-
col chitosan-5β-cholanic acid (GC-CA) which further encapsulates Gadolinium
(III) ion acting as a better probe and shows multimodal effect for NIR and MR
imaging in cancer therapy (Nam et al. 2010). Nanomaterial based probe shows
enhanced circulation time and more selective targeting in mice tissues. Cy5.5 also
conjugated with oleyl-chitosan (Lee et al. 2011), tumor homing chitosan based
nanoparticles (Kim et al. 2010), PEG conjugated iron nanoparticles (Cha et al.
2011) for enhanced NIR effect.
Inorganic nanomaterials including metal oxide nanoparticles and their quantum
dots revolutionize the imaging techniques and achieve tremendous heights in this
field. The nanostructures which are easily sensitized through different approaches
can be used as indifferent probes for bioimaging. The multimodal imaging as well
as real imaging of targeted drug can be ensured through different engineered nano-
materials. In early research iron oxide nanoparticles (IONPs) have been reported in
conjugation with amino-terminal fragment of urokinase plasminogen activator to
image breast cancer specifically (Yang et al. 2009b). According to a study by
Palmowski et al. molecular ultrasound imaging was performed by using VEGFR2-
and αvβ3-targeted microbubbles illustrating specific binding to angiogenic tumor
blood vessels (Palmowski et al. 2008). In another report, molecular ultrasound
imaging with the clinically translatable VEGFR2-targeted BR55 microbubbles was
applied to monitor antiangiogenic treatment responses in human colon cancer
xenografts (Pysz et al. 2010). Mulder et al. used 150 nm large paramagnetic RGD-
coated liposomes for molecular MR imaging of tumor angiogenesis and they
depicted various differences in the tumor accumulation pattern of RGD-coated
liposomes compared with nonspecific RAD-coated liposomes (Mulder et al. 2005).
Furthermore, hybrid silica nanoparticles (C dots) have diagnostic applications as
well. Phillips et al. showed its applications in patients with metastatic skin cancer
(Phillips et al. 2014). Many a times NPs are used for both imaging and treatment.
For instance, TiO2 NPs can be used both to enhance CT (computed tomography)
image contrast and as sensitizers for photodynamic therapy (Bae et al. 2011).
Magnetic NPs may be used for both improved MR imaging and hyperthermia
applications for advanced cancer treatment (Patitsa et al. 2017). IONPs can be
combined with methotrexate (Nosrati et al. 2018c), paclitaxel (PTX) (Tarantash
et al. 2018), or other anticancer drugs (Nosrati et al. 2018d; Shah and Dobrovolskaia
2018) for dual applications.
Gold nanoparticles have vast history spanning over imaging techniques in can-
cer therapy in comparison with other metal nanoparticles. The larger surface modi-
fication possibilities increase chances of cellular uptake and inter cellular trafficking
of gold nanoparticles. Similar studies were shown by Chunhai Fan et al. who had
reported DNA loaded gold nanoparticle for their dual fluorescent and plasmonic
effect and show the clustering inside cellular region and also long-lasting imaging
through plasmonic based dark field microscopy (DFM) (Liu et al. 2017). Single
crystal intercellular trafficking and null photobleaching of gold nanoparticles make
them more efficient in imaging than other fluorescent probes (Mayer and Hafner
2011). CT and MR imaging of hepatocellular carcinoma were also reported
recently by entrapping gold nanoparticles with lactobionic acid-modified multi-
functional polyethyleneimine (Li et al. 2017c). Quantum dots and CNTs have also
been formulated and used for potential theranostic (therapeutic and diagnostic)
applications.
38 S. Kumar et al.
2.4.2 Nanomaterials in Gene Therapy
In the 1980s, gene therapy emerged as a new approach for the treatment of diseases.
It involves modification of the genetic content of the cell through the introduction of
genes using delivering vectors. It helps to correct the defective gene and provide a
definitive cure. In the last few years, many researchers demonstrated the potential of
gene therapy with the help of nanotechnology be used in biomedical applications
(Hinrichs and Rosenberg 2014; Hidai and Kitano 2018; Lawler et al. 2017;
Yamamoto et al. 2017). When gene therapy is used to treat cancer, its aim is to
eliminate the cancerous cells selectively. It is designed to introduce genetic material
into patients’ cells to compensate for abnormal genes and to express specific pro-
teins which avoid the toxicity arising from chemotherapy (Wang et al. 2016b).The
efficiency of the transgenes to be expressed and the delivery vectors used decide the
success rate of gene therapy. Therapeutic transgenes can substitute their endoge-
nous counterparts that are unable to express certain proteins or which express a
non-functional form of the protein. Transgenes can also be used to inhibit expres-
sion of an active endogenous gene or to express a toxic protein which is able to kill
the cell in which it is expressed. Similar to this is the case of tumor cells that could
be modified to express suicide transgenes able to eliminate the tumor under the
stimulation of a prodrug. Gene therapy can be classified on the basis of dependence
on the delivery vector used. The vectors can be integrative or non-integrative.
Integrative vectors are viral vectors (gamma retroviruses, lentiviruses, adeno-
associated viruses, foamy viruses) and non-integrative vectors can be viral (adeno-
viruses, herpesviruses, poxviruses, vaccinia viruses) or nonviral vectors (Herranz
et al. 2011).
Gene therapy can be employed in two different ways. Firstly, gene augment to
upregulate tumor suppressor genes. For example, gene TP53 encodes the p53 pro-
tein and thereby known as a tumor suppressor gene (Xu et al. 2001). Secondly,
gene knockdown by means of agents, such as short interfering RNA, siRNA (Lee
et al. 2012). The siRNA agents have been encapsulated within the hydrophilic
cores of lipid or polymeric nanoparticles or attached to their cationic surfaces
(Lee et al. 2012). According to various studies, there is a possibility of toxicity
due to the cationic properties of such nanoparticles, which results into cell con-
traction, mitotic inhibition, aggregation in blood, and inflammatory response
(Akhtar and Benter 2007). Moreover, the low charge density of the nanoparticles
can result into instability. siRNA loosely attached to nanoparticles can undergo
rapid degradation by nucleases due to ease in release, before arriving at the target
site and cationic polymers such as polyethyleneimine can be cytotoxic to normal
cells. To overcome these issues, alternative strategies have been employed to
increase the molecular weight of siRNAs. This can help them in forming com-
pounds with cationic chitosan polymers that are more stable in the body and
would in turn enhance stability and delivery efficiency. Kim et al. used polymer-
ized siRNAs (poly-siRNA) and thiolated glycol chitosan polymers. The poly-
siRNA glycol chitosan nanoparticles (psi-TGC) showed better stability and gene
silencing efficacy (Lee et al. 2014).
2.4.3 Nanomaterials in Immunotherapy
Along with various trends in cancer treatment immunotherapy recently catches

much more attention in nondestructive techniques. The recent advancement in tech-
nology and understanding clinical trials make immunotherapy as next milestone in
cancer abolition (Sharma et al. 2019). Stimulated own body immune response
toward tumor cells can have long persistent effect which can be supported by nano-
materials. The nanoparticles based cancer immunotherapy has the potential to treat
cancer without side effects (Conniot et al. 2014; Shao et al. 2015; Park et al. 2013).
Vaccines are of a great use in cancer immunotherapy due to their ability to cause a
specific, strong, and persistent immune response. It has a fatal drawback as well due
to their low immune-stimulating capacity (Rosenthal et al. 2014). Gold nanoparti-
cles can be used as a vaccine vector due to their suitable payload (Niikura et al.
2013), low immunogenicity, and biocompatibility. Adoptive immunotherapy (AIT)
is another approach for cancer treatment, and the enrichment and expansion of
tumor-specific T cells has progressed with an iron-Dextran nanoparticle (Perica
et al. 2015). The use of dendritic cell (DC)-based therapeutic vaccines proved to be
a promising strategy against cancer (Janikashvili et al. 2010). Clinical trials on these
DC vaccine show better response and are effective in treatment (Constantino et al.
2016; Zabaleta et al. 2015). This is an expensive therapeutic and time-consuming
method (Eggermont et al. 2014). Therefore, to get through these limitations,
NP-based vaccines are being used for induction of an immune response. NP-based
active cancer immunotherapy has gained interest regarding DC maturation and acti-
vation in vivo which results in strong immunotherapeutic responses to cancer. DCs
are the most effective antigen-presenting cells (APCs), which present antigens to T
cells and secrete pro-inflammatory cytokines, which results in tumor-specific acti-
vation of cytotoxic T cells (Steinman 2008; Xiang et al. 2015; Yang et al. 2018b;
Han et al. 2016). So, in vivo maturation of DCs is an effective approach for NP-based
active cancer immunotherapy (Han et al. 2016).
2.4.4 Nanomaterials in Radiothermal Cancer Therapeutics
Radiation therapy (radiotherapy) (RT) is used to suppress tumor growth which is

alternatively important in localized retardation and abolition of cancerous cells.
This is one of largest used ablation techniques (Laperriere et al. 2002). RT can be
classified into internal radioisotope therapy (IRT) and external beam radiation ther-
apy (EBRT) (Sadeghi et al. 2010). IRT is done by the intrinsic radiation from thera-
peutic radioisotopes injected in vivo in a minimally invasive manner (Tian et al.
2017; Yi et al. 2015), which has the potential to treat both local and metastatic
tumors spread throughout the body. It is important to adopt tumor-targeted delivery
method of radioisotopes to avoid systemic radio-toxicity to normal organs (Chen
et al. 2017; D’Huyvetter et al. 2014). EBRT is excited by three types of external
ionizing radiation sources for precise localized treatment of deep-seated solid
tumors like breast, colorectal, lung, and brain tumors. EBRT can be classified on the
40 S. Kumar et al.
basis of ionizing radiation sources into proton therapy, heavy ion therapy, and X/γ-
ray therapy (Blattmann et al. 2015; Johnstone et al. 2015). X-ray therapy is mostly
used form in clinics which applies high-energy X-ray radiation to inhibit cancer cell
proliferation by damaging the DNA of rapid proliferating tumor tissues (Juzenas
et al. 2008). Some “green” radiosensitizers with high biocompatibility are highly
desirable due to the potential toxicity of chemotherapeutics and heavy metals. Some
gasotransmitters (NO) (De Ridder et al. 2008) and (H2S) (De Preter et al. 2016) can
act as “star” signaling molecules to regulate a number of physiological and patho-
physiological activities (e.g., neuronal communication, blood vessel modulation,
wound healing, etc.) and also as radiosensitizers to increase all of the hypoxic cells’
radiosensitivity which explains the phenomenon of gas radiosensitizers penetrating
and diffusing among all of the cancer cells (Cook et al. 2004).
2.4.5 Nanomaterials in Magnetic Hyperthermic Therapy (MHT)
This is based on the transformation of electromagnetic energy into heat (Fan and
Sommers 2013), MHT can perform several functions such as protein denaturation,
DNA damage, signaling interruption, cell growth inhibition, and apoptosis (Shi et al.
2009; Hayashi et al. 2013; Guardia et al. 2012). It can treat tumors seated at any
depth due to the large tissue penetration ability of the magnetic field. The contactless
use of alternating magnetic field (AMF) enables MHT to be a controllable remote
treatment to eliminate non-accessible tumors (Sanz et al. 2017; Barick et al. 2012).
The most commonly used magnetic nanomaterials for MHT are superparamagnetic
Fe3O4 NPs (Zhao et al. 2006). Fe3O4 NPs accumulate in tumors based on the mag-
netic attraction force and quickly increase the temperature to eradicate tumors ther-
mally with the assistance of an AMF (Bae et al. 2012), which also helps in protecting
the surrounding tissues from thermal damage. Zheng et al. constructed a phase-trans-
formation material to trap Fe implants within the tumor for repeated MHT treatment
to make MHT more effective (Yang et al. 2017). The designed liquid Fe/polylactic-
co-glycolic acid (PLGA)/Nmethylpyrrolidone (NMP) gel (denoted as L-Fe/PLGA)
instantly turned into solid Fe implants (denoted as S − Fe/PLGA) after contact with
body fluids or water. The Fe implants generated enough heat when exposed to AMF,
to cause a rapid increase in tumor temperature. The high temperature completely
ablated the tumor within 3 days. Hyperthermia caused by magnetic nanoparticles can
be explained by two mechanisms. Firstly, Brownian relaxation explains the heat
energy of magnetic nanoparticles coming from the rotation of entire particles within
their environment. Secondly, Neel relaxation describes the hyperthermia arising
from rotations of magnetic moments inside the magnetic core (Fortin et al. 2008).
Chitosan polymers proved to be an appropriate candidate to induce enhanced
hyperthermic therapy (Bae et al. 2012; Cervadoro et al. 2014). Cervadoro et al.
depicted magnetic nanoparticles made by confining multiple 20-nm nanocubes into a
deoxy chitosan polymer, with an overall diameter of about 156 nm (Cervadoro et al.
2014). Bae et al. demonstrated ferrimagnetic iron oxide nanocubes (FIONs) stabilized
by chitosan oligosaccharide for cancer hyperthermia (Bae et al. 2012). Handy et al.
developed ferromagnetic nanoparticles that were coated with biocompatible material

poly(methacrylic acid-co-hydroxy-ethylmethacrylate) using free-radical polymeriza-
tion. Antibodies were covalently attached to the surface of coated magnetic particles
for selective targeting (Yang et al. 2017; Praetorius and Mandal 2007).
2.4.6 Nanomaterials in Photodynamic Therapy

and Photothermal Therapy
Photodynamic therapy (PDT) with the help of reactive oxygen species has emerged
as an important cancer therapy. PDT uses photosensitizers to generate cytotoxic reac-
tive oxygen species (Zhou et al. 2016). A specific external light wavelength is
absorbed by photosensitizers, producing reactive oxygen species which destroy
tumors (Abrahamse and Hamblin 2016). Along with generating singlet oxygen, pho-
tosensitizers generate a fluorescence signal. Therefore, cancer-targeting nanoparti-
cles can be used for simultaneous cancer imaging and therapy. The nanoparticles
were also evaluated as potential PDT agents for pancreatic cancer (Li et al. 2015).
PDT has a number of advantages such as its localized effects, reduced cost, and the
fact that it is largely an outpatient therapy (Chatterjee and Yong 2008). It can also
induce immune responses even against tumors that are not particularly immunogenic
(Chatterjee et al. 2008). There are some limitations as well such as the limited pen-
etration depth of visible, infrared, and UV radiation into the patient (Allison and
Moghissi 2013; Yamaguchi et al. 2011) and the lack of effective dosimetry tech-
niques for PDT, making it difficult for dose distributions in the treatment volume to
be accurately measured (Allison et al. 2004).
Photothermal therapy (PTT) is another promising method for cancer therapy.
PTT utilizes various nanoscale heating agents which accumulate in tumor tissues
and are then used to induce local heating (Huang et al. 2008). The local temperature
is usually high enough to denature cell proteins or genes. Nanoparticles work to
enhance the heating effects of the laser light irradiation source by means of their
tuned absorption wavelengths. Wang et al. depicted gold nanorods stabilized by
thiolated chitosan polymer. They modified the surfaces of cetyltrimethylammonium
bromide (CTAB)-passivated gold nanorods with thiolated chitosan to deal with the
high cytotoxicity of CTAB (Wang et al. 2011). The chitosan-coated gold nanorods
were also modified with folic acid, which increase the specificity of internalization
by human colon HT-29 cancer cells, which overexpress the folate receptor (Key and
Park 2017). Guo et al. reported the limitations of PTT that they are applicable
mostly at the primary site, but not to metastatic cancer (Guo et al. 2014).
2.4.7 Nanomaterials in Chemotherapeutic Techniques
The size and pharmacokinetic properties of the NPs ensure accumulation at tumor
sites. This is achieved through passive targeting, which is the preferential accumula-
tion of NPs at tumor sites. Passive delivery can be enhanced by modification of the
42 S. Kumar et al.
nanomaterials’ surface using different hydrophilic spacers, such as poloxamer,

polyethylene oxide (PEO), PEG, lauryl ethers, and polysorbate (Soppimath et al.
2001). Active targeting is a molecularly driven process. It involves the attachment
of affinity ligands on the surface of an NP that identify uniquely overexpressed
molecules on the tumor cell surface to promote higher and more sustained drug
interactions with its target (Li et al. 2013). Antibodies (anti-Her2, anti-CD44, anti-
CD24), proteins (transferrins), small bioactive molecules (biotin, folate, galactose,
mannose and glucose), peptides (like L-arginine glycine L-aspartic acid (RGD))
and oligonucleotides (aptamers) have been utilized as ligands to recognize target
receptors on the tumor cells (Yu et al. 2012). Anti-HER2 targeting antibodies con-
jugated to liposome-grafted PEG chains increased the uptake of the NPs in HER2
expressing breast tumors.
In an ovarian carcinoma model, a luteinizing hormone-releasing hormone
(LHRH) peptide has been used to target cells which overexpress the receptor for this
hormone. Nano-polymers, such as dendrimers, micelles, and liposomes, that were
intercalated to doxorubicin and siRNA were conjugated with targeting peptides
toward CD44 or multidrug resistance protein-1 (MRP1) and showed the highest
tumor growth suppression (Minko et al. 2013). Aptamers are types of oligonucle-
otides with 15–40 bases that bind to various molecular targets, such as small mole-
cules, proteins, nucleic acids and even cells, tissues and organisms (Ireson and
Kelland 2006). AS1411 is an anti-nucleolin aptamer in phase 2 clinical develop-
ment with a 26-base guanine-rich oligodeoxynucleotide with affinity to nucleolin, a
nucleolar phosphoprotein that is overexpressed on the surface of various cancerous
cells (Bates et al. 2009; Mongelard and Bouvet 2010). Another aptamer which has
been widely used is the PSMA-specific A10 aptamer for engineering of targeted
NPs in multiple prostate cancer preclinical and clinical studies (Wu et al. 2011).
Table 2.1 exemplifies some of nanotechnological platform for their combinational
therapeutic action:
2.5 Fate of Nanomaterials in Biological System
The nanomaterials administered into body are not bare but consist of its supporting
components such as surface coated bio stabilizers and attached drug or other thera-
peutic agent. The bare nanomaterial considered as foreign particle in body and is
immediately countered by immune system. The organs involve in elimination of
nanoparticles are mainly liver, spleen, bone marrow, and kidneys (Seo et al. 2017).
The mononuclear phagocytic system (MPS) is mainly responsible for bioaccumula-
tion and excretion of NPs which works on protein corona based recognition system.
A protein coat generally opsonin protein is adsorbed through electrostatic interac-
tions on NPs surface and makes them viable for recognition for phagocytic cells
(Caracciolo et al. 2017). The internalization and further breakdown of NPs depend
upon the protein structures. Once they are internalized, they are stored in MPS
organs and further degradation is carried out by enzymes (Soenen et al. 2015).
Table 2.1 Nanomaterials used in different cancer therapy
Nano-technological platform Therapeutic action Target site/action Mode of action Reference
Cisplatin prodrug doped Photo Cisplatin drug delivery 2-Hydroxypropyl-γ-cyclodextrin/curcumin— Feng et al.
DiR dye thermal- chemotherapy liposome complex give strong NIR absorbance (2016b)
and fluorescence
MSN-HA-siRNA siRNA delivery/ Ovcar8-IP ovarian cancer cell siRNA against TWIST enhanced uptake to Shahin et al.
chemotherapy line ovarian cancer cells through MSN-HA complex (2018)
Transferrin template Cu Bio imaging/targeted TfR positive Daltons Luminescent properties of Tf-Cu NC–Dox NPs Goswami
NCs drug delivery lymphoma ascites (DLA) in are target specific and bio imaging et al. (2018)
mice
CsxWO3 NR@ PEM Bio imaging/ In vivo malignant HeLa cells NIR induced PT/PD ablation and better contrast Guo et al.
Photothermal/ agent for CT and PAT (2017b)
photodynamic therapy
Fe3O4@Arabic acid@DOX Imaging and drug Immunocompromised NOD/ Arabic acid as reversible binder with DOX and Patitsa et al.
delivery SCID mice MDA-MB-231 FeNPs as contrast agent for imaging (2017)
breast cancer cells
2 Emerging Nanomaterials for Cancer Therapy
MSN-Fe-AuNPs Photothermal/chemo/ WSU-HN6 cancer cells in pH sensitive drug release and H2O2 responsive Jin et al.
tumor therapy mice Fenton type ROS formation (2018)
Ce6@CaCO3 -PDA-PEG Imaging/PDT/ 4 T1 tumor cells in mice Mn+2 as imaging ions and Ce6 as therapeutic Dong et al.
chemotherapy agent and pH responsive PDT via lysosomal (2018)
breakdown
Gd-PEG-Bi NPs Imaging/PTT MTT cell in vitro and BALB/c Complex nanocomposite enhances MRI, CT, and Wu et al.
mice in vivo PEA imaging and NIR absorption leads to PTT (2018)
RGD-CuS-Cy5.5 Image guided PTT and MNK45 tumor lymph node Lymph node metastasis carried by dual imaging Shi et al.
multimodal imaging cells and complex shows strong NIR absorption for (2018)
PTT
MA-SPIOs@ MRI/PA imaging/PTT/ SCC-7 cell line Metalla-aromatic agent for PTT/PDT and SPIOs Yang et al.
Alkyl-PEI2k-PEG PDT transduction through micellar nanovehicles (2018a)
CoP NPs Triple-modal imaging 4 T1 mice cells Non-invasive imaging modality along with NIR Li et al.
and PTT based PTT (2018b)
(continued)
43
Table 2.1 (continued)
44
Nano-technological platform Therapeutic action Target site/action Mode of action Reference

DOX/ Fe3O4-alginate Chemotherapy/thermal MCF-7 cells SPION for hyperthermia and ACMs for Xue et al.
chitosan microsphere therapy controlled drug release (2018)
Chitosan- carbon dots Bioimaging/ drug 4 T1 breast cancer cells pH sensitive DOX drug release in cancer cells Wang et al.
hybrid nanogel (CCHNs) delivery/Photothermal and NIR activated photothermal activity (2017d)
Se@Au@mSiO2DOX Chemotherapy/ MCF-7 and MDA-MB-231 NIR irradiation triggers release of drug. Nano Ramasamy
Photoablation/ breast cancer cells composites contributes in hyperthermia functions et al. (2018)
hyperthermia
Graphene Oxide Quantum Drug delivery and Lung cancer cells A-549 and Nano drug vehicle and folic acid functionalized De et al.
Dots (FA-CH-GOQD) imaging neuroblastoma (SH-SY5Y) chitosan for site recognition and specificity of (2018)
in vitro target
Gold nanorods (Au@ Gene therapy/chemo/ Antioncogene p53/ hepatoma Au NRs for PTT enhanced chemotherapy and Chen et al.
HSN-PGEA, AHPs) photothermal therapy HepG2 and HEK293 cell lines CD-PGEA for gene transfection (2018)
Au NPs dendrimers Bcl2- siRNA/VEGF Glioma cell line Transfection efficiency and gene silencing Qiu et al.
entrapped modified with siRNA delivery enhanced (2018)
cyclodextrin (Au@
DENPs-β-CD)
Gold nanoclusters NGF-siRNA delivery Panc-1 cell line in vitro DNC-Cy5-siRNA complex traces the transfection Lei et al.
(GNC-siRNA) studies and complex is able to silence NGF gene in (2017)
pancreatic cell line
Nitrogen doped carbon Photothermal/ HePG2 cell line in vitro N-CDs as photothermal, hemin as photodynamic, Yang et al.
dots hemin/histidine photodynamic/gene and histidine as gene carrier in CC-NCs (2018c)
nanocomposite therapy
MoS2-ZnO Chemotherapy/ gene HeLa/HaCaT/HePG2 human Cell apoptosis inhibits angiogenesis, also Chacko et al.
therapy cancer cell line in vitro suppresses the mesenchymal gene expression (2018)
CAT@S/Ce6-CTPP/DPEG Phoodynamic/ 4 T1 mouse cancer cells Anti-PD-L1 treatment and PDT both Yang et al.
immunotherapy synergistically enhanced therapeutic action (2018d)
Fe-TBP metal organic Photodynamic/ CT26 colorectal Α-PD-L1 treatment increases CD4+ and CD8+ Lan et al.
framework immunotherapy adenocarcinoma hypoxic cell anticancer cells increasing immune response after (2018)
line PDT
S. Kumar et al.
The NPs are eliminated on the basis of their size. The size of the administered
NPs matters in terms of their transfection through the tissue cells. Hence smaller
sized NPs are more efficient and can be internalized deeper in tissues as compared
with large sized NPs. The surface coating prevents NPs from prompt immune clear-
ance. Otherwise, desired circulation time of nanomaterial inside system cannot be
achieved and therapeutic action may get weakened. To prevent this generally
PEGylation or other biocompatible protein or polymer materials are used (Bantz
et al. 2014). But hydrodynamic size of NPs after PEGylation increases which may
contrarily lower down the action (Hickey et al. 2015). NPs with larger size (<200 nm)
are eliminated by first accumulating them in spleen and NPs (50–150 nm) are
excreted through liver where Kupffer cells plays an important role. On the other
hand, NPs which are very small (<8 nm) are excreted through renal system (Wu
et al. 2017b).
2.6 Conclusion
Nanotechnology is playing tremendous role in cancer ablation techniques and con-

tributed many new approaches but it is only a tip of iceberg. Combination of various
therapies is future of cancer therapeutics where two or more combined approaches
can impinge on cancer. The nano-technological platform for cancer eradication is
explored well in case of chemo and radio and thermal techniques which are destruc-
tive in nature. On the other hand, immunotherapy for cancer attracts much attention
nowadays, which is a nondestructive technique and role of nanomaterials in this
technique needs to be explored yet. With the massive use of nanomaterials in thera-
pies one must also account for proper tracing of bio elimination and bio accumula-
tion of NPs. Since smaller size nanomaterials have different properties than bulk
materials hence they certainly have different impact on biological system.
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Application of Nanoparticles
in Dentistry: Current Trends 3
Subhashree Priyadarsini, Sumit Mukherjee,
Janmejaya Bag, Nibedita Nayak, and Monalisa Mishra
3.1 Introduction
An attractive smile with pearly white teeth in the oral cavity can enhance the per-
sonality to a certain extent. Teeth are an important part of the human body which has
its own fingerprints. Dental records are used in forensic investigations to determine
an individual’s age, sex, food habit, smoking habit, the practice of any chemical like
betel nut pan and gutkha within the mouth (Van der Geld et al. 2007; Gustafson
1950). Although the prime job of teeth is to break the food into pieces, it also acts
as a marker of food deficiency and infection. An infection in teeth may result in seri-
ous complications like an abscess, diabetes, heart diseases as well as neurodegen-
erative diseases (Allen 2003; Liljestrand et al. 2015; Ranjan et al. 2018). Being the
hardest part of the body, teeth can be stored for long and information can be col-
lected at any time even after death (Bergqvist 2003).
The maintenance of the oral cavity hygiene traces back to the ancient ages. In those
days people use animal bones, ashes, bird feathers, chew sticks, tree twigs, porcupine
quills and many other natural materials to clean the teeth (Jass et al. 2003). However,
these usual methods failed to protect the teeth and resulted in numerous dental com-
plications (Shipp 1997). With the advancement of medicine, a new term called
‘Dentistry’ emerged. Dentistry deals with the problems associated with teeth and den-
tal tissues surrounding it as well as the development of various dental materials. The
tooth is mainly comprised of four parts: enamel, dentin, cementum and dental pulp.
Of which, the hardest part is enamel which is highly mineralized part supported by
dentin, which occurs in between the enamel and the pulp chamber, with microscopic
channels known as dentinal tubules. Cementum is a specialized bone-like structure
that covers the dentin root whose principle role is to serve as a medium for attachment
of periodontal ligaments (Fig. 3.1). With the evolution of dentistry, various dental
Subhashree Priyadarsini and Sumit Mukherjee contributed equally with all other contributors.
S. Priyadarsini · S. Mukherjee · J. Bag · N. Nayak · M. Mishra (*)

Neural Developmental Biology Lab, Department of Life Science, NIT Rourkela,
Rourkela, Odisha, India
e-mail: mishramo@nitrkl.ac.in

https://doi.org/10.1007/978-981-13-8954-2_3
56 S. Priyadarsini et al.
Fig 3.1 Schematic Molars

diagram of a whole tooth
Enamel
Dentine
Crown
Pulp cavity
Neck Root canal
Gum tissue
Root Periodontal
ligament
Lateral canals
Cement
Bone
treatment methods and materials such as temporary dressings, dental restorative mate-
rials (such as crowns, bridges and dental fillings), endodontic materials (resin com-
posite and root canal treatment), a coating of the teeth and teeth polishing agents were
developed. These materials possess certain disadvantages such as the loss of teeth
integrity and whiteness, formation of bacterial biofilm on dental coatings and inability
to prevent dental erosion which made people think for the development of better mate-
rials to serve the treatment purposes without losing the teeth integrity.
By the late nineteenth century, the concept of nanotechnology came to existence
through the lecture of an American physicist, Richard Feynman, titled as ‘there is
plenty of room at the bottom (Feynman 1992)’. The fundamental concept of nano-
technology states that at atomic and molecular level, one can discover unlimited
possibilities and potentials. With the advancement of nanotechnology, various
materials or nanoparticles were developed which also included the development of
dental materials having superior properties compared to the bulk materials. Dental
materials along with nanoparticles (such as silica, zirconia, zinc oxide, titanium
oxide, silver and gold) enhance the physicochemical characteristics of these materi-
als under the name ‘nanodentistry’. Nanodentistry uses nanotechnological
approaches for diagnosing, treatment and prevention of oral diseases, improving the
health of the oral cavity (Mantri and Mantri 2013). Various nanoparticles with a
particle size of 10–100 nm have gained particular interests in the fields of dentistry
(Mantri and Mantri 2013; Priyadarsini et al. 2017). This chapter is the summariza-
tion of various applications of nanoparticles in the field of dentistry.
3.2 Nanoparticles used in Various Fields of Dentistry
‘Nanodentistry’ was born in the year 2000 and applied in diagnostics, therapy and
prevention of dental abnormalities and improvement of dental health (Mantri and
Mantri 2013). It is used for root canal irrigation, adhesives, implants, antibacterial
3 Application of Nanoparticles in Dentistry: Current Trends 57
Preventive Dentistry
Nanosized CaCO, HAP nanocrystals,
Mouthwash containing SNP and triclosan
Teeth Implant
loaded NP for biofilm prevention
Orthodontics Titanium NP, HAP, biophosponates in implant
Nanocomposite made of titanium disulfide coating to induce cellular differentiation, SNP in
(WS2) nanoparticles for corrosion reduction of the implant surface exhibit antimicrobial
archwire, reduction of tooth friction with properties.
olysulfone-embedded hard alumina NP
Endodontics Restorative Dentistry

Nanohydroxyapatite paste to promote Hybrid nanocomposite nanofillers to
periodontal ligament cell proliferation, decrease polymerization shrinkage,
collagen nanofibres to support endodontic hardness and wear resistance of
regeneration, Nanoparticles for disinfection restorative composite materials.
of root canal. Use of
nanoparticles
in Dentistry Bone and Tissue Regeneration
Oral and maxillofacial surgery Nano-HAP composite as bone tissue graft
Nanoparticles are used to cause scaffold having superior mechanical
local anesthesia. Nanorobots are properties and biocompatibility, CaSo4
used to numb the target dental nanocrystals improves longivity
tissues.
Oral medicine
NPs are used as effective agent for drug Prosthodontics
delivery to provide more precise delivery. Nanofillers for dental impression,
Nanomaterials for this include polymeric TiO2 or AI2O3 NPs as fillers to PMMA
Periodontics
nanoparticles, solid lipid nanoparticles, to reduce denture porosity and thus
Nanocarriers of drug to breach the
metallic and ceramic nanoparticles. bacterial attachment.
biofilm and able to deliver to the site
of inflammation, eg-triclosan loaded
poly-E-caprolactone
Fig. 3.2 Use of various nanoparticles in different fields of dentistry
medicine formulations, resin composite adhesives, mouthwash, cement and

bleaching agents (Padovani et al. 2015). They are way more effective than con-
ventional practices.
Various fields of dentistry that are currently using nanotechnological approaches
are summarized in Fig. 3.2. Various applications are described below.
3.2.1 Preventive Dentistry
Preventive dentistry deals with the protection of teeth against various biofilm-
forming bacteria on the tooth surface, resulting in tooth decay, caries and other
periodontal diseases. The formation of biofilm is due to bacterial attachment to a
thin acquired dental pellicle, composed mainly of proteins, lipids and sugars derived
from saliva within the oral cavity (Lindh et al. 2014). Consequently, there is a need
for nanotopograhy of favourable physicochemical properties that could resist bacte-
rial settlement on different structures. Recent studies have indicated that nanotech-
nology provides the tools for prevention and early treatment of these diseases by
using special varnishes, mouth washes and toothpaste containing nanoparticles with
antibacterial properties. A number of nanoparticles are known to prevent the forma-
tion of biofilm (Allaker 2010).
3.2.2 Restorative Dentistry
Restorative dentistry serves the main purpose of restoration of the hard tissues of
teeth, lost in the accident, due to some disease or dental caries. The materials used
include metals, ceramics, glass ionomers, cement, amalgam alloys, denture-based
resins with various physicochemical properties. Restorative dentistry not only aims
to restore teeth but also the complete regeneration of dental tissues desired by the
promotion of pulpal cell repair and differentiation. The conventional dental materi-
als such as amalgam alloys have some disadvantages and thus they are no longer
recommended. Dental fillers such as bonding resins undergo polymerization shrink-
age (reduction in size and shape due to polymerization) and thereby create small
gaps between the tooth and the filler materials which can be easily penetrated by
bacteria and other fluids causing marginal staining and dental caries. The incorpora-
tion of nanoparticles in the conventional restorative materials has enhanced their
biochemical properties, reduced polymerization shrinkage and more lively appear-
ance of that of the teeth (Bhardwaj et al. 2014). These materials also include antimi-
crobial nanoparticles that give protection to dental caries and resistant to oral
fractures. Composites with nanofillers also provide a better aesthetic outcome, as
the size of the nanoparticles is below the wavelength of light. Therefore nanofillers
are currently being added to the microcomposites, making them the ‘hybrid nano-
composites’, which exhibit advantages over micro- and nanocomposites
(Pokrowiecki et al. 2018).
3.2.3 Endodontic Treatment
Endodontics deals with the treatment of dental pulp. Dental caries in advanced stage
may cause inflammation of the dental pulp (pulpitis) which is irreversible in nature.
This irreversibility is due to the property of the tooth chamber, its blood irrigation
and lymphatic circulation and the molecular and cellular events that make the
inflammation process hard to control (Fioretti et al. 2011; Farges et al. 2015). The
first line of pulp protection aims at a process of regeneration. But this is rarely pos-
sible because there are not enough suitable biomaterials for such purpose (Fioretti
et al. 2011). Use of stem cells even failed pulp regeneration. With the evolution of
nanodentistry, new materials are now available to treat these diseases. Nano-
hydroxyapatite (nHAp) pastes promote proliferation of human periodontal ligament
cells (Kasaj et al. 2008). Thus it is widely used in clinical practice (Swarup et al.
2014).
Root canal treatment (RCT) is an alternative to pulp regeneration. It aims to
remove infected and inflamed dental pulp, decayed hard tissues, disinfection of the
root canals and filling them with proper materials. Materials that are used to disin-
fect root should be able to dissolve organic and inorganic tissue (the ‘smear layer’),
kill the microbes and present a non-toxic effect to the human tissues. NPs due to
their small size can penetrate dentine tubules in the root canal, seal them and protect
from bacterial leakage. Different NMTs use liquid irrigates with dispersed NPs such
as copper, gold and silver nanoparticles, with the latter being used widely
(Pokrowiecki et al. 2018). Iron oxide nanoparticles are used to eradicate Enterococcus
faecalis from the dentine root. Iron oxide nanoparticles could have additional
advantages compared with silver and other NPs as endodontic irritants, as they are
more biocompatible and demonstrate non-toxic effect to the soft oral tissues
(Bukhari et al. 2016).
3.2.4 Prosthodontics
Prosthodontics deals with tooth restoration with the help of prosthetic treatments
along with the diagnosis, treatment, rehabilitation and maintenance of oral health.
Oral treatment can be performed either by removable dentures or by fixed crowns or
bridges which are made based on the oral impressions taken from the oral cavity.
Resin-based products with good biocompatibility and acceptable aesthetics are sus-
ceptible to microbial adhesion and oral mucosal irritations. Metals like titanium or
cobalt alloys used for fixing crowns and bridges show poor resistance to corrosion
and questionable biocompatibility. Ceramics that are used for the production of
crowns and bridges, zirconium oxide (ZrO2), aluminium oxide (Al2O3), are of low
ductility and brittleness. Nanofillers such as polyvinylsiloxanes are added to the
materials for dental impressions, to make them flow better and enhance the preci-
sion details (Abiodun-Solanke et al. 2014). Incorporation of carbon nanotubes into
a heat cure monomers reduced the polymerization shrinkage, thereby improves the
mechanical characteristics of the dentine materials. Similar observations were
found after the addition of zeolites to polymethyl methacrylate (PMMA) (Hamouda
2012). Mixing of silver NPs to epoxy composites decreased the adhesion of Candida
albicans to the surface of the acrylic resin and suggests a way to prevent denture-
induced somatitis (Hamouda 2012). Use of TiO2 or Al2O3 NPas fillers to PMMA
reduces denture porosity and thus bacterial attachment (Acosta-Torres et al. 2011).
The artificial teeth that are embedded in the denture base, usually made up of
PMMA which can be replaced with nanocomposite denture teeth, exhibit the advan-
tages of nanocomposites (Pokrowiecki et al. 2018). Nanostructured ceramics are
four to five times harder and stronger than the conventional materials due to better
adhesion strength and low porosity. They also show excellent corrosion resistance
and transparency as compared to the resin-based materials that are being used
currently.
3.2.5 Orthodontics
Orthodontic treatment corrects the malocclusion of teeth by using removable or

cemented orthodontic appliances. Removable appliances share common disadvan-
tages as the removable dentures as they are made up of the same materials. Their
use reports many disadvantages from the biomechanical point of view (Littlewood
et al. 2001). Fixed orthodontic appliances (used in of archwires, brackets or
cement) are rather widely used besides the disadvantages of these materials (Batra
et al. 2016).
Nanotechnology overcomes these limitations by using composite nanocoatings
made of nickel phosphorus, fullerenes like NPs of tungsten disulphide (WS2)
(Redlich et al. 2008) and Co and fullerene-like WS2 NP (Friedman et al. 2007).
These NPs helped to improve the corrosion reduction of the archwires (Batra et al.
2016) and reduce the potential toxicity of WS2. The self-lubricating coatings of
molybdenum disulphide NPs were made. It reduces tooth friction and provides
strength by modifying brackets with polysulfone-embedded hard alumina NPs. To
overcome the problem of tooth decay around orthodontic brackets, antibacterial
adhesives with different nanoparticles such as TiO2, SiO2 or silver NPs were tested
and broadly discussed. It was seen that the incorporation of TiO2 NPs into an adhe-
sive material used in orthodontics increased the antibacterial effects; however, the
physical characteristics remain unchanged (Pokrowiecki et al. 2018).
3.2.6 Periodontal Treatments
Periodontal diseases are caused by dental plaque-forming bacteria which lead to an

immune and inflammatory response in the adjacent dental tissues. Prolonged inflam-
mation may cause the destruction of the supporting tissues, such as periodontium
and alveolar bone (Fig. 3.1). The aim of periodontal treatments is to
eradicate/minimize the impact of risk factors in disease progression, improve oral
hygiene and regenerate the lost tissues. Dental regeneration is of great interest in the
field of periodontology; however, from the clinical point of view, this is very chal-
lenging and results in frequent failure as the commonly used antibacterial agents
and antibiotics have poor penetration to the bacterial biofilm (Priyadarsini et al.
2017). Moreover, it is difficult to access the deep periodontal pockets due to root
anatomy and the lesion’s geometry. Nanotechnology provides fabrication of new
systems that can be incorporated into an active anti-infective part of the periodontal
treatments. The use of nanodelivery systems such as NPs, colloidal carriers or lipo-
somes could breach the hydrophobic barrier of oral biofilm and better penetrate the
inflamed tissues (Aminu et al. 2013). Nanoparticles due to its small size enable
penetration through the junctional epithelium, stability and increase bioavailability
(Aminu et al. 2013). Triclosan-loaded poly-ϵ-caprolactone NP showed potential
clinical application (Aminu et al. 2013). The use of ozone nano-bubble water
(NBW3) in the adjuvant therapy of periodontitis has significant clinical improve-
ment in the periodontal status of the treated patients (Hayakumo et al. 2013).
The regeneration of periodontal defects may be unsuccessful due to the relatively
fast rate of biodegradation of the biomaterials as well as their susceptibility of re-
infection (Osorio et al. 2016). The root cement regeneration is important as it is the
only tissue having the ability to anchor the tooth in the alveolus by insertion of
periodontal ligaments. However, this is barely achievable with the currently used
conventional materials (Arzate et al. 2015). Finding new techniques to overcome
these problems with the help of nanotechnology has been proposed in the last few
years, which can be targeted for alveolar bone repair and periodontal tissue regen-
eration (Osorio et al. 2016; Shimauchi et al. 2013). Nanomaterials also used for
cartilage regeneration. Materials such as nano-silicon dioxide with alginate and
poly (acrylic acid), biphasic CAN-PAC hydrogel and other nanofibrous scaffolds
showed the ability to mimic the collagen fibrils in the extracellular matrix of carti-
lage and promote differentiation of mesenchymal stem cells and chondrocytes (Li
et al. 2017; Liao et al. 2017).
3.2.7 Dental Implants
When tooth or teeth are lost, oral implants are ‘the last stand’ of bite reconstruction.
Titanium implants were introduced in the 1980s based on the concept of ‘osseointe-
gration’, a direct contact of a living bone with functionally loaded oral implants.
However, the quality of the implant-bone interconnection was not very good. The
‘rougher-the better’ implant surface hypothesis was rejected. The micro-rough sur-
face despite better stability in the beginning may initiate subsequent bone osteolysis
and favours biofilm formation and development of infection, known as ‘periimplan-
titis’. With the development of nanodentistry, the implant surface was modified
mimicking nature (Thakral et al. 2014). Implant surface which imitates bone-like
structures, properties and compositions at nanoscale level was made. Proper modi-
fication of the surface from macro to nanoscale may improve the process of implant
healing.
The process of implantation includes two phases:
(1) Primary interlock deals with the mechanical anchorage and design of the
implant
(2) Secondary anchorage relies on bone remodelling at implant interface and cre-
ation of biological bonding.
A reduction in the implant stability can be seen between the two anchorages, i.e.
primary and secondary as a natural consequence of bone tissue remodelling follow-
ing surgical injury. The nanostructured dental implants with peptides, stem cells and
preosteoblasts promote them to find a favourable niche for attachment, spreading
and differentiation (Thakral et al. 2014). For surface modification (1) direct surface
tailoring (e.g. etching and sandblasting) (2) functionalization (bio-glass coatings or
polymers, peptides, etc.) or combination of both the techniques are used to make the
implant surface rough at the micro level (Tomisa et al. 2011; Lavenus et al. 2010).
However, these techniques decrease surface hardness and cause a loss of superficial
material layer that damages the screw threads, negatively affecting proper implant
anchorage to bone (Duraccio et al. 2015). Surface etching with solutions of alkaline
sodium, alkaline potassium or hydrogen peroxide with or without heat treatment
forms stable oxide layers with nanostructures like nanotubes, nanoneedles and
nanowires (Pachauri et al. 2014; Tsimbouri et al. 2016). Nanostructures could
increase surface wettability which seemed to better stimulate protein adsorption,
blood clot formation, neoangiogenesis at the implant interface leading to the accel-
eration of the healing process (Kopf et al. 2015; Chen et al. 2017).
Implant with bioactive coatings with micro hydroxyapatite (HAp) or micro
β-tricalcium phosphate (β-TCP) surfaces appeared clinically undesirable. The poor
attachment of particles to the implant surface, which in turn resulted in their
mechanical peel off due to shear forces, initiates an inflammatory response and in
consequence removal of the implant. The problem with conventional ceramics was
replaced with nano counterparts with gradient micro nanosurfaces on the implant,
which can guide tissue regeneration. Nanostructured ceramics increase contact with
surrounding tissues, comparing to their bulk forms, and do not cause any inflamma-
tory response (Sharma et al. 2014). Nanocoatings on endosseous implants such as
nanocrystalline diamond, carbon nanotubes, bioactive glass, poly (lactide-co-
glycolide), hydroxyapatite nanocomposite or zinc-substituted n-HAp are the bio-
materials of the future implant (Pokrowiecki et al. 2018).
Besides the improvement of osseointegration, the antimicrobial properties
enhance the longevity of the implant. Formation of bacterial biofilm within the oral
cavity on the peri-implant space causes a serious threat to the longevity of implants.
The nanocoatings of the implant surface provide antibacterial properties, preventing
the implant loss due to infection. Various metallic NPs in combination with antibiot-
ics are embedded on the implant surface. The antibacterial effect can be obtained
either by contact killing or releasing the drug into the peri-implant space
(Pokrowiecki et al. 2018).
3.3 Common Nanoparticles Used in Dentistry
The wide use of nanoparticles in dentistry relies on its properties such as tiny size,
high surface area-volume proportion, less toxicity and good biocompatibility, which
is often absent from the micro and macro materials (Priyadarsini et al. 2017). These
properties make the nanoparticle to be potentially used in the regeneration of dental
tissue, treatment of oral bone fractures, cartilage repair of a temporomandibular
joint, repair of tooth pulp, ligament regeneration in periodontum, a coating of dental
implants, bone tissue grafts with better characteristics and so on. Various dental
nanofiller materials have been developed by incorporation of nanoparticles. Besides
that these particles have good strength and are resistant to corrosion and abrasion.
Nanoparticles are also useful for drug delivery for the treatment of periodontal dis-
eases. The nanoparticles have specific biological properties such as inertness, bio-
compatibility and antimicrobial property having definite surface topography for
which these can be used as a dental material and proved to be a promising approach
to induce dental restoration and repair dental tissues. Nanoparticles used in den-
tistry can be classified mainly into three classes (Fig. 3.3):
(1) Polymeric nanomaterial: chitosan, dendrimers, nanogels, polyethylene glycol

and solid lipids
(2) Metallic nanoparticles: silver, gold and copper
METAL CHANGE IN PROPERTIES

INORGANIC
1. MECHANICAL PROPERTIES
MINERAL
2. BIOLOGICAL PROPERTIES
3. PHYSICOCHEMICAL PROPERTIES
NANOPARTICLES
HYBRID 4. BIOCOMPATIBILITY PROPERTIES
5. CYTOCOMPATIBILITY PROPERTIES
NANOSTRUCTURED PARTICLES ORGANIC LIPID
DENTAL RESTORATIVES
POLYMERIC
TOOTH ADHESIVES
STRATEGIES AND ROLES
CEMENTS/SEALANTS
1. DRUG NANOCARRIER FOR CONTROLLED
AND SUSTAINED RELEASE OF DRUGS
COLLOIDS/PASTES/PATCHES, NANOGELS
2. MECHANICAL AND PHYSICOCHEMICAL
REINFORCEMENTSYSTEM
3. ANTIMICROBIAL NANOPARTICLES
4. PHARMACOLOGICAL AGENTS NANOMATERIAL OBJECTIVES
5. BIO-ADHESION AGENT
6. ANTI-ADHESIVE AGENTS 1. ORAL DISEASES/DENTAL
7. REMINERALIZING AGENT PATHLOGIES/TOOTH
8. ANTI-CORROSIVE AGENT EROSION/PERIODONTAL
9. NANOCOATING OF IMPLANT INFECTION
10. DENTAL NANOFILLERS METALS 2. DENTAL PLAQUE/CARIES/TEETH
INFLAMMATION/BACTERIAL
POLYMERS BIOFILM/HYPEERSENSITIVITY
3. ORAL CANCER DIAGNOSIS/
ORTHODONTIC
COMPOSITES
APPLIENCES/REMINERALIZATION/
FRICTION RESISTANCE
RESIN-COMPOSITES 4. DENTAL
NANOCOATING/DISINFESTION OF
ROOT CANAL/ TEETH WHITENESS
Fig. 3.3 Overview of the role of nanoparticles in various aspects of dentistry
(3) Inorganic nanoparticles: silica, zirconia, titanium dioxide, hydroxyapatite, zinc

oxide and carbon nanotubes.
3.3.1 Polymeric Nanoparticle
Chitosan
Chitosan is a natural polymer of N-acetyl-glucosamine and glucosamine residues
produced from chitin (Friedman et al. 2013). Chitosan is used frequently in the
fields of medicine and dentistry due to its antimicrobial and regenerative properties
as well as high biocompatibility. It has applications in different fields of dentistry
which includes restorative dentistry, endodontics, preventive dentistry, periodontol-
ogy, orthodontics, prosthodontics and oral surgery. Materials based on chitosan
have been used tremendously for wide applications in dentistry (Hayashi et al.
2007).
Nanogel
Nanogels are polymeric nanoparticles having a three-dimensional network. The
interests of using them have grown in the past few years due to their possible appli-
cability in the fields of biomedicine, such as bioimaging and drug delivery systems
(DDS). Nanogels stably entrap bioactive compounds like DNA/RNA, proteins or
drugs inside the nanospace within the polymeric network (Sasaki and Akiyoshi
2010). Moreover, nanogels are unaffected by the pH and temperature, which are
important for the restricted release of the bioactive compounds. They are used as
effective dental fillers along with the composite resin materials that help in the
reduction of polymerization shrinkage.
Dendrimers
Dendrimers are highly branched polymeric nanostructures that are radically sym-
metric, homogeneous and monodispersed. The three-dimensional structure allows
them a number of unique characteristics, such as nanoscaled globular shape, hydro-
phobic or hydrophilic cavities in the interior and functional groups at the periphery.
These properties make them a new class of macromolecular nanodelivery devices
(Abbasi et al. 2014). Most dendrimers are globular in shape having a molecular
diameters less than 10 nm, and their size and shape can be changed by varying the
production of them (Nanjwade et al. 2009; Noriega-Luna et al. 2014). This gives
dendrimers size and shape that is similar to specific proteins and other biomole-
cules, making them a perfect biomimic. The biomimetic property of dendrimers
helps in the regeneration of teeth enamel.
Polyethylene Glycol (PEG)

Polyethylene glycols (PEGs) are polymers of ethylene oxide, which are hydrophilic
in nature. The advantage of attaching a number of functional groups to the terminal
sites has greatly expanded their usage (Veronese and Pasut 2005). The covalent
attachment of the PEG derivative to the surface of molecules improves the solubility
and biocompatibility for drug development (Alcantar et al. 2000). Bifunctional PEG
derivatives are used frequently for the PEGylation of small peptides, proteins, oli-
gonucleotides, nanoparticles and various surfaces, whereas multi-arm PEG deriva-
tives are mostly employed in the formation of hydrogels, controlled drug release,
medical devices, regenerative medicine and many other applications (Hutanu et al.
2014). PEG is potentially used as an antifouling polymer. PEG coating on the tooth
surface prevent attachment of bacteria and thereby prevent the formation of bio-
films. Moreover, they immensely lower the demineralization of the enamel surface
and can solve the problems that arise due to bacterial accumulation around the den-
tal tissues (Buxadera-Palomero et al. 2015; Peng et al. 2017).
Solid Lipid Nanoparticles (SLN)

SLN act as a drug carrier system, an alternative to the traditional colloidal carriers
like polymeric or ceramic nanoparticles, liposomes and emulsions. They range from
50 to 1000 nm and are formed of biocompatible lipids (Saupe and Rades 2006;
Müller et al. 2000). The biocompatibility of the lipid matrix is a major factor that
allows SLN to be potentially used as a drug carrier. It combines the benefits of poly-
meric nanoparticles, liposomes and fat emulsions. The unique characteristics such
as nanosize and large surface area offer itself as an ideal nanoparticle for efficient
drug loading.
3.3.2 Metallic Nanoparticles
Silver Nanoparticles (AgNPs)

Silver nanoparticles are nano-sized particles with a size less than 100 nm and are
well known for its antimicrobial activities to have applications in various fields of
dentistry (Şuhani et al. 2018). The unique chemical, physical and biological proper-
ties of AgNPs make it effective even at a very low concentration. AgNPs bind to the
proteoglycans layer of bacterial cell membranes (Chaloupka et al. 2010). Moreover,
the silver ions interact with –SH groups at the time of translation, thus interfering
with the replication of bacterial DNA (Radzig et al. 2013). AgNPs have been applied
in prosthetic materials, dental adhesives and tooth implants to promote prevention
of dental caries, the formation of biofilms and osteogenic induction (Şuhani et al.
2018; Santos et al. 2013).
Gold Nanoparticles
Gold nanoparticles occur as a cluster up to 100 nm in diameter. The functional
versatility, biocompatibility, low toxicity, chemical stability and optical prop-
erties offer itself to be used in the field of dentistry (Chen et al. 2008). They are
easy to synthesize and have the ability to bind to a variety of molecules to their
surfaces in the biomedical field which includes drug delivery, chemical sens-
ing, biological imaging and treatment of various diseases. Gold NPs are ideal
candidates for carrying a large number of antibiotics without compromising
their activities. It is used as optical probes for the detection of oral cancers.
They are often conjugated to antibodies or signal receptors through coordinate
bonding or electrostatic interactions to act as a probe for cellular biomarkers
(such as EGFR), present in the cancer cells only. With these cellular markers
they can provide an optical signal for the early detection of oral cancers (Kah
et al. 2007). Gold nanoparticles are used in fixed prosthodontics before cement-
ing ceramic restorations which enhances adhesive properties and improve pol-
ish ability, aesthetics and wear resistance of moldable ceramics (Lboutounne
2017).
Copper Nanoparticles
Copper nanoparticles display unique catalytic, antifungal and antibacterial
properties. Copper nanoparticles are used in medicine, electronics, optics, nano-
fluids, antimicrobial agents and conductive films. The antibacterial action of
copper nanoparticles enhances its potential use in dental materials (Din and
Rehan 2017; Amiri et al. 2017). Copper NPs are incorporated with the resin
composites used as nanofiller. It provides reduced polymerization shrinkage,
higher dimensional stability, increased surface smoothness and thus reduce the
bacterial adhesion on these surfaces. Thereby copper NPs are considered as use-
ful orthodontic adhesives (Argueta-Figueroa et al. 2015). It is also used to coat
dental implants as well as in root canal treatments to produce antibacterial
activity.
3.3.3 Inorganic Nanoparticles
Silica Nanoparticles
Silicon dioxide or silica nanoparticles have potential biological applications due to
their excellent biocompatibility, low toxicity and thermal stability (Rahman and
Padavettan 2012). The particle size, porosity, crystallinity and shape can be pre-
cisely manipulated that make them suitable to be used for various applications. The
surface can be modified for drug loading, good dispensability and site-specific tar-
geting (Rahman and Padavettan 2012). Nanosilica filler synthesized by sol gel
method is typically used in dental composites. The extremely small size of the
nanoparticles allows making a wide range of shades and opacity of dental compos-
ites, thus providing tooth restorations that are highly aesthetic (Timpe et al. 2014;
Rezvani et al. 2016; Rahim et al. 2011). Spherical silica nanoparticles offer superior
polish and improved mechanical properties including hardness, flexural strength
and abrasion resistance to the teeth.
Zirconia Nanoparticles
Zirconia (or zirconium dioxide) is a ceramic material with polycrystalline nature,
biocompatible, high wear resistance, low reactive and good optical properties and is
an ideal material for extensive use in dental restorations and implantation. It is
widely used for implantation of dental crown although the toxicity of zirconia NP
recently came into account from animal studies (Mishra et al. 2017). In various
dental fillers it is used as a reinforcing agent as it enhances the adhesion of tissues
and increases the mechanical stiffness (Bona et al. 2015; Denry and Kelly 2008).
Zirconia-based nanoparticles are used as a dental adhesive to increase the tensile
strength and support mineralization after implantation. These nanoparticles inhibit
the growth of gram-negative bacteria. They bind to the bacterial cell membrane and
stop the metabolic exchange, thereby preventing the enamel corrosion (Fathima
et al. 2017). The outer coat of the zirconium oxide nanoparticles to the teeth pro-
vides external support to the teeth and enhances its lifespan.
Titanium Dioxide Nanoparticles

The photo activities and antimicrobial property of the TiO2 nanoparticles attract the
applications of these nanoparticles in the field of dentistry besides its toxicity report
from animal studies (Sabat et al. 2016). The whiteness, low toxicity and highly
stable nature along with its easy accessibility and low cost have made them good
dental material. They are used for tooth whitening, to increase flexure and wear
resistance (Sodagar et al. 2013).
Hydroxyapatite Nanoparticles
Hydroxyapatite is the main component of bone and teeth enamel and the most stud-
ied biomaterial in the medical field. In 1970, a Japanese company Sangi Co. Ltd first
took interest in hydroxyapatite after purchasing the rights from NASA. In 1978,
Sangi Co. Ltd first launched a toothpaste containing nano-hydroxyapatite that can
repair the tooth surface (Pepla et al. 2014). In 2006, the first toothpaste came to
market that contains synthetic hydroxyapatite biomimetic as a substitute of fluoride

for dental remineralization and repair of enamel.
Hydroxyapatite is an important source of phosphate and calcium essential for
enamel remineralization. The nano-HAp binds strongly with the proteins as well as
with the bacterial fragments of dental plaques when contained in toothpaste. The
ability is because of the size of the nanoparticles that considerably increase the sur-
face area to which proteins can bind. Nano-hydroxyapatite also acts as a filler, as it
repairs the depressions and small holes on the enamel surface. Hydroxyapatite
nanocrystals are used to generate tissue grafts which stimulate the proliferation of
the cells for periodontal tissue regeneration.
Nano-hydroxyapatite (nano-HAp) is useful in dental prosthetics as it has similar
size, crystallinity and chemical composition of the human hard tissues. Dental
implants are often coated with hydroxyapatite, thus reduces the chance of implant
rejection (Choi and Ben-Nissan 2016). Due to its chemical and crystallographic
affinity with the inorganic constituents of bone, hydroxyapatite is able to establish
chemical bonds and ensure a more rapid integration of titanium implants to bone
and surrounding tissues. Its bioactivity encourages the bone growth and thus restores
the defect. This process is an alternative to bone allografts and xenografts.
Hydroxyapatite nanoparticles are often incorporated in the health drinks taken by
the athletes to prevent teeth erosion (Min et al. 2015) although toxic reports were
found from various animal studies (Pappus et al. 2017).
Zinc Oxide Nanoparticles

Zinc has been used as a major dental filler for many years. Zinc oxide nanoparticles
(ZnO) show effective antibacterial activity (Padovani et al. 2015). This bactericidal
effect may be due to their contact with bacterial cell membrane; zinc binds strongly
to proteins and lipids, increasing membrane permeability (Hajipour et al. 2012).
Additionally, ZnO NPs induce oxidative stress in bacterial cells by generation of
reactive oxygen species (ROS). Zn2+ ions along with ROS reduce bacterial coloniza-
tion and growth (Huang et al. 2008). Furthermore, ZnO NPs are potentially biocom-
patible and non-toxic to the human cells as reported by several studies. ZnO
nanoparticles are used in dental coatings along with HAp nanoparticles. The addi-
tion of ZnO to bone composites can enhance the adhesion, differentiation and pro-
liferation of the bone cells (osteoblasts) (Memarzadeh et al. 2015).
The applications of various nanoparticles in dentistry are represented in Fig. 3.4.
Carbon Nanotubes
Carbon due to its unique properties occupies a wide variety of forms and struc-
tures. Out of which carbon nanotubes (CNT) are of particular interest. CNTs are
made up of hexagonal graphite sheets wrapped into single or multiple layers
(Collins and Avouris 2000). They are having unusual thermal, electronic and
mechanical characteristics, derived from their specialized carbon bonds, one-
dimensional nature and cylindrical symmetry. All these properties increase its
interest to be used as a material for reinforcement in various composites to ensure
new functions. CNT was introduced to dentistry in the early 1990s. The advantages
Nanomaterials Dental application of

nanomaterials
Calcicum phosphate
nanoparticles
Remineralisation
Dental resin
Zinc citrate
nanoparticle
prevent dental
plaque develpoment
PolyGA-
PEG-
cisplatin Oral cancer
management
Solid lipids
Periodontology
Hydrogels
Silver Dental carries prevention

nanoparticles
Dental polishing/surface coater

Gold
nanoparticles
Oral cancer
management
Quantum
dots
Titaninum
Corrosion resistant
nanoparticles
dental implants
Biomineralisation
Dendrimers Adhesive dentistry
Fig. 3.4 Overview of the current use of various nanoparticles in dental therapeutics
of CNTs include (1) improved strength of composite materials and implants, (2)
increased cell adhesion and proliferation, (3) nucleation of hydroxyapatite and (4)
protection against bacteria which have generated interest regarding their use in
dental fields (Bhattacharya and Seong 2013).
Functionalized single-walled carbon nanotubes (SWNT) incorporated into the

dental composite enhances the tensile strength thereby increases the durability of
the composite materials in the oral cavity (Zhang et al. 2008). In addition, SWNT
also increases the flexural strength of these composites by absorbing more stress.
CNT-reinforced composite resin has proved to be disadvantageous as CNT gives a
dark colour, a major drawback for the resin composites used for aesthetic purposes.
The microleakage developed in the dentin and composite resin interface due to
long-term use is reduced due to the application of CNT. These microleakages are
the major cause of failure of the tooth restorations. Once microleakage has devel-
oped in the interface of the tooth and composite resin, it acts as a niche for bacterial
colony formation, thus causing secondary tooth decay (Bhattacharya and Seong
2013). The presence of CNT at the interface reduces the chance of secondary decay
formation by providing protection against the decay-forming bacteria and initiating
the nucleation of hydroxyapatite on its surface. However, the major demerit of CNT
is that it produces grey colouration at the interface (Akasaka et al. 2009).
Quaternary Ammonium Compounds (QAC)

Quaternary ammonium compounds are contained in the nanomaterials, as they
show effective antibacterial activity against a number of species such as Streptococcus
mutans and Lactobacillus casei. Although the bactericidal mechanisms of QAC are
still unknown, it may be possible that it serves as cationic agents that are highly
active and results in adsorption of the cationic polymers present on the bacterial cell
membrane. These interactions result in a change of membrane permeability that
subsequently leads to the complete breakdown of the cell membrane (Zhang et al.
2018a; Makvandi et al. 2018).
3.4 Current Applications of Nanoparticles
3.4.1 Treatment of Bacteria
Many dental problems are associated with pathogenic microbes which colonize on
the enamel surface, holes of the dentine, and the delicate tissues that surround them
(Rao et al. 2011). The microbes produce acidic substances that affect the hard tis-
sues and causes dental caries. Filling of the root canal with an inert material enables
the pathogens to cause inflammation after teeth filling resulting in loss of teeth.
Thus dental materials should have antimicrobial property (Li et al. 2006). The resis-
tance of bacteria towards antibiotics warrants a new approach to treat teeth bacteria.
In this context various nanoparticles are growing its evidence to treat bacterial
infections and thus used in the field of dentistry. Dental materials with antimicrobial
activity are used for teeth filling materials, sealants, cement, temporary teeth restor-
ative materials, adhesives and teeth coating materials (Ahn et al. 2009). The metallic
nanoparticles like silver, copper and gold show antimicrobial activity (Monteiro
et al. 2009; Hannig et al. 2007), due to its large surface area–volume proportion,
size and shape (Morones et al. 2005). Compared to the antibiotics, nanoparticles
show their effects at much lower concentrations. Periodontal pathogens such as

Fusobacterium nucleatum, Porphyromonas gingivalis, Aggregatibacter actinomy-
cetemcomitans and Prevotella intermedia are susceptible to copper oxide and silver
nanoparticles. The nanoparticles-induced bacterial cell death occur via two separate
mechanisms. (1) Interaction of the nanoparticles to the bacterial cell membrane
resulted in electrostatic forces, by altering membrane potential, depolarization and
subsequently loss of membrane integrity. Furthermore, the major cell functions of
bacteria like respiration, nutrient transportation and disruption of energy transduc-
tion get affected resulting in death of the bacteria. (2) Reactive oxygen species
(ROS)-mediated cell death by inhibiting the protein function, damaging DNA and
resulting in an excess radical generation (Ibrahim et al. 2017). More research is
needed in this field to establish the bactericidal ability of metal nanoparticles.
3.4.2 Prevention of Biofilm
Bacterial biofilms are described as an aggregation of microbes that adhere to a liv-

ing or non-living surface (Fig. 3.5). Biofilms form on the dental surface causes gum
disease and tooth decay. Bacterial biofilm affects the individuals from all the age
groups. Biofilm can form on the teeth surface as well as on dental implants or
devices used for dental orthodontic applications. Conventional antibiotics are inef-
ficient to treat biofilms since they cannot penetrate the extrapolymeric layer cover-
ing the biofilm. Moreover, a high dose of antibiotic has many toxic effects on the
host and the bacteria also become resistant (Seil and Webster 2012; Beyth et al.
2015). Nanoparticles prevent biofilm formation by preventing the attachment of
bacteria to the teeth surface and by killing the microbes forming biofilms (Fig. 3.5)
(Allaker 2010). Metallic ions with positive charge alter the electrostatic interactions
between them and the bacterial cell membrane (Kim et al. 2007). Silver nanoparti-
cles inhibit DNA replication (Feng et al. 2000), loss of expression of ribosomal
proteins, inactivation of enzymes and cellular proteins required for the production
of ATP (Yamanaka et al. 2005). Silver ions also affect the membrane-bound respira-
tory enzymes (Bragg and Rainnie 1974). Ionic metal oxides like copper oxide
(CuO) show antimicrobial activity because of their significant crystal morphologies
and high surface areas having more number of potential reactive sites (Stoimenov
et al. 2002). It can be efficiently blended with polymers having minimum bacteri-
cidal concentrations (MBC), and investigated for their antimicrobial effects (Ren
et al. 2009) against E. coli and methicillin-resistant Staphylococcus aureus (MRSA).
Silica nanoparticles release nitric oxide (NO) and kill S. aureus, P. aeruginosa
and E. coli (Hetrick et al. 2009). NO enhances its penetration into the biofilm matrix
due to its rapid diffusion. Thus, it can enhance adequacy against microbes which are
embedded in oral biofilm. Mimic of natural surface like casein phosphopeptides
bound with amorphous calcium phosphate decreased bacterial attachment to the
surface (Cross et al. 2007). Nanoparticulate metal oxides or quaternary ammonium
poly(ethylene imine) antimicrobial nanoparticles were incorporated into dental
resin composites to prevent biofilm formation (Allaker and Ian Douglas 2015).
Steps for Biofilm formation
Attachment and Maturation and

colonisation Growth and proliferation detachment
Pellicle Layer
Enamel
Silver/copper/chitosan based
nanoparticle
Ferumoxytol nanoparticle
PEO-coated nanoparticle Iron oxide nanoparticle
Disruption of attached
biofilm survival Disruption of
Prevention of
adhesion formed biofilm
Steps to inhibit biofilm formation
Fig. 3.5 The top part represents the formation of biofilms and the bottom represents the preven-
tion by using nanoparticles
However, the process of manufacturing such a self-cleaning surface for the purpose
of dentistry is quite challenging.
3.4.3 Dental Implant
Tooth implants are useful for restoring lost teeth. However, its failure is related to
instability or poor fitting at the interface of implant-abutment connection. The den-
tal abutment, a connecting piece is generally implanted on top of dental implants for
holding and supporting dental crowns and to join dental crowns with implants. The
Crown
Abutment (Post)
Gum tissue
Bone
Implant (titaninum NPs)
Fig. 3.6 Schematic diagram of a Titanium implant
small gap at the interface of two-piece implants and bone is normally fluid filled
which leads to bacterial colonization and inflammation that result in bone loss
around this area (Parnia et al. 2017).
The biological fixation of dental implant is termed as osseointegration and occurs
due to the intimacy of bone growth onto implant surfaces after its surgical insertion
into the jaw bones (Fig. 3.6). Osseointegration is a two-step process—the first step
is known as primary stability which includes design and mechanical anchorage of
the implants to the bone. The secondary step of osseointegration is characterized by
covalent bonding between the teeth and the surface of the implant. A number of
surface modifications were employed to enhance the osseointegration of the
implants, which aim to generate implants with surface characteristics for protein
adsorption, attachment and differentiation of osteoblasts and integration of the tis-
sues. The biological abilities of an implant depend on the chemical nature, rough-
ness and wettability of the surface. Nanotechnology provides surface manipulations
that are helpful to understand cellular interactions to produce novel implant surfaces
with properties of tissue integration. Developments of new coating technologies are
applying hydroxyapatite and calcium phosphates (CaP) on the implant surface to
provide an osteoconductive surface to the titanium implants (Geesink 2002;
Leeuwenburgh et al. 2001).
Few minutes after the implantation, early bacterial colonization associated with
periodontitis can occur. Microorganisms can grow in the micro-gap of the fixture-
abutment interface that acts as a bacterial reservoir leading to inflammation of the
surrounding soft tissues. Currently, inadequate treatment may result in implant
removal after the onset of some chronic infections (Zitzmann and Berglundh 2008).
To avoid implant failure, implant surface modifications are done. As the implant
surface interacts continuously with bone and gingiva and partially to the oral cavity
where multiple species of microbes may occur, a coating of the implant surface with
a material with good biocompatibility as well as antimicrobial properties is impor-
tant to enhance the probability of implant success. Nanoparticles enhance
osseointegration of dental implants as well as an antimicrobial coating for the

implant surface to improve soft tissue integration and increased success of the den-
tal implant. Coating of Ti implant surface with bio-inert nanomaterials (such as
zirconium and aluminium) may alter the physical characteristics and also increase
the osteogenic properties of the implants (Chidambaranathan et al. 2016). To pre-
vent bacterial biofilms on the implant surface, Ti-based implants are modified by a
coating of antibacterial metal nanoparticles. The nanocoatings offer the chance to
leave the majority of the Ti-based surfaces uncoated and thus the surface is available
for osteoblasts colonization following osseointegration (Wood et al. 2015).
When a teeth implant is placed inside the oral cavity, it induces biological
responses mediated by implant and surface cells interaction. At the site of interac-
tion, specific genes get activated, adsorb specific lipids, sugar, proteins and ions
which can activate defence mechanisms of cells to either accept or reject the dental
implant by figuring out the type and population of cells on the implanted surface
(Elias 2014; Krishna Kumar et al. 2011). Surface alteration of nanoparticle pro-
motes cell adhesion and protein adsorption, mimics coatings by calcium phosphate
and incorporates growth factors to induce bone healing process (Le Guéhennec
et al. 2007).
3.4.4 Prevention of Tooth Erosion
Tooth erosion is the loss of hard tissues of oral cavity due to exposure to the acidic
substances produced by cariogenic bacteria as well as from diet such as acidic
foods, soft drinks and also from internal sources such as those originated from the
stomach during regurgitation. The reaction caused by acidic substances leads to a
permanent loss of dental hard tissue, followed by a subsequent softness of the sur-
face. Acid production is one of the main factors for the erosion of dental wear
(Magalhães et al. 2009). Tooth pastes having anti-erosive and repairing nature have
been marketed, which generally contain active substances like hydroxyapatite and
zinc–carbonate–hydroxyapatite nanoparticles to prevent dental erosion (Aykut-
Yetkiner et al. 2014). HApNPs provide a remineralization scaffold by filtrating
demineralized dentine (Besinis et al. 2012; Earl et al. 2009) and prevent enamel
erosion (Ganss et al. 2011).
Modern biomimetic nanoparticles prevent mineral loss and remineralize enamel
lesions (Hannig and Hannig 2010; Tschoppe et al. 2011). Enamel remineralizing
substances like HAp NPs and Zn-carbonate hydroxyapatite induce precipitation of
mineral on eroded dentine and enamel (Poggio et al. 2014). The addition of HAp
micro clusters to soft drink, mouthwashes and toothpaste can promote coating of a
carbonate-hydroxyapatite particle on eroded enamel and decreases erosiveness of
enamel because of the super saturation of calcium and phosphate (Poggio et al.
2010; Roveri et al. 2008; Min et al. 2015). Nanoparticles like casein phosphopeptide-
amorphous calcium phosphate (CPP-ACP) prevent demineralization by serving as a
reservoir of calcium and phosphate (Cai et al. 2007).
3.4.5 Drug Delivery
Nanoparticles have been evolved as an important strategy for therapeutic drug deliv-
ery, recombinant proteins, genetic material and vaccines. Most of the drug delivery
systems do not pin point the target cells precisely and thereby end up with harmful
side effects (Bhowmik et al. 2009). The discovery of the nanodrug delivery systems
greatly enhanced the functioning of the drugs at the right place (Gaur et al. 2014;
Ravichandran 2009).
The main focuses of these drug delivery systems are to:
1 . Build up a more specific and target oriented drug delivery system

2. Reduce toxic effects while maintaining therapeutic purpose
3. Provide increased safety and biocompatibility
4. Speed up the discovery of new and safe nanodrugs.
Nano-drug delivery systems are applied in the field of restorative dentistry, pre-
vention of caries, oral biofilm management, remineralization of the tooth, control of
hypersensitivity of the tooth, local anaesthesia, disinfection of root canal, periodon-
tal diseases, infection control, tissue engineering and therapy of oral cancer. The
application of nano-drug delivery systems in dentistry is generally classified into the
following categories:
Type of Drug Delivery System

1. Self-carrier nanodrugs: Control of caries and tooth remineralization, disinfection
of root canal.
2. Nanodrugs loaded nanocarriers: Vaccines for tooth caries, control of oral bio-
film, infection control, hypersensitivity of the tooth, tissue engineering, local
anaesthesia, treatment of oral cancer.
3. Nanodrugs loaded drug carriers: Dentin hypersensitivity, oral biofilm

management.
4. Drug loaded nanocarriers: Periodontal diseases.
Administration Route
The drug can be delivered via two different routes: (1) local route and (2) systemic
route.
1. Local route: Tooth remineralization, prevention of caries, oral biofilm manage-

ment, teeth hypersensitivity, local anaesthesia, disinfection of root canal.
2. Systemic route: There are three systemic routes of entry; oral (control of infec-
tion), nasal (vaccine for dental caries) and injectable (local anaesthesia, treat-
ment of oral cancer).
3.4.6 Aesthetics, Increase Whiteness
Consumption of coloured food and drinks such as tea, coffee, pan, gutkha and other
chromogenic substances affects the whiteness of teeth. Thus, people often opt for
teeth whitening treatments that bleach the teeth. Although teeth whitening is the
easiest way to brighten the teeth colouration, it is very difficult to accurately predict
the beneficial outcome of tooth bleaching (Joiner 2006; Sarrett 2002). The side
effects of teeth whitening are uncommon but do occur occasionally (Watts and
Addy 2001). They mainly include a sore throat, tooth sensitivity and gum ulcer
(Watts and Addy 2001; Goldberg et al. 2010). Keeping the negative impact in mind,
nanoparticles-based teeth whitening has been developed in dentistry.
Resin composites, dental fillers and non-heavy metals (silica, calcium phosphate,
titanium oxide and aluminium oxides) are used widely for teeth whitening (Wu et al.
2003). The resins in the composites are component of the acrylic resins. Recently
these acrylic resins are replaced with silane due to their toxic effects. The silane or
the silicate is the cementing substance that give rigidity and whitening to the teeth
and protect the tooth (García et al. 2006). The materials actually do not bleach the
teeth but the ions (e.g. Ca2+ ions) released from them form a calcium phosphate-
based layer (similar to hydroxyapatite) on the teeth surface. Teeth whitening become
more useful when there are white stains in the teeth caused by hypocalcification due
to loss of calcium in the tooth enamel. Due to exposure of the teeth to fluoride (fluo-
rosis), having high sugar or acidic substance in the diet and formation of heavy
plaque may lead to hypocalcification (Pulgar Encinas et al. 2001). The white spots
are also visible when brackets and orthodontic bands are removed from the tooth.
This spot can be marked due to the odd distribution of the whitening of the teeth due
to hypocalcification which appears as some parts are whiter than the other parts and
making the colour difference even more apparent (Pulgar Encinas et al. 2001).
Peroxidase has long been used as a teeth whitening agent (Baldión Elorza et al.
2012) although it is highly reactive. Teeth discolouration is mainly caused by the
electrons from the pigment molecules from different sources. During peroxide treat-
ments, these electrons are taken by peroxide oxidation and exposure of teeth to the
blue light accelerates this process (Dishman et al. 1994). However, a higher concen-
tration of hydrogen peroxide can disrupt the tooth enamel and thereby cause tooth
hypersensitivity (Lewinstein et al. 1994). Titanium oxide nanoparticles modified
with polydopamine (PDA) gets activated with blue light. The coating of the nano-
TiO2@PDA particles on the tooth surface can cause increased whiteness when
exposed to blue light (Zhang et al. 2018b). Besides the titanium nanoparticles, silica
nanoparticles are also used in the whitening of teeth (Bowen 1962). Peroxidase as
the solvent, doped with the nanoparticles are used in the aesthetic of teeth whiten-
ing. When these peroxides contact to the teeth surface, it oxidizes the teeth surface
both externally and internally and the nanoparticles are incorporated within it
(Wasyluch 2010).
3.4.7 Teeth Restoration
Dental restoration aims to restore the integrity, function and the morphological fea-
tures of teeth that may result from external trauma or caries as well as from teeth
replacements (Subramani and Ahmed 2011; Melo et al. 2013a). Teeth restoration is
divided into two types: indirect and direct type, which is further classified by the
size and location of the tooth (Manhart et al. 2004). Another type of restoration is
the filling of the root canal, where the restorative methods are used to fill up space.
The process and technique of direct and indirect restoration are as follows:
Direct Restorations
In direct technique tooth is filled by directly placing the soft or malleable substance
doped with nanoparticles. The filling material is set hard when comes in contact
with air and the tooth. It is an easy and quick process to restore the tooth. This filling
of the tooth is chosen by the location and size of the tooth cavity (Manhart et al.
2004). Therefore, the materials which are used to restore the tooth can be easily
found and when it passed through the cavity a limited heat is generated which main-
tain the tooth and cavity setting process.
Indirect Restoration
In the indirect technique, the tooth restoration is done outside of the mouth by using
the frame of the same tooth. In this technique, the technician fabricates the indirect
restoration from the previous record of the dentist. At last the final restoration is
done by the use of the dental cement, which held or bonded the tooth permanently.
This restoration is done by using the ceramic particles and the gold particles, which
enhance or increase the strength of restoration.
Resin-based composites (RBCs) like methacrylate resins were used earlier in
different regions of the oral cavity (Fig. 3.6). Along with nanoparticles such as car-
bon nanotubes RBCs enhance the brightness of surface, minimum surface rough-
ness, excellent colour density, tremendous attachment to dental tissue, reduced
polymerization shrinkage and durability of highly hydrophilic adhesive resins
(Curtis et al. 2008; Turagam and Prasad Mudrakola 2013; Morães et al. 2012). The
remineralization of enamel is enhanced when calcium phosphate, silver nanoparti-
cles and quaternary ammonium dimethacrylate nanoparticles are added to resin
composite (Melo et al. 2013b; Zhang et al. 2012) (Fig. 3.7).
To combat or prevent dental caries previously cement and metals were used as
filling materials but had reduced invasive and a traumatic restorative property. On
the other hand calcium phosphate nanoparticles and fluorinated hydroxyapatite
nanocrystals overcome this difficulty and emerged as a dental filler (Azami et al.
2011; Xu et al. 2011). Another way of tooth restoration is performed by the incor-
poration of nanoparticles like ammonium polyethyleneimine (PEI) doped with the
dental resin composite, which has a high malleable and long-lasting and also shows
antimicrobial properties. These PEI nanoparticles incorporate with the resin com-
posite and have a high potent antibacterial effect and on stress condition, it releases
and prevents the bacterial biofilm formation (Beyth et al. 2010).
E
n
a
m
e
l Resin
(Titanium dioxide/
ZrO2/
Hydroxyapatite (HA) NPs)
Adhesive
(Silver/
calcium phosphate/
Zinc oxide NPs)
Dentin
Fig. 3.7 Application of resin-based composites (RBC) with nanoparticles incorporated in dental
restorations
3.4.8 Prevention of Dental Caries
A dental disease that bothers people of all ages especially children is the formation
of tooth caries. The prevalence of the cariogenic bacteria in our mouth leads to the
development of this disease. The bacteria of mouth modify dietary carbohydrate to
generate acidic substances that cause damage to the tooth enamel. It possesses a
great threat to oral health and may also affect other systems of the human body (Dy
et al. 2011).
Once tooth decay is caused by bacterial biofilm, the treatment involves removal
of the decaying tooth and filling cavity with a restorative material. For filling the
tooth cavity, composite materials are used (Drummond 2008); however, bacteria
can still form biofilms over these materials. In addition, secondary caries develops
to the plaques over the restoration margins and compromise the durability/failure of
restoration. Thus, the composite materials used in restorative dentistry should have
antibacterial as well as remineralization capabilities (Cheng et al. 2015).
3.4.8.1 Antibacterial Approach

Metallic nanoparticles (such as zinc, silver, and gold) and some polymers such as
polyethylenimine show effective antibacterial activities. The high surface area–vol-
ume ratio of the nanoparticles enables a number of atoms on the surface, thus pro-
viding better contact with the bacterial cell membrane. Moreover, the size of these
particles is in nanoscale which allows them to penetrate easily to the bacterial cell.
Once these nanoparticles are inside the cell, they affect various cellular mechanisms
producing the bactericidal activity (Melo et al. 2013a). Silver nanoparticles (NAg)
have well-defined antimicrobial activity, which is proportional to the number of
silver ions (Ag+) comes in contact with the bacterial cell membrane (Zodrow et al.
2009). Zinc oxide (ZnO) nanoparticles also exhibit antibacterial activity against a
number of bacterial species, such as S. mutans (Kasraei et al. 2014). ZnO nanopar-
ticle’s bactericidal activity is associated with membrane modification and genera-
tion of oxidative stress by producing reactive oxygen species (Slman 2012). Beside
these metal nanoparticles, a polymeric nanoparticle and quaternary ammonium
polyethylenimine (QAS-PEI) particles are also incorporated in conventional restor-

ative materials to increase the bactericidal activity, which also provides mechanical
strength (Melo et al. 2013a). An advantage of using QAS-PEI is that it forms a
covalent bond with polymeric resin that leads to its immobilization in the resin
composite and thus not lost over time.
3.4.8.2 Remineralization Approach

The remineralization process includes the introduction of calcium or phosphorous
ions to the caries lesion to arrest or reverse the progression of dental caries
(Featherstone 2008). This process alters the surface chemistry and reduction of sus-
ceptibility of the tooth from acid attack and subsequent demineralization. Fluoride
used as a remineralizing agent (Hicks et al. 2005) is now replaced by nano silver
fluoride varnish due to their great remineralization capacity (Fung et al. 2013).
Amorphous calcium phosphate (ACP) stabilized by casein phosphopeptide
(CPP) nanocomplexes has anticariogenic property and acts as a material for remin-
eralization (Reynolds 2008). These materials maintain a high ionic concentration
gradient of calcium and phosphorus during the remineralization process and thus
result in ionic diffusion into the lesions caused by dental caries (Kumar et al. 2008).
ACP precipitates to form a supersaturated solution of calcium phosphate. It has
been widely used in the field of medicine due to their biocompatibility, biodegrad-
ability, enhanced cellular adhesion and high osteoconductivity (Zhao et al. 2011).
CPP forms a complex with ACP through multiple phosphorylated serine residues
that increase the stability of ACP (Poggio et al. 2009). The product is mainly used
to treat tooth hypersensitivity (Madhavan et al. 2012).
Tricalcium phosphate (TCP) nanofillers are also used as a remineralizing agent
by addition to fluoride (Zhang et al. 2015). After reaching the tooth surface, the
calcium complex of TCP is released and activated and protected by saliva and fluo-
ride ions, respectively. The high concentration of fluoride and calcium at the tooth
surface induces remineralization at the caries lesion (Amaechi 2015).
3.4.9 Reduced Corrosion
Corrosion is a physical phenomenon where the deterioration of the metal takes

place by the reaction between oxygen and water on the metal surface which reduces
the strength of that metal (Landolt 2007). The dentin of teeth possesses calcium,
which protects the tooth from decay and caries formation. Chewing of gutkha, paan
and zardas decreases the lifespan of the teeth and causes corrosion of the dentine,
forms caries and causes gum decay (Manuel et al. 2010). The acidic substances
released from these chemicals heat the tooth surface and cause the deterioration of
the tooth. Because enamel and dentin have no living cells, these cannot be repaired
again when they break (Mount et al. 2016).
Besides the chemicals, dental plaque often causes enamel corrosion by forming
a sticky film, made up of saliva, food particles and bacteria. This forms in between
our teeth and gets inside a small hole in the molars. Many times, the bacteria in
plaque changes the starchy food into acids and ultimately the plaque starts to dete-
riorate the healthy minerals in the tooth enamel. That leads to enamel wear down
appears as enamel corrosion:
1 . The edge of the teeth becomes more rough, jagged and irregular in shapes.
2. Indentation appear on the surface of the teeth.
3. Tooth hypersensitivity.
4. Teeth start to appear yellow, due to corrosion of enamels.
Silver and titanium NPs are used to reduce corrosion due to its antimicrobial prop-
erty and thus introduced to enhance the biocompatibility of the composite (Kim et al.
2007). Silica NPs are used for the tooth polishing, as well as to protect against damage
caused by carciogenic bacteria in a human being. Silver hydrosol nanoparticles when
released to the matrix of resin composite it reduces the tooth decay (Li et al. 2006). In
antimicrobial root canal cement, the silver hydrosol nanoparticle is used in the perma-
nent sealing of the root canal with the removal of infected caries and pulp.
3.4.10 NPs Used in Dental Prosthetics
Dental prosthetics or prosthodontics focuses mainly on treatments associated with

dental abnormalities and tooth loss, such as crowns, bridges and dentures. Dental
prosthetics also include the use of artificial prostheses for periodontal diseases, dis-
eases of temporomandibular joint and maxillofacial tissue defects (Wieckiewicz
et al. 2015). These materials restore dental function and facial appearance and main-
tain the health of the tooth. Mainly three types of materials are used in dentures: any
metal or ceramic material or materials based on resin (Blatz et al. 2003). Materials
used in the oral cavity for a long duration comes in contact with the mucosa, thus
the material must have good biocompatibility with comprehensive properties, such
as good mechanical strength, low thermal and electric conductivity, high fatigue
resistance and less shrinkage deformation (Narayan 2009; McCabe and Walls
2013). Chemical stability of material such as resistant to corrosion, breakage and
durability is essential. However, due to the properties of the material itself, continu-
ous use for a long period of time in the moist environment may result in a variety of
problems, such as pigment adhesion, discolouration and aging fracture.
Hydroxyapatite nanocrystals provide a platform for the development of nanomateri-
als with biomimetic properties. Nanomaterials used in prosthodontics are resin-
based denture material, polyvinyl siloxane materials for dental impression, ceramics,
maxillofacial materials, etc.
Approximately 70% of the removable denture wires are infected with stomatitis
caused by Candida albicans (Nobile et al. 2012). Incorporation of silver and plati-
num nanoparticles into these materials significantly reduces the bacterial growth.
Addition of metal nanoparticles to PMMA increases the surface hydrophobicity of
the denture to reduce microbial attachment (Acosta-Torres et al. 2012; Monteiro
et al. 2012; Li et al. 2016). Currently used dental crowns in prosthodontics mainly
include ceramics of alumina and zirconia. Ceramics of alumina have great aesthetic
properties, chemically stable, biocompatible and causing no inflammation, but the
biggest disadvantage is the low mechanical strength. The use of nanoceramic mate-
rials poses certain advantages such as super plasticity with hardness and ductility.
Nanotechnological approaches used in various aspects of removable and fixed
prosthodontics are as follows.
3.4.10.1 In Removable Prosthodontics

1. Incorporation of carbon nanotubes into heat cure monomer to reduce the polym-
erization shrinkage as well as improved mechanical characteristics.
2. Incorporation of metal oxide nanoparticles into PMMA has improved flexural
strength, antimicrobial property and reduced porosity.
3. Using polyhedral oligomeric silsesquiox as a reinforcing agent in maxillofacial
prostheses.
4. Nanocomposite denture teeth are stain and impact resistance with lively surface
texture (Gopinadh et al. 2015).
3.4.10.2 In Fixed Prosthodontics

1. Introduction of nanofillers into the resin matrix has led to the development of
newer nanocomposites with various advantages as, high mechanical strength,
low polymerization shrinkage, reliability, durability, low thermal expansion
coefficient, low water sorption and excellent marginal integrity (Kanaparthy and
Kanaparthy 2011).
2. Use of silica nanofiller provides higher bond strength. When used as coating
agent over aesthetic restorations, it produces stain and wears resistant surface
(Sasalawad et al. 2014).
3. Use of nanocare gold before composite/ceramic restorations enhances adhesive
and antibacterial properties (Robert and Freitas 2010).
4. Nanofillers in nano-optimized moldable ceramics enhance polish ability and
reduce wear (Schirrmeister et al. 2006). Nanopigments improve the aesthetics,
while nano modifiers improve the handling characteristics.
3.4.11 Reduction of Friction
Tooth friction is a principle regulation in orthodontic treatment in the alignment of

teeth. At the initial stage of alignment, a nickel-titanium (NiTi) archwire is generally
used. Once the archwire is placed into misaligned teeth due to its flexible shape, it
activates the wire, creating internal forces. After deactivation of the archwire, an inter-
nal force may straighten the wire, which applies force on the teeth. As a consequence,
the archwire replaced to the brackets of the neighbouring tooth causing tooth friction.
With the progress of treatment, the NiTi wires are altered by stainless steel archwires
(SS wires) which are more rigid in nature. During this time, the movement of the tooth
occurs and it slides over the archwire (Redlich and Tenne 2013). This force can be
counteracted by applying greater forces that might lead to an unwanted loss of
anchorage. There are other alternatives also which include varying the size and shape
of the wire, altering the design of the brackets or the wire surface coating with differ-
ent materials that might help in reducing resistance. These coatings are applied on the
surface of the brackets or NiTi wires or SS wires. Coating of Nickel-Titanium (NiTi)
wires with WS2 nanoparticles result in a reduction of friction (Samorodnitzky-Naveh
et al. 2009). Tungsten disulphide (WS2) is used as a surface lubricant. Coating of
stainless steel wires with a nickel-phosphorous and fullerene-like nanoparticles of
WS2 composite material was found to be effective (Redlich et al. 2008). Addition of
WS2 nanoparticles into Ni-W-P alloy coatings not only reduces the friction coefficient
but also shows improved corrosion resistance of the coating (Ranganatha et al. 2012).
WS2 nanoparticles may cause potential toxicity, thus coating of carbon nitride (gen-
eral formula-CNx) on SS wires was done (Wei et al. 2010; Kachoei et al. 2013).
Similarly, diamond-like carbon coatings, a coating of ZnO and inorganic fullerene-
like molybdenum disulphide nanoparticles are also used (Kachoei et al. 2013; Zhang
et al. 2016). Coating of the nanostructured DLC on S.S. wires also provides excellent
resistance to corrosion and elasticity (Batra et al. 2016).
3.4.12 Coating of Tooth
Dental implant often causes enamel demineralization due to adhesion of microbes

to the teeth appliances (Busscher et al. 2010). After the microbial adhesion, they
proliferate to develop pathogenic plaques, causing enamel demineralization. So it is
essential to control the adhesion of cariogenic microbes on orthodontic appliances
and to the enamel surfaces for successful orthodontic treatment. The reduction in
the bacterial adhesion can be done by using silver nanoparticles and nanofillers as a
substance for teeth coating. The restraint of bacterial attachment and its growth may
prevent demineralization of enamel around orthodontic devices (Ahn et al. 2009).
Also, teeth coated with ZnO or CuO NPs can inhibit bacterial growth, especially S
mutans. NP-coated teeth exhibit inhibition in biofilm formation by 85% and 70%,
respectively (Eshed et al. 2012).
3.4.13 Treatment of Oral Cancer
Nanoparticles play important roles in the early detection and treatment of oral can-
cers. Dendrimer nanoparticles have shown the capability of targeting a cancer cell
with high anticancer drug dosage (Poonia et al. 2017). Nanoshells with a silica core
and outer layer of metal can be modified to absorb infrared light and generate heat
to kill the cancer cells. It was reported by Christoph et al. that the magnetic nanopar-
ticles can carry mitoxantrone for the treatment of oral cancer (Tietze et al. 2010).
Single-walled carbon nanotubes (SWCNT) loaded with cisplatin and epidermal
growth factor (EGF) can kill cancer cells (Bhirde et al. 2009). Advance technologies
such as nanoscale cantilevers, nanopores, nanotubes, quantum dots made up of
nanocarbon material help in diagnosing oral cancer (Weng and Ren 2006).
Nano-electromechanical systems or nano-biosensors which can sense the biochemi-

cal change in the single molecule lead an electrical signal for better cancer diagno-
sis. Oral fluid nanosensor is used for detecting biomarkers of oral cancer in saliva,
while optical nano biosensor is used to detect intracellular components of cancer
such as cytochrome c (Kumar and Vijayalakshmi 2006; Pauwels et al. 2008).
3.5 atural Biomaterial Which Can Be Used to Protect

N
the Teeth
The natural products like chitosan nanoparticles, herbal tea, miswak, natural silk
fibroin nanoparticles, and propolis which have anticancer, antifungal, anti-
inflammatory and antimicrobial properties are introduced for oral health care due to
their biocompatibility and bioremediation properties. The applications of natural
biomaterials in the field of dentistry are summarized in great details. Various appli-
cations of natural biomaterial in the field of dentistry are summarized in Fig. 3.8.
Collagen
Natural biopolymer
extracted from ECM,
useful in dental Fibrin
Propolis regeneration processes
A resin like substances Component of blood plasma
from bee hives with with high biocompatibility,
antimicrobial properties, used as a scaffold for pulp
mainly used to treat regeneration, induce growth
dental caries of osteoblast
Silk fibroin Natural

Polymer of protein from Biomaterials
Alginate
silk producing organisms, in Dentistry
A natural polysaccharide,
silk nanosystems are used efficient in regeneration of
for regeneration in the dental pulp and periodontal
oromaxillofacial field and tissues
other dental applications
Chitosan
Natural polymer derives
Hyaluronic
from chitin. It has
Acid
antimicrobial properties
A component of
useful to prevent oral
ECM used as
microbes, also act as an
dental scaffold
effective drug delivery
system.
Fig. 3.8 Natural biomaterials used in dentistry

3.5.1 Collagen
Collagen is a natural biomaterial which can be used in dental regeneration and can
be extracted from structural proteins located in the ECM (extracellular matrix) of
connective tissues like bone, cartilage, muscle, skin and tendon which shares func-
tional and structural resemblance to the most prevalent structural protein found in
ECM of dental tissues (Sharma et al. 2014). Collagen is biocompatible, bioactive
and has high tensile strength and mild inflammatory response which promote cell
adhesion, cell migration, and cell growth required for pulp regeneration. The chemi-
cals like glutaraldehyde or diphenylphosphoryl azide, β-tcp/polyethylene and
hydroxyapatite can cross-link with collagen and increases its physical, mechanical
and bone conductivity properties (Wu et al. 2007; Wahl et al. 2007). Currently,
regeneration procedures of guided and defective bone or tissues, periodontal liga-
ment and alveolar bone, the collagen sponges and PRP (platelet-rich plasma)/PRF
(platelet-rich fibrin)-rich collagen membranes are extensively used in periodontics
and oral surgery.
3.5.2 Fibrin
The blood plasma consists of fibrinogen which is converted to fibrin by thrombin

and can be used as a biomaterial. Compared to synthetic polymers and collagens,
fibrin has more advantages such as low cost, biocompatible, no undesirable immune
response, high cell adhesion, easy reproducibility and easy availability as obtained
from autologous sources. However, it has some drawbacks such as quick degrada-
tion or shrinkage which can be overcome by crosslinking fixing agents like poly-L-
lysine or enzyme inhibitors with fibrin (Jockenhoevel et al. 2001). Fibrin is
mechanically less stiff and this property is greatly enhanced by cross-linking of
polyurethane, β-TCP or polyethylene and hyaluronic acid with fibrin and thus can
be used as a composite scaffold (Lee and Kurisawa 2013). The features of fibrin
hydrogel of being easily injectable or moulded into 3-D shape have rendered it as an
efficient scaffold material for regeneration of pulp. The platelets of blood, PRP
(platelet-rich plasma)/PRF (platelet-rich fibrin), consist of abundant of growth fac-
tors especially PDGF, TGFβ and cytokines which can initiate growth in capillary
when they bind to endothelial cells while binding to stem cells and osteoblast
induces mitosis and formation of bone when added with fibrin glue.
3.5.3 Alginate
Alginate is a natural polysaccharide which is used as a biomaterial for its non-toxic

and biocompatible feature but they lack mechanical stiffness and are rapidly
degraded in vivo. However, the mechanical stiffness of alginate hydrogels can be
enhanced by covalent crosslinking with a higher concentration of calcium. The
hydrogels of alginate are efficient in regenerating dentin pulp and periodontal tis-
sues. These hydrogels also form an appropriate drug delivery system for
enhancement of dental pulp regeneration by delivering growth factors like TGFβ to

it. Alginate hydrogels along with growth factors like TGFβ can effectively upregu-
late the secretion of regular tubular dentin matrix and also differentiation of odonto-
blast-like cells (Dobie et al. 2002). The nBGC (nano bioactive glass ceramic) along
with alginate composite scaffold increases the ALP (alkaline phosphatase) activity
of hPDLF (human periodontal ligament fibroblast cells) and forms a suitable
medium for hPDLF to adhere and proliferate (Srinivasan et al. 2012).
3.5.4 Hyaluronic Acid
The extracellular matrix (ECM) of connective tissue composed of a glycosamino-

glycan called hyaluronic acid which is an excellent dental material for regeneration
of dental tissues (Sharma et al. 2014). Hyaluronic acid is non-toxic, biocompatible,
has a low immunological reaction and thus emerged to be a potential material for
the dental scaffold. However, it is pulled down by some features such as poor
mechanical stiffness and rapid degradation in vivo but again these loopholes can be
repaired by cross linking and chemical modification of these polymers (Ouasti et al.
2011). The physical, mechanical, biological properties and cellular attachment, cel-
lular spreading and proliferation can be enhanced by incorporating alginate com-
posite and RGD peptides respectively in hyaluronic acid hydrogel (Ganesh et al.
2013; Park et al. 2003). Hyaluronic acid gels along with collagen sponge enhance
the proliferation and invasion of vessels in amputated dental pulp, forming a suit-
able scaffold for pulp regeneration (Inuyama et al. 2010).
3.5.5 Chitosan
The exoskeleton of the crustaceans (crab or shrimp) is the principal source of chitin
while the fungi, insects and certain plants such as mushrooms form the secondary
sources (Zhu et al. 2016; Nitschke et al. 2011). Chitosan has emerged as a potential
biomaterial for its favourable features such as biodegradability, biocompatibility,
tissue healing power, bioactivity, osteoinductive effects and antimicrobial nature
(Sarasam et al. 2008; Konovalova et al. 2017; Chen et al. 2006).
The chitosan-based materials have unique properties that led to exploring a wide
range of dental application, especially in the area of regenerative medicine, growth
factor and drug delivery and an excellent scaffold material in bone engineering
(Guo et al. 2006; Aksungur et al. 2004). A strong drug delivery system made up of
chitosan-based composites (CBCs) is designed to provide the drug to the oral
mucosa and it enhances mechanical properties, a sustained release profile, contact
time, bioavailability for treatment of oral pathologies. To treat the fungal infection
(Albasarah et al. 2010) and oral mucositis (Aksungur et al. 2004) in the periodontal
tissues, resorbable films containing chitosan nanoparticles deliver antibiotics such
as nystatin or metronidazole are used (Pichayakorn and Boonme 2013). The formu-
lation of thiolated chitosan-based biomaterials in the form of mucoadhesive patches
Oral Drug
Delivery
Antimicrobial Guided Tissue
activity Regeneration
Adhesion and Enamel repair and

Dentine Bonding Chitosan Remineralization
in
Dentistry
Prevention of Caries Dentrifrices
Modification of Tissue engineering

Glass Ionomer Scaffolds
Fig. 3.9 Use of chitosan in dentistry
is used to treat dental caries (Samprasit et al. 2015). The application of chitosan in
dentistry is summarized in Fig. 3.9.
Chitosan is further used to deliver antibacterial medicine to suppress the oral
microbes specifically S. mutans and effectively control dental plaque by destroying
oral pathogen (Actinobacillus actinomycetemcomitans, P. gingivalis) (Hayashi et al.
2007).
Effective regeneration of bone tissues is possible by hydroxyapatite or calcium
phosphate variants (tricalcium phosphate (TCP) α and β) together with chitosan
scaffold biomaterials (Fraga et al. 2011). Chitosan-based formulations as a restor-
ative method are used to deliver organic amelogen into the affected site of enamel
and thus can be regenerated successfully (Ruan et al. 2014).
Currently, the issue related to nano leakage (incomplete penetration of resin in
the hybrid layer) of dentine replacement materials was confronted by use of bio
adhesive polymers such as chitosan-based dentine replacement materials
(Perchyonok et al. 2013).
The glass ionomer cement (GICs) are used as a therapy for tissue related to pulp and
also used as a replacement material for a lost tooth, but GICs have a low mechanical
strength which is enhanced when CHS is added to it (Hewlett and Mount 2003; Petri
et al. 2007). Numerous studies suggested that chitosan interferes with the biological,
mechanical and morphological properties of surface and bone interface to improve the
osseointegration of dental implants (Luo et al. 2015; Bumgardner et al. 2007). Thus
chitosan holds a potential approach as a dental biomaterial to be used in dental restora-
tion, scaffolds for the alveolar bone and healing of periodontal complex.
3.5.6 Silk Fibroin Nanoparticles
The natural silk fibres are polymers of proteins obtained from silk-producing organ-
isms like spiders and silkworms (Zarkoob et al. 2004). The biocompatible, non-
toxic, high cell adherence, extensive proliferative properties and diverse physical
characteristics of natural silk have made it as an eminent biomaterial in dentistry
(Chiarini et al. 2003). Nanosystems of silk fibroin are used in tissue regeneration in
the oromaxillofacial field as nanofibres, nanospheres or nanoscaffolds (Virlan et al.
2014). Silk in combination with other composite materials like silver nanoparticles
has antibacterial properties which can inhibit oral bacterial growth (Kang et al.
2007). Chitosan and silk fibroin composite nanomembranes can lead to differentia-
tion of osteoblasts from human mesenchymal stem cells (Lai et al. 2014). Addition
of HAp nanoparticles to the chitosan/silk fibroin composite nanomembrane facili-
tates this osteogenesis process (Lai et al. 2015). Although silk fibroin nanoparticles
have never been used in treating oral cancer, these nanoparticles can be useful in
drug delivery and gene transportation to the tumorogenic cells (Numata et al. 2012;
Qu et al. 2014). Silk fibroin nanocarriers can efficiently load and deliver the antitu-
mor drug cisplatin to tumerogenic cells while causing no or very less cytotoxicity to
the surrounding normal cells (Qu et al. 2014). Thus it could be a potential candidate
for the future treatment of oral cancer. However, the limitation of such biopolymer
is its production, and characterization may vary from species to species.
3.5.7 Propolis
Propolis is a wax-cum resin substance used by bees for making hives. This chemical
protects the hive from pathogens, rain and foreign invaders (Wilson-Rich 2011).
Propolis is a natural, non-toxic resinous material with anti-inflammatory, antifun-
gal, antiviral, antimicrobial and anticancer properties and has a potential in medical
and dental applications (Deswal et al. 2016). Propolis has detrimental effects on the
Streptococcus mutans, glycosyl transferase activity, that limit plaque formation on
the tooth surface and also inhibit caries development in the rat models (Hayacibara
et al. 2005; Nam et al. 2016). The fatty acid component of propolis can efficiently
reduce the acid production by the cariogenic bacteria and reduce the spread of
microorganisms (Libério et al. 2009). Propolis as a natural therapeutic agent elimi-
nates the infection caused in the oral cavity (Nam et al. 2016; Choi et al. 2015). The
ethanolic content of propolis promotes demineralization of caries as reported by
Cordoso et al. (Cardoso et al. 2016).
Propolis extract is used in conventional root scaling and planning for periodontal
treatment (Coutinho 2012). Gebara et al. used propolis extracts in vitro, which
exhibits antimicrobial property by destroying microorganisms causing supra-
infection such as Staphylococcus aureus or Escherichia coli and also periodonto-
pathic bacteria such as Capnocytophaga gingivalis, Fusobacterium nucleatum and
Porphyromonas gingivalis (Gebara et al. 2002). Propolis ethanolic extracts when
used as mouth washes can act as an alternative to chlorohexidine and can arrest the
growth of gram-positive bacteria than gram-negative bacteria effectively (Akca

et al. 2016). Propolis containing mouth rinses can successfully cure buccal surgical
wounds, repair epithelial tissues damage due to tooth extraction and also have anti-
inflammatory action on orofacial pain (Rajoo et al. 2014). When propolis is present
in tooth paste it inhibits the formation of plaque and enriches the oral health
(Morawiec et al. 2013). Propolis reduces the dentine hydraulic conductance and
thus can reduce the dentine hypersensitivity (Hussain et al. 2017). The strong anti-
inflammatory property of constituents of propolis (flavonoids and caffeic acid)
allows it to use as a pulp capping agent (Ramos and Miranda 2007). Use of glass
ionomer cement (GIC) along with propolis enhances antibacterial and anti-biofilm
efficacy and mechanical strength leading to the development of a promising restor-
ative dental material (Altunsoy et al. 2016).
Natural products have always been a topic of interest in the field of medicine and
health. Though the use of natural biomaterial in dentistry holds a promising strategy
but the major drawback is the vulnerable extraction and processing techniques. Still,
extensive research is needed to understand the importance of natural products and
their application in biomaterial science in the field of dentistry.
3.6 Conclusion
Active research is going on in the field of nanotechnology to improve the quality of

life, which also includes the field of dentistry. The phase of dental materials will be
changed with a better understanding of the physicochemical and biological proper-
ties to be manifested at the nanoscale level. This will help in the creation of new
materials for restoration with better strength and durability. Future nanomaterials
will help in dental regeneration by taking knowledge from tissue engineering, cell
and molecular biology and developmental biology. Less regenerative dental tissue
incorporation of the nanomaterials to stem cells will facilitate the regeneration of
enamel, dentin and cementum. Nanoparticles with better surface nanotopography
and their addition to the dental composite materials enhance dental repair with bet-
ter characteristics. The small size, biocompatibility, bioinertness, antimicrobial
property and better surface interaction will help in tooth restorations. However, the
concentrations of these nanomaterials need to be controlled carefully as their higher
dosage can cause toxicity to the body. The behaviour of these nanoparticles in the
mouth cavity needs to be checked with various pH, acidic environment of the oral
cavity, buffering capacity of saliva, continuous contact with mucosa, etc. Future
nanomaterials will improve the oral health by the maintenance of the tooth proper-
ties, cost effectiveness and rigorous clinical trials are required before releasing these
nanomaterials to the market.
Acknowledgements SSP and SM is thankful for MHRD, JB is thankful to DBT nanobiotechnol-

ogy, NN is thankful to DST-INSPIRE for the financial support they received for their doctoral
work. MM lab is supported by BT/PR21857/NNT/28/1238/2017, SERB/EMR/2017/003054 and
Odisha DBT 3325/ST(BIO)-02/2017.
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Microbial Synthesis of Silver
Nanoparticles and Their Biological 4
Potential
Annuja Anandaradje, Vadivel Meyappan,
Indramani Kumar, and Natarajan Sakthivel
4.1 Introduction
The word ‘silver’ was originated from the Anglo-saxon word ‘siosulfur’ and the
symbol ‘Ag’ was derived from the Latin word ‘argentum’. Silver ions and silver
salts have been exploited for therapeutic purposes since the rise of human civiliza-
tion (Singh et al. 2017). Silver the lustrous transition metal exhibits good electrical
and thermal conductivity and therefore be considered as premier metal for therapeu-
tic applications. It is reported also that silver is more advantageous than risk factors
(Firdhouse and Lalitha 2015). Silver nanoparticles (AgNPs) are a class of zero-
dimensional materials that show innate morphology with the size ranging from 1 to
100 nm (Syafiuddin et al. 2017). Among metal nanomaterials, AgNPs possess thera-
peutic and clinical properties. AgNPs exhibit unique thermal conductivity, Raman
scattering and chemical stability (Krutyakov et al. 2008; Tran and Le 2013). AgNPs
have been synthesized by different approaches including physical, chemical and
biological methods. Among synthesis methods, biological synthesis method using
microbes is much preferred due to their cost-effectivity and reduced exposure to
hazardous chemicals. However, microbial-mediated method also has potential
drawbacks in the downstream processing during the separation of nanoparticles
from the microbial materials used (Schröfel et al. 2014). Copious bacteria, yeasts,
fungi and actinomycetes are exploited for the synthesis of AgNPs both intra- and
extracellularly (Narayanan and Sakthivel 2010). AgNPs have been synthesized
from cell-free extract and bacteria-derived components such as biosurfactants, acti-
norhodin pigment and polysaccharides (Gupta et al. 1998; Klaus et al. 1999; Oves
et al. 2013; Saifuddin et al. 2009; Satpute et al. 2010; Singh et al. 2013a). Due to
increase in morbidity and mortality by common bacterial and viral diseases, antimi-
crobial resistance has become a major threat. Hence, there is an urgent need to fight
A. Anandaradje · V. Meyappan · I. Kumar · N. Sakthivel (*)

Department of Biotechnology, School of Life Sciences, Pondicherry University,
Puducherry, India

https://doi.org/10.1007/978-981-13-8954-2_4
100 A. Anandaradje et al.
against the deadly and devastating multi-drug resistant pathogens with alternative
treatments. The non-traditional antibacterial agents such as silver are of great inter-
est to overcome the multi-drug resistance (Dos Santos et al. 2014). AgNPs have the
ability to suppress the emergence of multi-drug-resistant pathogens (Lazar 2011;
Taraszkiewicz et al. 2012). Unlike antibiotics, the silver ions lack selectivity and
therefore they are readily adsorbed to any moiety which has a higher affinity and
thus causing cell death (Mates 2000). Due to the interaction of silver ions with the
reductase enzyme or by scavenging their usual co-factor, intracellular glutathione,
the reactive oxygen species (ROS) are regulated (Mates 2000; Piao et al. 2011).
Likewise, AgNPs also possess antiviral activity against deadly viruses such as HIV-
1, monkeypox virus and Tacaribe virus (Galdiero et al. 2011; Leonard et al. 1990;
Rogers et al. 2008; Speshock et al. 2010). In addition to their therapeutic effects,
AgNPs have been employed in wound dressing, catheters, dental carries, bone
cements, cardiovascular implants and various other biomedical applications (Jiang
et al. 2017; Liang et al. 2016; Lotfi et al. 2011; Mansour et al. 2014; Zhou et al.
2011). Diabetes mellitus (DM) is a chronic metabolic complication of elevated glu-
cose levels because of insulin impairment. Currently available treatments for DM
are complicated due to side effects. In recent years, AgNPs were shown to effec-
tively regulate the glucose metabolism in streptozotocin-induced rats and therefore
considered as promising antidiabetic agents (Prabhu et al. 2018). Silver and AgNPs
have been reported as antimicrobial agent. Globally, per year approximately 800
megatons of silver/AgNPs are used because of their potent antimicrobial properties
(Geisler-Lee et al. 2014; Meyer et al. 2009; Wijnhoven et al. 2009). The antibacte-
rial properties of silver and AgNPs have been employed after discovery of bacteria.
Afterwards, silver-containing materials are used for purification of water
(Bandyopadhyaya et al. 2008; Jain and Pradeep 2005), wound dressing (Abboud
et al. 2014; Barillo et al. 2014), treatment of eye infection and dental hygiene (Xu
et al. 2013). In twenty-first century itself, silver was used for urinary tract and
catheter-related bloodstream infections (Barillo and Marx 2014). Also, silver and
AgNPs were employed for cardiovascular implants (Cipriano et al. 2014), bone
cements (Slane et al. 2015), dental carries (Divakar et al. 2018), antidiabetic (Prabhu
et al. 2018), antiviral (Gaikwad et al. 2013) and anticancer potential (Gurunathan
et al. 2015).
4.2 Microbial Synthesis of AgNPs
Divergence of nature provides exponential possibilities in the form of ‘mini’ nano-

factories in which microbes are used as economic and environment friendly tools.
Unlike other methods, toxic harsh chemicals and the immense energy are not required
for microbial nanosynthesis (Singh et al. 2016). Microbes have the ability to detoxify
heavy metals with the aid of reductase enzymes and reduce metal salts to nanomateri-
als. Microbes also facilitate the synthesis of nanomaterials with a narrow size distribu-
tion and less polydispersity (Hulkoti and Taranath 2014). Both intra- and extracellular
syntheses of nanomaterials have been reported using microbes. Metal-resistant
4 Microbial Synthesis of Silver Nanoparticles and Their Biological Potential 101
proteins, enzymes, peptides, reducing co-factors and genes play key role as reducing
agents and aid in providing natural capping and stability for nanomaterials (Singh
et al. 2015). Microbes such as bacteria (Fayaz et al. 2010a), fungi (Gudikandula et al.
2017; Zhao et al. 2018), actinomycetes (Otari et al. 2015) and yeasts (Li et al. 2018)
have been reported for the synthesis of AgNPs.
4.2.1 Bacteria
Bacteria have been widely used to synthesize metal nanoparticles and other inor-
ganic nanoparticles (Kumar et al. 2007). In the process of bacterial bioreduction of
silver, the nitrate reductase enzyme plays a key role in transforming nitrate to nitrite
(Prabhu and Poulose 2012). In this bioreduction, the lepton is transferred to the
silver ion, as nitrate is regenerated into cluster, resulting in the synthesis of AgNPs
(Rajput et al. 2017). Factors such as temperature, pH and species of bacteria decide
the properties and stability of synthesized AgNPs. Bacteria such as Arthrobacter
kerguelensis and Phaeocystis antarctica prefer different temperature parameters
(Shivaji et al. 2011). Bacteria such as Acinetobacter calcoaceticus, Bacillus amylo-
liquefaciens, B. flexus and Staphylococcus aureus have been reported for both intra-
and extracellular synthesis with various shapes such as spherical, disk, cuboidal and
triangular (Das et al. 2014; Nanda and Saravanan 2009; Priyadarshini et al. 2013;
Reddy et al. 2010; Saravanan et al. 2011; Shivaji et al. 2011; Wei et al. 2012).
Various other bacteria such as Pseudomonas stutzeri (Klaus et al. 1999), Plectonema
boryanum (Lengke et al. 2007), Enterobacter cloacae (Minaeian et al. 2008), E. coli
(El-Shanshoury et al. 2011), Bacillus licheniformis (Kalimuthu et al. 2008),
Lactobacillus fermentum (Sintubin et al. 2009), Klebsiella pneumoniae (Mokhtari
et al. 2009), Proteus mirabilis (Samadi et al. 2009), Brevibacterium casei
(Kalishwaralal et al. 2010) and cyanobacteria (Sudha et al. 2013) were also exploited
for microbial synthesis. AgNPs reported from bacteria are listed in Table 4.1.
4.2.1.1 Intracellular Synthesis of AgNPs

In general bacterial cells grown in medium are initially resuspended in water and
then mixed in silver salt to prevent the obstruction by media components (Gupta
et al. 1998). Small periplasmic space binding proteins that bind silver at surface are
reported to aid in detoxifying silver. The efflux pumps propel the incoming metals
and defend the cytoplasm from toxicity (Gupta et al. 1998). It has been perceived
that proteins that bind silver in organic matrix bear nucleation sites known as the
amino acid moieties for the formation of AgNPs. Similarly, the peptides of silver
precipitation involve in the reduction of silver ions and the synthesized AgNPs
exhibit crystalline nature. Different crystal (triangular and hexagonal) morphology
with a size range up to 200 nm in periplasmic space of P. stutzeri AG259 was dem-
onstrated (Klaus et al. 1999). Similarly, Lactobacillus sp. A09 was also reported for
the biosorption and bioreduction of anionic silver on surface of the cell (Narayanan
and Sakthivel 2010). The fundamental proof for microbial synthesis of AgNPs was
entrenched by P. stutzeri AG259 (Haefeli et al. 1984). This silver-tolerant strain in
Table 4.1 Bacteria-mediated synthesis of AgNPs

Time Size
Bacterium (h) Localization (nm) Reference
Acinetobacter 24 Extracellular 8–12 Singh et al. (2013b)
calcoaceticus
Acinetobacter – Extracellular 4–40 Singh et al. (2013b)
haemolyticus
MMC8
Aeromonas sp. SH10 – Extracellular and 6.4 Mouxing et al. (2006)
intracellular
Bordetella sp. 24 Extracellular 63–90 Thomas et al. (2012)
Enterobacter aerogenes – Extracellular 25–35 Karthik and Radha
(2012)
Escherichia coli 24 Extracellular 42.2– Deepak et al. (2011)
89.6
Geobacter – Extracellular – Law et al. (2008)
sulfurreducens
Gluconobacter roseus 24 Extracellular 10 Krishnaraj and
Berchmans (2013)
Idiomarina sp. 6–8 Intracellular 25 Seshadri et al. (2012)
Klebsiella pneumoniae 72 Extracellular 15–37 Kalpana and Lee (2013)
Morganella sp. 20 Extracellular 10–40 Parikh et al. (2008)
Proteus mirabilis 24 Extracellular and 10–20 Samadi et al. (2009)
intracellular
Pseudomonas 24 Extracellular 6.3 Srivastava and
aeruginosa SM1 Constanti (2012)
Rhodobacter – Extracellular 3–15 Bai et al. (2011)
sphaeroides
Shewanella oneidensis 48 Extracellular 2–16 Debabov et al. (2013)
MR-1
Stenotrophomonas – Extracellular 93 Oves et al. (2013)
maltophilia
Vibrio alginolyticus 4–12 Extracellular 50–100 Rajeshkumar et al.
(2013)
Xanthomonas oryzae 72 Extracellular 14.86 Narayanan and
Sakthivel (2010)
Yersinia enterocolitica – Extracellular 10–80 Pourali et al. (2012)
Bacillus flexus – Extracellular 12.65 Priyadarshini et al.
(2013)
Bacillus licheniformis 24 Cell-free extract 18.69– Shanthi et al. (2016)
Dahb1 63.42
Bacillus safensis 2 Extracellular 5–30 Lateef et al. (2015)
LAU 13
Bacillus 48 – 10–30 Wang et al. (2016)
methylotrophicus DC3
Bacillus subtilis 24 Extracellular – Kannan et al. (2011)
Bacillus thuringiensis 72 Extracellular 43–142 Banu et al. (2014)
Time Size
Bacterium (h) Localization (nm) Reference
Brevibacterium casei 24 Intracellular 10–50 Kalishwaralal et al.
(2010)
Enterococcus faecalis – Extracellular 10–80 Pourali et al. (2012)
Exiguobacterium sp. 12 Extracellular 5–15 Tamboli and Lee (2013)
Geobacillus – Extracellular 5–35 Fayaz et al. (2011)
stearothermophilus
Lactobacillus mindensis – Extracellular 2–20 Dhoondia and
Chakraborty (2012)
Rhodococcus sp. 24 Extracellular 10–15 Otari et al. (2014)
Staphylococcus – Extracellular 10–80 Pourali et al. (2012)
epidermidis
Thermoactinomyces sp. 72 Extracellular 20–40 Deepa et al. (2013)
Ureibacillus 24 Extracellular 10–100 Juibari et al. (2011)
thermosphaericus
silver mine intracellularly accumulates silver nanoparticles with size ranging from
35 to 46 nm along with silver sulphide (Slawson et al. 1992). Detoxification of and
bioreduction into elemental silver by P. stutzeri AG259 in the periplasmic space was
reported (Klaus et al. 1999). Intracellular synthesis of nanoparticles is less preferred
because of the requirement of additional step for the recovery of accumulated
nanoparticles (Kalishwaralal et al. 2010). Techniques such as autoclaving, deter-
gents usage and salts addition have been employed for cell lysis (Fesharaki et al.
2010; Krishnamurthy and Yun 2013).
4.2.1.2 Extracellular Synthesis of AgNPs

In the extracellular synthesis, AgNPs are secreted outside the bacterial cell. Metal
nanoparticles are obviously found extracellularly as the reductive enzymes or
secreted enzymes catalyse the metal ion reduction (Narayanan and Sakthivel 2010).
Various AgNPs of spherical, disk, cuboidal, hexagonal, triangular and other shapes
were synthesized by exploiting the biomass or the supernatant (Klaus et al. 1999;
Oves et al. 2013; Singh et al. 2013a; Srivastava and Constanti 2012). Generally
high-speed centrifugation at 10,000–20,000 rpm was employed to recover AgNPs
and pellet in the solvent.
Silver Nanoparticle Synthesis Using Culture Supernatant

In order to synthesis AgNPs, 24- to 48-h culture of bacterial supernatant, obtained
under sterile conditions, was incubated at optimal conditions after silver salt expo-
sure (Saifuddin et al. 2009). Microwave-irradiated culture supernatant supple-
mented with silver was utilized for the synthesis of AgNPs (Saifuddin et al. 2009).
Also, the cell-free supernatants enriched with medium and organic molecules were
used for the synthesis of AgNPs (Singh et al. 2015).
Silver Nanoparticle Synthesis Using Cell-Free Extract

The cell-free extract of bacteria was used for the synthesis of AgNPs extracellularly
(Singh et al. 2013a). In this process the cell-free extract was challenged with silver
salt under optimal conditions for the synthesis of AgNPs (Singh et al. 2013a). Cell-
free extracts subjected to solar or microwave radiation were also reported for rapid
nanoparticle synthesis (Boopathi et al. 2012; Wei et al. 2012). As these methods
completely remove biomass and media components, there is no requirement for the
downstream processing to recover nanoparticles. Various bacterial species such as
Aeromonas sp., SH10 (Mouxing et al. 2006), K. pneumonia, E. coli and E. coloacae
(Fayaz et al. 2010a; Shahverdi et al. 2007) and B. licheniformis (Kalishwaralal et al.
2008) are exploited to synthesize the AgNPs extracellularly. The extracellular
nanoparticle synthesis method is cost-effective when compared to the intracellular
synthesis.
Silver Nanoparticle Synthesis Using Bacterial-Derived Components

Microbes are efficient factories of biosurfactants, which are less toxic, biocompati-
ble and aid in biodegradation (Satpute et al. 2010). These bacterial biosurfactants
are utilized for silver nanoparticle synthesis as they aid in rapid synthesis and
enhance the stability of nanoparticles (Kiran et al. 2010). Other components derived
from bacteria such as bacterial enzyme (Deepak et al. 2011), actinorhodin pigment
(Manikprabhu and Lingappa 2013), polysaccharide bioflocculant (Sathiyanarayanan
et al. 2013), exopolysaccharides (Kanmani and Lim 2013), native and repolymer-
ized flagella (Gopinathan et al. 2013) were used for silver nanoparticle synthesis.
4.2.2 Fungi
Like bacteria, fungi also have been used to produce AgNPs. As compared to bacte-
ria, fungi synthesize more nanoparticles because of secreting more proteins
(Mohanpuria et al. 2008). This mycosynthesis system involves silver ion trapping at
the fungal cell surface (Mukherjee et al. 2001) and the reduction of silver ions to
AgNPs due to the catalysis by NADPH-dependent nitrate reductase enzyme (Tran
and Le 2013). Fungi are preferred over other microbes for the synthesis of nanoma-
terials (Tashi et al. 2016). Fungi namely Aspergillus flavus, A. fumigates, Fusarium
oxysporum, F. acuminatum, F. culmorum, F. solani, Metarhizium anisopliae, Phoma
glomerata, Phytophthora infestans, Trichoderma viride and Verticillium sp. have
been exploited intra- and extracellular synthesis of AgNPs (Bhainsa and D’Souza
2006; Durán et al. 2005; Durán et al. 2007; Siddiqi and Husen 2016; Vigneshwaran
et al. 2007a). AgNPs synthesized from fungi are listed in Table 4.2.
4.2.2.1 Intracellular Synthesis

The intracellular AgNPs are normally larger than those formed extracellularly. The
size of the nanoparticles is correlated to the nucleating particles within the organism
(Narayanan and Sakthivel 2010; Thakkar et al. 2010). The intracellular synthesis of
AgNPs was reported using Verticillium. In this process, the Ag+ ions interact with
Table 4.2 Fungus-mediated synthesis of AgNPs

Fungus Time (h) Localization Size Reference
Aspergillus flavus – Cell wall 8.92 Vigneshwaran et al. (2007a)
Aspergillus fumigatus 72 Extracellular – Bhainsa and D’Souza
(2006)
Aspergillus terreus 24 Extracellular 1–20 Li et al. (2011)
Cladosporium 78 – 10–100 Balaji et al. (2009)
cladosporioides
Coriolus versicolor – Extracellular 25–75 Sanghi and Verma (2009)
and intracellular and
444–491
Fusarium oxysporum 72 Extracellular 20–50 Ahmad et al. (2003)
Humicola sp. 96 Extracellular 5–25 Syed et al. (2013)
Macrophomina 24 Cell-free filtrate 5–40 Chowdhury et al. (2014)
phaseolina
Penicillium 24 – 58.35 Shaligram et al. (2009)
brevicompactum
Penicillium – Cell-free filtrate 25 Maliszewska et al. (2014)
nalgiovense AJ12
Phanerochaete 72 – 5–200 Vigneshwaran et al. (2006)
chrysosporium
Phoma glomerata – – 60–80 Birla et al. (2009)
Pleurotus ostreatus 24 – <40 Al-Bahrani et al. (2017)
Pleurotus sajor-caju 24 Extracellular 30.5 Vigneshwaran et al. (2007b)
Trichoderma 120 Extracellular 13–18 Mukherjee et al. (2008)
asperellum
Trichoderma reesei 120 Extracellular 5–50 Vahabi et al. (2011)
Trichoderma viride – Extracellular 5–40 Fayaz et al. (2010b)
negatively charged cell wall, where reduction occurred probably due to enzymes
present in cell wall. The size of synthesized AgNPs was around 25 ± 12 nm
(Mukherjee et al. 2001). The AgNPs were also synthesized using freeze-dried
mycelia of Phoma sp. 3.2883 with size of 71.06 ± 3.46 nm (Chen et al. 2003).
Intracellular synthesis of AgNPs using Fusarium oxysporum was also reported
(Korbekandi et al. 2013). In this process, the silver salt, fungal biomass and glucose
are acting as biotransformants, catalyst and electron donor for silver nanoparticle
synthesis. The synthesized spherical AgNPs were single or aggregates with a size
range of 25–50 nm or 100 nm, respectively (Korbekandi et al. 2013).
4.2.2.2 Extracellular Synthesis

The extracellular biosynthesis is an energy-saving and eco-friendly method that uti-
lizes cell-free filtrate of fungi as reducing agents (Narayanan and Sakthivel 2010).
During the synthesis of AgNPs by Coriolus versicolor, proteins within 1 h (Sanghi
and Verma 2009) catalysed the reduction of silver ions to AgNPs. In the extracel-
lular synthesis, the secreted biomolecules have been involved in the reduction of
cationic silver to AgNPs with a size range between 5 and 15 nm. These AgNPs were
found to be stabilized by the fungal proteins (Krutyakov et al. 2008). Moreover,
several fungi have been employed for extracellular synthesis of AgNPs (Gade et al.
2008). The cell filtrates of Penicillium oxalicum at different pH range between 5 and
13 were used for the extracellular synthesis of AgNPs. The smallest size of particles
was obtained at pH 12, and these particles were found to be spherical in shape with
a size of 4 nm (Du et al. 2015). Similarly, the AgNPs were obtained from cell-free
filtrate of Penicillium aculeatum Su1, and the synthesized particles were spherical
in morphology with a size range of 4–55 nm. FTIR analysis revealed that the protein
molecules act as both reducing and capping agents (Ma et al. 2017). The synthesis
of AgNPs was also reported using Arthroderma fulvum at optimum conditions such
as alkaline pH, 1.5 mM concentration of AgNO3, temperature at 55 °C and incuba-
tion time of 10 h. The synthesized AgNPs were found to be crystalline in nature
with the size of 15.5 ± 2.5 nm (Xue et al. 2016). Studies also reported the extracel-
lular synthesis of AgNPs from Penicillium polonicum isolated from marine green
algae, Chaetomorpha antennina (Neethu et al. 2018). The optimum synthesis of
AgNPs was observed using a concentration of 1 mM AgNO3, fungal biomass of
10 g at pH 7 with a duration of 60 min and 8:2 ratio of AgNO3. The crystalline
nanoparticles were spherical in morphology with a size range between 10 and
15 nm. The highly stable AgNPs were synthesized using fungus Guignardia man-
giferae, and the particles were of spherical shape with a size range between 5 and
30 nm (Tamboli and Lee 2013). The silver ions to AgNPs reduction were also
reported using culture supernatant of Aspergillus niger at room temperature. The
polydispersed AgNPs were found to be spherical in morphology with a size range
of 1–20 nm (Sagar and Ashok 2012).
4.2.3 Actinomycetes
Biosynthesis of AgNPs using actinomycetes as green chemistry approach offers

enormous benefits. Marine microbial biotechnology has opened up fascinating new
horizons for exploiting novel microbes to function as nano-factories. Oceans com-
prise nearly 70% of the earth’s surface and harbour diverse microbes that are meta-
bolically and physiologically unique from the terrestrial organisms (Selvakumar
et al. 2012). Actinomycetes are Gram-positive, filamentous spore formers with
>55% G + C content of DNA. These free-living saprophytic bacteria form a major
group of soil population where they decompose complex mixtures of polymers
present in dead plants. The majority of the secondary bioactive metabolites pro-
duced by actinomycetes are potent antibiotics, which made streptomycetes as potent
group to be exploited. Though actinomycetes are classified as prokaryote, they
share certain fungal characteristics. They are also known as ray fungi (Narayanan
and Sakthivel 2010; Reddy et al. 2010). Marine Streptomyces sp. MS26 has been
utilized for the synthesis of spherical AgNPs with a size range between 50 and
76 nm (Zarina and Nanda 2014). Extracellular synthesis of AgNPs using actinomy-
cete, Streptomyces sp., with particle size ranging from 70 to 100 nm has also been
reported (Saminathan 2015). Streptomyces fradiae—actinomycete and Actinomycete
Gordonia amicalis HS 11 were also utilized for the production of AgNPs of
Table 4.3 Actinomycetes-mediated synthesis of AgNPs

Actinomycete Time (h) Localization Size (nm) Reference
Streptomyces sp. 72 Intracellular 50–76 Zarina and Nanda (2014)
MS26
Streptomyces sp. 96 Extracellular 70–100 Saminathan (2015)
Streptomyces – – 100–200 Priya and Balasubramanian (2014),
fradiae Sowani et al. (2016)
Gordonia – – 25–50 Priya and Balasubramanian (2014),
amicalis HS 11 Sowani et al. (2016)
Streptomyces 96 – 19.5–20.9 Vijayabharathi et al. (2018)
griseoplanus
Rhodococcus sp. – Extracellular 5–50 Otari et al. (2015)
100–200 nm and 25–50 nm, respectively (Priya and Balasubramanian 2014; Sowani

et al. 2016). Airborne actinomycete such as Streptomyces sp. and Streptoverticillium
were exploited for AgNPs synthesis with a size range between of 5 and 8 nm and
rarely up to 70 nm (Prakash et al. 2014). Intracellular synthesis of AgNPs was
reported using Rhodococcus NCIM 2891. Microscopic observation confirmed the
synthesis occurred in the cytoplasm, and the synthesized nanoparticles were found
to be spherical in shape with a size range of 5–50 nm (Otari et al. 2015). AgNPs
synthesis using the cell-free extract of Streptomyces griseoplanus SAI-25 was also
reported (Vijayabharathi et al. 2018). Phytochemicals (alcohols, amines, phenols
and proteins) present in the cell-free extract act as reducing as well as stabilizing
agents. The synthesized particles were of spherical in morphology with a size range
between 19.5 and 20.9 nm. AgNPs synthesized from actinomycetes are listed in
Table 4.3.
4.2.4 Yeast
Yeasts are more beneficial than bacteria due to their ability for bulk production of
nanoparticles and their rapidity in growth by utilizing simple nutrients in the media
and easy to control in the laboratory circumstances (Kumar et al. 2011). It has been
studied that AgNPs of 2–5 nm in size were produced by exploiting the log state of
growth of silver-tolerant yeast strain MKY3 (Kowshik et al. 2002). Similarly,
AgNPs were synthesized using the psychrotrophic yeast Yarrowia lipolytica NCYC
789 (Debabov et al. 2013) and Saccharomyces cerevisiae (Sowbarnika et al. 2018).
Likewise, Cryptococcus laurentii and Rhodotorula glutinis were utilized for the
production of AgNPs with size ranging from 35 to 400 nm and 160 to 220 nm,
respectively (Fernández et al. 2016). Pleurotus florida was also exploited for syn-
thesis of spherical AgNPs with a size of 20 nm (Bhat et al. 2011). Synthesis of
AgNPs was reported using the supernatants of C. albicans and Saccharomyces sp.
XRD analysis further ensured the purity and crystalline nature. The synthesized
AgNPs were of spherical in morphology with a size range between 2 and 7.3 nm
(Ananthi et al. 2018). Further studies have reported the synthesis of AgNPs using
Table 4.4 Yeasts-mediated synthesis of AgNPs

Yeast Time (h) Localization Size (nm) Reference
Saccharomyces cerevisiae 24 Extracellular 2–5 Kowshik et al. (2002)
MKY3
Yarrowia lipolytica 120 – 15 Debabov et al. (2013)
Pleurotus florida 1 Extracellular 25 Bhat et al. (2011)
Rhodotorula sp. ATL72 24 – 8–21.4 Soliman et al. (2018)
Saccharomyces cerevisiae 1 – 2–7.3 Iravani et al. (2014)
Rhodotorula sp. strain ATL72 (Soliman et al. 2018). Microscopic analysis con-
firmed the spherical and oval morphology of AgNPs with a size range between 8.8
and 21.4 nm. The AgNPs were also synthesized using fresh and dried culture of
Saccharomyces cerevisiae. The synthesized AgNPs were of spherical morphology
with a size range between 2 and 20 nm and also found to be localized inside and
outside of the cells (Iravani et al. 2014). AgNPs synthesized from yeasts are listed
in Table 4.4.
4.3 Mechanism of Microbe-Mediated Synthesis of AgNPs
Bacterial cell wall plays a crucial role in the synthesis of AgNPs. The silver ion
nucleation into clusters results in electrostatic interaction between ions and nega-
tively charged carboxylate groups present in the cell wall during the initial phase of
AgNPs synthesis (Wang et al. 2012). The bioreduction of silver ions was catalysed
by nitrate reductases, and other redox proteins were released by the cell to synthe-
size AgNPs (Debabov et al. 2013; Mahdieh et al. 2012). The other synthesis mecha-
nism is the electrokinetic potential in which the transmembrane proton gradient is
generated by the bacteria. This active symport of Na along with neighbouring silver
ions is actively driven by the proton gradient (Prakash et al. 2011). The NADH-
dependent nitrate reductase is the prime enzyme in silver bioreduction machinery
(Kalimuthu et al. 2008). Initially, the catalyst nitrate reductase was induced by
nitrate ions in silver nitrate through which the electrons are supplemented to the
catalyst from NADH and gets oxidized to NAD+. Later, oxidation takes place to
reduce silver ions to AgNPs. Further, nitrate ions (NO3+) get converted to NO2, fol-
lowed by NO, N2O and finally to gaseous N2 (Karthik and Radha 2012). The nitro-
genase and hydrogenase enzymes are involved in NADH-independent synthesis of
AgNPs in Acinetobacter sp. (Brayner et al. 2007). It has also been reported that the
size of AgNPs are dictated by the concentration of the cellular nitrogenases (Brayner
et al. 2007).
Cell wall, cell membrane and other biomolecules such as protein and enzymes
play a significant role for the silver nanoparticle production. The fungal-mediated
mechanism of AgNPs synthesis is linked to the polymers of cell wall or the electron
shuttle quinones. During the bioreduction of Fusarium oxysporum by Ag+, the
naphthoquinones and anthroquinones act as electron shuttling compounds (Durán
et al. 2005). The Ag+ reduction to AgNPs were catalysed by NADPH-dependent
nitrate reductase enzyme (Durán et al. 2007; Kumar et al. 2007). The quinine deriv-
atives of anthraquinones and naphthoquinones also act as redox centres (Durán
et al. 2005). The synthesized AgNPs are stabilized by the fungal proteins and pep-
tides due to electrostatic interaction between free cysteine residues or amine groups
of proteins (Gole et al. 2001; Jain et al. 2011; Mukherjee et al. 2008; Sanghi and
Verma 2009). The electrostatic interaction in the cell surface trap the Ag+ and get
reduced to form the silver nuclei which were catalysed by the enzyme in the cell
wall and finally generates the metallic nanoparticles such as AgNPs on the mycelia
(Salunke et al. 2016). In the case of F. oxysporum, both intra- and extracellular syn-
theses occur in the presence of AgNO3 solution. The biomass accumulates the Ag+
ions in the cytoplasm and aggregates in the vesicles to ultimately secrete out of the
cells through membrane by exocytosis. The synthesized AgNPs were spherical or
aggregates in shape with a size range of 25–50 nm or100 nm, respectively
(Korbekandi et al. 2013).
4.4 Biological Properties of AgNPs
AgNPs exhibit antibacterial (Saravanan et al. 2018), antifungal (Rajeshkumar et al.

2014), antiviral (Gaikwad et al. 2013) and anti-inflammatory (Lopez-Esparza et al.
2016) properties.
4.4.1 Antibacterial Properties
The existing antibiotic therapies turn out to be ineffective because of the emergence
of multi-drug resistance in pathogen population (Seppala et al. 1997). Therefore, it
is essential to optimize and elucidate the pharmacokinetics and pharmacodynamics
of antibiotics or dug molecules to elicit the treatment outcomes and suppress toxic-
ity and resistance (Cassir et al. 2014). There is an urgent need to fight against the
deadly and devastating multi-drug resistant pathogens with alternative treatments
(Chen and Schluesener 2008). Silver-based nanoparticles are exploited to overcome
multi-drug resistance (Dos Santos et al. 2014). Due to its low cytotoxicity, particular
structure and different interaction modes with bacterial surface AgNPs were
exploited against Gram-positive and Gram-negative bacterial pathogens (Lazar
2011; Taraszkiewicz et al. 2012). AgNPs were employed as new class of antimicro-
bials to combat a wide range of multi-drug-resistant bacterial pathogens
(Chernousova and Epple 2013; Dos Santos et al. 2014; Galdiero et al. 2011; Lara
et al. 2011; Rai et al. 2014; Sweet et al. 2012; Sweet and Singleton 2011). The elec-
trostatic forces develop when AgNPs of positive zeta potential interact with nega-
tive zeta potential on the bacterial. Zeta potential is the key parameter for better
antimicrobial potential of AgNPs (Lazar 2011; Periasamy et al. 2012). A study
exploiting E. coli (Lazar 2011) confirmed that the accumulation of AgNPs on the
cell membrane weakens integrity of the bilayer that predisposes to the increase in
permeability and leading to bacterial cell death. Antimicrobial effect is influenced
by the size, shape and concentration of the AgNPs (Hashimoto et al. 2012; Periasamy
et al. 2012; Rolim et al. 2012). Several studies have proved that the toxicity of
AgNPs is size dependent (Tamayo et al. 2014; Wu et al. 2014). Silver due to its
active affinity with thiol groups inhibits essential enzyme activity, thus causing cell
death. Likewise, AgNPs upon cellular damage release the ROS such as O2−, −OH,
RCOO− and H2O2 and thus resulting in powerful bactericidal activity of free radi-
cals (Rice-Evans et al. 1997).
Varying degrees of bactericidal effects have been reported based on the concen-
trations and types of bacterial pathogens (Chernousova and Epple 2013). P. aerugi-
nosa and Vibrio cholerae were reported to be more resistant than E. coli and
Salmonella typhi (Zhang et al. 2014). AgNPs have been shown to be effective
against E. coli, S. typhi, S. epidermidis and S. aureus (Jain et al. 2009), P. aerugi-
nosa, Klebsiella and Proteus vulgaris (Sudha et al. 2013). Due to their greater posi-
tive charge, E. coli responded better to triangular AgNPs. The combinational effects
of AgNPs with lower antibiotic dosage have been exploited (Wu et al. 2014).
Synergistic action of AgNPs and antibiotics has intensified the antibacterial
potential against multi-drug-resistant pathogens. Studies suggested that positive
synergistic effect has been observed due to the bonding reaction between antibiotics
and AgNPs (Fayaz et al. 2010a). The silver–cysteine interaction generated dimer,
and it was related to the synergistic effects between Ag+ and UV-A/visible lights
(Akhavan and Ghaderi 2010; Hu 2010). Antibacterial activity of both AgNPs and
Ag–graphene oxide composites was found to be effective against both Gram-
positive and Gram-negative bacterial pathogens (Xu et al. 2011).
4.4.2 Antifungal Properties
In addition to their effective antibacterial activity, AgNPs are also act as potent fungi-
cides. They are highly toxic against a broad class of pathogenic fungi that includes the
genera of Aspergillus, Saccharomyces, Candida and Trichoderma (Reddy 2015).
AgNPs between 2 and 100 nm size range with the minimal inhibitory concentration of
0.4–100 ug/ml showed antifungal properties (Reddy 2015). Even though the fungicide
activity of AgNPs is not fully understood, studies report that their mechanism of action
is similar to their antibacterial activity (Wright et al. 1999). AgNPs synthesized from
Alternaria alternata showed the antifungal activity against Phoma glomerata, Phoma
herbarum, Fusarium semitectum, Trichoderma sp., and C. albicans. Thus synergistic
antifungal effects of AgNPs with fluconazole were observed (Gajbhiye et al. 2009).
Similarly, AgNPs synthesized using Amylomyces rouxii showed the antifungal activity
against F. oxysporum and C. albicans (Musarrat et al. 2010).
4.4.3 Antiviral Properties
AgNPs also have potent activity against viruses such as HIV-1 (Elechiguerra et al.
2005; Lara et al. 2011; Sun et al. 2005), Hepatitis B virus (Lu et al. 2008),
monkeypox virus (Rogers et al. 2008), Tacaribe virus (Speshock et al. 2010) and
influenza virus (Papp et al. 2010). The size-dependent antiviral activity of AgNPs
was reported (Galdiero et al. 2011). AgNPs creep into the host cell and thus block-
ing viral replication and also get adhered to the viral genome ensuring the lack of
polymerase action and hence no production of progeny virions (Bryaskova et al.
2011). AgNPs with a size range of 10 nm showed with potent antiviral activity
against the bursal disease virus infecting the embryonated chicken eggs (Pangestika
and Ernawati 2017). Likewise, AgNPs with cytotoxic potential towards poliovirus
were reported (Huy et al. 2017). Recently, tannic acid-modified AgNPs were shown
to exhibit antiviral activity against HSV-2 virus (Orłowski et al. 2018).
4.4.3.1 Antiviral Activity of AgNPs
Antiviral Activity Against HIV-1

Robust interactions of AgNPs were observed with cysteine residue (Elechiguerra
et al. 2005). The outer part of HIV-1 was gp120 subunit, which is exposed to the
peripheral, and gp41—a transmembrane glycoprotein. The gp120 docks with CD4+
on the TH cells in the host organism (Leonard et al. 1990). Three disulphide bonds
in CD4+ cells are the dire striking sites for virus interaction (Elechiguerra et al.
2005). The High Angle Annular Dark Field (HAADF) is utilized to determine regu-
lar spatial arrangements. AgNPs interact with the HIV-1 virus by means of efficient
binding to the gp120 glycoprotein knobs. AgNPs with a size range of 1–14 nm
interact with the glycoprotein knob and therefore able to attach to the virus envelope
(Elechiguerra et al. 2005).
Antiviral Activity Against Monkeypox Virus

Monkeypox virus is a big threat to human life. Studies confirmed that poxvirus
creeps into the host cell through endocytosis or by plasma membrane fusion,
succeeded by viral replication. AgNPs with a size range of 10, 25 and 80 nm
exhibit the inhibition of monkeypox virus. The internalization of AgNPs dis-
rupts the intracellular pathway that eventually blocks the viral replication
(Rogers et al. 2008).
Antiviral Activity Against Tacaribe Virus

AgNPs effectively bind to thiol groups and easily interact with the cysteine-rich
residues in the viral membrane (Iapalucci et al. 1991; Kim and Kwon 2009). This
interaction results into three phenomena (i) prevention of internalization of the viral
particle by interfering in receptor binding, (ii) inhibition of the viral replication of
Tacaribe virus L protein by internalizing the silver nanoparticle and (iii) prevention
of the viral coating in the endosome by binding the AgNPs to the viral glycoprotein.
Studies proved that AgNPs inactivate virus before cellular entry (Speshock et al.
2010). The internalization of Tacaribe virus was projected by confocal microscopy,
and results confirmed that the10-nm-sized AgNPs effectively enter into the cells and
endosome, whereas the 25-nm-sized AgNPs enter into the individual endosomes
(Speshock et al. 2010).
4.4.4 Anti- Inflammatory Properties
AgNPs possess potent anti-inflammatory properties. Studies have reported the anti-
inflammatory and wound-healing properties of topically applied AgNPs (Chaloupka
et al. 2010). Likewise, AgNPs were evidenced to downregulate the photolytic metal-
loprotease enzymes that are of prime importance in repair and inflammatory pro-
cesses (Kirsner et al. 2001). AgNPs play a major role in achieving scarless wound
healing by modulating both local immune response as well as systemic immune
responses (Tian et al. 2007). Likewise, AgNPs are also efficacious in reducing the
post-operative adhesion formation, which would prove to be an effective therapeutic
and clinical approach (Wong et al. 2009).
4.5 Toxicity of AgNPs
There have been exponential evolutions for the field of metal nanoparticles in areas
such as drug delivery, medical imaging, diagnostics, therapeutics and engineering
technology. As there is a meagre information on the impact of metal nanoparticles
on human health and environment, it is necessary to comprehend the accumulation
and bio-distribution of AgNPs in living systems (Braydich-Stolle et al. 2005). The
prime feature of any metal nanoparticle includes their size and dimension that is
amidst of individual atoms or molecules and their corresponding bulk materials.
The surface area and the size of the particle alter the nanomaterials’ physicochemi-
cal properties and also influence the reactivity of the nanomaterial with the cellular
environment, resulting in variations in cellular uptake causing adverse biological
effects. In addition, as the size of a nanomaterial decreases, the toxicity also
increases. Hence, in terms of safety, the toxic effect of AgNPs is a major concern.
The cytotoxic effects of metal nanoparticles have been demonstrated (AshaRani
et al. 2008a; Braydich-Stolle et al. 2005; Hussain et al. 2005; Kawata et al. 2009).
In mammalian cells, defective cell morphology, lactate dehydrogenase leakage
from the membrane and mitochondrial dysfunction were reported (AshaRani et al.
2008a; Braydich-Stolle et al. 2005; Hussain et al. 2005; Kawata et al. 2009). At cel-
lular levels, AgNPs react with defence mechanism of antioxidants, resulting in the
production of ROS. Oxidation of biomolecules including lipid, protein and DNA
takes place in the presence of excess ROS. This oxidative stress mediates through
upregulation of redox-sensitive transcription factors (NK-kappa B), activator pro-
tein, and kinases induces inflammation (Aillon et al. 2009; Lanone and Boczkowski
2006; Rahman 2000; Rahman et al. 2005). Moreover, the genotoxicity, carcinoge-
nicity and teratogenicity occur because of nanoparticles. Some nanoparticles bypass
the blood–brain barrier, which lead to brain toxicity (Tse et al. 2011; Unrine et al.
2010). Also, the nanoparticles have the ability to cross the digestive tract because of
increase in absorption due to high nanoparticle loading. Indeed, hypersensitivity in
small proportion of burn patients has been reported in many clinical conditions (Hill
et al. 2000; Muhlfeld et al. 2008). The toxicity was found to be related with smaller
size of nanoparticle. The bioavailability of AgNPs for cellular uptake is influenced

by size-dependent mechanisms. Hence, it is safe to use the low doses of AgNPs (Nel
et al. 2006; Rothen-Rutishauser et al. 2007).
AgNPs are efficacious antimicrobials. Hence, it has been exploited to have inim-
ical effects on human reproductive system in various commercial products includ-
ing contraceptive devices and feminine hygiene (Elechiguerra et al. 2005; Gong
et al. 2007; Sondi and Salopek-Sondi 2004). AgNPs are utilized in various cosmetic
products and textile materials. Based on fabric quality, pH, sweat formation and
quantity of silver coating (Ahamed et al. 2010), the AgNPs could be released and
enhanced skin exposure to toxic silver nanomaterials.
Silver ions show elevated toxicity towards microorganisms. Studies report that
free silver ions are more toxic to microbes than silver nitrate (Choi et al. 2008).
Moreover, interaction of silver ions with thiol groups in proteins leads to bacterial
cell wall inactivation (Morones et al. 2005). Also, the silver ion in micromole con-
centration uncouples electrons involved in photophosphorylation and thus inhibit-
ing the process of microbial respiration (Feng et al. 2000). AgNPs disrupt the
bacterial DNA replication on interaction with the nucleic acid (Feng et al. 2000).
Many studies have also proved the extreme toxicity of silver ions and AgNPs
towards bacterial species such as E. coli (Gogoi et al. 2006; Kim et al. 2007; Mohan
et al. 2007) and yeast (Kim et al. 2007). Antimicrobial toxicity of AgNPs has been
confirmed by proteomic analysis (Lok et al. 2006). Nanoparticles lead to the disrup-
tion of the bacterial membrane and alter the cellular integrity due to unceasing leak-
age of intracellular potassium from the cell which further lowers the ATP and
declines the cell viability.
Due to defective impacts of AgNPs, pit formation occurs in fungal cell wall and
thus leading to efficient transfer of larger AgNPs by membrane perturbation. This
further results in the release of cellular components such as glucose and trehalose.
AgNPs with a size range between 1 and 10 nm exhibit toxicity, and nanoparticles of
size <5 nm have been proven to be toxic against nitrifying bacteria (Choi and Hu
2008; Reidy et al. 2013). After disintegration of AgNPs, the silver ions generated
tend to interact with sulphur proteins in the bacterial cell wall leading to defective
functionality (Levy and Marshall 2004; Reidy et al. 2013).
Some defence mechanisms are adopted by bacterial cells in order to defend
themselves from silver toxicity. The first line of defence against silver toxicity
includes the thick peptidoglycan membrane and the phospholipids and the activa-
tion of molecular chaperons (Sedlak et al. 2012). By means of efflux pumps, bacte-
ria resist silver ions and subdue toxicity (Jung et al. 2008). The plasmid-borne
cassettes encode the efflux pumps in bacteria. The reports further suggest that bac-
teria accumulate the dense electron light particles in the centre of cell as defence
strategy (Feng et al. 2000; Jung et al. 2008). The thick peptidoglycan layer of Gram-
positive bacteria poses effective inhibition against the toxic effects of silver ions
(Jung et al. 2008). Soon after the exposure of bacteria to the silver salts or silver
ions, genes of plasmid and chromosomes show a high degree of resistance (Silver
2003).
4.5.1 In Vitro Toxicity
The hallmarks of silver nanotoxicity include the oxidative stress, DNA damage and
modulation of cytokine production. ROS production enhanced by the cellular uptake
of AgNPs results in oxidative stress and genotoxic effects. Also, cell death by either
apoptotic or necrotic pathways is induced by ROS (Ahamed et al. 2008; Almofti
et al. 2003; Asharani et al. 2008b; Carlson et al. 2008; Hussain et al. 2005; Hwang
et al. 2007). Studies indicated that the toxicity is dependent upon both shape and size
of nanoparticles (Carlson et al. 2008). However, data also suggest that such toxicity
effects may either be case- or species-dependent (Hussain et al. 2005). AgNPs are
known to induce genotoxic effect by damaging DNA of human lung fibroblasts
(IMR-90) and human glioblastoma cells (U251) through indirect boost or decrease
in the ATP production leading to mitochondrial damage and thus impairing the DNA
repair mechanisms (AshaRani et al. 2008a). Similarly, direct DNA damage by silver
ions or AgNPs was also reported (Ahamed et al. 2008; Cha et al. 2008; Chi et al.
2009; Yang et al. 2009). The alveolar macrophages exposed to AgNPs responded
with an increase in the production of proinflammatory cytokines (TNF-α, MIP-2 and
IL-1β) (Carlson et al. 2008), and human epidermal cells exposed to AgNPs resulted
in increased production of IL-1β, IL-6, IL-8 and TNF-α (Samberg et al. 2009).
Whereas, human mesenchymal stem cells exposed to AgNPs exhibited both descent
(IL-6 and IL-8) and ascent (IL-8) in proinflammatory factors (Greulich et al. 2009).
4.5.2 In Vivo Toxicity
In contrast to in vitro studies, limited information is available on the potential

in vivo toxicity mechanisms of AgNPs. Upon exposure of laboratory rodents to
AgNPs, toxicological responses including effects on circulatory, respiratory, central
nervous and hepatic systems have been observed (Ji et al. 2007). On topical admin-
istration of AgNPs, impacts on dermal tissues have also been reported. Likewise,
ingestion or inhalation of AgNPs resulted in their translocation to the circulatory
system (Ji et al. 2007; Sung et al. 2008b). Reports on animal study suggest that mice
injected with AgNPs responded with a decrease in platelet aggregation (Shrivastava
et al. 2009). The mechanistic response remains exotic (Sung et al. 2008a). A phar-
macokinetic study revealed that the AgNPs injected into the bloodstream affirmed
the translocation of AgNPs through the blood–brain barrier, accumulation in the
brain and neuronal degeneration and necrosis (Tang et al. 2008). Evidences too
indicate that liver and bile ducts are targets of AgNPs toxicity where Ag+ found to
accumulate in liver after exposure to AgNPs (Ji et al. 2007; Kim and Kwon 2009;
Kim et al. 2008; Sung et al. 2008b). Histopathological analyses of liver and bile
ducts of mice upon exposure to AgNPs affirmed vacuolization and hepatic focal
necrosis, and hyperplasia of bile ducts (Cha et al. 2008). Upregulation of genes
involved in apoptotic and inflammatory pathways was confirmed in mice livers
when exposed to AgNPs (Cha et al. 2008). Toxic effects of AgNPs in a dose-
dependent manner were observed during the development of zebrafish that resulted
in mortality, declined heart rate and hatching rate. Further, morphological changes,
edema, uniform distribution of the nanoparticles in the zebrafish embryos and

overall decline in development were also observed (Asharani et al. 2008b). Studies
suggested that morphological alterations in pigs’ skin cells were observed during
the exposure of AgNPs of 20–50 nm, 0.34–34 μg/ml, for 14 days and noticed that
the highest doses resulted into edema, epidermal hyperplasia and focal inflamma-
tion (DiVincenzo et al. 1985). Moreover, animal and human studies suggested that
it is difficult to expel the AgNPs completely; once it gets deposited in the body,
they get excreted through sweat, urine and faeces (DiVincenzo et al. 1985).
4.6 Biomedical Applications of AgNPs
The silver and silver-based nanoparticles are used in various biomedical applica-
tions such as wound dressing (Liang et al. 2016), cardiovascular implants (Cipriano
et al. 2014), catheters (Thomas et al. 2015), bone cements (Slane et al. 2015), dental
carries (Divakar et al. 2018) and bio-diagnosis (Zhou et al. 2011).
4.6.1 Wound Dressing
Wound dressing that protects the wound from infections should not be toxic or aller-
gic but should be biocompatible for effective wound healing (Liang et al. 2016).
Development of AgNPs and AgNPs-based materials for wound dressing was mainly
focused on antibacterial potential (Hernández-Rangel et al. 2019). It is well known
that AgNPs are broadly used as antibacterial agents due to their large surface area to
volume ratio. Wound dressing not only protects the wound from microorganisms
but also enhances the wound-healing process (Khil et al. 2003; Zhang et al. 2005).
The dressing materials developed with size-dependent AgNPs increase the wound-
healing potential (Zamani et al. 2013). The extracellular synthesis of AgNPs using
A. niger was used to regulate the cytokines that participate in wound healing in rat
(Gade et al. 2010). The AgNPs-based dressing material such as Acticoat Flex 3
applied to 3D fibroblast cell culture significantly reduces the mitochondrial activity
without affecting the cell viability. The cellular staining techniques confirmed the
nuclear integrity (Rigo et al. 2013). Various biocomposites modified with ionic sil-
ver such as Acticoat™ and Bactigras™ (Smith & Nephew), Aquacel™ (ConvaTec),
PolyMem Silver™ (Aspen) and Tegaderm™ (3 M) have been approved by the US
Food and Drug Administration (FDA) and used for wound dressing applications
(Burdușel et al. 2018). Hebeish et al. (Hebeish et al. 2014), prepared wound dress-
ing material using various concentrations of AgNPs such as 60, 125 and 250 ppm.
They have found that the 250 ppm of AgNPs coated with cotton fabrics is more
potent against clinical pathogens such as E. coli, S. aureus and C. albicans. Further,
more effective wound healing was seen at 250 ppm of AgNP with cotton fabrics in
rat. Liang et al. (Liang et al. 2016) obtained the wound dressing material by using
Konjac glucomannan or silver nanoparticle composite sponge. These composite
sponges exhibited the potent antibacterial activity against S. aureus and E. coli and
also showed enhanced wound healing in rabbit. The synergistic effects of Ag
nitroprusside complex nanoparticles on wound-healing property and antibacterial

activity in mice were reported (Rao et al. 2018).
4.6.2 Cardiovascular Implants
Different properties of nanomaterials such as physiochemical properties, surface

topographies and surface energy promote the cellular responses in the cardiovascu-
lar system (Jiang et al. 2017). Nanomaterials directly interact with biomolecules
inside the human body and hence provoke the desirable cellular and tissue level
responses to cardiovascular implants (Cipriano et al. 2014). First cardiovascular
devices such as prosthetic silicone heart valves are coated with silver cement to
avoid bacterial infections as well as to reduce the inflammation response of the heart
(Grunkemeier et al. 2006). Moreover, silver-coated heart valves were found to cause
the hypersensitivity, prevent the normal function of fibroblast and ultimately lead to
paravalvular leakage (Jamieson et al. 2009). The medical devices coated with
AgNPs are safe and non-toxic unlike metal silver. The multilayer film containing
AgNPs was developed. These nanoparticles have antibacterial, hydrodynamic and
mechanical properties and therefore used in cardiovascular implant coatings (Fu
et al. 2006; Ghanbari et al. 2009).
4.6.3 Catheters
Catheters-associated urinary tract infections are one of the most common sites in
the human being for bacterial infections that causes the morbidity and mortality
(Cooper et al. 2016; Mansour et al. 2014). Urinary tract infections are more com-
mon in women than men. Approximately, 80% of urinary tract infections are due to
the presence of an indwelling urinary catheter (Apisarnthanarak et al. 2007; Mansour
et al. 2014; Tambyah 2004). Thomas et al. (Thomas et al. 2015) reported the synthe-
sis of AgNPs from Bacillus sp. SJ 14, and these AgNPs have been used to coat on
the surface of urinary catheters (Silicone-Foley Balloon Catheter). The AgNPs-
coated catheter prevents the attachment and colonization of coagulase-negative
Staphylococci such as Staphylococcus epidermidis and Staphylococcus haemolyti-
cus. Antimicrobial urinary catheters have been developed by photochemical deposi-
tion of silver solution on the outer and inner surfaces under UV radiation, as a result
of in situ synthesis of AgNPs. These developed catheters showed potent antibacte-
rial activity towards urinary tract-associated bacteria such as E. coli, K. pneumoniae
and P. mirabilis (Cooper et al. 2016).
4.6.4 Bone Cements
In general, bone cements are made out of self-polymerizing biomaterials and

broadly employed for the orthopaedic, traumatology, oncological and spinal
treatments (Rau et al. 2017; Tanner et al. 2010; Yu et al. 2017). Bone cements must
be biocompatible with good mechanical properties, and it should prevent the infec-
tions caused by pathogenic microorganisms (Cui et al. 2017; Goto et al. 2005).
Mainly, bone cements are loaded with antibiotics used for antibacterial potential.
But these materials are not ideal as these have reduced mechanical properties, short
duration of antibacterial effect and may not be effective against antibiotic-resistant
bacteria (Koh et al. 2015; Zhu et al. 2017). AgNPs loaded with acrylic bone cement
are used to evaluate the mechanical, material and antibacterial properties. They
observed that mechanical and material properties that were not greatly different
from the standard cement. Further, it has been shown that acrylic bone cement mod-
ified with AgNPs reduced the biofilm formation on cement surface (Slane et al.
2015). Bone cement composites have been developed with silver-containing bioac-
tive glass powder and compared with commercial bone cements based on polymeth-
ylmethacrylate (PMMA) for mechanical and antibacterial properties. The bone
cement composites showed the good mechanical and antibacterial properties as
compared to commercial bone cements (Miola et al. 2014).
4.6.5 Dental Carries
The AgNPs and AgNPs-based nanomaterials have been used in dental applications
such as endodontics (Lotfi et al. 2011; Samiei et al. 2013), periodontics (Flores et al.
2010; Zhao et al. 2011), orthodontics (Ahn et al. 2009; Moreira et al. 2014) and
restorative dentistry (Durner et al. 2011; Jia et al. 2008) due to their antimicrobial
potential. Divakar et al. (2018) prepared the chitosan-conjugated AgNPs where chi-
tosan was obtained from Aspergillus flavus Af09. These nanoparticles exhibited the
antimicrobial potential against dental pathogens such as Streptococcus mutans and
Porphyromonas gingivalis. Also, these nanoparticles inhibited the biofilm forma-
tion along with production of quorum sensing molecules. The hybrid dental resin
containing AgNPs was developed and used for the treatment of periodontal disease
caused by S. mutans and Streptococcus sobrinus (Lee et al. 2007). The AgNPs were
evaluated for antimicrobial potential against oral pathogens such as S. mutans
(MTCC 497), S. oralis (MTCC 2696), Candida albicans (MTCC 183), Lactobacillus
acidophilus (MTCC 10307) and L. fermentum (MTCC 903) (Panpaliya et al. 2018).
The chitosan membrane containing AgNPs was used against dental pathogen P.
gingivalis (Lee et al. 2018).
4.6.6 Bio-Diagnosis
Nanoparticles-based bio-diagnosis is more advantageous than conventional diagno-

sis method. Nanoparticles enable fast, sensitive and cost-effective diagnosis due to
their unique properties and surface-coated biomolecules (Colino et al. 2018).
AgNPs-based biosensor was developed for clinical detection of serum p53 in head
and neck squamous carcinoma cell (Zhou et al. 2011). Also, using photothermal
therapy, AgNPs were used to obtain dual-imaging/therapy-immunotargeted

nanoshells to detect and destroy specific targeted cancer cells (Loo et al. 2005).
AgNPs can identify the interaction between amyloid-derived and anti-amyloid-
derived diffusible ligands antibody, which might be responsible for the cause of
Alzheimer’s disease (Haes et al. 2004). Au–Ag nanorods were employed for the
early detection of cancer. The label surface-enhanced Raman spectroscopy (SERS)
functionalized with monoclonal antibodies, incorporated with AgNPs, has been
employed for the detection of Salmonella and Staphylococcus aureus and other
bacteria (Lin et al. 2014).
4.6.7 Anticancer Potential
Cancer is a group of diseases through diverse signalling mechanisms including cell

proliferation, angiogenesis and metastasis generating various pathological and met-
abolic changes in cellular environments (Seigneuric et al. 2010). Anti-neoplastic
drugs lack chemotherapeutic efficacy to fight effectively against multi-drug-resistant
cancer cells, which further increases the death rates of cancer patients (Abdel-Fattah
et al. 2017; Raghunandan et al. 2011). Currently used chemotherapeutic drugs fail
to promote apoptosis as well as possess enormous side effects (Thorley and Tetley
2013) and therefore exploitation of metallic drugs deserves merit.
Bacillus tequilensis has been reported for the synthesis of AgNPs that exhibit
apoptotic potential against human breast cancer cell (Gurunathan et al. 2015).
AgNPs from marine bacterium E. coli VM1 showed potent anticancer activity
against human cervical cancer cell line (HeLa) (Maharani et al. 2016). AgNPs pro-
duced by Enterococcus sp. showed anti-neoplastic activity against human liver can-
cer cell lines (HepG2) and lung cancer cell lines (A549) (Rajeshkumar et al. 2016).
Similarly, AgNPs synthesized from Penicillium brevicompactum showed cytotoxic-
ity towards breast cancer cell line (MCF-7) (Majeed et al. 2018). Likewise, AgNPs
synthesized by Klebsiella sp. showed broad-spectrum cytotoxicity towards human
breast (SKBR3 and 8701-BC) and colon (HT-29, HCT 116 and Caco-2) cancer cell
lines (Buttacavoli et al. 2018). Recently, AgNPs synthesized by Trichoderma atro-
viride have been reported for their anticancer potential against human breast cancer
cell line (MDA-MB-231) (Saravanakumar and Wang 2018). Thus, the innate cyto-
toxic potential of AgNPs against human cancer cell lines has been documented.
This broad-spectrum cytotoxic property of AgNPs could be exploited for cancer
therapeutic treatment.
4.6.8 Theranostic Applications
Nanomaterials have been employed for the treatment of cancer theranostic. The
theranostic approach is mainly directed towards diagnosis and therapy (Warner
2004; Ahmed et al. 2012). This new technological area is used for diagnosis, tar-
geted delivery of therapeutic agents, monitoring the therapeutic response and also
determining the effectiveness of therapeutic agents (Svenson 2013). Nanomaterials

improved the theranostic applications through targeted delivery of specific site of
the affected organ (Jabir et al. 2012). Among nanomaterials, AgNPs have excellent
SPR that supports for their potent theranostic applications. Colloidal AgNPs that
possess anti-neoplastic activity against A549 and MCF-7 have been reported
(Mukherjee et al. 2014). AgNPs have also been reported for multifunctional ther-
anostic applications such as antibacterial, antileishmanial, anti-inflammatory and
anticancer potential (Ovais et al. 2018).
4.7 Conclusion and Future Challenges
Among metal nanoparticles, AgNPs are considered to be important as they exhibit

innate antimicrobial and anticancer potentials. Apart from this, AgNPs have been
explored in various biomedical applications such as wound dressing, cardiovascular
implants, catheters, bone cements, dental carries, bio-diagnosis and anticancer
potential. Increasing awareness towards green chemistry and biological processes
has led to a desire to develop the synthesis of biological nanoparticles using
microbes. Other processes in physical and chemical methods involve hazardous
chemicals. In contrast, microbes-based biological methods are simple, clean, non-
toxic, cost-effective and eco-friendly in nature. Therefore, synthesis, characteriza-
tion and applications of microbial silver nanomaterials have received attention.
Microbes serve as promising candidates for the synthesis of AgNPs using cell and
supernatant extracts. Since polydispersity is the major concern, it is imperative to
optimize the conditions to synthesize monodispersed AgNPs.
Furthermore, it is well known that the microbial synthesis of nanomaterials is
utmost slow reduction process as compared to physiochemical approaches. By
varying the reaction parameters, microbial synthesis time can be minimized and
the required dispersity, particle size and stability of AgNPs can also be achieved.
Therefore, further research is required to improve the microbial synthesis process,
stability and control of the size and shape of nanomaterials. Studies have con-
firmed that higher concentrations of AgNPs may cause health problems and are
toxic to the environment. Hence, more research efforts are required to minimize
the toxicity, and the silver nanotoxicity should be monitored carefully. Moreover,
an array of enzymes, proteins and metabolites of microbes is involved in the reduc-
tion of Ag+ ions to AgNPs; the cellular, biochemical and molecular mechanisms of
synthesis of nanoparticles by various microbes are not fully understood. Hence,
further research is required to identify precise synthesis parameters, the genes,
enzymes and other biomolecules that mediate the mechanisms of microbial synthe-
sis of AgNPs.
Acknowledgement We thank the University Grants Commission (UGC) and Department of

Biotechnology (DBT), New Delhi, for financial support through M.Sc. Biotechnology fellowship
to Annuja Anandaradje and Ph.D. research fellowship to Indramani Kumar, respectively. We also
thank UGC-SAP and DST-FIST programs coordinated by Prof. N. Sakthivel for providing infra-
structure facilities.
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Fluoride Nanoparticles for Biomedical
Applications 5
M. S. Pudovkin and R. M. Rakhmatullin
5.1 Introduction
Fluoride nanoparticles have very unique and distinguishable physical and chemical
properties such as transparency of fluorides in a broad spectral range (0.2–6 μm),
low energy of phonons which prevents undesirable multiphonon relaxation in elec-
tron levels of doping ions, high resistance to moist, high thermal conductivity, and
unique magnetic properties. Some fluorides can contain relatively high concentra-
tion of doping ions which allows obtaining desired chemical composition of the
nanoparticles (Fedorov et al. 2011; Kuznecov et al. 2006; Li et al. 2017; Alakshin
et al. 2016; Pudovkin et al. 2014, 2018, 2019; Shcherbakov et al. 2015; Naccache
et al. 2015; Ptacek et al. 2010; Rahman and Green 2009; Li and Lin 2010; Ye et al.
2013; Yi and Chow 2005; Bao et al. 2013; Tan and Jin 2018; Liu et al. 2014a).
Materials such as CaF2 and LnF3 (Ln = lanthanides) have very low solubility (around
10−5–10−6 mol/L) (Pudovkin et al. 2018) and as consequence low toxicity
(Shcherbakov et al. 2015). In case of LnF3 and NaLnF4 fluoride nanoparticles, the
synthesis procedures are relatively simple and cost-effective which allow obtaining
nanoparticles having desirable size and morphology (Fedorov et al. 2011). Due to
the above-mentioned properties, the doped fluoride nanoparticles demonstrate
excellent photostability, long luminescent lifetimes, and sharp emission bands
(Naccache et al. 2015; Ptacek et al. 2010; Rahman and Green 2009; Li and Lin
2010; Ye et al. 2013; Yi and Chow 2005; Bao et al. 2013; Tan and Jin 2018). Also
these nanoparticles can operate into a broad spectral range from ultraviolet (UV) to
near infrared (IR). These excellent physical and chemical properties of doped fluo-
ride nanoparticles make them highly promising not only for conventional bioimag-
ing but in a significantly broader list of application including dentistry, thermometry,
magnetic resonance imaging, and photodynamic therapy (Fedorov et al. 2011;
M. S. Pudovkin (*) · R. M. Rakhmatullin

Kazan Federal University, Kazan, Russia

https://doi.org/10.1007/978-981-13-8954-2_5
136 M. S. Pudovkin and R. M. Rakhmatullin
Kuznecov et al. 2006; Liu et al. 2014a; Pudovkin et al. 2014, 2019). In particular,
rare earth doped fluoride nanoparticles are utilized in luminescence thermometry of
living cells (Kucsko et al. 2013). In this case the temperature is extracted from
temperature-dependent luminescence features of doping ions into fluoride matrix.
For example, Pr3+ and Nd3+ have thermally coupled electron levels which lead to the
phenomenon that the intensities of emissions of these levels are temperature depen-
dent. In turn, Yb3+, Er3+:NaYF4 up-conversion nanoparticles also demonstrate nota-
ble temperature sensitivity via the similar mechanism. The required spatial
resolution is achieved via using visible light in temperature reading. Temperature-
sensitive nanoparticles are very promising in hyperthermia with simultaneous tem-
perature control of the heating area (Yang et al. 2011; Balabhadra et al. 2015;
Wawrzynczyk et al. 2012; Pudovkin et al. 2017; Jaque and Vetrone 2012; Brites
et al. 2012, 2018; Dramićanin et al. 2014; Nikolić et al. 2014; Bu et al. 2015; Lojpur
et al. 2013; Zhou et al. 2014; Gharouel et al. 2018; Weber 1968; Caspers et al. 1965;
Collins et al. 1998). For example, highly doped Yb3+:LaF3@Nd3+:LaF3 core@shell
nanoparticles effectively convert light into heat via luminescence quenching pro-
cesses. In turn, the temperature-dependent luminescence signal of these nanoparti-
cles is used for temperature control (Balabhadra et al. 2015; Wawrzynczyk et al.
2012; Jaque and Vetrone 2012). The high brightness and ability to operate into
broad spectral range make rare earth doped fluoride nanoparticles very promising in
bioimaging (Wolfbeis 2015; Ren et al. 2011; Wang et al. 2009; Dong et al. 2011;
Jaque et al. 2016; Villa et al. 2015; Henderson and Imbusch 1989; Yang et al. 2012).
In particular, Yb3+, Er3+ or Yb3+, Tm3+:NaYF4 up-conversion nanoparticles serve as
fluorescence agents in bioimaging of living cells under infrared 980 nm excitation.
It allows avoiding undesired autofluorescence and obtaining high-contrast image
without photobleaching. For in vivo bioimaging usually Nd3+ doped LaF3 or SrF2
nanoparticles are used. These nanoparticles operate into the first and second bio-
logical windows (700–950 nm and 1000–12,000 nm, respectively) where the tissues
are partially transparent (Rocha et al. 2014a). In dentistry fluoride nanoparticles are
of importance primarily as perspective F reservoirs (Lellouche et al. 2012;
Kulshrestha et al. 2016; Bapat et al. 2018; Silva et al. 2015; Sun and Chow 2008;
Xu et al. 2008; Kanazawa et al. 1991; Azami et al. 2011; Prentice et al. 2006). For
example, CaF2 nanoparticles exhibit increased reactivity and solubility compared
with its macro counterpart. These unique properties are highly required in caries
prevention (Bapat et al. 2018) where CaF2 nanoparticles are added in glass-ionomer
cement. The next important application is photodynamic therapy of cancer. In pho-
todynamic therapy rare earth doped fluoride nanoparticles can increase the depth of
this treatment. In this case rare earth doped fluoride nanoparticles can activate pho-
tosensitizers conjugated on the nanoparticles’ surface under X-ray or infrared light
(Dolmans et al. 2003; Bekah et al. 2016; Lucky et al. 2015a, b; Pelaez et al. 2012;
Lipovsky et al. 2009; Kamkaew et al. 2016; Cooper et al. 2014; Förster 1960; Selvin
2000; Nie et al. 2017; Sanders et al. 2012; Hou et al. 2015; Zhang et al. 2015; Dou
et al. 2015; Kostiv et al. 2017; Qian et al. 2009; Khaydukov et al. 2016; Chen and
Zhang 2006; Tang et al. 2015; Clement et al. 2016). In magnetic resonance imaging
5 Fluoride Nanoparticles for Biomedical Applications 137
fluoride nanoparticles containing trivalent lanthanide ions that exhibit paramagnet-

ism such as Tb3+, Dy3+, Ho3+, and Er3+ can serve as contrast agents. This approach
allows improving visibility of pathologic areas (Zheng et al. 2016; Dong et al. 2015;
Geraldes and Laurent 2009; Hu and Zhao 2012; Blow 2009; Fries et al. 2014; Kattel
et al. 2012; Alakshin et al. 2018; Das et al. 2012). Unique antioxidant properties of
CeF3 nanoparticles were also found (Shcherbakov et al. 2015). According to the
many papers rare earth doped NaYF4, NaGdF4, CeF3, LaF3, CaF2 nanoparticles are
very low toxic toward cells and small animals at applied concentrations (approxi-
mately 1 μM–10 mM) (Alakshin et al. 2018; Das et al. 2012; Pudovkin et al. 2016;
Wysokińska et al. 2016; Chen et al. 2014; Jang et al. 2014; Bala et al. 2017; Wang
et al. 2013). The nanoparticles are taken up by spleen and liver mainly (Cheng et al.
2011; Cao et al. 2013; Xing et al. 2012; Yang et al. 2013). However, the toxicology
concerns are still under the intense investigation (Pudovkin et al. 2018; Shcherbakov
et al. 2015; Wysokińska et al. 2016; Chen et al. 2014; Jang et al. 2014).
Here we review the application of fluoride nanoparticles in biomedicine espe-
cially focusing on luminescent thermometry, photodynamic therapy, bioimaging,
and dentistry. A brief review of toxicity of fluoride nanoparticles in vitro and in vivo
is also given.
5.2 oxicity and Biological Activity of Fluoride

T
Nanoparticles
5.2.1 Toxicity of Fluoride Nanoparticles
Although rare earth doped or undoped fluoride nanoparticles are considered very
promising materials for a broad range of biomedical applications, the deep under-
standing of the toxicity of nanomaterials is still insufficient (Wysokińska et al. 2016;
Chen et al. 2014; Jang et al. 2014). This knowledge is of great importance in the
light of growing use of the biofunctionalized nanoparticles. Some questions about
the safety of these nanomaterials are raised since the very same properties of
nanoparticles that are desirable and potentially useful from a technological or bio-
medical perspective may also give rise to unexpected and hazardous toxicities
(Wysokińska et al. 2016; Chen et al. 2014; Jang et al. 2014; Bala et al. 2017). It
should be noted that the most applicable and promising nanomaterials for biomedi-
cal applications are rare earth doped or undoped NaYF4, NaGdF4, NaLuF4, LnF3
(Ln = lanthanides), and CaF2 nanoparticles. Hence the toxicity of these nanomateri-
als is intensively studied. It is noteworthy to say that a special attention is paid
toward such important nanomaterials in bioimaging and photodynamic therapy as
the up-conversion nanoparticles. Particularly, in Wysokińska et al. (2016) cytotoxic-
ity of bare, silica coated, and polyethylene glycol (PEG) coated Yb3+,Er3+:NaGdF4
up-conversion nanoparticles toward mouse macrophage (RAW264.7) and fibroblast
(NIH3T3) cells was studied via MTT assay. The exposure time was 48 h. The 300-
nm non-spherical bare Yb3+:Er3+:NaGdF4 nanoparticles were relatively low toxic
toward both cell lines into 1–100 μg/mL range. The maximum viability loss of
NIH3T3 cells was around 60% at 100 μg/mL. The viability loss was accompanied
by an extensive apoptosis observed in both RAW264.7 (at 10 μg/mL nanoparticles
concentration) and NIH3T3 cells (at 100 μg/mL nanoparticles concentration).
However the 29 nm polyethylene glycol (PEG) coated Yb3+:Er3+:NaGdF4 nanopar-
ticles demonstrated a negligible cytotoxicity (viability loss of NIH3T3 cell line was
around 15% at 100 μg/mL) and lack of apoptosis. The viability loss of NIH3T3 cells
after exposing by 61 nm silica coated Yb3+:Er3+:NaGdF4 nanoparticles demonstrated
the similar concentration dependence as for bare Yb3+:Er3+:NaGdF4 nanoparticles.
Therefore it was concluded that the PEG coating of Yb3+:Er3+:NaGdF4 nanoparticles
significantly reduced the cytotoxic effect comparing with bareYb3+:Er3+:NaGdF4
nanoparticles or silica coated ones. However the sizes of the nanoparticles were
notably different between each other. Thus the size effect was not taken into account.
The size-dependent cytotoxicity of Eu3+:NaYF4 nanoparticles toward endothelial
cells (EC) was studied via MTT assay in Chen et al. (2014). The Eu3+:NaYF4
nanoparticles with average sizes of 50, 158, and 354 nm were synthesized via dif-
ferent variations of the hydrothermal method. After cells and nanoparticles incuba-
tion, the viability of ECs was decreased in dose- and time-dependent manners. All
the nanoparticles demonstrated relatively low cytotoxicity into 20–100 μm/mL
range. The values of cells’ viability after 72 h exposure for 50, 158, and 354 nm
Eu3+:NaYF4 nanoparticles were around 60% at 100 μm/mL. However in case of
354-nm Eu3+:NaYF4 nanoparticles slightly lower viability was observed. However,
the size did not affect the viability strongly.
In Jang et al. (2014) the cytotoxicity of 16.7 nm spherical hexagonal phased
Ce3+,Tb3+:NaYF4 nanophosphors toward Capan-1 cells was studied into
0.5 pM–40 nM concentration range via MTT assay. After 48-h incubation the via-
bility assays revealed the dose-dependent almost linear decreasing of viability from
~100% at 0.5 pM to ~30% at 40 nM. The size did not affect the viability strongly.
The toxicity of a widespread host-matrix CaF2 is intensively studied (Shcherbakov
et al. 2015). Specifically, the undoped 20–60 nm CaF2 nanoparticles do not demon-
strate any cytotoxic effect toward mammalian Vero cells after 24 h incubation at 31.25,
62.5, 125, 250, 500, and 1000 mg/mL (Bala et al. 2017). In this work nontoxic nature
of CaF2 nanoparticles was attributed to the slow and sustained release of fluoride ions.
Finally it was shown in Pudovkin et al. (2018) that the cytotoxicity of ~16 nm Pr3+:LaF3
nanoparticles did not reveal any significant toxic effect up to 5 mM concentration
toward COLO-320 cells. At this value the cells’ viability was 62% (Fig. 5.1).
The toxicity in vivo of fluoride nanoparticles is also a field of intensive study. In
particular, toxicity of the above-mentioned hexagonal phased Ce3+,Tb3+:NaYF4 nano-
phosphors toward zebrafish models was studied in Jang et al. (2014). The treatment of
zebrafish embryos with the nanoparticles into the 0.5–500 pM concentration range did
not reveal any abnormalities even at 24, 48, and 72 h post-fertilization. Only after 48 h
of post-fertilization 10.9% and 28.6% embryos treated with 500 pM and 750 pM of
nanophosphors, respectively, exhibited the slight growth retardation. Toxicity of
7–10 nm Yb3+, Er3+:LaF3 nanoparticles toward the zebrafish embryos is studied into
5–400 μm/mL range in Wang et al. (2013). The zebrafish embryos do not demonstrate
100% 96%
100 91%
80 71%
62%
Survival (%)
60
40
20
0
Control 0.5 mM 1 mM 2.5 mM 5 mM
Fig. 5.1 MTT assay data of survival rate of COLO-320 cells after 24 h of COLO-320 cells and
Pr3+:LaF3 nanoparticles exposition (Pudovkin et al. 2018). The publisher is Hindawi. It is open
access. Open-access authors retain the copyrights of their papers, and all our articles are distributed
under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided that the original work is properly cited
any significant changes in frequency of movement and the heart rate after incubation
with the nanoparticles. No any significant adverse effects into 5–100 μm/mL range of
Yb3+,Er3+:LaF3 nanoparticles were observed on embryonic survival and development.
The notable toxic effects were at 200 μg/mL and at 400 μg/mL (the survival is around
50% for both concentrations). Here the mechanism of toxicity is attributed to La3+
ions binding with DNA resulting in possible DNA damage.
The toxicity and biodistribution of PEG coated Tm3+:NaYF4 nanoparticles
toward Kunming mice with average weight of 20 g were studied by Xing et al.
(2012). Similarly, the nanoparticles were mainly taken up by the liver and spleen
even after 0.5 h after injection (Figs. 5.2 and 5.3). According to Xing et al. (2012)
after 30 days the nanoparticles escaped these organs almost completely. Histological
analysis showed no noticeable tissue damages. Any other toxic effects on organs
were not observed, indicating the good biocompatibility of the PEG coated
Tm3+:NaYF4 nanoparticles in vivo.
In vivo study of 25- to 30-nm 153Sm3+,Yb3+,Tm3+:NaLuF4 (153Sm is radioactive)
up-conversion nanoparticles modified with oleic acid (OA) and 6-aminohexanoic
acid (AA) on 4-weeks-old Kunming mice was conducted in Yang et al. (2013).
153
Sm3+,Yb3+,Tm3+:NaLuF4 nanoparticles were injected into Kunming mice through
the tail vein, and in vivo single-photon emission computed tomography (SPECT)
images were detected by Nano SPECT plus at 1 h and 24 h after injection. Intense
radioactive signals were detected exclusively in the liver and spleen, confirming that
the 153Sm3+/Yb3+/Tm3+:NaLuF4 nanoparticles were rapidly taken up by these two
organs. At 24 h, a much larger uptake by the spleen compared to the liver was
observed, which is partially due to the spleen being the largest organ of the immune
system. Rapid accumulation of nanoparticles was found in the liver (w56% ID/g of
the injected dose) and spleen (w20% ID/g of the injected dose) at 1 h, while at 24 h
the liver uptake had decreased to w30% ID/g, and the spleen accumulation had
increased to w140% ID/g. Uptake in the heart, lung, kidney, and other organs
(including bone) was very low (<5% ID/g). Since free 153Sm3+ has an innate bone-
targeting capability, the observation of low radioactivity in bone suggests that most
of the 153Sm3+ remained in the nanocrystals.
Cheng et al. (2011) conducted a comprehensive in vivo study on polyacrylic
acid-PAA and polyethylene glycol-PEG coated β-Tm3+,Er3+:NaYF4 nanoparticles
on Balb/c mice. A total of 50 healthy female Balb/c mice were injected with
200 μl of 2 mg/mL PEG coated β-Tm3+,Er3+:NaYF4 nanoparticles or PAA coated
β-Tm3+,Er3+:NaYF4 nanoparticles (a dose of 20 mg/kg) and scarified at various
time points after injection (3, 7, 20, 40, and 90 days, five mice per time point). The
blood circulation curves for both nanoparticles were fitted via double exponential
function followed. The first and second phase blood circulation half-lives for PEG
coated β-Tm3+,Er3+:NaYF4 nanoparticles were 5.1 ± 2.5 min and 13.1 ± 6.2 min,
respectively, in marked contrast to the very short blood half-lives of PAA coated
β-Tm3+,Er3+:NaYF4 nanoparticles at 0.13 ± 0.11 min and 3.5 ± 0.4 min. The pro-
longed blood circulation time of PEG coated β-Tm3+,Er3+:NaYF4 nanoparticles
was attributed to the biocompatible PEG coating on the nanoparticle surface and
consistent to its improved stability in physiological solutions. However, the
nanoparticle residues would stay inside the mouse body for long periods of time.
Within 90 days, the liver uptake of both types of nanoparticles decreased by
approximately 50%; the spleen uptake of PAA coated β-Tm3+,Er3+:NaYF4 nanopar-
ticles decreased by approximately 40% from an extremely high level, while that
of PEG coated β-Tm3+,Er3+:NaYF4 nanoparticles failed to show a drastic change.
Compared with PAA coated β-Tm3+,Er3+:NaYF4 nanoparticles, PEG coated
β-Tm3+,Er3+:NaYF4 showed similar uptake in the liver but significantly reduced
accumulations in the spleen and lung. Interestingly, PEG coated β-Tm3+,Er3+:NaYF4
nanoparticles seemed to accumulate more to a lower extent in other organs, where
PAA coated β-Tm3+,Er3+:NaYF4 nanoparticles could not accumulate due to the
rapid reticuloendothelial system clearance. No apparent toxic effect of the
nanoparticles to treated Balb/c mice was observed in a relatively large-scale time-
course toxicology study.
◄
Fig. 5.2 In vivo up-conversion luminescence (UCL) imaging of Kunming mice after intravenous
injection with UCM (150 mg Yb/kg) at different time points (power density = 150 mW/cm2 on the
surface of mouse). Row 5 and 6: UCL images of dissected mouse and organs, respectively, after
intravenous injection with UCM for 0.5 h (power density = 100 mW/cm2 on the surface of dis-
sected mouse and organs): 1 heart, 2 liver, 3 spleen, 4 lung, 5 kidney, 6 stomach, 7 intestines. Xing
et al. (2012) with permission from the Elsevier
a 140 b
0.5 h 140
Feces
120 24 h
7d 120 Urine
30 d
Yb excretion(µg/g)
Yb uptake (%ID/g)
100
100
80
80
60 60
40 40
20 20
0 0
Heart Liver Spleen Lung Kidney 1 2 3 4 5 6 7
Time (day)
Fig. 5.3 (a) Biodistribution of nanoparticles (Yb uptake % ID) in various organs of mice har-
vested after intravenous injection of UCM (150 mg Yb/kg) at different time points (n = 3). (b) Yb
contents in mice excretions (feces and urine) in the first week after intravenous injection (N = 3).
Xing et al. (2012) with permission from the Elsevier
In turn, Cao et al. (Cao et al. 2013) conducted a comprehensive in vivo study on
8 nm PEG coated Yb3+,Er3+,153Sm3+:NaYF4 nanoparticles (153Sm3+ is a radioactive
ion) on Kunming mice (female, 6 weeks old, 20–23 g/animal). The blood circula-
tion of PEG coated Yb3+,Er3+,153Sm3+:NaYF4 nanoparticles followed a typical two
compartment biexponential model also. After a rapid decay with the first-phase
half-life of 0.4 ± 0.1 h for biodistribution, these PEG coated Yb3+,Er3+,153Sm3+:N
aYF4 nanoparticles in circulating blood exhibited a long second-phase half-life of
4.3 ± 0.6 h for elimination. Because of condensed PEG molecules coating on the
nanoparticles, this long blood circulation of the nanoparticles provides a potential
application for enhanced tumor targeting by the enhanced permeability and reten-
tion effect. The biodistribution of PEG coated Yb3+,Er3+,153Sm3+:NaYF4 nanoparti-
cles (10 mCi injection/mouse) was investigated by γ-counter analysis over 48 h at 5
intervals (1, 6, 12, 24, and 48 h) and expressed as the percentage injected dose per
gram tissue. Although SPECT imaging and γ-counter data showed that nanoparti-
cles were mainly taken up by the liver (36.93 ± 5.80%ID/g) and spleen
(25.71 ± 9.40%ID/g), radioactive signals were also observed in other organs such as
the heart, bladder, kidney, and in urine. It was concluded that the SPECT imaging
and the γ-counter analysis clearly indicated that PEG coated Yb3+,Er3+,153Sm3+:N
aYF4 nanoparticles with an ultrasmall size (<10 nm) had a long blood retention time
in vivo and provide a potential route to make these nanoparticles renal excretion for
elimination from the body of small animals.
In can be concluded that studied fluoride nanoparticles do not demonstrate sig-
nificant toxicity in vitro and in vivo. The 10–30 nm nanoparticles are easily internal-
ized by cells. The nanoparticles mainly localized in cytoplasm and did not damage
organelles. The surface modification of the nanoparticles by biocompatible poly-
mers (for example, PEG and PAA) slightly reduces toxic effect toward eukaryotic
cells. Fluoride nanoparticles did not demonstrate any toxic effect in vivo toward
zebrafish embryos and mice. After injection the nanoparticles are uptaken in spleen
and liver predominantly. The circulation half-life of polymer coated nanoparticles is
around 30 min. It decreases with the increasing of the size of the nanoparticles.
Such good properties and biocompatibility make the fluoride nanoparticles very
promising in different biomedical applications (Semashko et al. 2018; Yu et al.
2016). However, some fluoride nanoparticles behaved differently toward some bac-
teria. Specifically, antibacterial activity of nanomaterials such as YF3 and CaF2 was
clearly observed (Lellouche et al. 2012; Kulshrestha et al. 2016). Indeed bacteria
have higher susceptibility to fluoride ions in comparison to eukaryotic cell (Li et al.
2013). Thus some applications in dentistry are based on the approach that fluoride
nanoparticles exhibit higher antibacterial activity with non-toxic nature.
Based on many modern research data it can be concluded that the fluoride
nanoparticles do not demonstrate any significant toxicity toward eukaryotic cells
and small animals. However, the nature of interaction between nanoparticles and
cells is still a multidimensional issue involving many biochemical processes.
Activation of these processes by nanoparticles can lead to unexpected effect such
as activation of cancer cells growth (Huang et al. 2010; Cui et al. 2012; Unfried
et al. 2008; Jiang et al. 2008; Liu et al. 2014b). In this case, the nature of interac-
tion of nanoparticles with eukaryotic cells can be based on interactions between
nanoparticles and transmembrane signal receptors. In particular, this interaction
depends on many factors including size and shape of the nanoparticles, chemical
composition, and reactivity (Nel et al. 2009). The above cited works (Huang
et al. 2010; Cui et al. 2012; Unfried et al. 2008; Jiang et al. 2008; Liu et al.
2014b) are devoted to studying of interactions of silica, gold nanoparticles, and
carbon nanotubes with eukaryotic cells. In addition to the above-mentioned
nanomaterials the work by Semashko et al. (2018) proposed a theory that the tiny
rare-earth fluoride nanoparticles (PrF3 and LaF3) activate the tumor cell growth
via electrical polar interactions. Indeed, in this work it was reliably revealed that
A549, SW837, and MCF7 cancer cells exposed by PrF3 or LaF3 nanoparticles
demonstrated cell overgrowth, especially with the SW620 cell line at 5 mM. The
WST viability assay histograms of three different cancer cell lines (A549,
SW837, and MCF7) treated with different concentrations of PrF3 and LaF3
nanoparticles in biological media are shown in Fig. 5.4a. At higher concentration
(5 mM), for both RE suspensions, an ascending growth for all cell lines was
obtained (Fig. 5.4b, c).
This work is based on the idea that these rare-earth fluoride nanoparticles may
interact with specific domains such as metal ion-dependent adhesion sites (MIDAS),
adjustment to MIDAS (ADMIDAS), synergistic metal ion binding sites (SyMBS),
and ligand adhesion binding site (LABS), located in the ανβ3 subunit or other integrin
subunits. These processes occur via electrical dipole interactions (Dong et al. 2012;
Dickeson et al. 1998). Indeed, fluoride anions are the most reactive electronegative
elements, and the mean radii extension of the unscreened 4f electronic configuration
of La and Pr trivalent ions is relatively large, which leads to high electric surface
charges. It was suggested that the interaction occurs between the nanoparticles and
LABS of integrins on a cell. The interaction between nanoparticles and transmem-
brane signal receptors may activate the cancer cell growth. Herein, tiny size (<10 nm)
LaF3 and PrF3 nanoparticles in DMEM+FBS suspensions stimulated the tumor cell
a 2.5
b
A549 SW837 MCF7
2.0
1.5
Viability
1.0
0.5
0.0
CTRL 5mM 1mM 0.5mM 5mM 1mM 0.5mM
PrF3 LaF3
c d
A 549 SW 837
pAKT pAKT
pERK1/2 pERK1/2
Tubulin Tubulin
CTRL LaF3 PrF3 CTRL PrF3 LaF3
Fig. 5.4 (a) WST viability assay histograms of three different cancer cell lines (A549, SW837,
and MCF7) treated with different concentrations of PrF3 and LaF3 nanoparticles in biological
media. (b) AFM image of a single A549 cancer cell. (c) AFM image of a divided A549 cancer cell
in RE-nanoparticles in DMEM+FMS. (d) Western blot phosphorylation analysis of the A549,
SW837 cells with AKT and ERK pathways (Semashko et al. 2018). Open-access authors retain the
copyrights of their papers, and all our articles are distributed under the terms of the Creative
Commons Attribution License, which permits unrestricted use, distribution, and reproduction in
any medium, provided that the original work is properly cited
growth in three different human cell lines (A549, SW837, and MCF7). The size dis-
tribution of nanoparticles, activation of AKT and ERK signaling pathways, and via-
bility tests pointed to mechanical stimulation of ligand adhesion binding sites of
integrins and EGFR via a synergistic action of an ensemble of tiny size nanoparticles.
Specifically, direct activation of AKT and ERK signaling pathways was confirmed
by specific antibodies and Western blot assays in the A549 and SW837 cell lines
grown in DMEM+FBS with 5 mM of LaF3 and PrF3 nanoparticles for 24 h. High
phosphorylation activity of ERK1/2 and AKT in treated cells as compared to control
cells was obtained (Fig. 5.4d).
The process of tumorigenesis via activation of AKT and ERK pathways is sche-
matically shown in Fig. 5.5.
It seems that although fluoride nanoparticles demonstrate low toxicity toward
eukaryotic cells, the nature of interaction between fluoride nanoparticles and cells is
still a matter of deep concerns.
5.2.2 Antioxidant Properties of Fluoride Nanoparticles
Reactive oxygen species can cause serious damage to an organism due to oxidative
stress (Finkel and Holbrook 2000; Kannan and Jain 2000; Mittler 2002; Celardo
et al. 2011; Popov et al. 2018). Antioxidant therapy is the novel frontier to prevent
Fig. 5.5 AKT and

ERK1/2 signal
transduction pathways
activated by
RE-nanoparticles via
EGFR stimulation. EGFR
is activated only by tiny
size of ~5 nm nanoparticles
(Semashko et al. 2018).
Open-access authors retain
the copyrights of their
papers, and all our articles
are distributed under the
terms of the Creative
Commons Attribution
License, which permits
unrestricted use,
distribution, and
reproduction in any
medium, provided that the
original work is properly
cited
and treat an impressive series of severe human diseases, and the search for ade-
quate antioxidant drugs is fervent (Celardo et al. 2011). Cerium oxide nanoparti-
cles (nanoceria) are redox-active owing to the coexistence of Ce3+ and Ce4+oxidation
states and to the fact that Ce3+ defects and the compensating oxygen vacancies are
more abundant at the surface (Celardo et al. 2011; Popov et al. 2018). However, in
Shcherbakov et al. (2015) it was proposed that CeF3 nanoparticles demonstrate
antioxidant activity via the same mechanism as CeO2 nanoparticles do. Moreover
in this work the antioxidant activity of CeF3 and CeO2 nanoparticles was com-
pared. It was revealed that CeF3 and Tb3+:CeF3 nanoparticles are capable of hydro-
gen peroxide inactivation in a 0.08- to 2.5-mM concentration range of the
nanoparticles. In the experiment ST cells were treated with H2O2 and CeF3 nanopar-
ticles, H2O2 and Tb3+:CeF3 nanoparticles, and H2O2 and CeO2 nanoparticles. The
both untreated ST cells (100% survival) and H2O2-treated ST sells (~65% survival)
served as control. The maximum protective ability of CeF3, Tb3+:CeF3, and citrate
stabilized CeO2 nanoparticles was achieved into 0.078–2.5 mM concentration
range. However, both CeF3 and Tb3+:CeF3 nanoparticles displayed notably higher
antioxidant activity comparing with citrate stabilized CeO2 nanoparticles.
5.3 Photodynamic Therapy
Photodynamic therapy (PDT) is a treatment that uses a drug, called a photosensi-

tizer (PS) or photosensitizing agent, a particular type of light, and oxygen. After
dissolution into oxygen-containing medium photosensitizers exposed to a specific
wavelength of light can create reactive oxygen species which can cause irreversible
damage to living organisms. However, this combination of PSs, light, and oxygen
can be used therapeutically. Indeed, the PS molecules collected into the tumor can
kill it under light exposure (Dolmans et al. 2003; Bekah et al. 2016). There is a
broad list of different PSs differing between each other at least by physicochemical
properties, toxicity, and PDT efficiency. At the same time, the PSs can be commonly
classified into organic (porphyrin, chlorin, phthalocyanine) (Lucky et al. 2015a) and
inorganic (TiO2, ZnO) nanoparticles (Pelaez et al. 2012; Lipovsky et al. 2009).
Usually the PSs are activated via ultraviolet (UV) or visible light which cannot
penetrate through tissues deeply (<1 mm for UV and < 1 cm for visible light).
However there is a possibility of indirectly activating the PS by luminescent
nanoparticles using light at wavelengths that the PS normally does not absorb. There
are first and second biological windows (700–950 nm and 1000–12,000 nm, respec-
tively) which are classified as spectral regions where tissues are partially transparent
(Rocha et al. 2014a). In turn, X-ray irradiation has weak tissue absorption. In order
to enlarge PDT depth two approaches are proposed: up-conversion and down-con-
version nanoparticles which can convert infrared (IR) light (up-conversion) or X ray
(down-conversion) into visible or UV light. These types of light are capable of acti-
vating the PS conjugated to the surface of the nanoparticles (Kamkaew et al. 2016).
The mode of action of nanoparticles-based PDT is schematically shown in Fig. 5.6.
The energy transfer efficiency from the nanoparticles to the PS is a very critical
parameter requiring very good overlap between the emission peaks of the nanopar-
ticles and the absorption or excitation peaks of the PS. For this approach the most
effective energy transfer occurs via Forster resonance energy transfer (FRET)
(Cooper et al. 2014; Förster 1960; Selvin 2000).
Infrared irradiation Activation of

in case of up conversion photosensitizer
nanoparticles Shell loaded with and singlet oxygen
photosensitizer generation
1
O2
Or Visible light
1
O2
Up conversion or
Down conversion
nanoparticle
O2
1
X ray in case of
down conversion
nanoparticles
Fig. 5.6 Functional principle of photodynamic therapy based on up-conversion or down-conver-

sion nanoparticles (by authors)
The nanoparticles for PDT are usually comprised of host materials, embedded
with transition metal, actinide, or rare earth ions. The most promising ions are Yb3+,
Er3+, and Tm3+ (Lucky et al. 2015a) for up-conversion and Ce3+ and Tb3+ for down-
conversion. For up-conversion nanoparticles the most usable hosts are NaYF4 and
NaLnF4 (Ln, lanthanides). These hosts demonstrate low phonon energy (for exam-
ple, 360 cm−1 and 290 cm−1 for NaYF4 and NaLaF4, respectively) and as conse-
quence low multiphonon decay probability (Nie et al. 2017). On the other hand
down-conversion nanoparticles which are capable of converting X-ray into visible
light are highly relevant for the so-called hybrid radiation-PDT (Bekah et al. 2016).
Usually down-conversion nanoparticles for such applications are based on Ce3+ or
Tb3+ emission overlapping with absorption or excitation peaks of PS such as chlorin
(Bekah et al. 2016).
Usually PS molecules are delivered intravenously. They are collected in inflamed
or malignant areas due to the enhanced permeability and retention effect. There is
some suggestion that such “PS + nanoparticle” composites can be maintained for a
longer time in the body in comparison with traditional organic PS. This feature of
nanoparticles can enhance the therapy efficiency (Bekah et al. 2016).
5.3.1 U
p-Conversion Fluoride Nanoparticles
for Photodynamic Therapy
For inorganic PSs such as TiO2 and ZnO the UV activation is required (Sanders
et al. 2012). In Hou et al. (2015) the photodynamic effect is achieved by using
(Yb3+,Tm3+:NaYF4@ Yb3+:NaGdF4core@shell)@TiO2core@shell nanocomposites.
The obtained samples with good dispersibility emit light into UV/visible regions
under 980 nm NIR laser excitation. The UV emission peaks of Tm3+ ions can be
derived from 1I6–3F4 (348 nm) and 1D2–3H6 (365 nm) radioactive transitions. The
visible emission can be assigned to 1D2–3F4 (453 nm) and 1G4–3H6 (480 nm) transi-
tions of Tm3+ ions. Such system is capable of killing cancer cells via mitochondria-
involved apoptosis pathway. In Zhang et al. (2015) folic acid (FA)-targeted
Yb3+,Tm3+:NaYF4@SiO2@TiO2 nanocomposites (FA–Gd–Si–Ti nanoparticles)
were constructed for both in vivo magnetic resonance imaging (MRI) and near
infrared (NIR)-responsive inorganic PDT, in which TiO2 component could be
excited by NIR light due to the up-conversion luminescence performance of
Yb3+,Tm3+:NaGdF4 converting NIR to UV light. The results showed that the as-
prepared FA–Gd–Si–Ti nanoparticles had good biocompatibility in vitro and
in vivo. Moreover, MR study indicated that FA–Gd–Si–Ti nanoparticles were good
T1-weighted MRI contrast agents with high longitudinal relaxivity (T1) of
4.53 mM−1 s−1, also in vivo MRI of nude mice showed “bright” signal in MCF-7
tumor. Under the 980-nm laser excitation at the power density of 0.6 W/cm2 for
20 min, the viability of HeLa and MCF-7 cells incubated with FA–Gd–Si–Ti
nanoparticles could decrease from about 90% to 35% and 31%, respectively.
Furthermore, in vivo PDT of MCF-7 tumor-bearing nude mice model showed that
the inhibition ratio of tumors injected with FA–Gd–Si–Ti nanoparticles reached up
24 980 nm laser 4
F5/2 Tn
excitation
22 4
H11/2
20 4
S3/2 S1
18
Energy (103 cm-1)
4
F9/2
16
14 4
I9/2
12 T1
4
I11/2
Singlet
10 4
F5/2 660 nm
660 nm oxygen
8 1
O2
6
4
I13/2
4
2 S0
F7/2
4
4
I15/2
0 O2
3
Yb3+ Oxygen
Er3+
Energy transfer
Fig. 5.7 The schematic illustration of NIR-triggered photodynamic therapy using Yb3+/Er3+:NaYF4
up-conversion nanoparticles (by authors)
to 88.6% after 2-week treatment compared with that of nude mice in control group.
In Dou et al. (2015) the Yb3+,Tm3+:NaYF4@ZnO core@shell nanoparticles demon-
strate PDT efficiency toward breast cancer cells (MDA–MB–231 and 4 T1). In
Lucky et al. (2015b) the system TiO2@ Yb3+,Tm3+:NaYF4 nanoparticles proved its
efficiency for in vitro and in vivo PDT.
Probably the majority of publications are devoted to the systems based on organic
PS and up-conversion nanoparticles. In Kostiv et al. (2017) aluminum
carboxyphthalocyanine-conjugated up-conversion Yb3+, Er3+NaYF4@SiO2 nanopar-
ticles are studied. A schematic illustration of NIR-triggered PDT is shown in
Fig. 5.7. The bare nanoparticles demonstrate intensive red emission upon 980 nm
excitation. But after modification with SiO2, branched polyethyleneimine, and PS
(aluminum carboxyphthalocyanine) the emission completely vanished, indicating
efficient energy transfer from the nanoparticles to PS, which leads to the generation
of singlet oxygen (1O2).
The therapeutic efficiency of such PS + Yb3+, Er3+:NaYF4 nanoparticles was
tested in a pilot study lasting for 30 days on the outbred athymic nude mice with
subcutaneously growing human mammary carcinoma (MDA–MB–231 cell line).
Intratumorally injected PS + Yb3+, Er3+:NaYF4 nanoparticles after irradiation with
980 nm excitation laser caused the development of necrotic areas in all the five
tested animals. Bare nanoparticles did not show photodynamic effect under the
same conditions. In Qian et al. (2009) the system based on the mesoporous-silica-
coated up-conversion Yb3+, Er3+:NaYF4 nanoparticles showed its efficiency. In this
work the photosensitizer, zinc phthalocyanine, is incorporated into the mesoporous
silica. The photosensitizers encapsulated in silica are protected from degradation in
the harsh biological environment. The efficiency of this system was proved via a
special dye called 9,10-anthracenediyl-bis (methylene)dimalonic acid (ABDA).
The ABDA reacts with 1O2 to form an endoperoxide, and the decrease in amount of
ABDA can be estimated by measuring the fluorescence intensity at the wavelength
of 431 nm when excited at 380 nm.
The energy transfer processes in the system of core/shell Yb3+, Tm3+:NaYF4@
NaYF4 nanoparticles conjugated with riboflavin PS are studied in detail in the work
by Khaydukov et al. (2016). It is remarkable that the decrease in the luminescence
lifetime of the Tm3+ ions was used to estimate the contribution of the FRET pro-
cesses into the energy transfer from a donor to an acceptor. In particular, it was
determined that FRET process was present in case of 1G4–3H6 (475 nm) and 1D2–3H6
(360 nm) transitions of Tm3+ ions and its efficiency was 11% and 3%, respectively.
In case of 1D2–3F4 (450 nm) and 1I6–3F4 (345 nm) transitions the FRET process did
not manifest itself. As it was stated earlier, the efficiency of the FRET energy trans-
fer process depends mainly on the distance between the exited ion and the PS. This
distance should not be greater than 10 nm, which limits the thickness of the shell.
Thus, it is possible to achieve the high FRET energy transfer efficiency optimizing
the architecture of a composite in order to achieve effective energy transfer and
decrease the number of luminescence quenchers.
5.3.2 D
own-Conversion Fluoride Nanoparticles
for Photodynamic Therapy
In this case the PDT serves as additional treatment during the radiotherapy which leads
to a new term “hybrid” radiotherapy-PDT. A study published in 2006 (Chen and Zhang
2006) proposed that the efficacy of radiation therapy could be improved by conjugating
a dye used in photodynamic therapy to a scintillating nanoparticle. For these cases rare
earth doped fluoride nanoparticles showed their high efficiency. One of the common
hosts used for lanthanide-doped down-converting nanoparticles is the lanthanum fluo-
ride (LaF3) as it is optically transparent, has a large band gap and relatively low phonon
energy. As before, if the absorption spectrum of the PS overlaps with the emission
spectrum of the nanoparticle, the dye should produce singlet oxygen as in PDT. The
possibility of using of such nanoscintillators in “hybrid” radiotherapy-PDT approach
has attracted a considerable attention over the last decade (Villa et al. 2015).
As it was mentioned above, effective energy transfer between the exited ion and
the PS in a crucial factor in “hybrid” radiotherapy-PDT. In many “nanoparticle +
PSs” systems the high energy transfer efficiency was achieved. In particular in
Bapat et al. (2018) the LaF3:Ce@ LaF3 and CeF3@LaF3 core@shell nanoparticles
conjugated to deuteroporphyrin IX 2,4-disulfonic which serves as a PS demonstrate
energy transfer efficiency around 80%. In Tang et al. (2015) mesoporous
LaF3:Tb3+nanoparticles conjugated to Rose Bengal PS demonstrate the energy
transfer efficiency around 85%.
In Förster (1960) the system based onCeF3 and verteporfin conjugates is used
toward Panc 1 cells in 0–6 Gy radiation dose range. It is noteworthy that bare CeF3
nanoparticles also demonstrate slight photodynamic activity under 8 keV X-ray exci-
tation. However, it is notably weaker in comparison to “nanoparticle + PSs” system.
In addition, interesting conjugation method was proposed in the work (Tang et al.
2015). It was shown that the ratio between Ln3+and F−(Ln = La3+, Tb3+) in
mesoporousTb3+:LaF3 nanoparticles is significantly less than 3:1 and consequently
there are lots of unsaturated bonds of Ln3+. This property is common for some fluo-
ride and oxide nanoparticles (Shcherbakov et al. 2015; Schubert et al. 2006). Paired
with the mesoporous structure of the nanoparticle this property allows creating stable
composites of these nanoparticles with rose bengal dye due to both loading of the
nanoparticles into the pores of the nanoparticles and electrostatic forces. This system
shows a big FRET efficiency of ~85%. The system based on Tb3+:LaF3 nanoparticles
demonstrate an attractive noninvasive option on glioma cell line by using Tb3+:LaF3
scintillating nanoparticles in combination with photosensitizer, meso-tetra(4-car-
boxyphenyl)porphyrin (MTCP), followed by activation with soft X-ray (80 kVp)
(Chen et al. 2017). Scintillating 25 nm Tb3+:LaF3 nanoparticles were synthesized via
hydrothermal method. The nanoparticles had good dispersibility in aqueous solution
and possessed high biocompatibility. However, significant cytotoxicity was observed
in the glioma cells while the Tb3+:LaF3 nanoparticles with MTCP were exposed
under X-ray irradiation. It was reported that approximately 56.7% of energy can be
transferred from X-ray to the adjacent photosensitizers via Tb3+:LaF3 nanoparticles.
In conclusion there are many systems based on PS and nanoparticles demonstrat-
ing good high energy transfer efficiency. Some systems also showed good therapeu-
tic efficiency in vitro and in vivo. However looking for new highly effective systems
for therapies as well as irradiation regimes is a very challenging task.
5.4 luoride Nanoparticles for Luminescent

F
Thermometry in Biomedicine
Temperature measurement at nanoscale is a very important task for some biological

and medical applications. In case of biological applications measurement of local
temperature response inside living cells upon external chemical and physical stim-
uli is a very important and highly developing branch of knowledge requiring new
approaches in thermometry (Kucsko et al. 2013; Yang et al. 2011; Balabhadra et al.
2015; Wawrzynczyk et al. 2012). Indeed the average size of a eukaryotic cell is in
the range from 10 to 50 μm. Hence temperature measurements inside the single cell
or into its local parts cannot be performed via conventional methods: thermal cam-
eras, medical thermometers, etc. However, these measurements can be performed
via special nanosized thermometers which can be placed into the cell. Temperature-
sensitive nanoparticles serving as such thermometers are very promising tools for
these measurements. Such nanosized thermometers should have some temperature-
dependent physical properties into the so-called physiological temperature range
20–60 °C (Pudovkin et al. 2017). Usually temperature-dependent luminescent prop-
erties are used. This approach is called luminescent thermometry which is based on
the temperature sensitivity of luminescence features of nanomaterials (intensity,
lifetime, band shape, band width, polarization, and spectral position) which are ana-
lyzed and which are used for the temperature measurement (Jaque and Vetrone
2012; Brites et al. 2012). The use of visible or near IR luminescence signals allows
obtaining submicrometer spatial resolution in temperature mapping. The nanoscale
dimensionality of the thermometers and a special modification allow them localiz-

ing in the chosen cellular areas providing targeting.
In this field fluoride nanoparticles are very promising candidates. Indeed the
luminescence features of some rare earth doped fluoride nanoparticles are highly
sensitive to temperature. The developed methods of synthesis allow obtaining
nanoparticles having desirable morphology. The nanoparticles surface can be modi-
fied in order to provide targeting.
Usually in the luminescence thermometry the temperature dependence of the
band shape and luminescence lifetime are used (Dramićanin et al. 2014; Nikolić
et al. 2014; Brites et al. 2018; Bu et al. 2015; Lojpur et al. 2013). In case of the band
shape the temperature sensitivity can stem from an intensity ratio of two emissions
(fluorescence intensity ratio (FIR)) from the thermally coupled electron levels or
from the temperature dependence of multiphonon decay probability. Here, rare
earth fluoride nanoparticles demonstrate the higher temperature sensitivity in com-
parison to rare earth oxide nanoparticles. Indeed conventional fluoride hosts such as
LaF3 and NaYF4 have phonon energy around 300–400 cm−1 (Pudovkin et al. 2017;
Zhou et al. 2014). Some oxide hosts have phonon energy around 1000 cm−1
(Gharouel et al. 2018). This low phonon energy is a great advantage of fluoride host
not only because of the lack of undesirable multiphonon decay, but it also leads to
better temperature sensitivity of some systems based on fluoride hosts. For example
in LaF3 host 3P0 and 3P1 electron states of Pr3+ (Fig. 5.8) have energy gap of ~500 cm−1
(Weber 1968; Caspers et al. 1965).
bulk LaF3:Pr3+(1%) P2
3
P0→3H4
lex=444nm nano LaF3:Pr3+(12%) 1

0
3
P0→3H6
Normalized intensity (a.u.)
1
D2
3
487nm
537nm
523nm
601nm
580nm
672nm
P1→3H5
1
G4
3
P0→3H5
3
F4
P1→3H6
P0→3F2
P0→3F4
3
P1→ H4
P1→3F4
2
P1→3F3
3
H6
3
3
3
3
3
3
H5
3
3
H4
450 500 550 600 650 700 750
Wavelength (nm) Pr 3+
Fig. 5.8 Emission spectrum of the Pr3+:LaF3 microcrystalline powder and the nanoparticles under
444 nm excitation (Pudovkin et al. 2017). The publisher is Hindawi. It is open access. Open-access
authors retain the copyrights of their papers, and all our articles are distributed under the terms of
the Creative Commons Attribution License, which permits unrestricted use, distribution, and
reproduction in any medium, provided that the original work is properly cited
After excitation of 3P0 state of Pr3+ ions 3P0 and 3P1 states share their electronic
populations via Boltzmann law from which the thermal sensitivity is extracted
(Pudovkin et al. 2017; Zhou et al. 2014; Collins et al. 1998). However the tempera-
ture sensitivity of Pr3+-based systems can stem from multiphonon decay as well.
Indeed multiphonon decay from 3P0 to 1D2 states of Pr3+ is observed for some oxide
hosts. The multiphonon decay from 3P0 to 1D2 does not occur. Hence the emission
from 1D2 state is not found at least for LaF3 and NaYF4 hosts. In these hosts the
emission from 1D2 is not observed because of the lack of non-radiative relaxation of
3
P𝑗 to 1D2 due to low cutoff phonon frequency in LaF3 or NaYF4 (350–400 cm−1),
which for example is two times less than the one for YAG (700–865 cm−1). Indeed
to bridge the 3P0–1D2 energy gap, the 9 phonons are required in case of LaF3 and
only 4 or 5 for YAG; thus, the multiphonon relaxation is not observed. On the other
side oxide hosts such as MIPr(PO3)4 (MI=Na, Li, K) and NaPrPO4 have phonon
energy around 1200 cm−1. For such systems multiphonon decay from 3P0 to 1D2 is
clearly observed. Hence the temperature sensitivity of such oxides as MIPr(PO3)4
(MI=Na, Li, K) and NaPrPO4 stems from temperature-dependent multiphonon
decay from 3P0 to 1D2 electron states (Gharouel et al. 2018). Finally in the FIR ther-
mometry based on Boltzmann law the Pr3+:LaF3 system demonstrates the absolute
temperature sensitivity around 160 × 10−4 K−1 at 300 K (Pudovkin et al. 2017). In
turn the FIR thermometry based on multiphonon decay demonstrates the absolute
temperature sensitivity of NaPrPO4 system around 41 × 10−4 K−1 at 300 K which is
notably less than the temperature sensitivity of Pr3+:LaF3. In this case the fluoride
hosts are more attractive in terms of luminescent thermometry.
In particular, the temperature dependence of the luminescence spectra of the
Pr3+:LaF3 nanoparticles is shown in Fig. 5.9.
The increasing of 3P1–3H5 emission and simultaneous decreasing of 3P0–3H5
emission upon warming is a consequence of thermally coupled nature of 3P1 and 3P0
states of Pr3+ ions.
In case of luminescent thermometry there are several classes of materials such as
quantum dots, organic dyes, and rare earth based materials which are utilized for the
measurement of cellular temperature (Jaque and Vetrone 2012; Brites et al. 2012).
In this field the rare earth doped fluoride nanoparticles demonstrate excellent tem-
perature sensitivity, photostability, and brightness. Moreover as it was mentioned
above these materials are significantly less toxic comparing with quantum dots and
some organic dyes (Kattel et al. 2012). One of the pioneer work devoted to acquir-
ing the temperature of a single living cell (Alakshin et al. 2018) demonstrates the
possibility of using up-conversion 18 nm polyethylenimine-capped α-Yb3+,
Er3+:NaYF4 nanoparticles for this purpose. The two-photon process occurs via
energy transfer from Yb3+ ions to the fluorescent Er3+ ions. The green emission con-
sists of distinct bands between 515 and 535 nm (centered at 525 nm) and 535 and
570 nm (centered at 545 nm) emanating from transitions from two excited states
(2H11/2 and 4S3/2, respectively) to the ground state. These two states are in close prox-
imity, essentially separated by only several hundred wavenumbers, leading to a ther-
mal equilibrium governed by the Boltzmann factor. This fact explains the temperature
sensitivity of such nanoparticles. After calibration the nanoparticles were used for
P0→3H4
60000
3
50000
P0→3H6
40000
Intensity (a.u.)
3
30000 P0→3H0
P0→3F4
P1→3H5
P1→3F4
20000
3
3
3
3
10000 100
(K)
150
200
ure
250
rat
0 300
pe
500 550 600 650 700 750
Tem
Wavelength (nm)
Fig. 5.9 Temperature-dependent spectra of the Pr3+:LaF3 nanoparticles, recorded from 80 to

320 K under 444 nm excitation (Pudovkin et al. 2017). The publisher is Hindawi. It is open access.
Open-access authors retain the copyrights of their papers, and all our articles are distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted use, distribu-
tion, and reproduction in any medium, provided that the original work is properly cited
temperature sensing of HeLa cervical cancer cells. The nanoparticles were internal-
ized by the cells after 1.5 h incubation. The temperature of the cell is varied by
changing the applied voltage. The experimental set-up based on confocal micro-
scope was used. The experiment revealed that at room temperature (25 °C), the
cancer cells show an irregular shape, typical of such cells. A subsequent tempera-
ture increase to 35 °C does not produce any relevant changes in its morphology.
However, after increasing the temperature to 45 °C, a small membrane fragment
was observed, which is indicative of cell death. In all the three cases the temperature
was measured correctly.
Another important application of nanoparticles-based luminescence thermome-
try is the photothermal therapy (or hyperthermia). Photothermal therapy (PTT) is a
therapeutic strategy in which the photon energy is converted into heat to cause irre-
versible damage at the cellular level and that could efficiently treat a great variety of
diseases including cancer (Pekkanen et al. 2014). The net effects caused on cancer
tumors during PTTs strongly depend on both the magnitude of the heating as well
as the treatment duration. In this regard, in order to achieve an efficient treatment
and keep the collateral damage at minimum it is extremely necessary to have a tem-
perature reading during PTT. As a consequence, there has been an increasing inter-
est in the design of multifunctional luminescent nanoparticles capable of
simultaneous heating and thermal sensing under single power excitation as they
would constitute significant building blocks toward the achievement of real con-
trolled PTTs as well as subcutaneous studies.
For PTT the efficiency of light-to-heat conversion plays a crucial role. In this
case the rare earth doped fluoride nanoparticles have big advantages among other
nanomaterials. One of the most remarkable properties of some fluoride matrices is
that they can contain high concentration of doping ions. Hence the high efficiency
of light-to-heat conversion can be achieved by using highly doped fluoride nanopar-
ticles. Indeed for highly doped fluoride nanoparticles for some ions such as Pr3+,
Nd3+ the probability of non-radiative transitions due to different luminescence
quenching processes is much higher comparing with fluoride nanoparticles having
low concentration of doping ions. High concentration of Pr3+ or Nd3+ ions leads to
high probability of cross-relaxation processes which are also responsible for light
into heat conversion. However in such highly doped systems the luminescence sig-
nal is still observable. If this luminescence signal is temperature-dependent the tem-
perature reading can be achieved by means of luminescent thermometry.
Consequently the simultaneous heating and temperature reading can be achieved by
varying of chemical composition and structure of nanoparticles. The schematic
illustration of this approach is shown in Fig. 5.10.
On the other hand one of the main requirements for such system is that the lumi-
nescence nanothermometers should operate into the biological window (Cerón et al.
Infrared excitation
Temperature control via

Heat induced by Temperature dependent
light and Luminescence signal
Infrared luminescence
nanoparticles
Subtissue area where the

nanoparticles are
injected
Tissue
Heat
Fig. 5.10 Functional principle of subcutaneous heating and thermal sensing in the second
biological-window (by authors)
2015). Such system was developed by Ximendes et al. (2016). For the purpose of
subtissue temperature reading and controlled heating the Nd3+:LaF3@
Yb3+:LaF3core@shell nanoparticles were used. The Nd3+:LaF3 shell serves as a
heater under 808 nm CW laser excitation. For high concentration of Nd3+ ions
(~10%) in the shell, the non-radiative de-excitations become dominant over radia-
tive ones and, consequently, the shell becomes a heater surrounding the core.
Simultaneously, energy transfer processes at the core/shell interface lead to relax-
ation of Nd3+ ions down to their ground state and to a simultaneous excitation of
Yb3+ ions from the ground state to the 2F5/2 excited state, from which infrared emis-
sion is produced. The temperature sensing of such system is based on temperature
dependence of the intensity ratio Δ = INd/ΔIYb where INd and IYb are defined as the
emitted intensities at 1350 (Nd3+: 4F3/2–4I13/2) and 1000 nm (Yb3+:2F5/2–2F7/2), respec-
tively. The relative thermal sensitivity of such nanoparticles defined by Pudovkin
et al. (2017) is Sr = 0.74 ± 0.02% °C−1 at 20 °C. In Rocha et al. (2014b) the possibil-
ity of Nd3+:LaF3 nanoparticles to accomplish heating and simultaneous thermal
sensing ex vivo on chicken breast is demonstrated. The temperature sensitivity
stems from the FIR 887 and 866 nm emission lines generated by the high and low
energy Stark levels of the 4F3/2 state of Nd3+, respectively.
The very interesting thermometry approach was proposed in Jang et al. (2014).
In this work, Nd3+: LaF3 nanoparticles have been used to provide the optical con-
trol over subtissue temperature in a single-beam plasmonic-mediated heating
experiment. In this experiment, gold nanorods were used as nanoheaters while
thermal reading was performed by the Nd3+: LaF3 nanoparticles. The possibility
of a real single-beam-controlled subtissue hyperthermia process was, therefore,
pointed out. The Nd3+: LaF3 nanoparticles displayed a remarkable luminescence
thermal sensitivity with a value of ±2 °C. Temperature variations affected both
the spectral position of emission lines as well as the relative intensities of the
different lines.
In conclusion, important advantages such as low phonon energy, low toxicity,
possibility of operating in a broad spectral range, and possibility of creating highly
doped systems make fluoride nanoparticles very promising in thermometry for bio-
medical applications (Rocha et al. 2013).
5.5 Bioimaging
Luminescent imaging is a noninvasive diagnostic manner for visualizing various bio-

logical processes, which might be crucial for the pathogenesis and progression of
many diseases (Dong et al. 2015). Moreover, benefiting from the high-sensitivity and
capacity of real-time monitoring, luminescence imaging has been adopted as an
excellent approach to detect the morphological, anatomical, and physiological details
in tissues as well as the full range of biosamples from cells to animals (Dong et al.
2015; Hao et al. 2013; Zhang et al. 2010; Yu et al. 2017). Conventional biomarkers
such as quantum dots and organic biomarkers have some disadvantages: poor photo-
stability, low signal-to-noise ratio, cross-talked emissions, limited penetration depth,
and prominent biotoxicity (Liu et al. 2014c; Jin et al. 2017). These facts hinder their
generalization in bioimaging studies (Wolfbeis 2015; Dong et al. 2015). As it was
mentioned above some rare earth doped fluoride nanoparticles operate into “biologi-
cal windows.” This fact allows using them as bioimaging probes. Such probes based
on the rare earth doped fluoride nanoparticles have apparent advantages comparing
with traditional bioimaging probes usually operating in UV and/or visible spectral
range (Kuznecov et al. 2006; Wolfbeis 2015; Dong et al. 2015). Moreover the pos-
sibility of surface modification paves the way toward precise targeting.
In the field of bioimaging the cellular imaging is a high developing scientific
field offering an intuitionistic visualization for the physiological processes at the
cellular or subcellular level (Dong et al. 2015). In this case the luminescent nanopar-
ticles are internalized by cells via endocytosis process (Rocha et al. 2014a). In par-
ticular, in Ren et al. (2011) folic acid coupled Ce3+, Tb3+, Gd3+:NaYF4 nanoparticles
were successfully used in bioimaging of C6 glioma cells. In vitro bioimaging stud-
ies were conducted in live C6 glioma cells which were treated by folic acid conju-
gated with Ce3+, Tb3+, Gd3+:NaYF4 nanoparticles for 12 h. As it is shown in Fig. 5.11,
the comparison between fluorescence and bright field images suggested the signal
distributions strongly correlated with the C6 glioma cells, proving the fine attach-
ment of nanoparticles on the surface of cells. No conspicuous cell death was
observed, which further indicated that the nanoparticles were not cytotoxic to the
cells. The results demonstrated that the Ce3+, Tb3+, Gd3+:NaYF4 nanoparticles could
be used as an efficient probe for fluorescence bioimaging.
In Celardo et al. (2011) Yb3+, Er3+:NaYF4 nanoparticles were used for imaging of
HeLa cells. The successful conjugation of antibody to the nanoparticles was found
to lead to the specific attachment of the nanoparticles onto the surface of the HeLa
cells, which further resulted in the bright green up-conversion fluorescence from the
NP-labeled cells under 980 nm near-infrared (NIR) excitation and enabled the
a b
Fig. 5.11 (a) Brightfield and (b) fluorescence images of C6 glioma cells incubated with folic acid
coupled with Ce3+, Tb3+, Gd3+:NaYF4 nanoparticles. The fluorescence image was collected at 500–
560 nm. The scale bar is 20 μm. Figure is taken from Ren et al. (2011). The publisher is Hindawi.
It is open access. Open-access authors retain the copyrights of their papers, and all our articles are
distributed under the terms of the Creative Commons Attribution License, which permits unre-
stricted use, distribution, and reproduction in any medium, provided that the original work is prop-
erly cited
fluorescent imaging and detection of the HeLa cells. However, the use of visible
emitted wavelengths restricts their real application in bioimaging due to their short
tissue penetration depths (caused by tissue scattering and specific absorptions of
tissue components such as melanin and hemoglobin). To overcome this problem,
other emitting ions could be used, with excited fluorescence bands lying within the
“biological window.” In the work by Dong et al. (2011) remarkable two-photon
excited fluorescence efficiency in the “biological window” of Yb3+,Tm3+:CaF2
nanoparticles was demonstrated. On the basis of the strong Tm3+ ion emission (at
around 800 nm), tissue penetration depths as large as 2 mm have been demon-
strated, which are more than four times those achievable based on the visible emis-
sions in comparableYb3+, Er3+:CaF2 nanoparticles.
For example in Rocha et al. (2014a) the Nd3+:LaF3 doped nanoparticles operating
into the first and second biological windows were used for in vitro cellular imaging.
Excitation at 808 nm allows obtaining three main emission channels of Nd3+ ions:
4
F3/2 → 4I9/2,4F3/2 → 4I11/2, and 4F3/2 → 4I13/2 that lead to emissions at around 910, 1050,
and 1330 nm, respectively. By systematically comparing the relative emission
intensities, penetration depths and subtissue optical dispersion of each transition it
was proposed that optimum subtissue images based on Nd3+:LaF3 nanoparticles are
obtained by using the 4F3/2 → 4I11/2 (1050 nm) emission band. For in vitro experi-
ments HeLa cancer cells were incubated with Nd3+:LaF3 nanoparticles. After incu-
bation, the HeLa cells were examined in a confocal microscope illuminated with a
Ti:Sapphire CW laser tuned to 808 nm as the pump source and the fluorescence was
detected with an InGaAs array (1000–1200 nm range). The 808 nm laser excitation
radiation was focused with a 50× microscope objective down to a 1 μm spot size. As
can be observed, the intracellular fluorescence displays the characteristic emission
band of neodymium ions centered at 1050 nm.
Rare earth doped fluoride nanoparticles demonstrated applicability in bioimaging
of small animals after injecting into the animal intravenously (Wolfbeis 2015). In this
case the working into “biological window” is crucial because of strong attenuation of
the blue-green (short wavelength) emission by tissues (Jiang et al. 2008). Moreover
excitation into the “biological window” reduces or even excludes undesirable auto-
fluorescence. In particular in Villa et al. (2015) Nd3+:SrF2 nanoparticles demonstrated
excellent bioimaging potential. For all the in vivo experiments, 50 μL of a solution of
Nd3+:SrF2 nanoparticles dispersed in phosphate-buffered saline at a concentration of
0.3 wt.% were intravenously injected through the retro-orbital sinus into 9-week-old
female CD1 mice. The optical images of a mouse are shown in Fig. 5.12.
Fluorescence in vivo imaging was performed by using a Peltier-cooled AsGaIn
camera (XEVA 1.7, Xenics Corp.) coupled to a C-Mount objective. The excitation
intensity of 808 nm irradiation was always kept below 1 W/cm2. It was proved that
among emission bands centered at around 900, 1060, and 1340 nm (4F3/2–4I9/2, 4F3/2–
4
I11/2 and 4F3/2–4I13/2, respectively) (Henderson and Imbusch 1989) the emission at
1340 nm demonstrates lack of autofluorescence. In most hosts this emission is weak
however in SrF2 this emission is strong. For these reasons the Nd3+:SrF2 nanoparti-
cles were chosen. The nanoparticles were collected predominantly in liver and
spleen (Fig. 5.13).
Fig. 5.12 Left column:

optical images of a mouse
food pellet and an
Eppendorf tube containing
a colloidal solution of
Nd:SrF2 nanoparticles (a),
a living mouse after
intravenous administration
through the retro-orbital
venous sinus of 50 μL of a
colloidal solution of
Nd:SrF2 nanoparticles in
phosphate-buffered saline
(d), and the same mouse
after being sacrificed and
opened to obtain direct
access to organs (g).
Middle column:
corresponding fluorescence
images of the three
systems obtained under
808 nm illumination and
recording the fluorescence
in the 900–1500 nm range.
Right column: fluorescence
images were also obtained
under 808 nm but in this
case the fluorescence
intensity was recorded in
the 1300–1500 nm range.
Villa et al. (2015) with
permission from the
Springer Nature
It was demonstrated that 1340 nm emission band of Nd3+: SrF2 nanoparticles

can be used to produce autofluorescence-free, high-contrast in vivo fluorescence
images.
Up-conversion nanoparticles are very useful tool for cellular imaging. The exci-
tation around IR spectral range leads to autofluorescence-free images. However as
it was mentioned above the visible light is not suitable for in vivo imaging. On the
other hand there are systems based on Yb3+/Tm3+ ion pair. These systems are capa-
ble of operating into the biological window (Cheng et al. 2011; Huang et al. 2010;
Cui et al. 2012) as well. For example Yb3+, Tm3+:NaLuF4 nanoparticles demonstrate
strong emission band at 800 nm (3H4–3H6 transition of Tm3+) after excitation at
a Optical
900–1,500 nm
1,300–1,500 nm
Liver Spleen Intestine &
stomach Pancreas Brain Heart &
lungs Kindney
b c
Emitted intensity (a.u.)
Pancreas
Pancreas
Liver
Liver
Kidneys
Kidneys
Brain
Brain
Spleen
Intestine
Lungs
Stomach
Spleen
Intestine
Lungs
Stomach
Heart
Heart
Fig. 5.13 (a) Optical images and fluorescence images in the 900–1500 and 1300–1500 nm spec-
tral detection ranges of the organs extracted from a sacrificed mouse 1 h after an intravenous injec-
tion of Nd:SrF2 nanoparticles. (b) 900–1500 nm integrated fluorescence intensity obtained from
the different organs. (c) 1300–1500 nm integrated intensity obtained from the different organs. In
all cases, the integrated fluorescence intensity has been normalized by the organ weight. Villa et al.
(2015) with permission from the Springer Nature
980 nm (2F7/2–2F5/2 transition of Yb3+) (Yang et al. 2012). These nanoparticles dem-
onstrated their applicability in bioimaging of mice. Near-infrared to near-infrared
(NIR-to-NIR) up-conversion emission of polymer-coated Yb3+, Tm3+:NaLuF4
nanoparticles can penetrate >1.5 cm tissue of pork with high contrast.
It can be concluded that the rare earth doped fluoride nanoparticles serve as opti-
cal imaging agents for many scientific purposes very successfully. Cellular imaging
is performed mostly via up-conversion nanoparticles into visible spectral range
under NIR excitation. Small animals imaging is performed into biological windows
fully via either up-conversion or down-conversion nanoparticles. The chemical
composition of nanoparticles can be adopted in order to obtain high-contrast
autofluorescence-free images at suitable wavelengths by varying hosts and/or ion(s).
The suitable surface modification with antibodies can provide targeting.
5.6 Fluoride Nanoparticles in Dentistry
The antibacterial activity of some fluoride nanoparticles and at the same time nontoxic
nature is a very promising property for some medical applications. Fluoride nanoma-
terials have some important advantages comparing with some counterparts in terms of
antibacterial activity. In particular, such materials as antibiotics, metal nanoparticles,
and quaternary ammonium compounds are associated with concerns about antibiotic
resistance, complex chemical synthetic process, environmental pollution, high cost,
low heat resistance, high decomposability, and short life expectancy. So, there is
always a need for developing new antibacterial agents which can overcome the disad-
vantage of these traditional methods (Bala et al. 2017; Wang et al. 2013; Semashko
et al. 2018). The fluoride nanoparticles providing slow fluoride ions release are very
promising candidates. For example, in Lellouche et al. (2012) the antibacterial activity
of YF3 nanoparticles toward E. coli and S. aureus is related to release of fluoride
anions. In this work the YF3 nanoparticles were deposited on catheter wall. These YF3
NP-modified catheters were investigated for their ability to restrict bacterial biofilm
formation. Antimicrobial activity was observed at millimolar concentrations and was
strongly dependent on particle size for both bacteria, with smaller sized nanoparticles
having more efficient antibacterial activity than larger nanoparticles. In Kulshrestha
et al. (2016) the antibacterial activity of CaF2 nanoparticles toward Streptococcus
mutans was studied. Streptococcus mutans is known to produce biofilm which is one
of the primary causes of dental caries. In vitro studies revealed 89% reduction in bio-
film formation at 4 mg/mL of CaF2 nanoparticles.
As it was mentioned above in spite of the antibacterial activity of some fluoride
nanoparticles they are still low toxic toward eukaryotic cells. This remarkable anti-
bacterial activity with nontoxic nature is highly demanded in dentistry firstly.
In dentistry fluoride nanoparticles are of importance primarily as perspective F
reservoirs (Bapat et al. 2018; Silva et al. 2015). In the majority of publication
devoted to F reservoirs the CaF2 nanoparticles and composites based on CaF2 are
studied. The CaF2 nanoparticles exhibited increased reactivity and solubility com-
pared with its macro counterpart (Bala et al. 2017); because of the higher solubility
of nano-CaF2 compared with macro-CaF2, an increased amount of fluoride absorp-
tion in the apatite product occurred. These unique properties are highly required in
caries prevention (Bapat et al. 2018). Indeed it was shown in Sun and Chow (2008)
that CaF2 nanoparticles can act as anticaries agents by increasing the labile fluoride
levels in fluids of the oral cavity, enhancing tooth remineralization. The authors also
concluded that CaF2 nanoparticles are very useful for reduction of dentin permeabil-
ity (Sun and Chow 2008). In the work by Xu et al. (2008), the authors incorporated
CaF2 nanoparticles into dental resin in order to develop stress-bearing, F-releasing
nanocomposite. The composite containing 20% CaF2 had a cumulative F− release of
2.34–0.26 mmol/L at 10 weeks. The initial F− release rate was 2 mg/(h cm2), and the
sustained release rate after 10 weeks was 0.29 mg/(h cm2). Flexural strength
(mean ± SD; n = 6) was 110 ± 11 MPa for the composite containing 30% CaF2 and
35% whiskers by mass.
The composite based on CaF2 is also much required in dentistry. In particular,
synthetic hydroxyapatite (HAp) has been used extensively as a bone implant
material. Apatite containing fluoride, the “CaF2-like” salts are of significant interest
in dentistry for their roles as labile fluoride reservoirs in caries prevention. Fluoride
HAp with formulation of Ca10(PO4)6(OH)2 doped with F− substituting OH−groups
has unique physical and biological properties (Gharouel et al. 2018). In particular
the study (Azami et al. 2011) aimed at preparing biphasic CaF2/FHAp solid solution
through a continuous precipitation method especially for dental applications, which
could be used not only as an osteoconductive material due to the nature of FHAp but
also as a labile F− reservoir for developing potentially more effective F−regimens
and as an agent for use in the reduction of dentin permeability and dental caries
prevention (Silva et al. 2015).
The work (Prentice et al. 2006) evaluated the effects of the addition of YbF3
nanoparticles on the strength and reactivity of commercial glass-ionomer cement.
YbF3 nanoparticles were incorporated into the powder component of Riva SC (SDI
Ltd., Bayswater, Australia) at 1, 2, 5, 10, 15, and 25% by weight. Working and ini-
tial setting times were reduced with the addition of YbF3 nanoparticles. Compressive
strength decreased with the addition of either YbF3 nanoparticles, while surface
hardness was slightly but insignificantly higher at 1–2% nanoparticles and then
decreased with increasing the nanoparticles’ concentrations.
5.7 Fluoride Nanoparticles as MRI Contrast Agents
Magnetic resonance imaging (MRI) is a noninvasive imaging technique which has

exceptional advantages, including deep penetration, high spatial resolution, and
avoidance of radiochemicals. However, the sensitivity of MRI is relatively low
(Zheng et al. 2016). As in early stage diagnosis, the difference between normal and
abnormal regions is usually subtle, and additional contrast is required to highlight
the abnormal region. Contrast agents (CAs), namely exogenous substances that can
shorten the proton relaxation time, are often utilized to improve the image contrast
(Zheng et al. 2016; Dong et al. 2015). Paramagnetic Gd-chelates act as T1CAs,
which are the most frequently used in clinics (Geraldes and Laurent 2009). Super
paramagnetic iron oxide nanoparticles act as T2CAs (Hu and Zhao 2012). The now-
adays trend of MRI development is working under an ultrahigh magnetic field
(>3 T) in order to achieve a better signal-to-noise ratio and better temporal and
spatial resolution (Blow 2009). However, Gd-based CAs are low field CAs, and suf-
fer from reduced efficiencies with increased magnetic field. Iron-based CAs are also
known to have saturated magnetization below 1 T, which limits their efficiencies at
higher magnetic fields (Fries et al. 2014). Therefore, novel contrast agents with high
performance under an ultrahigh magnetic field are required. Trivalent lanthanide
ions that exhibit paramagnetism, such as Tb3+, Dy3+, Ho3+, and Er3+, can enhance
transverse proton relaxation at high magnetic fields via the Curie mechanism (Zheng
et al. 2016; Kattel et al. 2012; Alakshin et al. 2018).
Particularly, Tb3+, Dy3+, Ho3+, and Er3+ based fluoride nanoparticles can serve as
very promising CAs due to low toxicity and high chemical stability. It is worth
noted that rare earth based fluoride nanoparticles have excellent optical properties.
These facts pave the way toward creating of multifunctional nanoparticles.
Specifically, in Zheng et al. (2016) MRI imaging was performed under ultrahigh
magnetic field (>3 T), using polyethylenimine (PEI) coated TbF3 nanoparticles as
T2-weighted MRI CAs in Kunming mice. Here, the dosage was set to be 0.1 mmol Tb/
kg body weight in accordance to the clinically used dosage of Gd-based CAs. After
administration of the TbF3 nanoparticles through tail vein injection, the variation in
signal intensity by T2-weighted MRI was carried out. As compared to those before
injection, the signal intensities in the liver, spleen, and kidneys dramatically
decreased 15 min after injection, suggesting that the nanoparticles had been distrib-
uted in these organs. About 32 h after injection, both the cortices and the medullas
of the kidneys were brighter than they were 15 min after injection, suggesting that
the nanoparticles were gradually eliminated from kidneys.
The work by Ren et al. (2011) demonstrates the possibility of using
Ce3+,Tb3+,Gd3+:NaYF4 nanoparticles on white Sprague-Dawley (SD) rats (3–60 g).
Health SD rats were injected with Ce3+,Tb3+,Gd3+:NaYF4 nanoparticles (4.17 mg/
kg) through tail veins. Three-dimensional T1-weighted images were acquired at the
time points of 1.5, 3, and 48 h after injection of the probes.
As shown in Fig. 5.14c, obvious enhancement of the MR signal intensity was
observed within1.5 h after injection. The maximum relative enhancements were
58%, 36%, and 37% in the liver, spleen, and kidney, respectively. After 3 h after
injection, the MR signal decreased gradually. After 48 h after injection, few probe
MR signals were observed only in the intestinal tract, indicating the almost clear-
ance of the injected probes. All the treated rats had survived for more than 2 months
without any obvious toxicity response.
The β-NaDyF4 nanoparticles as T2CAs suitable at ultrahigh fields (9.4 T) are
studied by Das et al. (2012). These nanoparticles effectively enhance T2 contrast at
9.4 T, which is tenfold higher than the clinically used T2CA (Resovist). Evaluation
of the relaxivities at 3 and 9.4 T shows that the T2 contrast enhances with an increase
in NP size and field strength. Specifically, the transverse relaxivity (r2) values at
9.4 T were ∼64 times higher per NP (20.3 nm) and ∼6 times higher per Dy3+ ion
compared to those at 3 T, which is attributed to the Curie spin relaxation mecha-
nism. These results and confirming phantom MR images demonstrate their effec-
tiveness as T2CAs in ultrahigh field MRI.
In can be concluded that rare earth based fluoride nanoparticles successfully
serve as CAs for in vivo experiments on small animals and cells. The nanoparticles
allow obtaining high contrast MRI image without toxic effect. However, these
nanoparticles are still not clinically approved and further investigations are required.
Fig. 5.14 Series of in vivo 3D-T1 MR images at a few representative time points of different
organs in rats. On the top of arrays (a) were axial images of liver and at the bottom (b) were coronal
images of spleen and kidney. (c) Mean values of relative signal intensity of different organs col-
lected from before and after Ce3+, Tb3+,Gd3+:NaYF4 nanoparticles solution injection (Ren et al.
2011). The publisher is Hindawi. It is open access. Open-access authors retain the copyrights of
their papers, and all our articles are distributed under the terms of the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided
that the original work is properly cited
Before 1.5 h 3h 48 h
4480
a
Intensity (a.u.)
745
c
1.6
Relative signal intensity
1.4
1.2
1
0.67 1.5 3 9 24 48
Time after injection (h)
Spleen Error bars: 95% CI
Kidney
Liver
5.8 Composite Fluoride/Oxide Nanoparticles
Composite nanoparticles combining fluoride nanoparticles like CeF3 and oxide

nanoparticles like ZnO or CeO2 may be promising for biomedical application.
Recently CeF3/ZnO nanoparticles were proposed to use for self-lighted photody-
namic therapy (SLPDT) of cancer (Rimoldi et al. 2016). The SLPDT presents a
novel approach for the treatment of deep cancers by low doses of X-rays, and this
technique extends the traditional PDT to deep cancer tissues, which cannot be
directly reached by visible light. A composite nanostructure reported by authors of
research work (Rimoldi et al. 2016) is based on a nano-CeF3 as scintillator material,
coupled to nano-ZnO, with size in the range of 100–500 nm. ZnO has the ability to
generate ROS and to have photocatalytic activity. Thus as a result of the X-rays
irradiation at low doses the CeF3 nanostructures generate the UV radiation causing
ZnO structures release highly cytotoxic reactive oxygen. Orsi et al. (2018) shows
that these CeF3–ZnO nanoparticles can be used to kill cancer cells with less side
effects as compared with usual X-ray therapy.
Recently the CeO2/CeF3 composite structure was proposed to use for SLPDT in
the similar matter as CeF3/ZnO nanoparticles (Pavlov et al. 2018; Morozov et al.
2018). In this composite nanoparticles cerium dioxide, CeO2, is the subject of inten-
sive studies for biomedical application (see, e.g., Tarnuzzer et al. 2005; Das et al.
2007; Kim et al. 2012; Rubio et al. 2018; Ferraro et al. 2017; Dutta et al. 2016).
The application of cerium dioxide is based on the capacity of ceria to store and
release oxygen. In ceria the reversible oxygen storage is associated with a change in
the oxidation state of cerium between Ce3+ and Ce4+ depending on environmental
conditions (Sun et al. 2012). The releasing oxygen or absorbing oxygen is the most
important for antioxidant or pro-oxidant properties of ceria (Tarnuzzer et al. 2005).
The suggestion of application of CeO2/CeF3 composite nanoparticles for bio-
medical application is based also on enhanced photocatalytic activity of CeO2/
CeF3 nanoparticles reported in a recent work by Xiang et al. (2016). In that work
CeO2/CeF3 composites are synthesized through a facile one-pot hydrothermal
method. Obtained nanoparticles had with different morphologies, and varying
dimensions from 40 to 500 nm. The evaluation of the photocatalytic performance
of synthesized composites was conducted under UV light irradiation by measur-
ing the photocatalytic degradation of methylene blue. The enhancement of the
photocatalytic activity of CeO2/CeF3 polyhedrons was supposed to be attributed to
the formation of the special morphologies, the highly reactive facets exposed and
the effective separation of photo-generated electron-hole pairs at hetero-junction
interfaces.
The different way of synthesis of CeO2/CeF3 composite nanoparticles was used
in the recent work by Pavlov et al. (2018). To obtain CeO2/CeF3 composite nanopar-
ticles authors of Pavlov et al. (2018) applied the fluorination of CeO2 nanoparticles
in CF4 atmosphere. According to the results of this work an annealing of CeO2
nanoparticles in CF4 atmosphere at high temperature induces the formation of CeF3
crystalline phase on the surface of CeO2. The average size of synthesized nanopar-
ticles was varying from 100 to 400 nm. The unit-cell parameters and optical
properties of the CeF3 crystalline phase in CeO2/CeF3 composite nanoparticles and

CeF3 single crystal are the same. It was found that the concentration of the CeF3
phase increases with an increase in annealing temperature. It was concluded that the
high concentration of Ce3+ ions in the CeO2/CeF3 composite nanoparticles will have
a positive effect on catalytic activity and antioxidant properties. The composite
nanoparticles fabricated with the similar methods were studied with the EPR and
magnetization measurement techniques (Morozov et al. 2018). It was found a con-
siderable enhancement of saturated magnetization in the composite nanoparticles
compared to the initial CeO2 sample as well as with the air annealed sample at
1000 °C while the vacuum annealed at 1000 °C ceria sample displays mainly para-
magnetic contribution to the magnetization curve at room temperature. The knowl-
edge of magnetic properties of such nanoparticles is important for the biomedical
application (Pankhurst et al. 2003).
It is interesting to note that the excellent photocatalytic activity for the organic
contaminants degradation under UV-light and visible light irradiation was also dis-
covered for F-doped CeO2 nanoparticles (Miao et al. 2016). F-doped CeO2 nanopar-
ticles are synthesized by the solution combustion method using citric acid as fuel.
The authors found that F-doping leads to smaller particle size and the formation of
CeO2 nanocubes. The CeO2 nanoparticles featured the irregular spherical morphol-
ogy and had an average diameter of about 100 nm, whereas F-doped CeO2 nanopar-
ticles were approximately nanocubes with 50 nm in dimensions. Miao et al. (2016)
supposed that the enhanced photocatalytic activity of F-doped CeO2 nanocubes
originates from the fact that F-doping induces the small size, the highly reactive
facets exposed, the intense absorption in the UV–vis range, and the narrowing of the
band gap.
Composite nanoparticles, TbF3@CeF3@SiO2, were studied recently as perspec-
tive material for biological applications (Grzyb et al. 2013). The nanocrystals, cov-
ered by a shell, composed of cerium fluoride were synthesized by a co-precipitation
method. Their complex structure was formed spontaneously during the synthesis.
The surface of these core/shell nanocrystals was additionally modified by silica.
The hydrodynamic diameter and f-potential of the obtained nanoparticles in water
were measured with a Malvern Nano Zeta Sizer, Dynamic Light Scattering (DLS)
instrument. According these measurements the average size distribution of the
obtained nanostructures was approximately 49 nm for TbF3@CeF3, and approxi-
mately 1411 nm for TbF3@CeF3@SiO2. Results of this work suggest the safety and
adequacy of TbF3@CeF3@SiO2 composite nanoparticles for future use in medical
or biological applications.
5.9 Conclusion and Future Perspectives
It was revealed that among a huge variety of fluoride nanomaterials the most useful
ones for biomedical applications are doped metal fluoride nanoparticles (the dopants
are lanthanides or transition metals, the host matrices are metal fluorides). Such
chemical compositions are chemically stable and demonstrate excellent physical and
chemical properties as well as unique biological activity. It seems that the most
demanded areas for such nanoparticles are photodynamic therapy, bioimaging, and
magnetic resonance imaging. According to the literature data, doped fluoride
nanoparticles successfully proved their applicability in the above-mentioned areas on
eukaryotic cells and small animals. However, it seems that the experiments on bigger
animals were still not carried out. Indeed, the linear sizes of tissues of such animals
are big enough and the resolution and sensitivity of imaging are significantly lower.
More importantly, the nanoparticle transport and their fate in organism are still mat-
ters of deep concerns. For these reasons further investigations are highly required.
Nevertheless, one of the possible ways to circumvent existing limitations of
imaging technique is involving several methods of imaging simultaneously. The
targeting can be provided by using antibodies which can specifically interact and
with the tissues of interest. If such “smart” system has also a function of treatment
(for example by photodynamic therapy) the process combining diagnostics and
treatment can be realized. Indeed, the nowadays trend is creation of multifunctional
nanoparticles for theranostics (Melancon et al. 2011). Theranostics is a new field of
medicine which combines specific targeted therapy based on specific targeted diag-
nostic tests. It can combine, for example, photodynamic therapy, bioimaging, and
magnetic resonance imaging in order to diagnose and treat. Indeed, for such com-
plex tasks special multifunctional nanoparticles are required. These nanoparticles
should be luminescent in desirable range of spectrum. They should serve as contrast
agents in magnetic resonance imaging. Furthermore, such nanoparticles should be
capable of carrying and activating specific drugs. Finally, such nanoparticles should
provide targeting. Such multifunctionality can be provided by using special chemi-
cal composition and surface modification.
For example, Gd3+,Er3+:BaYbF5 up-conversion nanoparticles with combination
of the merits of multiple molecular imaging techniques, such as up-conversion
luminescence imaging, X-ray computed tomography, and magnetic resonance
imaging, could significantly improve the spatial resolution in imaging (Li et al.
2017). PEG coated Yb3+, Er3+@Ba2GdF7 nanoparticles also demonstrated their
applicability in in-vivo multi-modality imaging including up-conversion lumines-
cence, X-ray computed tomography, and T1-weighted magnetic resonance (Feng
et al. 2017). In Sui et al. (2019) Yb3+,Er3+Ag@NaGdF4 core-shell nanocomposites
applied on bioimaging and photothermal therapy.
It can be concluded that the future of using of fluoride nanoparticles in biomedi-
cine lies in the development of non-toxic multifunctional nano-platforms. These
nano-platforms should be capable of finding the place of interest in the organism
and treat it. More importantly, the approbation of such nano-platforms should be
performed for mice, rats, and bigger animals. The further experiments concerning
toxicity and transport of the nanoparticles are also needed.
Acknowledgments The works was supported by the subsidy allocated to KFU for the state
assignment in the sphere of scientific activities [3.1156.2017/4.6] and [3.5835.2017/6.7]. Maksim
Pudovkin was supported by the research grant of Kazan Federal University.
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Gold Nanostructures in Medicine
and Biology 6
Siavash Iravani and Ghazaleh Jamalipour Soufi
6.1 Introduction
Gold nanostructures and nanoparticles (NPs) showed advantageous chemical,

physical, and optical properties which make them appropriate for novel medi-
cal, biological, and biomedical applications (Fig. 6.1) (Huang et al. 2007). The
biocompatibility, corrosion resistance, immunity, and high photo-bleaching of
nano-gold contrast properties were used to diagnose and treat disorders (Dykman
and Khlebtsov 2012). The optical properties of gold-based nanostructures are
Diagnostics & bioimaging
Drug delivery & gene therapy
Photothermal and photodynamic therapy

Gold nanostructures can be
conjugated with various Au
functional moieties:
Immunoassays
- Poly(ethylene glycol)
- Drug molecules Electron microscopy
- Targeting groups
- DNA or RNA Biosensors
- Charged polymer
- Smart polymer Cancer diagnosis & treatment
Fig. 6.1 Important applications of gold nanostructures and NPs
S. Iravani (*)
Faculty of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Sciences,
Isfahan, Iran
G. J. Soufi
Radiology Department, Isfahan University of Medical Sciences, Isfahan, Iran

https://doi.org/10.1007/978-981-13-8954-2_6
176 S. Iravani and G. J. Soufi
highly sensitive to their morphologies and sizes (Huang et al. 2007). Absorption
and diffusion properties of gold nanostructures might be mediated from the
infrared region, which supply valuable nanomaterials for biomedical applica-
tions. Surface plasmon resonance (SPR) of gold-based nanostructures is
extremely sensitive to physicochemical changes. Gold nanostructures have been
successfully applied for imaging and diagnosing the important diseases (e.g.,
cancers) with the standard clinical methods like X-ray, computed tomography
(CT) scanning, and magnetic resonance imaging (MRI) (Kojima et al. 2010;
Rand et al. 2011).
Gold nanostructures have been suggested for use in the diagnosis and remedy of
diseases like cancers (Liu et al. 2015). Cancer is a major disease affecting the whole
world, and in this regard numerous methods for detection, diagnosis and treatment
were expanded using gold nanostructures. For instance, folate-conjugated gold NPs
hold significant promises in targeted cancer therapy (Samadian et al. 2016).
Additionally, it was reported that gold nanorods can be used for efficient conversion
of photon energy into heat, resulting in hyperthermia and suppression of tumor
growth in vitro and in vivo (Turcheniuk et al. 2016). Nanostructures based on gold
having optical ownership between 600 and 1000 nm were used as instruments for
applying heat locally and contrast factors in organisms (Hosomi et al. 2008). In
nanomedicines, theranostic NPs have been reported to have the potentials to signifi-
cantly improve treatment outcome of drug therapy, and result in the development of
personalized medicine, where the device may be adapted for the treatment of
patients on the basis of their DNA profiles. Indeed, there are numerous biomedical
and medical applications for gold nanostructures and NPs; in this chapter, some
important ones in biology and medicine (including diagnostics, gene therapy, drug
delivery, and cancer diagnosis and therapy) are highlighted.
6.2 Diagnostics (Imaging)
Gold NPs can be simply visualized by photometry and electron microscopy because
of their high electron density, characteristic absorption, and scattering in the visible
and NIR region, which make these NPs as a potential candidate for cellular label-
ling, imaging, and biosensing (Murali et al. 2018). Metal NPs have very tunable
optical attributes that can be adjusted toward desired wavelengths by changing the
shape and combination of it (Conde et al. 2012). Consequently the metal NPs are
extensively used as activating agents of imaging techniques, for monitoring or for
imagery of the cells in situ cancer diagnostics (Kim et al. 2011). In numerous parts
of the biological tissue, the light absorption is minimized in the NIR or if the nano-
structures may be designed to be actuated in the area for in vivo imaging and hyper-
thermic treatment (Conde et al. 2012). Probes of metal NPs can face many
restrictions of usual NIR organic dyes, including poor hydrophilic and photostabil-
ity, low quantum efficiency, and detection sensitivity and is unstable in organic sys-
tems (Conde et al. 2012). Zhang et al. (2010) developed fluorescent metal nanoshells
as a molecular imaging agent for the detection of single molecules microRNA
6 Gold Nanostructures in Medicine and Biology 177
(miRNA) in cells of lung cancer. Metal nanoshells were composed of silica spheres
with encapsulated Ru(bpy)32+ complexes as cores and thin silver layers as shells.
These metal nanoshells showed large emission intensity (up to six times), emissions
of longer lifetime, and prolonged photostability (up to two times). This strong and
long emission lifetime only to the nanoshells clearly isolated cellular auto-
fluorescence images of cells. By measuring alterations in the levels of expression of
miRNA, it could provide a baseline to the early diagnosis of lung cancer and other
diseases (Zhang et al. 2010). Outside of its use in imaging techniques need of near-
infrared wavelengths, the gold NPs were occupied in surface-enhanced Raman scat-
tering (SERS), or as contrast agents for CT, the MRI, as optical coherence
tomography and imaging photoacoustic. Wang et al. (2004) applied photoacoustic
imaging to monitor the distribution of poly(ethylene glycol) coated with gold
nanoshells and silica in the vascular circulation of a rat brain system and found that
these improved nanoshells of optical absorption in the vessels of the brain are a
maximum of 63%, which had a more detailed image of the vasculature structure at
greater profundity (Wang et al. 2004). SERS using gold or silver NPs with a reporter
molecule attached to a specific Raman signing might be considered to demonstrate
cell structures and to provide details on molecular structure of the cell environment
in living cells (Kneipp et al. 2006).
6.3 Drug Delivery
A problem encountered in the delivery of drugs is about the specificity to the tissue,
which generally means that the drugs become uniformly distributed in the body, and
they have a short half-life in blood as well as a high overall clearance rate. Actually
just small drug quantities managed reach target site, which can be a major problem
for certain diseases like cancer (Conde et al. 2012). Additionally, the uniform drug
delivery in the body can lead to serious side effects. To solve these problems, lots of
researches have been conducted on the use of NPs as carriers for drug delivery. In
the event of the metal NPs, the performance can be easily adjusted to control the rate
of drug release and the disintegration of the particles by altering the size and surface
functionalization of these NPs. Until now, investigations on the use of metal NPs for
drug delivery showed that these systems can provide delivery of unstable drugs and
a more focused dispensing and the ability to avoid or circumvent the barriers
organic. However, their nanoscale size means they can easily get into various cells,
making it more difficult to be a specific tissue. To solve this issue, the metal NPs
were conjugated with different ligands and biomolecules that produce new
approaches to targeted drug delivery (Tiwari et al. 2011). The gold NPs for delivery
of pharmaceutical agents and drugs include anticancer drugs like paclitaxel,
platinum-based drugs and 5-fluorouracil (Selvaraj and Alagar 2007). Additionally,
the use of metal NPs for the administration of drugs permits external control of the
delivery of a drug. These intelligent dispensing systems have been used successfully
for the encapsulated enzyme, a single request with nanosecond laser pulse release
(Tiwari et al. 2011).
In one study, the double-walled Au nanocage/SiO2 nanorattle was prepared (Hu

et al. 2015). Consequently, the whole nanorattle served as a high efficient drug car-
rier because of the structural characteristics of gold nanocage and SiO2 shell with
hollow interiors and porous walls. Moreover, gold nanocage with large electromag-
netic enhancement acted as a sensitive surface-enhanced Raman scattering (SERS)
substrate to track the internalization process of the nanorattles by human MCF-7
breast cancer cells, as well as an efficient photothermal transducer for localized
hyperthermia cancer therapy due to the strong near-infrared absorption (Hu et al.
2015). Additionally, controlled drug delivery from low-temperature-sensitive lipo-
somes mediated was reported by photothermal heating from multi-branched gold
nanoantennas in triple-negative breast cancer cells in vitro (Ou et al. 2016). The
unique geometry of multi-branched gold nanoantennas enables the generation of
mild hyperthermia (approximately 42 °C) by converting near-infrared light to heat
and effectively delivering doxorubicin from the low-temperature-sensitive lipo-
somes in breast cancer cells. Imaging of fluorescent live/dead cell indicators and
MTT assay outcomes both showed substantial reductions in cellular viability when
cells were treated with the combination therapy. Findings demonstrated that the
synergistic therapeutic effect of photothermal hyperthermia of multi-branched gold
nanoantennas with drug delivery from the low-temperature-sensitive liposomes can
successfully eradicate aggressive breast cancer cells with higher efficacy than free
doxorubicin by providing a controlled light-activated approach and minimizing off-
target toxicity (Ou et al. 2016).
6.4 Gene Delivery and Therapy
Giljohann et al. (2009) synthesized and characterized RNA-conjugated gold NPs

which efficiently knocked down the production of luciferase in HeLa cells-
transfected luciferase versatile. Furthermore, they proved that the resulting conju-
gates have a lifetime six times longer than the double-stranded RNA, cells penetrate
easily without using transfection agents and had a upper capacity gene knockdown
in their model cellular (Giljohann et al. 2009). For the spatial and temporal control
of gene delivery, Braun et al. created a functionalized gold nanoshell with TAT-lipid
layer for transfection and selective releasing of the siRNA, wherein the lipid coating
of TAT-mediated cellular uptake of the nanomaterial while the output of the siRNA
was addicted near-infrared (NIR) laser pulses (Braun et al. 2009). Furthermore,
researchers demonstrated an effective delivery system and cytoplasmic siRNA gene
silencing using NPs of gold (Guo et al. 2010). In another study, gold nanorods were
functionalized with ssRNA and reduced virus replication of H1N1 (Chakravarthy
et al. 2010).
According to time and temperature used, hyperthermia may cause various effects
of reducing the metabolism and apoptosis of tumor cells in the immediate physical
destruction of cells. Because of the properties of metals, metal NPs may be used to
heat the cancer cells beyond their tolerance limits of temperature, and selectively
kill if NPs are functionalized to target tumor cells in particular. The heating of the
NPs is generally obtained by exposing the patient or all of targeted area to a mag-
netic field of alternating current, a radiofrequency source or intense light which
causes the NPs to heat and induce thermal ablation tumor (Conde et al. 2012). Super
paramagnetic iron oxide NPs coated with gold (SPIONs) were developed which
showed an increase of a factor of 4 to 5 in a resulting heat release on demand of the
low frequency oscillating magnetic fields. SPIONs have been applied for hyperther-
mia treatment of brain cancer. Hyperthermia using NPs in conjunction with a
reduced dose of radiation has been found to be effective and safe, and could lead to
more overall survival after diagnosis of the first recurrence of the tumor, in compari-
son to conventional treatment in the current treatment of recurrent cancer (Maier-
Hauff et al. 2011).
In one study, multifunctional hetero-nanostructures of cellulose nanocrystal
(CNC)-gold nanoparticle hybrids were prepared which has been wrapped with low-
toxic hydroxyl-rich polycations to integrate versatile functions for effective cancer
therapy. Biocompatible CNCs with the superior rod-like morphology for high cellu-
lar uptake were employed as substrates to flexibly load spherical gold NPs or
nanorods via gold-thiolate bonds, producing hetero-layered nanohybrids of CNC-
gold NPs or CNC-gold NRs. Profound hydroxyl-rich cationic gene carrier, CD-PGEA
(comprising β-cyclodextrin cores and ethanolamine-functionalized poly(glycidyl
methacrylate) arms), was then assembled onto the surface of CNC-gold nanohybrids
via host–guest interaction and gold-thiolate bonds, where PEG was employed as the
intermediate and spacer. The resultant CNC-Au-PGEA hetero-nanostructures dem-
onstrated outstanding performances as gene carriers. In addition, CNC-gold nanorod-
PGEA comprising gold nanorods showed promising optical absorption characteristics
and was validated for photoacoustic imaging and combined photothermal/gene ther-
apy with significant antitumor influences (Hu et al. 2017).
6.5 Cancer Diagnosis and Therapy
Gold nanostructures showed exciting potential for effective photothermal hyper-

thermia therapy. These gold nanomaterials are applied for thermal destruction of
cancer because of their ease of surface functionalization and photothermal heating
capability (Abadeer and Murphy 2016). In one report, palladium-gold nanostruc-
tures containing multiple gold nanocrystals on highly branched palladium seeds
have been prepared. Consequently, palladium-gold heterostructures irradiated with
an 808-nm laser light caused destruction of HeLa cancer cells in vitro, in addition
to complete destruction of tumor xenographs in mouse models in vivo for effective
photothermal hyperthermia (McGrath et al. 2015). Furthermore, ultra-small disso-
ciable gold nanorod@poly(ethylene glycol)/poly(lactic-co-glycolic acid) was
assembled from small gold nanorod. As a result, some important features have been
reported, such as prolonged circulation and prominent tumor accumulation, rapid
excretion from the body as gold nanorod@poly(ethylene glycol) after therapy,
accelerated photoacoustic and photothermal characteristics, and high photothermal
cancer therapy efficacy (Song et al. 2015).
Different shapes of gold nanomaterials (approximately 50 nm in size) including

spherical gold NPs, nanospikes, and nanorods were prepared and functionalized
with poly(ethylene glycol) molecules (Ma et al. 2017). Interestingly, all of these
gold nanostructures were coated with the same poly(ethylene glycol) molecules, but
their cellular uptake behavior differed significantly. As a result, gold NPs demon-
strated the highest cellular responses as compared to the gold nanospikes and
nanorods (based on the same gold mass) after incubation with KB cancer cells for
24 h. It was reported that all of these gold nanostructures could induce accelerated
cancer cell-killing rates more or less upon X-ray irradiation, and the gold NPs
showed a higher anticancer efficiency than both gold nanospikes and nanorods upon
X-ray irradiation. It was concluded that in order to gain efficient and strong radio-
sensitization effect in cancer radiotherapy, it is essential to apply gold-based nano-
materials with a high cellular internalization. More investigations on the
radiosensitization mechanisms revealed that reactive oxygen species generation and
cell cycle redistribution triggered by gold nanostructures played essential roles in
enhancing radiosensitization. Findings showed that the shape of gold-based nano-
materials had an important effect on cancer radiotherapy (Ma et al. 2017).
Biodegradable plasmon resonant liposome gold NPs were able to kill cancer
cells via photothermal therapy (Rengan et al. 2015). Pharmacokinetic analysis of
liposome gold NPs done in a small animal model demonstrated in situ degradation
in hepatocytes and further getting cleared through the hepatobiliary and renal
route. Moreover, liposome gold NPs were tested in mouse tumor xenograft model
using NIR laser (750 nm) illumination, and it was shown that complete ablation of
the tumor mass was achieved, and therefore prolonging disease-free survival
(Rengan et al. 2015). Furthermore, the potential of polyethylene glycol functional-
ized reduced graphene oxide enrobed gold nanorods for the photothermal destruc-
tion of human glioblastoma astrocytoma (U87MG) cells in mice was reported
(Turcheniuk et al. 2016). It was shown that prepared nanocomposites, as ideal
multifunctional theranostic nanostructures, might exert efficient photothermal
destruction of tumors in mice upon low doses of NIR light excitation and might act
as fluorescent cellular markers because of the existence of a NIR dye integrated
onto the reduced graphene oxide shell. In vivo investigations in mice showed that
upon irradiation of the tumor implanted in mice at 800 nm under low doses,
U87MG tumor growth has been suppressed (Turcheniuk et al. 2016). Additionally,
gold nanodendrites were prepared in organic solvent using long chain amines as a
structural directing agent. As a result, in vitro and in vivo investigations demon-
strated that gold nanodendrites with a higher degree of branching were more effec-
tive for photothermal tumor destruction under a lower wavelength NIR irradiation
(Qiu et al. 2016). In another study, near-infrared-absorbing gold nanopopcorns
containing a self-assembled iron oxide cluster core were produced through a seed-
mediated growth approach (Bhana et al. 2015). The prepared hybrid nanostructures
were superparamagnetic and demonstrated significant photothermal conversion
efficiency under near-infrared irradiation. As compared to the combination treat-
ment without applying a magnetic field and the two treatments alone, it was shown
that the dual mode photothermal and photodynamic therapy with the assistance of
magnetic-field-guided drug delivery significantly enhanced the therapeutic value

of cancer cells (Bhana et al. 2015). Moreover, bifunctional gold-coated polypyr-
role nanostructure with Raman and photothermal activity was prepared, in which
polypyrrole spheres were applied as the core and coated with small gold NPs to
form polypyrrole-gold nanostructures. It was shown that the nanostructures could
specifically bind to the membrane protein MUC1, a kind of tumor-related bio-
marker which is overexpressed on the surface of human breast cancer cells (MCF-
7). Consequently, photothermal investigations revealed that MCF-7 cells incubated
with these nanostructures nearly died under 808 nm irradiation (about 20 min), and
the tumors bearing on the mice were magnificently inhibited after photothermal
therapy as well (Luo et al. 2017).
Thermally triggered gold nanoparticle assembly was reported for the develop-
ment of innovative and intelligent platforms for multimodal imaging and cancer
photothermal therapy (Sun et al. 2017). Site-specific conjugation of a thermally
sensitive elastin-like polypeptide to gold NPs yielded thermally sensitive NPs.
Interestingly, elastin-like polypeptide-gold NPs showed significant near-infrared
light absorption and great photothermal influence. These thermally responsive
characteristics of these NPs enabled simultaneous photothermal/photoacoustic/
X-ray computed tomographic imaging and photothermal therapy of melanoma
after single intra-tumoral injection of them (Sun et al. 2017). In another study,
gold nanoraspberries (as the plasmonic nanostructures) were prepared with tun-
able size and localized surface plasmon resonance. These nanoraspberries dem-
onstrated significant selectivity to tumor cells, and therefore, enabling
loco-regional therapy at the cellular level. Consequently, tumor selectivity of
gold nanoraspberries by photothermal ablation of tumor cells was shown (Gandra
et al. 2015).
6.6 Conclusion
Gold nanostructures have attracted particular attentions due to some important fea-
tures, including unique optical and physicochemical properties, biocompatibility,
functional flexibility, tunable monolayers, controlled dispersity, and high surface
area for loading the density of drugs. Moreover, stability and non-toxicity make
these nanomaterials an efficient and valuable nanocarriers for drug delivery. These
nanostructures can be applied as appropriate candidates for cancer therapy modali-
ties because of simple synthesis, biocompatibility, functionalization, physiochemi-
cal stability and optical tunable properties. Moreover, it appears that plasmonics-active
gold NPs offer significant potentials in molecular imaging and cancer therapy. The
multifunctional gold nanostructures (e.g., multifunctional gold nanostars) can be
applied for future pre-treatment cancer diagnostics, image-guided therapy, and
intraoperative imaging. Furthermore, thermally stimulated assembly of various NPs
with optical, electronic, and magnetic properties into nanoparticle assemblies can
be used for the establishment of intelligent platforms for different biomedical
applications.
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Fungus-Mediated Nanoparticles:
Characterization and Biomedical 7
Advances
7.1 Introduction
7.1.1 Nanobiotechnology
Nanobiotechnology is an inventive branch of technology, which impacts all features

of the human environment. Nanoparticles are used in a variety of industries. Their
main applications are in biomedicine (Vigneshwaran et al. 2007). Nanoparticles are
particles that measure 100 nm or less in at least one dimension. The properties of
materials change when they are produced in the form of nanoparticles (Balaji et al.
2009). Nanoparticles have a larger surface-to-volume ratio than the bulk form of the
same particles, which leads them to be more reactive to some other molecules
(Kathiresan et al. 2009). Polymeric micelle nanoparticles are used to deliver drugs
for treatment of tumors. Polymer-coated iron oxide nanoparticles can be used to
divide clusters of bacteria, allowing more potential for treatment of chronic bacterial
diseases (Basavaraja et al. 2008). Protein-filled nanoparticles have the ability to stim-
ulate an immune response. Researchers are developing ways to use carbon nanotubes
based cancer drugs fastened to carbon-based materials such as nanodiamonds (which
are invisible to the naked eye) to treat brain tumors and leukemia (Vigneshwaran
et al. 2006). Nanoparticles that can be synthesized using fungi include silver, gold,
cadmium sulfide, copper oxide, selenium, calcium oxide, titanium oxide, titanium
dioxide, copper acetate, copper sulfate, and zinc nanoparticles (Basavaraja et al.
2008), and the synthesis can be done using extracellular and intracellular methods. A
fungus is a eukaryotic organism biomass employing and very effective secretors
(Jaidev and Narasimha 2010). Nanomedicines act against bacterial and fungal infec-
tions and disease (Elahian et al. 2017). There are new alternatives for synthesis of
nanoparticles through use of plant extracts, live plant biomass, fungi, bacteria, and
S. Rajeshkumar (*) · D. Sivapriya

Nanobiomedicine Lab, Department of Pharmacology, Saveetha Dental College, Saveetha
Institute of Medical and Technical Sciences (SIMATS), Chennai, Tamil Nadu, India

https://doi.org/10.1007/978-981-13-8954-2_7
186 S. Rajeshkumar and D. Sivapriya
yeasts (Gajbhiye et al. 2009). Fungal species are mostly used because fungi are able
to give a greater yield in production of highly stable nanoparticles, and molecular
aggregation is prevented even after prolonged storage (Vigneshwaran et al. 2006).
Also, silver nanoparticles are used as preservatives in the cosmetic industry because
they inhibit primary microbial contamination during formulation and production of
cosmetics. In recent years the significance of nanoparticle technology in different
industries has extended considerably. Nanoparticles possess unique physicochemical
properties (Barabadi et al. 2014). These parameters are an indicator identifying the
size and structure of the nanoparticles. Most silver nanoparticles are synthesized
from fresh boiled plant leaves that have medicinal properties. The nanotechnology
used in reproductive drugs gives patients a better chance of a safe pregnancy and
delivery of a healthy baby (Andries et al. 2016) (Fig. 7.1).
7.1.2 Biosynthesis of Nanoparticles using Fungus
In recent years, various fungi have been used in the synthesis of nanoparticles,
such as Fusarium oxysporum, Colletotrichum sp., Trichothecium sp., Trichoderma
asperellum, Trichoderma viride, Phanerochaete chrysosporium, Fusarium solani,
Fusarium semitectum, Aspergillus fumigatus, Coriolus versicolor, Aspergillus
niger, Phoma glomerate, Penicillium brevicompactum, Cladosporium cladospori-
oides, Penicillium fellutanum, and Volvariella volvacea (Fatima et al. 2015). Metal
nanoparticles such as silver, gold, copper, and platinum nanoparticles are widely
produced by physical, chemical, and biological methods. Green production of
Fig. 7.1 Biosynthesis of nanoparticles

7 Fungus-Mediated Nanoparticles: Characterization and Biomedical Advances 187
nanoparticles is achieved using naturally occurring reagents such as vitamins, sug-

ars, plant extracts, biodegradable polymers, and microorganisms. Nanostructured
synthesis of silver vanadate is used to manufacture an antibacterial addictive for
water-based paints for potential application in bathrooms, kitchens, and hospital
environments (Krishnan et al. 2016). Also, ultraviolet (UV) irradiation, aerosol
production (which is used in food products such as cream and cooking sprays),
printed circuits, removal of materials from solids (using laser beams, pressure, and
velocity), and solar energy conversion into carbon dioxide have been used success-
fully to produce nanoparticles, but they remain expensive and involve use of haz-
ardous chemicals. Thus, there is significant interest in development of
environmentally friendly and sustainable methods of nanoparticle production
(Shankar et al. 2003). Silver hydroxyapatite (HA) used to makes it a popular
replace the bone material by patient own body material as well as a coating layer
on the metal used for manufacturing implants like Fe, Ti by silver HA nanocoat-
ings and silver HA nanosized biomaterials (Joshi et al. 2017). Nanomaterials are
also used as medicines for fish infections, as metal nanoparticles act against fish
pathogens (Castro et al. 2014).
7.2 Characterization of Nanoparticles
Characterization is essential to determine the average size, shape, and features of

synthesized nanoparticles. Figure Figure 7.2 shows the different characterization
techniques used for analysis of nanoparticles. These bioactive nanomaterials can be
easily analyzed using color changes in the prepared materials on ultraviolet–visible
(UV-vis) spectrophotometry. X-ray powder diffraction analysis (XRD) is used for
identification and quantification of crystalline phases. It is a nondestructive tool for
analysis of all types of matter from fluids to powders (Shantkriti and Rani 2014).
Thermogravimetric analysis is an essential tool used to analyze changes over time,
with increases in temperature, and as a function of time. Fourier-transform infrared
Fig. 7.2 Characterization of nanoparticles

Fig. 7.3 Biomedical applications of fungus-mediated nanoparticles
spectroscopy (FTIR) is used to determine functional groups and structural compo-

nents present in synthesized nanoparticles (Patel et al. 2016). Atomic force micros-
copy (AFM) and transmission electron microscopy (TEM) are also used (Shantkriti
and Rani 2014).
7.3 Applications of Nanoparticles
Figure 7.3 shows applications of nanoparticles synthesized using fungi. Fungus-

mediated nanoparticles are mainly used in biomedical applications for their antimi-
crobial properties (such as antibacterial, antifungal, and antiviral activities) and
their anti-inflammatory, anticancer, dye degradation, photocatalytic, drug delivery,
and antitumor activities.
7.4 Silver Nanoparticles
Studies of fungus-mediated synthesis of silver nanoparticles are summarized in

Table 7.1.
7.4.1 Applications of Silver Nanoparticles
Fungi are recognized as eukaryotic organisms and are a type of decomposer organisms.
They are applied for synthesis of nanoparticles by a combination of biological and
chemical methods. They are used in many fields of nanoscience (Tripathi et al. 2014).
Table 7.1 Studies of fungus-mediated synthesis of silver nanoparticles
Fungus Localization Characterization Shape FTIR Reference
Fusarium Extracellular SEM: 8–60 Spherical –N–H stretching bond Basavaraja et al. (2008)
semitectum XRD: (1 1 1), (2 0 0), (2 2 0), and vibrations in amide linkages of
(3 1 1) proteins
Phanerochaete Extracellular SEM: 50–200 Homogeneous – Vigneshwaran et al.
chrysosporium TEM: 100 spherical (2006)
XRD: (1 1 1), (2 0 0), (2 2 0), and
(3 1 1)
Cladosporium Extracellular SEM: 10–100 Mostly –N–H stretching bond Balaji et al. (2009)
cladosporioides XRD: (1 1 1), (2 0 0), (2 2 0), and spherical vibrations in amide linkages of
(3 1 1) proteins
Aspergillus On cell wall SEM: 20 um Spherical C–N stretching vibrations of Vigneshwaran et al.
flavus surface TEM: 50 nm (8.92 ± 1.61 nm) aromatic and aliphatic amines (2007)
XRD: (111), (200), and (220)
Penicillium Extracellular SEM: 5–25 Mostly – Kathiresan et al. (2009)
fellutanum spherical
Aspergillus Extracellular TEM: 3–30 Spherical Carbonyl groups of amino acid Jaidev and Narasimha
niger residues and peptides (2010)
Coriolus Extracellular XRD: (111), (200), (220), and (311) Spherical –SH stretching vibration Sanghi and Verma
versicolor and intracellular TEM: 25–75 (2009b)
Alternaria Extracellular SEM: 20–60 Polydisperse –N–H–, C–C, and C–N Gajbhiye et al. (2009)
alternata spherical stretching functional groups
suchas–COO,–NO3,and–C–O–C–
Pichia pastoris Intracellular TEM: 70–180 Spherical – Elahian et al. (2017)
AFM: 50
7 Fungus-Mediated Nanoparticles: Characterization and Biomedical Advances
Verticillium Intracellular TEM: 100–200 Quasispherical – Sastry et al. (2003)

SEM: 1 m and 500 nm
Penicillium – SEM: 90–120 Spherical C–N stretching vibration of Barabadi et al. (2014)
citrinum amines
(continued)
189
190
Fungus Localization Characterization Shape FTIR Reference

Agaricus – TEM: 8–20 Spherical – El-Sonbaty (2013)
bisporus
Aspergillus Intracellular XRD: 13.80–2.0 nm Spherical – Velhal et al. (2016)
terreus SEM: 4
Neurospora – SEM: 5–20 Quasispherical – Quester et al. (2016)
crassa TEM: 10–100
Aspergillus – SEM: 200 Circular – Elgorban et al. (2016)
versicolor TEM: 0.1 mm
Fusarium solani Extracellular TEM: 5–35 Irregular Functional groups such as C–N, Ingle et al. (2009)
C–O–C, amide, and –COO–;
these may be between amino
acid particles in proteins
AFM atomic force microscopy, FTIR Fourier-transform infrared spectroscopy, SEM scanning electron microscopy, TEM transmission electron microscopy,
XRD X-ray powder diffraction analysis
Because they have a sufferance and biocompatibility capacity, fungi are used in the
preliminary stages of studies on green production of metallic nanoparticles (Shankar
et al. 2003). The biosynthetic method can involve use of a plant extract or microorgan-
ism for synthesis of nanoparticles. These approaches are used in industrial and medici-
nal applications (Barabadi et al. 2014). The fungus acts as a stabilizing agent for
synthesis of silver nanoparticles. Biosorption of silver by the bio-molecules present in
the fungus Aspergillus flavus shows good results. These nanoparticles have been found
to be constant in water for more than 3 months, which can be attributed to surface
attachment of stabilizing material produced by the fungus (Vigneshwaran et al. 2007).
In F. semitectum–mediated extracellular synthesis of silver nanoparticles, a protein
coating may play an important role in stabilization of the nanoparticles (Basavaraja
et al. 2008). P. chrysosporium is a white rot fungus used to synthesize silver nanopar-
ticles. This method is employed for medical uses and in the textile industry for its
efficient antimicrobial function. The nonpathogenic character of white rot fungi allows
abundant production of silver nanoparticles (Vigneshwaran et al. 2006). C. cladospo-
rioides–mediated synthesis of silver nanoparticles has been used to control the size
and shape of biogenic nanoparticles. Proteins, organic acids, and carbohydrates
excreted by the fungus are suspect to cable for distinguish the various type of crystal
structure and growth of enlarged spherical crystals (Balaji et al. 2009).
P. fellutanum is isolated from coastal mangrove sediment. Silver nanoparticle synthe-
sis by use of this fungal culture occurs rapidly within minutes when silver binds with the
cell filtrate. The main applications of these nanoparticles are in spectrally selective coat-
ings for solar energy absorption, optimal receptors in intercalation materials for electri-
cal batteries, polarizing filters, catalysts in chemical reactions, biolabeling, and
antimicrobial agents (Kathiresan et al. 2009). Extracellular synthesis using A. niger pro-
duces nanoparticles with antibacterial activity against pathogenic bacteria such as
Staphylococcus sp., Bacillus sp., and Escherichia coli (Jaidev and Narasimha 2010).
Silver nanoparticles can be biosynthesized using fungal proteins from C. versi-
color. Co-operation between amino groups in the fungus and silver ions is important
for stabilization of the silver nanoparticles. These protein-bound nanoparticles have
potential uses as water-soluble metal catalysts and also for labeling of living cells
and tissues (Sanghi and Verma 2009a). Alternaria alternata is used to synthesize
silver nanoparticles with antifungal activity against Phoma glomerata, Phoma her-
barum, F. semitectum, Trichoderma sp., and Candida albicans (Gajbhiye et al.
2009). A genetically modified strain of Pichia pastoris has been used to synthesize
silver and selenium nanoparticles with reduced cytotoxicity (Elahian et al. 2017).
Silver nanoparticles synthesized using Verticillium sp. in an intracellular method
have various applications. Verticillium is an acidophilic fungus isolated from Taxus
plants maintained in potato dextrose agar. The logical use of controlled intracellular
environments such as the periplasmic space and cytoplasmic vesicular parts (e.g., mag-
netosomes) to modulate nanoparticle size and shape is an exciting possibility but is yet
to be seriously explored (Sastry et al. 2003). Penicillium citrinum isolated from soil has
been used to synthesize silver nanoparticles. The silver nanoparticles have in impact on
energy consumption and resolving the health problems due to the intake of the very
effective drug. The fungal biomolecules responsible for the capping and efficient stabi-
lization of silver nanoparticles, according to the FTIR result (Barabadi et al. 2014).
Silver nanoparticles have been biologically synthesized from an aqueous extract of

Agaricus porous and were found to be effective against tumorigenicity. The antitumor
activity of silver nanoparticles was suggested to stimulate toxicity via oxidative
depression and inflammation by generating reactive oxygen species (ROS) involved
in a variety of different cellular processes ranging from apoptosis and necrosis (cell
lysis) to cell replication and carcinogenesis (El-Sonbaty 2013). Aspergillus terreus–
mediated synthesis of silver nanoparticles plays an important role in production of
antibacterials and cotton fabric textiles. These nanoparticles inhibit multidrug-resis-
tant microorganisms. Silver metal is very well known for its antibacterial activities
and could have uses in bacterial-resistant fabric. It acts against Gram-positive Bacillus
cereus. Staphylococcus aureus is more susceptible to silver nanoparticles than Gram-
negative Pseudomonas aeruginosa, E. coli, and Proteus vulgaris (Velhal et al. 2016).
Neurospora crassa extract has been used to synthesize silver nanoparticles under
various environmental conditions to produce nanoparticles of a small size with a nar-
row size dispersion. It was responsible for stabilizing the nanostructures that were
produced (Quester et al. 2016). Silver nanoparticles have been synthesized using
Aspergillus versicolor and act against plant pathogenic fungal strains. The antifungal
activity of biogenic silver nanoparticles was evaluated against white mold (Sclerotinia
sclerotiorum) and grey mold (Botrytis cinerea) in strawberry (Fragaria × ananassa)
(Elgorban et al. 2016). Extracellular synthesis of silver nanoparticles was achieved
using F. solani (USM-3799) isolated from infected onion. The silver nanopartilces
helps to identify the plant pathogenic infections (Ingle et al. 2009).
7.5 Gold Nanoparticles
Studies of fungus-mediated synthesis of gold nanoparticles are summarized in

Table 7.2.
7.5.1 Applications of Gold Nanoparticles
The nonpathogenic and agriculturally useful fungus Trichoderma harzianum has

been used to mediate synthesis of gold nanoparticles. Potential mechanisms involved
in biosynthesis and colorimetric detection have been described. The specificity and
selectivity of biosynthesized gold nanoparticles have also been investigated in the
presence of other heavy metal ions (Tripathi et al. 2014). Gold nanoparticles synthe-
sized using a simple method involving Aspergillum sp. have been effectively
employed in degradation of aromatic pollutants. This catalytic activity was ana-
lyzed using various nitroaromatics and azo dyes. These are all very toxic refractory
pollutants (Qu et al. 2017). The myco-nanotechnology interface between mycology
and nanotechnology has been employed for extracellular synthesis of gold nanopar-
ticles, using the phosphate-solubilizing fungus Bipolaris tetramera isolated from
rhizosphere soil, which has antibacterial efficacy against Bacillus subtilis, B. cereus,
S. aureus, E. coli, and P. aeruginosa; antifungal efficacy against A. niger and
Trichoderma sp.; and cytotoxic effects (Fatima et al. 2015).
Table 7.2 Studies of fungus-mediated synthesis of gold nanoparticles
Fungus Localization Characterization Shape FTIR Applications Reference
Trichoderma Extracellular TEM: 20–50 nm and Short and plump N–H stretching Colorimetric detection Tripathi et al.
harzianum 20–40 nm (2014)
Aspergillum sp. Extracellular SEM: 50–100 Club – Aromatic pollutant Qu et al. (2017)
degradation by catalytic
activity
Bipolaris tetramera Extracellular TEM: 58.4 nm, Triangular, – Antifungal, antibacterial, Fatima et al.
110.13 nm, and spherical, and and cytotoxic activities (2015)
261.73 nm hexagonal
Candida Extracellular TEM: 100–500 Spherical C–N stretching Biomineralization Krishnan et al.
parapsilosis XRD: (111) and (2016)
(100)
Endophytic fungus Extracellular TEM: 50–100 Triangular C–O stretching Proteins stabilize gold Shankar et al.
(Colletotrichum sp.) XRD: (111), (200), nanoparticles synthesized (2003)
(220), and (311) using Colletotrichum sp.
Cladosporium Extracellular SEM: 60 nm – O–H groups Antimicrobial and Joshi et al.
cladosporioides XRD: [111], [200], antioxidant activities (2017)
[220], and [311]
Botrytis cinerea Extracellular TEM: 100 Triangular, – Denaturation Castro et al.
hexagonal, (2014)
spherical,
decahedral, and
pyramidal
Penicillium Extracellular AFM: 100 nm Spherical Amide linkages and Industrial production Barabadi et al.
crustosum –COO–, which may (2014)
be between amino
7 Fungus-Mediated Nanoparticles: Characterization and Biomedical Advances
acids
(continued)
193
194
Fungus Localization Characterization Shape FTIR Applications Reference

Fusarium Extracellular TEM: 10–35 Spherical Carbonyl stretching Medical applications Sawle et al.
semitectum XRD: (111), (200), and –N–H stretching (2008)
(220), and (311) vibrations in amide
linkage bonds
Candida albicans – TEM: 20–40 Nonspherical Hydroxyl stretching Diagnostics and Chauhan et al.
AFM: 40 nm vibrations in therapeutics (2011)
phenolic and
alcoholic compounds
Chrysosporium Extracellular TEM: 2–15 – – Activity against Aedes Soni and
tropicum XRD: (111), (200), aegypti Prakash (2012)
and (211)
Phanerochaete – TEM: 10–15 Club – Role in cosmetic industry Andries et al.
chrysosporium (2016)
AFM atomic force microscopy, FTIR Fourier-transform infrared spectroscopy, SEM scanning electron microscopy, TEM transmission electron microscopy,
XRD X-ray powder diffraction analysis
A cell-free extract of Candida parapsilosis has been used in biological synthesis

of gold nanoparticles. This biosynthesized gold nanoparticles with pH 12 solution
make a particle mono-dispersity which is identified by size-dependent catalytic
activity for reduction of 4-nitrophenol (Krishnan et al. 2016). Colletotrichum sp.
isolated from the surface of disinfected leaves of Pelargonium graveolens has been
used for synthesis of gold nanoparticles. It is possible to produce secondary metabo-
lites through symbiotic systems (Shankar et al. 2003). Gold nanoparticles have been
biosynthesized from C. cladosporioides, an endophytic fungus on the seaweed
Sargassum wightii. Marine endophytes are the most untapped group of microorgan-
isms. These nanoparticles have been shown to have antimicrobial activity, such as
antibacterial activity, by a well-diffusion method. These nanoparticles are active
against S. aureus (MTCC 7443) but less active against B. subtilis (MTCC 441).
They also have antioxidant activity (Joshi et al. 2017).
The phytopathogenic fungus B. cinerea has been used to produce gold nanopar-
ticles in an extracellular synthesis method, for applications in drug delivery, medical
diagnostics, imaging, and therapy (Castro et al. 2014). Penicillium crustosum, iso-
lated from soil, has been used for biosynthesis of gold nanoparticles. Extracellular
synthesis of gold nanoparticles is important in industries where experiments can be
very expensive and time consuming (Barabadi et al. 2014). F. semitectum has been
used to synthesize gold nanoparticles in an aqueous system and was responsible for
reducing and stabilizing them (Sawle et al. 2008). C. albicans–mediated synthesis
of gold nanoparticles plays an important role in anticancer activity. Cell surface–
specific antibodies (generated in mice with induced liver cancer) conjugated with
synthesized gold nanoparticles can differentiate between normal and cancerous
cells (A. Chauhan et al. 2011). Synthesis of gold nanoparticles mediated by the
pathogenic fungus Chrysosporium tropicum has been studied for use against
mosquito-related health problems. This fungus is very effective against Aedes
aegypti mosquito larvae (Soni and Prakash 2012). The environmental fungus
Phanerochaete chrysosporium has been used to mediate synthesis of gold nanopar-
ticles for use in pharmaceutical and biomedical applications (Andries et al. 2016).
7.6 Other Metallic Nanoparticles
Studies of fungus-mediated synthesis of other metallic nanoparticles are summa-

rized in Table 7.3.
7.6.1 Applications of Other Metallic Nanoparticles
Synthesis of CdS nanoparticles mediated using the immobilized fungus C. versi-

color has been employed for biomedical applications. Another important potential
benefit of this process is semiconductor CdS nanoparticles synthesized extracellu-
larly is very stable and synthesized more amount. This is thus a very important
application in comparison with other production methods in which the nanoparti-
cles are entrapped within a cell matrix in limited quantities, whereby additional
Table 7.3 Studies of fungus-mediated synthesis of other metallic nanoparticles
196
Chemical
name Fungus Localization Size Shape FTIR Application Reference
CdS Coriolus Extracellular SEM: 100–200 Spherical N–H and O–H Waste Sanghi and Verma
versicolor XRD: (1 1 1), (2 2 0), stretching treatment (2009a)
and (3 1 1) processing
CdS Fusarium Extracellular TEM: 100 Well- – – Ahmad et al.
oxysporum XRD: (111), (101), dispersed, (2002)
(002), and (220) individual
TiO2 Aspergillus – SEM: 62–74 Spherical N–H stretching Antimicrobial Rajakumar et al.
flavus TEM: 40–60 activity (2012)
XRD: 1 0 0, 0 0 2, and
1 0 0
ZnO Pichia Extracellular SEM: 10 um Rectangular – Antimicrobial Chauhan et al.
fermentans XRD: (111), (200), and activity (2015)
(220)
ZnO Candida diversa Extracellular SEM: 73 nm Rectangular – Bioassays Chauhan et al.
XRD: (1 1 1), (2 0 0), (2014)
and (2 2 0)
ZnS Saccharomyces Intracellular TEM: 30–40 – – Biomedicine Sandana Mala and
cerevisiae XRD: (1 1 1), (2 2 0), Rose (2014)
and (3 1 1)
CuO Stereum Extracellular TEM: 50 Spherical C–O–C pyranose – Cuevas et al.
hirsutum XRD: [110], [111], ring stretching (2015)
[200], [220], and [222]
FTIR Fourier-transform infrared spectroscopy, SEM scanning electron microscopy, TEM transmission electron microscopy, XRD X-ray powder diffraction
analysis
processing is essential to expose them from the matrix (Sanghi and Verma 2009a).
Enzyme-mediated synthesis of cadmium sulfate nanoparticles using the fungus
F. oxysporum uses an extracellular method. It has many important significances
such as bioleaching, bioremediation, and microbial corrosion, as well as synthesis
of nanoparticles (Ahmad et al. 2002). A. flavus–mediated synthesis of titanium
oxide (TiO2) nanoparticles has been used to observe their antimicrobial activity. The
antimicrobial activity of synthesized TiO2 nanoparticles was evaluated against
S. aureus (MTCC-3160), E. coli (MTCC-1721), P. aeruginosa (MTTCC-1034),
Klebsiella pneumoniae (MTCC-4030), and B. subtilis (MTCC-1427), using a well-
diffusion method (Rajakumar et al. 2012). Pichia fermentans JA2 has been used for
synthesis of zinc oxide nanoparticles. The antimicrobial effects of extracellularly
biosynthesized ZnO nanoparticles against pathogenic organisms such as Gram-
positive bacteria (Enterococcus sp. and S. aureus), Gram-negative bacteria (E. coli,
Salmonella sp., Shigella sp., Proteus mirabilis, K. pneumoniae, and P. aeruginosa),
and fungal strains (Candida tropicalis, Fusarium sp., Scedosporium sp. JAS1,
Ganoderma sp. JAS4, and A. terreus strain JAS1) has been measured using a well-
diffusion method (Chauhan et al. 2015).
Fungus-mediated synthesis of ZnO nanoparticles has been used to study their
antimicrobial properties. Candida diversa strain JA1 has been isolated from waste-
water from a milk processing unit. Gram-negative organisms (E. coli, Salmonella
sp., Shigella sp., P. mirabilis, K. pneumoniae, and P. aeruginosa), Gram-positive
organisms (Enterococcus sp. and S. aureus), and fungal strains (C. tropicalis,
Fusarium sp., Scedosporium sp. JAS1, Ganoderma sp. JAS4, and A. terreus strain
JAS1) have been used to study the antimicrobial properties of biologically synthe-
sized metallic nanoparticles (Chauhan et al. 2014). Saccharomyces cerevisiae
MTCC 2918 has been used in synthesis of ZnS nanoparticles. Yeasts have been
commercially exploited for several industrial applications (Sandana Mala and Rose
2014).
7.7 Conclusion
Green synthesis of fungal nanoparticles has major advantages in comparison with

traditional chemical synthesis methods. Various applications are available for reduc-
ing the risk factors in each field. The synthesized bioactive molecules have simulta-
neous actions—for example, they may function as both reducing and stabilizing
agents. As summarized in this review, these biologically synthesized nanoparticles
have promising applications in drug delivery, biomedicines, agriculture, and
pharmaceutics.
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Precautions to Avoid Consequences
Leading to Nanotoxification 8
Sharda Sundaram Sanjay
8.1 Introduction
As a relatively new field of science and technology, nanomedicine has recently

received a great boost and attracted considerable attention. Nanotechnology is the
art of creation and utilization of materials and devices at the atomic or molecular
level and supramolecular structures in the size range of 0.1–100 nm. Through
exploitation of the unique properties of materials and devices developed in this size
range, hundreds of consumer products incorporating nanoparticles are now on the
market, including cosmetics, sunscreens, sporting goods, clothing, electronics, baby
and infant products, and food and food packaging. Because of their specific charac-
teristics, nanoparticles may penetrate cell walls and become involve in intra- and
intercellular molecular processes. This has resulted in intensive and diverse applica-
tions of nanoparticles and nanomaterials in human medicine, known as nanomedi-
cine. The aims of nanomedicine are to monitor, control, construct, repair, defend,
and improve human biological systems at the molecular level, with the help of
nanodevices and nanostructures that operate massively in parallel at the cell level,
in order to achieve medical benefit.
The specific definition of nanomedicine that was compiled by the Medical Standing
Committee of the European Science Foundation (ESF) is “the science and technology
of diagnosing, treating, and preventing disease and traumatic injury, of relieving pain,
and of preserving and improving human health, using molecular tools and molecular
knowledge of the human body” (ESF 2004). The five main disciplines of nanomedi-
cine that the committee defined are (1) analytical tools; (2) nanoimaging; (3) nanoma-
terials and nanodevices; (4) novel therapeutics and drug delivery systems; and
(5) clinical, regulatory, and toxicological issues. The US National Institutes of Health
Roadmap for Medical Research in Nanomedicine (National Institutes of Health 2006)
S. S. Sanjay (*)
Department of Chemistry, Ewing Christian College, Allahabad, Uttar Pradesh, India

https://doi.org/10.1007/978-981-13-8954-2_8
202 S. S. Sanjay
defined nanomedicine as “an offshoot of nanotechnology, [which] refers to highly

specific medical interventions at the molecular scale for curing disease or repairing
damaged tissues, such as bone, muscle, or nerve.” The European report simplified the
terms as “the focus [of nanomedicine] is always on nanointeractions within a frame-
work of a larger device or biologically with a sub-cellular (or cellular) system” (ESF
2004). This means that nanomedicine has emerged from nanotechnology, which is
generally defined as construction and use of materials at the atomic and molecular
levels, specifically <100 nm. The gist of this is that nanomedicine is maintenance and
upgrading of human health using molecular gears and molecular knowledge of the
human body.
The study of the interactions of nanomaterials and nanodevices with biomole-
cules, both in vitro and in vivo, is advancing. Exploitation of the physical properties
generated by the enhanced surface-to-volume ratio of materials at the nanoscale is
now becoming operational in different fields related to medicines too. Gold
nanoshells for use in diagnosis and treatment of cancer are ready for human trials,
as are liposomes for use as vaccine adjuvants and as vehicles for drug transport
(Boisseau and Loubaton 2011).
In comparison with larger-sized devices, medical technologies are now progress-
ing for smaller devices, which are less hostile and can be implanted inside the body
with greater efficiency. Because of their smaller size, their biochemical reaction
times are much shorter. Nanodevices make crucial drug delivery faster and more
sensitive (LaVan et al. 2003).
It is important to mention some applications of a few nanomaterials that are
entering the human biological system for medicinal purposes such as cancer therapy
(Stephen et al. 2012; Nie et al. 2007; Zheng et al. 2005; Loo et al. 2004). Carbon
nanotubes (CNTs), which have a diameter in the range of 0.5–3 nm and a length of
20–1000 nm, are used for detection of DNA mutations and for detection of disease
protein biomarkers. Dendrimers <10 nm in size are used as image contrast agents
and for controlled-release drug delivery. Nanocrystals 2–10 nm in size are used for
improved formulation of poorly soluble drugs and for labeling of breast cancer
marker HeR2 on the surface of cancer cells. Nanoparticles 10–1000 nm in size are
used as contrast agents in magnetic resonance imaging and ultrasound imaging. As
permeation enhancers and reporters, these are used for targeted drug delivery, apop-
tosis, and angiogenesis. For tumor-specific imaging and deep tissue thermal abla-
tion, nanoshells are used. Nanowires have applications in detection of disease
protein biomarkers, DNA mutations, and gene expression. Quantum dots 2–10 nm
in size may be useful in optical detection of genes and proteins in animal models
and cell assays, for tumor and lymph node visualization.
Metallic nanoparticles—such as gold, nickel, silver, iron oxide, zinc oxide, gado-
linium, cerium, and titanium dioxide particles—are the ones most commonly used.
They are extremely small (~1–100 nm) (Doria et al. 2012) and thus have a large
surface area, facilitating integration of relatively higher doses of drugs (Tourinho
et al. 2012; Wang et al. 2012). A number of nanodevices have been developed to be
used for medicinal purposes. A categorization of nanomaterials used in medicine
and biosciences (Thomas et al. 2011) is shown in Figs. 8.1 and 8.2.
8 Precautions to Avoid Consequences Leading to Nanotoxification 203
Naoemuision
Nanoparticle
Nanodevices formulations
Nanoparticles
Nanomicelle
Silica nanoparticles
1D Quantum dot Metal nanoparticles
Nanostructures Fullerene Metal oxide nanoparticles
Nanocantiliver Magnetic nanoparticles
Nanoparticles Nanorod, Super magnetic nanoparticles
Nanoarray Nanotube, Hydrogel nanoparticles
Nanoprobe Nanomaterial Nanohorn,
Polymeric nanoparticles
Nanowire
Nanorobot 2D Nanopipette
Sherical nanoparticles
Nanobomb Nanofibre Fluorescent nanoparticles
Nanostructures Nanostructures
Nanofibre Hydrogel nanoparticles
Nanoboat Lipid nanoparticles
Nanotube
Nanosensor Nanofilm Lipid coated nanoparticles
Nanochannel Nanosheet Gelatin nanoparticles
PLGA nanoparticles
Nanochip
Nanoshell Hydroxyapatite nanoparticles
Nanopumps Nanodevices
Nanocage Polymer, chitosan, etc. coated
3D nanoparticles
Nanorespirocytes Nanocell
Nanostructures Drug, gadolinium etc. loaded
Nanocapsule
Nanobud nanoparticles
Fig. 8.1 Categorization of nanomaterials used in medicine and biosciences
Fig. 8.2 Nanodevices and nanomaterials (including nanorobots, a nanoprobe, gold nanoshells,
phosphorene quantum dots, red blood cells (RBCs), and a DNA probe) and morphologies of some
nanoparticles
We can easily assume that use of nanoscale particles as nanomedicines may be

helpful in the treatment different diseases and in different surgeries. However, in
addition to their numerous benefits, they have a number of disadvantages, which
cannot be ignored. Let us see what disadvantages nanomedicines may have.
204 S. S. Sanjay
Since nanoparticles are able to cross biological membranes and access cells, tis-
sues, and organs that larger-sized particles normally cannot, they can gain access to
the bloodstream after inhalation or ingestion. Some of them may penetrate the skin.
Once they get into the bloodstream, they can be transported around the body and
taken up by organs and tissues, including the brain, heart, liver, kidneys, spleen,
bone marrow, and nervous system. Unlike larger particles, they may be taken up by
cell mitochondria and cell nuclei. Studies have demonstrated that they have the
potential to cause DNA mutation and can induce major structural damage to mito-
chondria or even result in cell death.
The European Union (EU) Scientific Committee on Emerging and Newly
Identified Health Risks (SCENIHR) (http://europa.eu.int/comm/health/ph_risk/
committees/04 scenihr/04scenihr_en.htm) has pointed out the following:
Existing toxicological and ecotoxicological methods may not be sufficient to address all of
the issues arising with nanoparticles. Exposure evaluation of the dose requires information
on the number of nanoparticles and/or their surface area in addition to traditional mass
concentration characterization. Equipment for routine measurements for representative
exposure to free nanoparticles in various media is inadequate. . . . Very little is known about
the physiological responses to nanoparticles . . . existing methodologies may require modi-
fications regarding hazard evaluation, including the assessment of whether nanoparticles
can exacerbate pre-existing medical conditions, and the detection of nanoparticle distribu-
tion in the human body and in environmental compartments. . . . There are major gaps in the
knowledge necessary for risk assessment. These include nanoparticle characterisation, the
detection and measurement of nanoparticles, the dose-response, fate, and persistence of
nanoparticles in humans and in the environment, and all aspects of toxicology and environ-
mental toxicology related to nanoparticles. Of special importance are the questions con-
cerned with the transport of nanoparticles in the human body and the mechanisms of
interaction at the sub-cellular and molecular levels.
The physical and chemical properties of a substance are derived from its atomic
and molecular origin in an intricate way. At the nanoscale level, particle–particle
interactions are mostly dominated by weak van der Waals forces, which make them
sticky in nature. Stronger polar and electrostatic interactions or covalent interac-
tions also play roles. Particle aggregation is generally determined by the interparti-
cle interaction, which depends on the viscosity and polarizability of the fluid. The
tendency of colloidal particles to agglomerate or coagulate can be enhanced or hin-
dered by modification of the surface layer.
The dimensions of biological molecules such as proteins and nucleic acids are
almost the same as those of nanoparticles. Many of these, such as DNA, consist of
long, macromolecular, folded chains and are shaped by cooperative and weak
hydrogen bond interactions between side groups. Mostly, all nanoparticles, on
exposure to the tissues and fluids of the body, are adsorbed immediately onto their
surfaces, thus gaining easy entry into cells. This adsorption process depends on the
surface characteristics of the particles, including their surface chemistry and surface
energy, and the surface can be modulated by certain modifications or by functional-
ization (Schellenberger et al. 2004).
With active functionalization and interaction of nanoparticles with biomolecules,
the dose and dose rate become important with regard to their ability to proliferate
within the body and within the ecosystem. It should be remembered that the extent
and severity of toxicity depend largely upon the dose and the nature of the cellular
organelles that are affected by the nanoparticles. It is very interesting that the very
same properties that are responsible for the broad utility of nanoparticles are also
the major cause of their severe toxicity.
In nanoparticles with considerable solubility, the interaction with living systems
remains similar to that of their bulk forms. If they are biodegradable, the particle com-
position and degradation products will influence their biological effects. Materials
with very low solubility or degradability may accumulate within biological systems
and remain there for longer durations. Such nanoparticles are of the greatest concern,
as their persistence affects metabolic and cellular activities within the target host.
It should be remembered that solubility may be improved by surface-active sur-
factants. However, when experimenting with soluble nanoparticles, it is important
to consider the physics of exposure, i.e., aerodynamic and hydrodynamic or diffu-
sive processes, which depend on the size of the particles and can affect their ability
to penetrate different tissues—for example, penetration of airborne particles into the
respiratory system.
8.2 Factors Causing Toxicity
The characteristics of nanoparticles depend mainly on their structural features, such

as their size, shape, composition, and surface coating; accordingly, they are referred
to as fine nanomaterials (Kuhn et al. 2015).
As these characteristics vary, the possibility of finding these nanoparticles in dif-
ferent site-specific regions of the gastrointestinal tract changes. Through recent
research it has been realized that no particles are completely inert; even at low con-
centrations, they can have unintended effects on health (Ferin 2004). During the
course of inhalation, nanoparticles are deposited throughout the entire respiratory
tract, from the nose and pharynx down to the lungs (Oberdörster 2001; Elder et al.
2006) (Fig. 8.3).
A brief description of each of these factors is given in the following sections.
Fig. 8.3 Factors causing

nanotoxicity
Area
Size ace
Surf
Nanotoxicity Chemical
Solubility
composition
Mor
e pho
rfac logy
Su rge
a
Ch
206 S. S. Sanjay
8.2.1 Size
The use of nanoparticles in medical applications is mainly accredited to their

extrasmall size. This nanosize makes them able to overcome our defense mecha-
nisms and display appealing properties and promising results in imaging, remedial,
and therapeutic applications. However, the very same property leads to increased
deleterious effects because at the nanoscale level, as a result of the dramatically
reduced size of the particles, there is an enormous increase in the surface-to-volume
ratio, which in turn increases the relative number of the molecules of the chemical
on the surface and results in increased biological reactivity, thus leading to enhanced
intrinsic toxicity. Smaller particles may cause greater toxicity than larger particles
of the same composition, morphology, and crystalline structure, generating greater
inflammation in the lungs (Oberdörster et al. 1994). Evidence provided by animal
studies has shown rapid translocation of metal nanoparticles from the lungs into the
circulatory system and then to other organs. The nanoparticles that were found had
diameters of 30 nm (gold) (Oberdörster et al. 2005) 22 nm (titanium dioxide in
pulmonary capillaries), and 15 nm (silver) (Takenaka et al. 2001) in the blood, liver,
kidney, spleen, brain, and heart.
Donaldson et al. (2001, 2002, 2004) reported potential cardiovascular and pul-
monary toxicity of ultrafine particles. Gliga et al. (2014) experimentally verified the
size-dependent cytotoxicity of silver nanoparticles in vitro. Liu et al. (2010) reported
different levels of toxicity in the rat pulmonary system with different-sized TiO2
nanoparticles; inhaled finer nanoparticles that accumulated in the alveolar region
rather than in the upper lung airways proved to be more damaging than larger par-
ticles (Braakhuis et al. 2014).
After inhalation, nanoparticles are deposited throughout the entire respiratory
tract, starting from the nose and pharynx and extending down to the lungs
(Oberdörster 2001). Small nanoparticles measuring less than 100 nm cause adverse
respiratory health effects and more inflammation than larger particles of the same
material (Ferin et al. 1992). Particles larger than 100 nm fail to reach the bone mar-
row, while particles larger than 300 nm are absent from the blood (Jani et al. 1990).
8.2.2 Surface Area
In research on differently sized particles with the same mass, the same chemical
composition, and the same crystalline structure, greater toxicity was observed with
nanoparticles than with their larger, bulkier forms. This clearly implied that the
inflammatory effect was also dependent on the surface area of the nanoparticles.
Smaller nanoparticles have a larger surface-to-volume ratio, and so the particle
number per unit of mass is increased in comparison with larger particles. The body,
therefore, will react differently to the same dose administered in the form of billions
of nanoparticles versus several microparticles. A larger surface area leads to
increased reactivity (Roduner 2006) and thus becomes an increased source of reac-
tive oxygen species (Donaldson and Stone 2003).
With regard to surface chemistry, metallic nanoparticles are of considerable

chemical reactivity, and ionic crystal nanoparticles have been observed to accumu-
late in the protein layer during cellular exposure via the cytoplasm or lymphatic
fluid. This protein layer is possibly involved in the interaction of the nanoparticle
with the cellular system. The interaction of nanoparticles with living systems is
affected by their characteristic dimensions. As mentioned above, nanoparticles that
are a few nanometers in size may reach well inside biomolecules—a situation that
is not possible with larger particles. Oberdörster et al. (1994, 2000, 2002, 2004,
2005) reported that inhaled nanoparticles reach the blood and other target locations
such as the liver, heart, and blood cells.
It has been observed that agglomeration of nanoparticles is an important phe-
nomenon that influences their toxicity. Larger particles (such as nanoparticle aggre-
gates larger than 100–200 nm) have lower toxicity than smaller ones, which can
more easily evade the macrophage clearance defense mechanism (Oberdörster et al.
2005). It was demonstrated by Gurr et al. (2005) that a high concentration of
nanoparticles could promote particle aggregation, thereby reducing their toxic
effects in comparison with lower concentrations (Takenaka et al. 2001). Nanoparticles
may translocate through membranes. There is little evidence for an intact cellular or
subcellular protection mechanism (Fig. 8.4).
8.2.3 Chemical Composition
The toxicity of chemical nanoparticles largely depends on their chemical composi-

tion (Donaldson et al. 2004). Renwick et al. (2004) showed that the effect of carbon
black is more severe than that of titanium dioxide, although both types of
NANOPARTICLES INTERNALIZED IN CELLS

Mithocondrion
Nucleus
Cytoplasm
Membrane
Lipid vesicle
Lungs Nanoparticle inhalation Nanoparticle inhalation Brain

Skin/Gut/Blood Thrombogenesis
Nanoparticles Cardiovascular Neurogenerative
ingestion deaths diseases
Liver/Spleen
Asthma Health disorder's
Lung Cancer Orthopedic implants
wear debris Hospitalizations
Chronic obstructive -
Atheromatous Autonomous
pulmonary diseases plaques nervous
system
Fig. 8.4 Effect of nanoparticle internalization in cells

208 S. S. Sanjay
nanoparticles induced lung inflammation and epithelial damage in rats to a greater

extent than their larger, bulkier forms. While studying the effects of several different
types of nanoscale particles (polyvinyl chloride, TiO2, SiO2, Co, and Ni), Peters
et al. (2004) found that only Co induced toxicity in endothelial cells, which was
accompanied by production of the proinflammatory cytokine interleukin-8 (IL-8),
and TiO2 and SiO2 induced minor and major IL-8 releases, respectively. These
observations may have been due to differences and/or size differences at the
nanoscale, as the mean particle diameters ranged from 14 nm to 120 nm, and clus-
ters as large as 420 nm were associated with the number of ultrafine particles.
Sufferers with the same type of blood disorder show fibrous tissue clots embed-
ding nanoparticles with different compositions: gold, silver, cobalt, titanium, anti-
mony, tungsten, nickel, zinc, mercury, barium, iron, chromium, nickel, silicon,
glass, talc, and stainless steel. The common feature of the particles is their size,
ranging from tens of nanometers to a few microns (Gatti et al. 2004). Absorption of
particles in the gastrointestinal tract depends on their size; uptake diminishes with
larger particles. In the intestinal tract there is a complex mix of compounds,
enzymes, food, bacteria, etc., which can interact with ingested particles and some-
times reduce their toxicity. It has been reported that particles in vitro are less cyto-
toxic in a medium with a high protein content. Diseases associated with
gastrointestinal uptake of nanoparticles (such as Crohn’s disease and ulcerative coli-
tis) have no cure and often require surgical intervention (Jani et al. 1990).
One should be very clear in differentiating the composition and the chemistry of
the particles. Particles may have the same composition but different chemical or
crystalline structures. The type of crystalline structure may also introduce toxicity;
for example, rutile and anatase are both allotropes of titanium dioxide with the same
chemical composition, but because they have different crystalline structures, they
develop different chemical and physical properties. In the absence of light, rutile
nanoparticles (200 nm) were found to induce oxidative DNA damage, but anatase
nanoparticles of the same size were found to be safe (Gurr et al. 2005). Silver
nanoparticle aggregations have been found to be more toxic than asbestos, whereas
titanium oxide, alumina, iron oxide, and zirconium oxide were found to be less toxic.
Like nano-organisms, such as viruses, nanoparticles are capable of penetrating
cells and interacting with subcellular structures, depending on the size and shape of
the nanoparticles (Xia et al. 2006).
Both in vivo and in vitro studies have shown that nanoparticles of various com-
positions (fullerenes, CNTs, quantum dots, and automobile exhaust) generate more
free radicals and reactive oxygen species than larger particles, likely because of
their larger surface area (Stone et al. 1998; Wilson et al. 2002). Reactive oxygen
species damage cells by peroxidizing lipids, altering proteins, disrupting DNA,
interfering with signaling functions, and modulating gene transcription (Brown
et al. 2004). Oxidative stress causes many diseases, including cardiovascular and
neurological diseases, pancreatitis, and cancer.
A few decades ago, a demonstration with polio viruses (measuring 30 nm) and
silver-coated gold nanoparticles (measuring 50 nm) showed that these particles
migrated to the olfactory nerves and bulb in monkeys. Silver-coated gold nanopar-
ticles that reached the olfactory bulb were located in the mitochondria, raising major
concern about their toxicity (Oberdörster et al. 2005).
8.2.4 Shape/Morphology
For assessment of the toxicity of various nanomaterials, their toxic effects should be
compared with those of known toxic particles. There are a number of methods used
for synthesis of nanoparticles. Accordingly, their shapes can also be varied, from
zero-dimensional quantum dots to three-dimensional structures. Thus, the shape of
these particles also becomes an important factor in their toxicity. For example, there
is significant evidence of the toxicity of two-dimensional fibers, especially in rela-
tion to inhalation, as their thinness and length appear to be important physical
parameters determining their respirability and inflammatory potential. A number of
in vitro and in vivo studies have shown that the shape of nanoparticles has an impact
on their cellular uptake (Gorka et al. 2015). Cells are more accessible to rod-like and
needle-like nanoparticles than to cylindrical nanoparticles, and nanoparticles of
other shapes, such as spherical nanoparticles, show increased toxicity in the lung
epithelium (Gratton et al. 2008; Barua et al. 2013; Hsiao and Huang 2011).
Nanofibers—a special category of nanotubes—range from a few nanometers in
diameter to a length of several micrometers. The carcinogenic effects of asbestos
fibers are well known. Fibers with a small diameter may penetrate deep into the
lung, although fibers with a very long aspect ratio will remain in the upper airways.
Single-walled CNTs (SWCNTs) have been shown to induce lung granulomas after
intratracheal administration during in vivo studies (Lam et al. 2004; Warheit et al.
2004), which specifically indicates that these nanotubes cannot be categorized as a
new form of graphite on material safety data sheets.
Studies have shown the extreme toxicity of CNTs, which damage lungs more
than carbon black or silica (Muller et al. 2005). Some CNT aggregates and carbon
blacks have shown cytotoxicity comparable to that of asbestos (Soto et al. 2005).
8.2.5 Surface Charge
Often being of a colloidal nature, nanoparticles have an important chemical charac-

teristic—a surface charge—which determines their fate in biological systems, spe-
cifically in cells. Being negative charged, the plasma membrane has shown greater
affinity for positively charged nanoparticles in a number of experiments, resulting
in enhanced toxic effects in comparison with negative or neutral nanoparticles (Feng
et al. 2015). Panyam et al. (2002) found that poly(lactic-co-glycolic acid) (PLGA)
nanoparticles were ingested by cells through endocytosis. In a report published by
Konan et al. (2003), escape of nanoparticles from endosomes into the cellular cyto-
plasm was suggested to be caused by a change in the surface charge from negative
to positive during cytoplasmic delivery of the incorporated drug. Data obtained
using negatively charged polystyrene nanoparticles have shown the influence of the
positive surface charge on their escape from endosomes. Negatively charged
nanoparticles measuring 50 and 500 nm were found to permeate the skin during
dermal administration, while positively charged and neutral particles of all sizes
failed to do so. It was suggested that the total charge of the particle is more impor-
tant than the particle size. Moreover, both 50-nm and 500-nm latex particles showed
permeation through the membrane, whereas 100-nm or 200-nm negatively charged
210 S. S. Sanjay
particles did not (Kohli and Alpar 2004). It was suggested that a greater concentra-
tion of charge was responsible for overcoming the skin barrier. The 50-nm nanopar-
ticles permeated it because of their small size and large surface area, while the
500-nm nanoparticles permeated it because of the large number of charged groups.
Chen et al. (2004) reported that cationic dendrimers were more cytotoxic and
hemolytic than anionic or PEGylated dendrimers.
During evaluation of the surface characteristics of nanoparticles, Lockman et al.
(2003) observed that neutral nanoparticles and anionic nanoparticles at low concen-
trations had no effect on blood–brain barrier (BBB) integrity, but high concentra-
tions of anionic nanoparticles and cationic nanoparticles were toxic to the BBB. The
rates of brain uptake of anionic nanoparticles at lower concentrations were higher
than those of neutral or cationic formulations at the same concentrations. Therefore,
the surface charges of nanoparticles cannot be ignored with regard to toxicity and
brain distribution patterns.
The charge of nanoparticles plays an essential role in their uptake by platelets
and their influence on blood clot formation. Uncharged polystyrene particles do not
have an effect on blood clot formation. Negatively charged nanoparticles signifi-
cantly inhibit thrombus formation, while positively charged nanoparticles enhance
platelet aggregation and thrombosis. The interaction between platelets and posi-
tively charged particles seems to be due to the net negative charge that platelets
carry on their surface (Nemmar et al. 2002). Positively charged nanoparticles inter-
act with negatively charged platelets and reduce their surface charge, making them
more prone to aggregation.
The kinetics of particles in the gastrointestinal tract depend strongly on the charge
of the particles. Positively charged latex particles are trapped in negatively charged
mucus, while negatively charged latex nanoparticles diffuse across the mucus layer
and became available for interaction with epithelial cells (Hoet et al. 2004) (Fig. 8.5).
Fig. 8.5 Nanovesicles for

use in drug delivery via
nanotubes, liposomes,
dendrimers, etc.
8.2.6 Dose-Dependent Effects
The dose is defined as the amount or quantity of a substance—or, more appropri-

ately, a medicine—that reaches a biological system. The dose is directly associ-
ated with exposure or the concentration of the medicine in the relevant medium
(air, food, water) multiplied by the duration of the contact. Dose-dependent effects
were seen in a study performed in trout by Smith et al. (2007), who observed rises
in the ventilation rate and gill pathologies (edema, alteration of mucocytes, hyper-
plasia) due to precipitation of SWCNTs on gill mucus at concentrations between
0.1 mg/L and 0.5 mg/L, showing that SWCNTs can act as a respiratory toxicant
in trout.
In a comparison of the health effects of inhaled TiO2 nanoparticles of different
sizes, it was observed that low-dose (10-mg/m3) exposure to particles 20 nm in
diameter resulted in a greater lung tumor incidence than high-dose (250-mg/m3)
exposure to particles 300 nm in diameter (Hoet et al. 2004).
Nanoparticles with molecules of a compatible size, geometry, bonding, and
charges may interact with receptors. In such cases, nanoparticles may be engulfed
by phagosomes (which protect cellular organelles from the nanoparticles) and,
according to Porter et al. (2006), nanoparticles such as C60 molecules, after entering
the cell, may be found on the nuclear membrane and within the nucleus. Because of
such uptake and free movement of nanoparticles within the cell, they can be found
in various locations inside the cell, making them very dangerous, as they may have
direct access to cytoplasmic proteins and organelles (Stefani et al. 2005; Garcia
et al. 2005), mitochondria, lipid vesicles, the nuclear membrane, or the inside of the
nucleus; moreover, they may damage DNA. These different types of damage may
ultimately cause cell death. Nanoparticles may also be internalized by various other
cells such as endothelial cells, pulmonary epithelial cells, gastrointestinal epithelial
cells, red blood cells, platelets, and nerve cells (Oberdörster et al. 2004; Stefani
et al. 2005; Garcia et al. 2005; Li et al. 2003; Penn et al. 2005; Hopwood et al. 1995;
Peters et al. 2006; Rothen et al. 2006). These internalizations depend on the size of
the nanoparticles; for example, smaller nanoparticles (measuring <100 nm) localize
in organelles such as mitochondria, causing disruption of mitochondrial architec-
ture (Li et al. 2003), and C60 molecules (with a diameter of 0.7 nm) may penetrate
cells via different mechanisms through ion channels or pores in the cell membrane
(Porter et al. 2006).
Detailed study of the adverse effects of engineered nanoparticles is needed,
including nanoparticles of different compositions and particle sizes, and well-
defined target organisms or cell types, to study their toxicokinetics (including trans-
location, excretion dynamics, acute versus chronic toxicity, and toxicity mechanisms)
to establish dose–response relationships (Table 8.1).
212 S. S. Sanjay
Table 8.1 Consequences of health toxicities due to cell permeation by certain nanoparticles
Nanoparticles Consequences of health toxicities
Carbon nanotubes Apoptosis, decreased cell viability, lung toxicity, oxidative stress,
retarded cell growth, skin irritation, etc.
Fullerenes Retarded cell growth, decreased cell viability, oxidative stress,
apoptosis, etc.
Nanosilver Alterations in nonspecific immune responses, altered cell signaling,
apoptosis, necrosis of cells, oxidative stress, etc.
TiO2 nanoparticles Excessive exposure in humans may result in increased oxidative
stress, retarded cell growth, slight changes in the lungs, etc.
Silicon-based Cardiovascular effects, cytotoxicity, increased oxidative stress, etc.
nanoparticles
Polymeric Oxidative stress, inflammation, alterations in cellular morphology and
nanoparticles functioning, etc.
Multifarious Arrest of cell growth and sometimes even cell death, chromatin
nanostructures condensation, free radical formation
Nanostructured flame Oxidative stress, fibrosis, cardiovascular effects, cytotoxicity,
retardants carcinogenic effects, etc.
Kreyling et al. (2002, 2004) reviewed and proposed hypotheses to explain the
adverse health effects of nanoparticles and mainly emphasized the interactions and
particle characteristics summarized below:
• The importance of a large surface area for interactions with cells and tissues
• Complex formations with biomolecules
• Formation of increased levels of radical species in comparison with larger
particles
• Increased induction of oxidative stress
• Induction of cellular DNA damage
• Induction of oxidative stress by lipid peroxidation
• Distribution
• Deposition characteristics dependent on size
• Uptake by cells of the respiratory epithelium
• Increased access to interstitial spaces
• Access to systemic circulation
• Organ system effects, including effects on immune and inflammatory systems
• Reduced function of macrophages, reduced phagocytosis of the particles them-
selves, reduced macrophage mobility, and cytoskeletal dysfunction
• Increased proinflammatory activity, and induction of cytokines and other
mediators
• Adverse effects on cardiac function and vascular homeostasis
8.3 Green Solutions to Nanotoxicity
As we have seen, research in the field of nanoscience has proliferated worldwide,

with little consideration of nanomaterial safety and toxicity. This raises the question
of whether nanotechnology is a boon or a curse. Intellectuals, scientists, and policy
makers have therefore started thorough and intensive deliberations to address envi-
ronmental health and safety (EHS) concerns, which have developed as a consequence
of this proliferation of nanotechnology, to minimize the hazards that it may cause
(Hussain et al. 2016). The ultimate solution to this contentious issue is the practice of
safe, green nanoscience. For commercialization, improvement of low-waste methods
and high precision of nanoproduction are crucial. As a new tool for green chemistry,
green nanoscience is providing novel design strategies for synthesis of new nano-
structured materials, with improved control of their chemical and physical structures
(Zhao et al. 2014). Green nanoscience can be considered an applicatory approach to
green chemistry to yield safer nanotechnology (Sanjay 2019). Implementation of the
12 principles of green chemistry—avoidance of waste generation, atom economy,
less hazardous chemical synthesis, design of safer materials, safer solvents and aux-
iliaries, design for energy efficiency, use of renewable feedstock, reduced use of
derivatives, use of catalysis to improve the selectivity of reactions and reduce the
amount of energy necessary to initiate a reaction, design for degradation, real-time
analysis for pollution prevention, and inherently safer chemistry for accident preven-
tion (Anastas and Warner 1998)—has already paved the way to greener nanoscience
to develop potentially safer synthetic strategies for manufacture of nanomaterials
with reproducibility of a definite structure, composition, and purity; development of
safer and greener substitute materials by avoidance of production of hazardous prod-
ucts and by-products; and increases in potential nanomaterial production.
To practice these principles, it is indispensable to acquire thorough knowledge of
mechanistic understanding, synthetic methods, characterization tools and strategies,
and biological testing procedures, so that proper judgment for selection of safer
materials can be exercised.
A large number of researchers are now practicing green chemistry and synthesiz-
ing green nanoparticles of well-defined chemical composition, size, and morphol-
ogy with the help of various green methods, and are utilizing them for different
progressive technologies (Vinothkannan et al. 2015; Saini et al. 2015; Mashwani
et al. 2015; Emmanuel et al. 2014).
Ample examples are available to demonstrate applications of principles to show
how greener approaches can improve production of materials by reducing costs,
improving output, and avoiding waste (Yan et al. 2016). A review by Nath and
Banerjee (2013) discussed new hope for medical biology in consistency with univer-
sal efforts to minimize generation of toxic waste, and appealed for integration with
contemporary developments in science and technology to develop energy-efficient
production, green chemistry, and biochemical processes.
Yong et al. (2015) synthesized nanocomposite biodegradable polymers with
physical and mechanical characteristics equal to those of engineering plastics, based
on biopolymers such as chitosan and starch, intercalated with nanolayered clay. To
develop design rules for greener and safer nanomaterials, Kumar et al. (2015)
selected compositionally, structurally, and well-defined nanomaterials to study
hypotheses regarding the influence of nanostructures on biological impacts. The
impacts of synthesized nanomaterials can be interconnected to structural features by
systematic characterization and tests of purity (Ghodake et al. 2016). Before appli-
cation of a nanomaterial, testing for its biocompatibility is necessary and, accord-
ingly, the mechanisms of its action for completion of a reaction can be predicted.
214 S. S. Sanjay
New hypotheses have been proposed that may contribute to material design. The
subsequent iterations of the data lead to understanding of the activity or structure
relationships, as well as “design rules” for greener and safer nanomaterials (Balbus
et al. 2007). For designing new nanomaterials, the approaches should be purely
judicial: firstly, total avoidance of compositions that are toxic in nature or that pro-
duce toxic products; and, secondly, mapping out of the hazards of materials at the
micrometer, nanometer, and molecular scales. Even reduction of production of haz-
ardous by-products should be given priority.
In minimizing adverse impacts and improving manufacturability, efficiency
plays an important role. Material efficiency can be increased by precise product
control and avoidance of waste (Kaur et al. 2014).
Functionalized nanoparticles show considerable interactions with biomolecules.
The amount/concentration and dose rate of a particulate nanomedicine display its
ability to be distributed within the human body. Nanoparticles with considerable
solubility intermingle with living systems and remain similar enough to the bulk
material for their toxicological testing procedures and approaches to be the same as
those applicable to their bulk forms. The biodegradability of particles, their compo-
sition, and their end products may influence biological processes. Nanomaterials
with very low solubility or poor degradability may accumulate within biological
systems and remain there for long durations. Such nanoparticles warrant the great-
est concern and attention.
8.4 Health Concern Organizations
Some health concern organizations have also issued warnings about novel risks
posed by nanoparticles, nanomaterials, and nanodevices. Some of these health orga-
nizations, who are pioneers in this field, are:
1. The Organization for Economic Co-operation and Development (OECD) evalu-

ates risk assessment approaches for manufactured nanomaterials, which is done
by information exchange and identification of opportunities to strengthen and
enhance risk assessment capacity.
2. The US National Institute for Occupational Safety and Health (NIOSH) devel-
ops and implements commercial nanotechnology by conducting strategic plan-
ning and research to provide national and world leadership for incorporation of
research findings on the implications and applications of nanotechnology into
good occupational safety and health practice.
3. The EU NanoSafety Cluster works on projects addressing all aspects of nano-
safety, including toxicology, ecotoxicology, exposure assessment, mechanisms
of interaction, risk assessment, and standardization. Moreover, it conducts work-
shops and seminars to educate people, particularly nanotechnology workers.
4. The German Federal Institute for Materials Research and Testing (BAM) is
involved in the following EU-funded FP7 (7th Framework Programme for
Research and Technological Development) projects:
a. Nano-Define: The aim of Nano-Define is to resolve the governance chal-

lenges associated with implementation of nanomaterial legislation by address-
ing the issues of the availability of suitable measuring techniques, reference
material, and validated methods acceptable to all stakeholders; and by deliv-
ering an integrated and interdisciplinary approach.
b. Nano-Valid: The main objective of Nano-Valid is development of new refer-
ence methods and certified reference materials, including methods for charac-
terization, detection/quantification, dispersion control, and labeling, as well
as hazard identification and assessment of exposure to and risks of engineered
nanoparticles.
5. The German Federal Ministry of Education and Research (BMBF) focuses on
safe handling of manufactured nanomaterials and studies the effects of engi-
neered nanomaterials on humans and the environment.
6. The responsibilities of the German Federal Institute for Risk Assessment (BFR)
are:
a. To demonstrate and establish new principles and ideas based on data from
value chain implementation studies
b. To establish safety-by-design as a fundamental pillar in the validation of
novel manufactured nanomaterials
c. To establish nanomaterial grouping/classification strategies according to tox-
icity and biological effects for supporting risk assessment
d. To group nanostructured materials for protection of workers, consumers, and
the environment, and for risk minimization
7. The main responsibility of the German Federal Institute of Occupational Safety
And Health (BAuA) is grouping of nanostructured materials for protection of
workers, consumers, and the environment, and for risk minimization.
8. The German Federal Research Institute of Nutrition and Food (Max Rubner
Institut (MRI)) detects and characterizes nanomaterials in complex matrices
such as food; it supports and performs research on nanosized carrier systems for
bioactive compounds and studies interactions of nanomaterials with compounds
in the food matrix.
9. The specific objectives of Modelling Nanomaterial Toxicity–European

Cooperation in Science & Technology (MODENA-COST) are to study synthesis
of engineered nanomaterials with controlled composition, size, area, and nano-
texture; to develop strategies to immobilize engineered nanomaterials in matri-
ces on substrates with a minimal effect on the desired properties and surface
reactivity; and to identify the relevant data sets for quantitative nanostructure–
toxicity relationship (QNTR) modeling.
8.5 Conclusion
Nanomedicines undoubtedly have vast applications but may be quite expensive. It

is interesting that the very same properties that make them supermedicines are also
responsible for their hazards to health. Nanoparticles may behave differently from
216 S. S. Sanjay
their bulk forms as a result of their size, composition, surface area, shape, morphol-
ogy, and dose within the body. Each type of nanoparticle has different characteris-
tics, and a combination of two or more types may behave in different ways, causing
nanotoxicity and giving rise to various diseases such as cancer; benign-tumors; or
cardiovascular, respiratory, gastrointestinal, neural, Alzheimer’s, or Parkinson’s dis-
eases. Solutions to nanotoxicity lie only in green nanotechnology. As a ray of hope,
to minimize nanotoxicity, many health concern organizations have come forward to
combat problems caused by nanoparticles.
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Nanoparticles

Ashutosh Kumar Shukla

ISBN 978-981-13-8953-5 ISBN 978-981-13-8954-2 (eBook)

© Springer Nature Singapore Pte Ltd. 2020

This collection intends to highlight the potential applications of nanoparticles in

Allahabad, India Ashutosh Kumar Shukla

1 Theranostic Applications of Lysozyme-­Based Nanoparticles������������������ 1

A549 Adenocarcinomic human alveolar basal epithelial cells

S. Das · C. R. Patra (*)

© Springer Nature Singapore Pte Ltd. 2020 1

MALDI Matrix-assisted laser desorption/ionization

Lysozyme is an enzyme (anti-bacterial) generally distributed in divergent biological

1.1.1 Lysozyme and Its Background

Lysozyme is a ubiquitous anti-microbial enzyme generally found in fluids, secre-

1.1.2 Biological Activity

Lysozyme shows catalyzing activity by hydrolyzing the peptidoglycan residues

lysozyme and showed their mechanisms for enhanced anti-microbial activity

1.1.3 Application in Different Fields

Lysozyme has various biological applications like anti-microbial, anti-proliferative,

1.1.3.1 Anti-Microbial Activity

1.1.3.2 Anti-Proliferative Activity of Lysozyme

concentrations, whereas at concentration lower than that it triggered the prolifera-

1.1.3.3 Amyloid-Forming Ability

marine lysozyme hen egg lysozyme

or without SAP (serum amyloid p component), member of pentraxins (Helmfros

1.2 Interaction of Lysozyme with Nanoparticles

1.3  herapeutic Application of Lysozyme-Based

Lysozyme-based nanoparticles have several applications including anti-microbial,

1.3.1 Anti-Microbial Activity

Owing to the rampant growth of drug-resistant microbes, alternative approaches

for the synthesis of lysozyme-coated silver nanoparticles through various methods

Fig. 1.4 Bactericidal effect of lysozyme-stabilized Ag NPs against P. aeruginosa 3. Zones of

mentioned that in the presence of methanol lysozyme took an amphiphatic form

1.3.2 Drug Delivery

a Control Lysozyme ZNPs Lysozyme-ZNPs

Pristine ND clusters ND-COOH ND-PEI Timolol-embedded ND-nanogel

1.3.3 Wound Healing

Day 0 Day 3 Day 6 Day 9 Day 12 Day 15 Day 18 Day 21

Dressing * *** ***

1.3.4 Catalytic Activity

1.4  iagnostic Application of Lysozyme-Based

Lysozyme-based nanoparticles have been developed for diagnostic purposes such as

1.4.1 Cell Imaging

Cell imaging is one of the foremost important subsets in biological applications.

1.4.2 Sensing Applications

Viable cell (%)

1.5 Future Prospects of Lysozyme-Based Nanoparticles

Certainly, there is a drift in combining the therapeutic as well as diagnostic applications

GENE THERAPY/ CHEMOTHERAPY/

Nanotechnology includes the orchestrating development of small-sized devices that

© Springer Nature Singapore Pte Ltd. 2020 25

Among many advantages of nanomaterials in cancer treatment, the fate of adminis-

2.2 Nanotechnology and Cancer Therapy

by different ligands encapsulated drug molecules which are transferred to target

2.3 Nanomaterials as Cancer Therapeutic Agents

2.3.1 Metal Nanoparticles

Transition metal compounds such as arsenic compounds have historical background

The first platinum-containing complex to be used in cancer therapy is cisplatin.

2.3.1.1 Plasmonic Metal Nanoparticles

2.3.1.2 Magnetic Metal Nanoparticles

micro molar concentrations in tumor site is maintained by combining it with mag-

2.3.1.3 Semiconductor Metal Nanoparticles

2.3.2 Carbon Dots

Allahabad, India Ashutosh Kumar Shukla

1 Theranostic Applications of Lysozyme-Based Nanoparticles�� 1

1.1.1 Lysozyme and Its Background

1.1.2 Biological Activity

1.1.3 Application in Different Fields

1.1.3.1 Anti-Microbial Activity

1.1.3.2 Anti-Proliferative Activity of Lysozyme

1.1.3.3 Amyloid-Forming Ability

1.2 Interaction of Lysozyme with Nanoparticles

1.3 herapeutic Application of Lysozyme-Based

1.3.1 Anti-Microbial Activity

1.3.2 Drug Delivery

1.3.3 Wound Healing

Dressing * * *

1.3.4 Catalytic Activity

1.4 iagnostic Application of Lysozyme-Based

1.4.1 Cell Imaging

1.4.2 Sensing Applications

1.5 Future Prospects of Lysozyme-Based Nanoparticles

2.2 Nanotechnology and Cancer Therapy

2.3 Nanomaterials as Cancer Therapeutic Agents

2.3.1 Metal Nanoparticles

2.3.1.1 Plasmonic Metal Nanoparticles

2.3.1.2 Magnetic Metal Nanoparticles

2.3.1.3 Semiconductor Metal Nanoparticles

2.3.2 Carbon Dots

2.3.3 Carbon Nanotubes (CNTs)

2.3.4 Organic-Based Nanomaterials

2.3.4.1 Solid-Lipid Nanomaterials

2.3.4.4 Polymeric Micelles

2.3.4.5 Polymeric Nanoparticles

2.4 Role of Nanomaterials in Cancer Ablation Techniques

2.4.1 Nanomaterials as Imaging Probes

2.4.2 Nanomaterials in Gene Therapy

2.4.3 Nanomaterials in Immunotherapy

2.4.4 Nanomaterials in Radiothermal Cancer Therapeutics

2.4.5 Nanomaterials in Magnetic Hyperthermic Therapy (MHT)

2.4.6 Nanomaterials in Photodynamic Therapy

2.4.7 Nanomaterials in Chemotherapeutic Techniques

2.5 Fate of Nanomaterials in Biological System

3.2 Nanoparticles used in Various Fields of Dentistry

3.2.1 Preventive Dentistry

3.2.2 Restorative Dentistry

3.2.3 Endodontic Treatment

3.2.6 Periodontal Treatments

3.2.7 Dental Implants

3.3 Common Nanoparticles Used in Dentistry

3.3.1 Polymeric Nanoparticle

3.3.2 Metallic Nanoparticles

3.3.3 Inorganic Nanoparticles

3.4 Current Applications of Nanoparticles

3.4.1 Treatment of Bacteria

3.4.2 Prevention of Biofilm

3.4.3 Dental Implant

3.4.4 Prevention of Tooth Erosion