Materials Horizons: From Nature to Nanomaterials

Amit Kumar Nayak

Md Saquib Hasnain Editors

Biomedical
Composites
Perspectives and Applications
Materials Horizons: From Nature
to Nanomaterials

Series Editor
Vijay Kumar Thakur, School of Aerospace, Transport and Manufacturing,
Cranfield University, Cranfield, UK
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Amit Kumar Nayak · Md Saquib Hasnain
Editors

Biomedical Composites
Perspectives and Applications
Editors
Amit Kumar Nayak Md Saquib Hasnain
Department of Pharmaceutics Department of Pharmacy
Seemanta Institute of Pharmaceutical Palamau Institute of Pharmacy
Sciences Chianki, Daltonganj, Jharkhand, India
Mayurbhanj, Odisha, India

ISSN 2524-5384 ISSN 2524-5392 (electronic)

Materials Horizons: From Nature to Nanomaterials
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Preface

Composites designed for the uses in various biomedical applications are usually well-
known as “biomedical composites”. There are numerous prospective biocomposites,
which can be classified into various overlapping categories. The most important
applications of biomedical composites include drug delivery, gene delivery, cancer
therapy, antimicrobial, tissue regeneration, wound healing, dentistry, prosthesis,
bioimaging, biosensors, etc.
The current book entitled Biomedical Composites: Perspectives and Applications
covers the recent innovations in the developments of various biomedical composites
and their applications in different biomedical uses like drug delivery, gene delivery,
cancer therapy, antimicrobial, tissue regeneration, wound healing, dentistry, pros-
thesis, bioimaging, biosensors, etc. The current book is a collection of 9 chapters
presenting different key topics along with emphasis on recent advances in the biomed-
ical field by academicians and researchers across the world. A concise account on
the contents of each chapter has been described to provide a glimpse of the book to
the readers.
The Chapter entitled “Biomedical Applications of Nanosilicate Composites”
describes different uses of nanosilicate composites in tissue engineering and regen-
erative medicine, dentistry, drug delivery, wound healing, etc. In addition, a brief
introduction of biomedical composites has been presented.
The Chapter entitled “Polysaccharide-Based Composites for Biomedical
Applications” presents the most relevant and more recent advances in the devel-
opment of polysaccharide-based composites and their uses in biomedical fields like
drug delivery, biosensors, wound dressing, tissue engineering, etc.
The Chapter entitled “Biomedical Nanocomposites” offers the most recent
significant researches and developments on various nanocomposites for their poten-
tial uses in drug delivery, gene delivery, antimicrobial, tissue regeneration, wound
healing, dentistry, prosthesis, bioimaging, and biosensors. In addition, nanocompos-
ites and biomedical nanocomposites have been defined with a brief introductory
understanding.

v
vi Preface

The Chapter entitled “Antimicrobial Nanocomposites” presents a comprehen-

sive review on the nanocomposites as antimicrobial agents in food packaging, wound
dressings, water treatment, anticancer, and dental applications.
The Chapter entitled “Potential Application of Silver Nanocomposites
for Antimicrobial Activity” highlights the pathogenic strains responsible for the
microbial infections, the limitations of the conventional treatment strategies, emer-
gence of silver nanocomposites as the new treatment strategy. Various synthesis
methods of the silver nanocomposites and their applications in the area of antimicro-
bial activity and its ultimate mechanism of action behind it are discussed. In addition,
the challenges and future perspectives to be met in order for the silver nanocompos-
ites as excellent antimicrobial agents in the upcoming period of commercial market
have been addressed.
The Chapter entitled “Cellulose and Chitin Nanofibers: Potential Applications
on Wound Healing” discusses the uses of cellulose, chitin, and their derivatives to
prepare cellulose nanofibers and chitin nanofibers for wound healing applications.
The Chapter entitled “Bionanocomposites for In Situ Drug Delivery
in Cancer Therapy: Early and Late Evaluations” highlights the recent advances
in the development of bionanocomposites for in situ drug delivery, their efficacy
and impact on cancer therapy, how to select the right polymer, and how to study
polymeric-drug interactions, in silico.
The Chapter entitled “Tumor Microenvironment and Intracellular Signal-Ac-
tivated Nanocomposites for Anticancer Drug Delivery” summarizes the recent
advances in various tumor microenvironments and intracellular signal-activated
nanocomposites for anticancer drug delivery. Future perspectives of design consid-
eration were also discussed in detail.
The Chapter entitled “Nanocomposites for Cancer Targeted Drug Delivery
Therapeutics” deals with the materials that can be combined to form the most inter-
esting nanocomposites will be summarized by making a classification depending on
the nature of one of the constituent materials. Then, polymer-based nanocomposites,
clay-based nanocomposites, metal-based nanocomposites, silica-based nanocom-
posites, magnetic-based nanocomposites, and carbon-based nanocomposites will be
described in relation to their application in nanomedicine for cancer therapy.
We would like to convey our sincere thanks to all the authors of the chapters
for providing timely and valuable contributions. We thank the publisher—Springer
Nature. We specially thank Dr. Vijay Kumar Thakur (Series Editor, Materials
Horizons: From Nature to Nanomaterials, Springer Nature), Swati Meherishi,
Sushmitha Shanmuga Sundaram, Vindhya H Pillai, and Ashok Kumar for their
invaluable support in the organization of the editing process right through the begin-
ning to the finishing point of this book. We gratefully acknowledge the permissions to
reproduce copyright materials from various sources. Finally, we would like to thank
our family members, all respected teachers, friends, colleagues, and dear students for
their continuous encouragements, inspirations, and moral supports during the prepa-
ration of the current book. Together with our contributing authors and the publishers,
Preface vii

we will be extremely pleased if our endeavor fulfills the needs of academicians,

researchers, students, biomedical experts, pharmaceutical students, and drug delivery
formulators. In a nutshell, it will also help the health professionals in academia as
well as in the industries.

Mayurbhanj, India Amit Kumar Nayak

Daltonganj, India Md Saquib Hasnain
Contents

Biomedical Applications of Nanosilicate Composites . . . . . . . . . . . . . . . . . . 1

Ashwini Kumar and Awanish Kumar
Polysaccharide-Based Composites for Biomedical Applications . . . . . . . . 19
Patrícia Alves, Filipa Gonçalves, and M. H. Gil
Biomedical Nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Amit Kumar Nayak, Saad Alkahtani, and Md Saquib Hasnain
Antimicrobial Nanocomposites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
El-Refaie Kenawy, Mohamed M. Azaam, Syed Anees Ahmed,
and Md Saquib Hasnain
Potential Application of Silver Nanocomposites for Antimicrobial
Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
Shagufta Haque, Mamatha Julappagari, and Chitta Ranjan Patra
Cellulose and Chitin Nanofibers: Potential Applications on Wound
Healing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Athira Johnson, M.S. Neelakandan, Jiya Jose, Sabu Thomas,
and Nandakumar Kalarikkal
Bionanocomposites for In Situ Drug Delivery in Cancer Therapy:
Early and Late Evaluations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145
Luiza Steffens Reinhardt, Pablo Ricardo Arantes,
Jeferson Gustavo Henn, and Dinara Jaqueline Moura
Tumor Microenvironment and Intracellular Signal-Activated
Nanocomposites for Anticancer Drug Delivery . . . . . . . . . . . . . . . . . . . . . . . 167
Yilan Huang, Yiheng Huang, Yuefei Zhu, Xiaowen Zhu, and Zhiqing Pang
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics . . . . . 201
Francisco N. Figueroa, Dariana Aristizabal Bedoya,
Miriam C. Strumia, and Micaela A. Macchione

ix
About the Editors

Dr. Amit Kumar Nayak (M. Pharm., Ph.D.) is currently an Associate Professor at
Seemanta Institute of Pharmaceutical Sciences, India. He has earned his Ph.D. from
IFTM University, Moradabad, India. He has over 12 years of research experiences
in the field of pharmaceutics, especially in the development and characterization of
novel biopolymeric and nanostructured drug delivery systems. He has authored over
123 research and review publications in various high impact peer-reviewed journals
and 82 book chapters. He has edited/authored 9 international books. He has earned
highly impressive publishing and cited record in Scopus (h-index: 35) and in Google
Scholar (h-index: 40, i10-Index: 110). He has received University Foundation Day
Research Award-2019 by Biju Patnaik University of Technology, Odisha. Dr. Nayak
is life member of Association of Pharmaceutical Teachers of India (APTI) and a
Registered Pharmacist.

Dr. Md Saquib Hasnain (M. Pharm., Ph.D.) has over 9 years of research expe-
rience in the field of drug delivery and pharmaceutical formulation analyses,
especially systematic development and characterization of diverse nanostructured
drug delivery systems, controlled release drug delivery systems, bioenhanced drug
delivery systems, nanomaterials, and nanocomposites employing Quality by Design
approaches and many more. He has authored over 45 publications in various high
impact peer-reviewed journals, 80 book chapters, and 10 books to his credit. He is
also serving as the reviewer of several prestigious journals. He has an impressive
publishing and cited records in Google Scholar (h-index: 18, i10-Index: 35). He has
also participated and presented his research work at over ten conferences in India,
and abroad. He was also the member of scientific societies i.e., Royal Society of
Chemistry, Great Britain, International Association of Environmental and Analytical
Chemistry, Switzerland, and Swiss Chemical Society, Switzerland.

xi
Biomedical Applications of Nanosilicate
Composites

Ashwini Kumar and Awanish Kumar

1 Biomedical Composites: Definition and Applications

The term “composite” used either by general material scientists or by biomedical

engineers can in general be defined as a material, not existing naturally, composed
of two or more chemically and physically distinct materials that are arranged in
a manner that the new property of the composite is completely different from the
individual components. Generally, a composite has a continuous bulk phase called
matrix and a non-continuous phase known as reinforcement. The bulk phase or matrix
generally has lower Young’s modulus and higher elasticity while the reinforcement
has high load capacity, physical, and mechanical characteristics. In biomedical engi-
neering, the matrix of a composite is generally a biopolymer while an inorganic bioac-
tive material is considered for reinforcement. These composites, that have specific
applications in biomedical engineering, are termed as “Biocomposites” [1, 2]. In
composites, matrix or the continuous bulk phase supports the reinforced material
and improves the mechanical and physical properties needed for specific biological
application. Matrix has also been reported to improve the processing parameters
and characteristics of the biocomposites [1]. Biocomposites can contain polymer,
ceramic, or metals that individually or together have certain bioactivity or should be
bio-inert but should always be biocompatible. The term biocompatibility includes
biofunctionality, bioinertness, bioactiveness, and biodegradability. Another impor-
tant consideration today for choice of materials to be biomaterials is that they should
avoid biofouling. Biofouling of biomaterials is one of the biggest medical threats
that can result in infections that are emergency conditions [3].
In commercial terms, the global market for biomaterials is promising with greatest
demand for heart valves (around 1 lakh per year globally), and orthopedic implants

A. Kumar · A. Kumar (B)

Department of Biotechnology, National Institute of Technology, Raipur 492010, Chhattisgarh,
India
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2021 1

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_1
2 A. Kumar and A. Kumar

(around 1 million per year globally) [3]. This figure consists of both biocomposites
and metallic implants. A growing interest in natural biopolymers-based biocom-
posites owe to great biocompatibility of these biopolymers with the human body
and are biodegradable too (if there is need). These polymers include silk, collagen,
chitosan, alginate, hyaluronic acid, and starch. There are synthetic polymers such as
polyethylene glycol (PEG), poly glycolic acid (PGA), poly lactic acid (PLA), poly-L-
Lactic acid (PLLA), poly (lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL),
polyanhydrides, and poly propyl fumarate that are biocompatible but have compar-
atively slower biodegradation rate. These polymers have been extensively studied
for various biomedical applications such as drug delivery, tissue engineering, and
medical implants. Out of these PEG, PLA, PLLA, and PLGA are FDA-approved
biopolymers with Generally Regarded As Safe (GRAS) status [3].
Biocomposites have found substantial applications in medical science, primarily
as the biomedical implant materials. Starting with orthopedic implants, UHMWPE
(ultra high molecular weight polyethylene) and PEEK (poly ether ether ketone) poly-
mers have been the mainstay in this application to from composites. These are high
molecular weight polymers exhibiting extreme resistance to chemicals, pressure, and
temperature. Researchers across the globe have been trying to reinforce these poly-
mers with certain inorganic materials for wide application purpose [3]. In case of
load bearing bone implants, the traditional use of 316L stainless steel and Ti-6Al-V4
titanium alloy has witnessed an induction of composite materials such as carbon fiber
reinforced PEEK or polysulfone matrices. These can be much better tailored as per
the mechanical properties of the load bearing bones. The issue of biocompatibility
from the carbon debris in the implants has been addressed by coating with hydrox-
yapatite or the titanium-based alloy. For fracture fixation surgeries, it was necessary
that the bone fixation plates are slowly biodegradable so that the need for second
surgery to remove the plates can be averted. In this regard, PLA was chosen as the
matrix and reinforced either with carbon fiber or the calcium phosphate-glass fiber
to make composites. The former inorganic filler is half biodegradable while the latter
is completely resorbed gradually [1]. An Interpenetrating-Polymer Network (IPN)
formed by mixing Bis-Phenol A-Glycidyl Dimethacrylate (Bis-GMA) and Triethy-
lene Glycol Dimethacrylate (TEGDMA) displayed remarkable biomineralization
characteristics and have been used as restorative composite filler in dentistry. Dental
composites have been more successful as compared to bare metals and ceramics in
dentistry. These composites easily match the physical and mechanical properties of
the natural teeth. Dental composites have been used in the preparation of crowns and
even as dental fillers. Dental crowns are usually all-ceramic composites. An impor-
tant example is In-Ceram that is a composite of alumina and glass. Orthodontic arch-
wires are another example of dental composite application. Orthodontic brackets are
also formed from a composite structure composed of polyethylene matrix reinforced
with HAp nanoparticles [1, 3]. Various other composites such as PCL-Polyurethane,
Poly Ethylene Oxide-PCL (PEO-PCL), and Poly (ether) Urathane-Poly Dimethyl
Siloxane (PEtU-PDMS) have been tested for synthetic vascular scaffolds for the
purpose of arterial and venous grafting. Biocomposites of biopolymer and inorganic
substances such as hydroxyapatite (HAp) and tricalcium phosphate (TCP) have been
Biomedical Applications of Nanosilicate Composites 3

tested extensively for bone tissue engineering and dental applications. This kind of
composite is said to mimic the natural bone/cartilage and imparts bone/cartilage-like
characteristics to the prepared scaffold [3].
Apart from the various inorganic reinforcement materials used in various biocom-
posites, the use of clay minerals has recently gained momentum. These are usually
also termed biomedical clays based on their unconventional application in biomed-
ical field. Clays are the naturally occurring minerals that are important and major
constituents of sedimentary rocks. They are primarily composed of silica (layered
silicates) mostly in the form of aluminosilicates. The general chemical formula for
these aluminosilicates is (Ca, Na, H)(Al, Mg, Fe, Zn)2 (Si, Al)4 O10 (OH)2 xH2 O, where
x represents the amount of water present in the mineral unit. Thus, we can say that
clay minerals are hydrated aluminum silicates with variable amount of magnesium,
zinc, iron, and other alkali and elements. Clay minerals are broadly classified into two
categories: natural and synthetic clays based on the occurrence of alternating tetrahe-
dral SiO2 and octahedral AlO6 sheets with varying ratios. This ratio could be 1:1 (one
octahedral layer linked to a tetrahedral one), 2:1 (two tetrahedral sheets on either side
of an octahedral layer), and 2:1:1 or 2:2 (a positively charged brucite sheet between
the layers that restrict the swelling ability). Common biomedical clays are grouped as
kaolinite (KAO; 1:1 organization), bentonite (BEN; 2:1 organization), laponite (LAP;
2:1 organization), montmorillonite (MMT; 2:1 organization), and saponite (SAP; 2:1
organization). Most of these biomedical clays are used as nanoclays. Nanoclays are
the clay minerals with at least one dimension in the nanometer range (1–100 nm).
Nanoclays are either cationic or anionic depending upon their surface charge. For
various biomedical applications, nanoclays have mostly been used as reinforcement
in the biopolymer matrix. Because of their complete absence of toxicity, they have
found their use in the research related to tissue engineering, drug delivery, and wound
healing. They have also been tested as bone cement [4]. Figure 1 shows the common
classification of clay minerals. The coming sections deal with various biomedical
clays and their applications in detail.

2 Clay Minerals and Nanosilicates

Clay minerals or clays have an interesting history of being eaten by primitive

humans, Homo habilis, around 2 million years ago. This mode of nutrition was called
Geophagy. Even with Homo sapiens, the clays have remained in active use for more
than 25,000 years ago. Moraes et al. has also reported that primitive humans used
ocher clay mixed in water for wound healing and other skin ailments. Ancient Egypt
called the present-day clay minerals as medicinal earth and used them for wound
healing and hemostasis. Egyptians also used Nubian-earth as anti-inflammatory
material [5, 6]. As stated in the section above, clays are the natural minerals that
are chemically hydrous aluminum silicates with a variety of other doped cations
such as magnesium, iron, and few others. Clays are considered to be environmental
friendly and safe for human applications. The versatility of clay minerals for human
4 A. Kumar and A. Kumar

Fig. 1 Clay mineral classification (from the information provided in https://pubs.usgs.gov/of/2001/

of01-041/htmldocs/clay.htm and reference # 8); The ratio mentioned shows the ratio of SiO2 and
AlO6

application owe to their properties such as high adsorption quality, high surface area,
colloidal property, thixotropy, swelling capacity, and chemical inertness. Secondly,
they have very low toxicity upon oral administration (as also revealed by the geophagy
culture). Among the naturally occurring clay minerals, kaolinite (kaolin, halloysite),
smectites (MMT, SAP), and sepiolite are the most used for pharmacological applica-
tions. Though considered very safe on oral administration, the concern of formation
of renal stone on long term consumption has also been raised. Secondly, adsorption of
gastric enzymes could also take place that might disturb normal digestion process [7].
Clay minerals show different water solubility. Clays such as illite, sepiolite, and paly-
gorskite do not swell in water while montmorillonite swells easily and forms a gel-like
structure. A brilliant comprehensive review by Kotal and Bhowmik on various prepa-
rations and characteristics of nanoclays and polymer composite described various
preparations, structures, and properties of such composites [8]. They have described
the polymer-based nanocomposites of both natural and modified clays. Among all
the clays, MMT, kaolin, and saponite have found most applications in fields such
as electronics, biomedical engineering, optics, and aeronautics. The nanoscale form
of clays has improved physico-mechanical characteristics such as tensile strength,
modulus, and lower thermal expansion coefficient. Various methods such as solution
intercalation, melt mixing, and in situ polymerization have been applied to form the
polymer-based nanocomposites. Polymers with more polar groups are reported to
interact well with the nanoclays [8].
To start with very basic medical applications, clay minerals have long been used
in various semi-solid formulations such as gels, ointments, lotions, shampoo, and
Biomedical Applications of Nanosilicate Composites 5

pastes. A review by Viseras et al. has mentioned various cosmetic and health-
care semi-solid preparations that consist of clays such as montmorillonite, kaolin,
saponite, and sepiolite [9]. Khurana et al. [10] have reviewed the pharmaceutical
properties and applications of various clays. Clay minerals find their first use as
excipients in pharmaceutical industry for various purposes such as enhancing color,
flavor, increasing viscosity, emulsifying agent, lubricating agents, and binding agents.
Apart from the application as excipients, various clays have shown to have certain
protective action over pathological conditions. Clays have shown to be effective
in protecting gastric ulcers by acting as antacid agents (smectite, sepiolite), anti-
diarrhoeal agents (smectite, sepiolite, kaolinite), dermatological protection (kaoli-
nite, smectite), and anti-inflammatory (kaolinite). The details of these works shall
be discussed in the section specific to the application. In terms of use, Moraes et al.
has reported that approximately 30 clay minerals are being used currently in various
biomedical and cosmetic industries. Apart from the widespread common use of kaoli-
nite and montmorillonite, halloysites have recently gained prominence in the field of
nano-therapeutic delivery. As per the US and European pharmacopoeias, kaolinite,
montmorillonite, saponite, talc, sepiolite, and palygorskite are approved for various
pharmaceutical and cosmetic purposes [6]. Since the clay minerals are phyllosil-
icates (silicate-based minerals), these nanoclays have been described as nanosili-
cates by Gaharwar and team in various researches focusing multifaceted biomedical
applications of clay minerals [11–13].
The wide application of Kaolinite can be seen from an extensive review by Awad
et al. in 2017 [14]. Kaolinite which is chemically Al2 Si2 O5 (OH)4 is one of the most
abundant and inexpensive clay mineral in the earth’s crust. Other members of kaoli-
nite family are halloysite, dickite, and nacrite. Kaolinite is hydrous aluminum silicate
with 1:1 ratio of alumina (octahedral surface) and silica (tetrahedral siloxane surface)
arranged in dioctahedral fashion. The average platelet size of kaolinite (pseudohexag-
onal platelets) is reported to be <2 μm which is much lesser in size than average
human RBCs. The edges of these alumina and silica layers contain oxygen atoms and
hydroxyl (OH) groups. In solutions or environment with acidic range (pH < 3.6), the
hydroxyl groups become protonated, while deprotonation happens at pH > 3.6. Thus,
kaolinite surfaces experience two types of charges: the permanent negative charge on
the tetrahedral face and the variable pH-dependent charge caused by the protonation
or deprotonation of hydroxyl groups on the amphoteric sites, at the edges and the
octahedral faces. Pharmaceutical grade kaolin has been in application for long as an
excipient with usage such as binder, diluent, particle film coating, and disintegrant.
Kaolin has been reported to be a good disintegrant in solid dosage forms due to
its high porous nature and swelling ability. Kaolin has also been reported for drug
delivery applications. Kaolin, in combination with other materials such as PEG and
alginate has been shown to have modified dug release activity. Apart from its use
as pharmaceutical excipients, the kaolin has found its commercial use as an active
heamostatic agent. Because of the negative charge of kaolinites surface in the pH
range of human blood, the kaolin nanoplatelets instantly coagulate the bleeding as
soon as it comes in contact with the blood. Specifically, kaolin is seen to enhance the
6 A. Kumar and A. Kumar

activity of coagulation factor XII that in turn activates coagulation factor XI and pre-
kallikrein. Therefore, kaolin has been used in various commercially available wound
dressings and hemostatic dressings such as QuikClot Combat Gauze™ (QCG), Quik-
Clot Combat GauzeXL (QCX), QuikClot Combat Gauze TraumaPad (QCTP), and
QuikClot® Interventional which are FDA approved products [7, 14]. Kaolinite has
also been reported to be used in preparations and formulations concerning inflamma-
tion (Caloplast, KL kaolin), dermatological concerns (kerodex, REINOL Aquagard,
CALMA mask, Lactocalamine lotion), gastrointestinal and anti-diarrhoeal agents
(ASDA Tablets, Entrocalm, Boots Kaolin, Kaopectate, Kaomix suspension, Kaolin
Antacil), anti-bacterial material, antiviral agents, and anti-tumor compounds [14].
Montmorillonite (MMT), which is chemically [(Na, Ca)0.33 (Al, Mg)2 (Si4
O10 )(OH)2 ·nH2 O], is a 2:1 dioctahedral silicate mineral having two tetrahedral and
one octahedral sheet. A typical MMT particle or nanoplatelet is approximately 1 nm
thick and 0.2–2 μm in diameter. The layered structure and the spaces between the
layers provide excellent adsorption characteristics. Depending upon the geological
location, MMT is either sodium MMT or calcium MMT. It has been reported that
Na-MMT shows more swelling ability than Ca-MMT, therefore is more applied for
drug delivery applications. The biomedical usefulness of MMT can be seen from
the fact that hardly any oral toxicity in mice was seen at a dose of 1000 mg/kg
body weight. MMT has much higher swelling capacity as compared to kaolinite.
MMT has been extensively evaluated as a drug carrier for novel sustained delivery
formulations with drugs such as gentamicin, metronidazole, praziquantel, chlorpro-
mazine, metformin, and insulin [7, 15]. A recent review has mentioned and referred
vast literatures suggesting that MMT has been extensively used for wound healing,
tissue engineering, and regenerative medicine [16]. In reports published in 2014 and
2017, researchers have reported that ingestion of Uniform Particle Size NovaSil and
ACCS100 (pharmacological grade refined Ca-MMT used in adults) in aflatoxin-
affected children significantly reduced the urinary metabolite of aflatoxin (AFM1)
without having any secondary adverse effect [17, 18]. This further confirms the
excellent biocompatibility of MMT.
Laponite is a synthetic clay belonging to the Smectite group due to its chemistry
and structure. Being synthetic clay, Laponite enjoys having minimal impurities, low
toxicity, and controlled nanosized structure. It is available in various grades, but
Laponite XLG is the medical grade version. Laponite crystals are disc shaped with
approximate diameter of 25 nm and a height of 0.92 nm with a chemical formula
Na+ 0.7 [(Si8 Mg5.5 Li0.3 )O20 (OH)4 ]−0.7 . Biocompatibility and biodegradation are two
important parameters for any material to be used in biomedical applications. Laponite
has been shown to degrade in acidic condition generating aqueous silica, sodium,
magnesium, and lithium. Laponite XLG has been shown to be cytocompatible with
human mesenchymal stem cells till a concentration of 1 mg/mL [19].
Halloysite, a natural kaolinite family member, is a 1-dimensional hollow
nanotubular structure nanosilicate. Thus, it is a naturally occurring nanotube and
hence referred to as halloysite nanotube (HNT). It has a chemical formula of
Al2 Si2 O5 (OH)4 .nH2 O. The wall of the HNTs consists of 10–15 bilayered struc-
ture with an approximate spacing of 0.72 nm between the walls. HNT surface shows
Biomedical Applications of Nanosilicate Composites 7

positive charge in the inner surface while negative charge on the outer surface on a
wide pH range. Typical length of HNTs is in the range of 0.2–1.5 μm while the inner
and outer diameters are in the range of 10–30 nm and 40–70 nm, respectively. Since
the surfaces of the HNTs can be widely modified and HNTs are reported to be highly
biocompatible, there has been a growing interest among biomedical researchers to
use the natural nanotube as a drug delivery carrier. Apart from the small molecule
drugs, researchers have even tested the loading and release of insulin and found that
insulin is released in a sustained manner for 7 days and stability assay confirmed that
the released insulin is in native state [7, 20]. The unique tubular structure of HNTs
has enabled their successful application in enzyme immobilization too apart from
its investigated role in drug delivery, tissue engineering, and wound healing [20].
Further insight into the characteristics and application of HNTs can be referred from
reference number 20. Figure 2 is a pictorial representation of various biomedical
applications of nanoclays.

Fig. 2 Various biomedical applications of nanoclays/nanosilicates

8 A. Kumar and A. Kumar

3 Nanosilicate Composites in Tissue Engineering

and Regenerative Medicine

Since there is a huge gap in the number of tissues/organs available and the number of
recipients waiting for the transplant, tissue engineering research is perhaps the only
ray of hope for replacement of diseased or damaged tissues/organs. In the seminal
paper on tissue engineering, Langer and Vacanti have clearly stated that “Tissue
engineering is an interdisciplinary field that applies the principles of engineering
and the life sciences toward the development of biological substitutes that restore,
maintain, or improve tissue function” [21]. In correlation to the natural extra-cellular
matrix of the tissues/organs, tissue engineering approach requires the fabrication of
a suitable scaffold as the primary step. Though tissue engineering approach has been
in concept since early 90s and clay minerals are used in cosmetic products for long,
the application of nanoclays or nanosilicates as a component of scaffold has gained
recent attention and interest. The scaffold fabrication is largely dependent upon
biopolymers. A lot of importance is given to doping these polymers with suitable
inorganic components to render them with better biocompatibility and functionality.
Nanosilicates have gained recognition because of their compatibility with a wide
range of biopolymers and their ability to be easily chemically modified. Because of
their considerably good biocompatibility and bioactivity, they have been used as a
dopant with biopolymers in the preparation of suitable scaffolds using either tradi-
tional way of scaffold fabrication or 3D printing. Important to mention here again,
nanosilicates are disc-like 2D nanoplatelets that are approximately 1 nm in thick-
ness and can have diameter ranging from 25 nm to over 100 nm [22]. The structure
of nanosilicates is such that there are permanent negative charges on the surfaces
while positive charges on the edges. The high surface area (>900 m2 /g) and effec-
tive charge distribution makes them attractive materials for chemical modification
and make beautiful interaction with the biopolymers. Since there has been a lot of
attention over bioink fabrication after the advent of 3D bioprinting, the remarkable
cytocompatibility of nanosilicates with the mammalian cells has made these materials
highly significant in recent bioink fabrication. Small amount of nanoclays or nanosil-
icates can significantly improve the mechanical behavior of the hydrogel network
that is supposed to be used for scaffold fabrication. Another important feature of these
nanosilicates is their applicability in drug delivery that is often an important consid-
eration of tissue engineering [22]. Naumenko et al. [23] used halloysites nanotubes
(HNTs) as the dopant in the chitosan-agarose-gelatin blend to create a biocompat-
ible and mechanically superior printable bioink. Halloysite tubes have been reported
to have length range of 0.3–1.5 μm while the inner and outer diameter is approxi-
mately 10–20 nm and approximately 40–70 nm, respectively. The innermost alumina
surface of the HNTs is positively charged while the outermost silicate surface is
negatively charged. The inner lumen of HNTs can be used as nanocontainers for
loading drugs and other pharmaceutically important agents. Animal studies have
shown that these HNT doped chitosan-agarose-gelatin scaffolds displayed complete
biodegradation and restoration of complete blood supply in 6 weeks with significant
Biomedical Applications of Nanosilicate Composites 9

biocompatibility [23]. Paul et al. [24] in 2016 reported the osteogenic differentia-
tion of human mesenchymal stem cells (without any osteogenic growth factor such
as bone morphogenic protein or BMPs) when seeded on nanosilicate doped GelMa
(methacrylated gelatin) polymer as the scaffold polymer. There were no signs of
cellular apoptosis and no evidence of inflammatory signals. Such bioactive hydro-
gels have a high scope of being used as viable bioink for tissue engineering applica-
tions, particularly bone tissue engineering. In support of this MSCs differentiation
capacity of nanosilicates, Carrow et al. [25] analyzed the transcriptome of the MSCs
upon differentiation under the influence of Laponite. Laponite XLG is a synthetic
nanosilicate finding a wide range of biomedical applications recently. The intracel-
lular dissociation of Laponite into Si, Mg, and Li is reported to be the best possible
reason for the osteogenic and chondrogenic differentiation of MSCs without any
specific growth factor. Carrow et al. [25] also mentioned that Si ions induced cWnt
pathway leading to the chondrogenic differentiation of MSCs. Similarly, an upregu-
lation of Col1A1 and VEGF has been shown to be triggered by magnesium ions. The
effectiveness of Laponite in osteogenic and chondrogenic differentiation of MSCs
was also evident from the fact that there was a significant upregulation of Col1A1
expression (osteogenic), alkaline phosphatase (ALP) expression (osteogenic), miner-
alization (osteogenic), glycosaminoglycan (chondrogenic), and aggrecan (chondro-
genic). Nojoomi et al. [26] has reported that Laponite doped imine-functionalized
cross-linkable polyethylene glycol (PEG) injectable hydrogel was quite effective
as a cytocompatible biocomposites hydrogel. Balakrishnan et al. also reported the
formation of composite of high-density polyethylene (HDPE) with hydroxyap-
atite and MMT for the purpose of fabrication of bone scaffold. They, however,
concluded that doping of MMT has a favorable effect on the mechanical properties
of HDPE/HA composite but there was a significant loss of bioactivity [27]. Recently,
Wang et al. [28] have fabricated electrospun Laponite XLG reinforced polycapro-
lactone (PCL) nanofibres for bone tissue engineering application and found positive
results. Recently, Carrow et al. [29] fabricated Laponite XLG doped poly(ethylene
oxide terephthalate)/poly(butylene terephthalate) (PEOT/PBT) copolymer for bone
tissue engineering using 3D printing. Earlier, PEOT/PBT copolymer has been shown
to have calcification and bone bonding ability in vivo, while 2D nanosilicates
have the property of osteogenic differentiation of human mesenchymal stem cells
(hMSCs) even in the absence of osteoinductive agents or growth factors. Therefore,
nanosilicate doping increased the bioactivity of the PEOT/PBT scaffold that was
confirmed by observing significant upregulation of osteo-proteins and mineraliza-
tion. Very recently, Nadernezhad et al. [30] successfully developed agarose-Laponite
RD composite shear-thinning bioink for fabrication of scaffold for bone tissue engi-
neering using 3D printing. Laponite was also mixed with methacrylated chitosan and
methacrylated gelatin to create a suitable bioactive bioink for bone scaffold fabri-
cation by Cebe et al. [31]. Methacrylated chitosan-Laponite composite displayed
better cell adhesion, osteogenic differentiation, and mineralization as compared to
the gelatin counterpart. Addition of Laopnite to human adipose-derived stem cells
resulted in approximately 70-fold increase in Runx2 (osteo-specific marker), nine-
fold increase in osteocalcin, and 45-fold increase in the expression of osteopontin.
10 A. Kumar and A. Kumar

Secondly, there was a significant production of collagen 1 which is a hallmark of

mineralized matrix [32]. Apart from the conventional biocomposites-based scaf-
folds, Laponite has also shown to be a great dopant in formulation of injectable
self-healing hydrogel for skeletal tissue engineering [33]. HNTs have been reported
to be combined with a variety of biopolymers such as agarose, gelatin, chitosan,
and gellan gum to fabricate bioinks for relevant scaffold fabrication. These HNT
doped biopolymeric scaffold displayed significant cytocompatibility [34]. Ahlfeld
et al. have fabricated Laponite XLG reinforced alginate-methylcellulose bioinks for
tissue engineering. They successfully demonstrated that hMSCs remained viable
(70–75%) for 21 days within the fabricated construct. Moreover, the shape of the
construct was preserved over longer period too. Secondly, they also demonstrated
that addition of Laponite resulted in sustained release of model proteins (BSA and
VEGF) as compared to the construct without Laponite [35]. Very recently, Cidonio
et al. fabricated VEGF loaded Laponite-GelMA bioink as a prospective scaffold
candidate for bone tissue engineering. They have demonstrated significant angiogen-
esis through VEGF-LAP-GelMA hydrogel as compared to that from VEGF-GelMA
hydrogels suggesting a promoting role of Laponite in retention of VEGF [36]. In
a recent study, Cui et al. have developed photocrosslinkable methacrylated glycol
chitosan reinforced with MMT as an injectable in situ polymerizing hydrogel for
bone regeneration without any growth factor or other osteogenic proteins. The most
important characteristic highlighted in this research was the ability of the modified
chitosan-MMT blend to recruit native osteoblast cells from the injected environment
to generate de novo osteogenesis [37]. As we know, 3D printing and 4D printing
has gained momentum since the last few years, preparation of bioinks has become a
completely separate field of research for material and biomedical scientists. Nano-
materials such as graphene, carbon nanotubes (CNTs), nanoclays, HA nanoparticles,
tricalcium phosphate nanoparticles, and iron oxide nanoparticles have been doped
to display favorable results. Since nanoclays or nanosilicates are much cheaper and
abundant than CNTs and have much better biocompatibility, they have gained better
response as reinforcement in biocomposites. These biopolymer-nanoclay composites
have shown improved mechanical and physico-chemical characteristics. Till date,
the nanosilicates have been used as dopant with alginate, chitosan, GelMA, and
hyaluronic acid. Because of their surface charge, nanosilicates form effective chem-
ical crosslinks with the polymer chain providing suitable characteristics. Nanosili-
cates can bind with multiple polymer chains simultaneously providing balanced and
even stress dissipation. Overall, even small amounts of nanosilicates can be applied
to significantly increase the stiffness, fracture energy, extensibility, and printability
of biopolymeric hydrogel networks. The electrostatic interactions between nanosil-
icates and biopolymers confer the bioinks with a revocable network structure that
collapses during flow though the extruder (nozzle) and then quickly reforms after
printing [22].
Biomedical Applications of Nanosilicate Composites 11

4 Nanosilicate Composites in Dental Research

Dental restorative materials are used in dentistry to replace the lost part (dental caries
or trauma, etc.) of the crown or the cavity made during root canal treatment. The
restorative materials should have mechanical properties similar to the natural tooth
and provide aesthetic features too. The materials should be highly biocompatible and
should not be degradable. Though silver-mercury amalgam has been used for ages,
the possible leakage of mercury still remains the biggest health concern. Use of dental
composites has largely replaced amalgam, though is a bit expensive as compared to
the latter. Composites (also called dental resin or dental adhesive) made of polymer
matrix are now widely used in restorative dentistry to fill cavities (diseased or thera-
peutic), restore fractured teeth, and to replace missing teeth. The most popular dental
composites typically consist of a polymer matrix along with a photo initiator chem-
ical (for photo-curing), a small amount of pigment, and stabilizers along with a high
percentage of inorganic filler material (mostly silica). Most common composition
use monomers such as bisphenol A glyceryldimethacrylate (Bis-GMA), triethylene
glycoldimethacrylate (TEGDMA), and urethane dimethacrylate (UDMA). Though
composites are widely used, they face the issue of polymerization shrinkage creating
a minute gap between the composite and the natural enamel. This space (microcavi-
ties) is a suitable place for bacterial adhesion and subsequent infection. Triethyleng-
lycoldimetachrylate (TEGDMA) is frequently added to reduce the viscosity of Bis-
GMA polymer matrix. But if the photo-curing is not proper, there is a high possi-
bility of leakage of TEGDMA which is cytotoxic. Other challenges in conventional
polymer composites are mechanical stability, biocompatibility, carries inhibition
activity, and marginal leakage. Since polymerization shrinkage is a serious clin-
ical failure, it has been reported that addition of inorganic filler materials such as
nanoclays reduces the shrinkage and renders better mechanical properties to the
composite matrix [38]. It has been reported that addition of nanoclays grafted filler
as the adhesive resin increases the mechanical properties of the composite along with
improved biocompatibility. In this regard, more than a decade ago, Atai et al. [39] used
PMMA grafted MMT as the restorative filler for dental adhesive and demonstrated
significantly higher mechanical properties. de Menezes and da Silva also improved
the mechanical properties of the dental resin by using synthetic MMT (Dellite 67G
and Viscogel B8) as the nanofillers by using only 0.2% w/w as the optimum concen-
tration [40]. Menezes et al., in a separate work, showed that the nanoclays modified
composite was much better in all aspects compared to the composites containing
hydrophilic silica and organomodified silica. The results were best when the concen-
tration of nanoclays was just 2.5% [41]. The features of nanoclays are so attractive
that very recently Nikolaidis et al. used various modified MMTs as nanofillers in the
dental polymeric composites [42]. Glass Ionomer Cement (GIC) are conventional
filling materials in restorative dentistry. The clinical GIC consists of two mixing
components viz. the liquid component (aqueous solution of polyacrylic acid stabi-
lized with 5% tartaric acid copolymers) and the powder component that is a fluoro-
aluminosilicate glass. GIC is preferred in dentistry due to good chemical bond to the
12 A. Kumar and A. Kumar

tooth structure, anti-cariogenic properties, and excellent biocompatibility. But, even

GIC faces the same problem of low mechanical strength and high wear chance. Fareed
and Stamboulis prepared MMT linked GIC polymer and improved the mechanical
properties of the GIC composite that could be even better utilized in dentistry [43].

5 Nanosilicate Composites in Drug Delivery Research

Sustained or controlled drug delivery is required for reasons such as effective dose at
required site for longer time, reduced drug dose, reduced administration frequency,
and overall reduced adverse effects. With this kind of delivery pattern, the drug
remains at the plasma therapeutic level for a longer time. Secondly, such delivery
systems can also be engineered to have a targeted delivery of the drug. Nanopar-
ticles (or microparticles) have been used for almost last 30 years for drug delivery
starting with the use of liposomes as the vehicles. Though there are commercially
available nanoformulations, often the need arises to have a material that could
be easily engineered for drug loading and targeting, and is extremely biocompat-
ible, non-immunogenic, low cost, and easily available. Nanoclays fit all the above-
mentioned criteria. They even have unique properties such as intercalation, swelling,
and non-toxic degradation products [44].
The general preparation of clay–drug complex is usually carried out by mixing a
proper volume ratio of an aqueous dispersion of clay with a suitable solution of drug.
The solid phase is filtered off, washed several times with an appropriate solvent
so that the physically adsorbed drug is washed off, and dried under vacuum. But
the chemical reason behind the formation of nanoclay-drug complex is a simple
chemistry. Most of the biomedically relevant and used nanosilicates such as kaolin,
MMT, and halloysite are cationic clay, i.e., they have an overall permanent negative
charge on the surface. Therefore, they are able to interact with the basic drugs. But
for controlled drug release, the drugs are also intercalated between the nanosilicate
layers. For example, the common anti-cancer drug doxorubicin (DOX) has been
loaded in both kaolin (methoxy intercalated) and HNT with a loading efficacy of
approximately 54% and 80%, respectively. It was further showed that at pH 5.5
(approximate pH of cancer tissue), cumulative release of approximately 32% DOX
was observed in 30 h while the release was only about 9.5% at neutral pH. This pH
responsive delivery shows to be an effective characteristic for targeted drug delivery
that could minimize the adverse effects of the anti-cancer drugs [44, 45].
MMT has been one of the most investigated natural nanosilicate for biomedical
applications. It is highly resistant chemically and is stable under acidic conditions
too. It has an appreciable swelling capacity and is a potent detoxifier too. PLGA is
an FDA approved GRAS polymer for biomedical applications. Jain and Datta devel-
oped an MMT-dexamethasone-PLGA composite for sustained release of the drug
since dexamethasone (DEX) is hydrophobic with low bioavailability. Comparison
with DEX-PLGA system, the introduction of MMT in the MMT-DEX-PLGA system
showed no burst release with a much controlled release of the drug for over 30 h
Biomedical Applications of Nanosilicate Composites 13

[46]. Venlafaxine is an anti-depressant that has a short half-life of approximately 4 h.

Thus, the medicine is required to be administered 2 to 3 times a day. Jain and Datta
in yet another experiment reported the formulation of MMT-alginate microspheres
for effective loading (~97%) of Venlafaxine while the release displayed minimal
burst release with approximately 20% release in 26 h (in gastric fluid) and 22%
release in 29 h (in intestinal fluid). They, therefore, concluded that the presence of
MMT nanoclays prevented the loss of drug from burst release and also rendered a
controlled release feature to the formulation [47]. A very comprehensive review of
application of MMT in drug delivery has been reviewed by Jayrajsinh et al. [15].
Laponite, a synthetic nanoclay, has gained a huge momentum and interest in various
biomedical applications. The stacked structure, charge distribution over surface, and
no impurity have been the major forces behind using Laponite as a drug delivery
vehicle. For example, Doxorubicin was shown to be intercalated in the LAP layers
and displayed release at the acidic pH (gastric pH) while minimal release at neutral
pH. In vitro anti-cancer activity demonstrated much better cytotoxicity with LAP-
DOX complex as compared to the free DOX alone. Being a highly charged nanos-
tructure, LAP is susceptible to form aggregates at high polyelectrolyte environment,
such as human blood. Therefore, surface coating or encapsulation of the drug-LAP
complex is essential to avoid such aggregations. The ability of LAP to solubilize water
insoluble compounds has initiated an interest in LAP assisted photodynamic therapy
for cancer. LAP was reported to dissolve aluminum phthalocyanine hydroxide (a
photosensitizer) which could generate highly toxic singlet oxygen ion [48]. In a
recent report, Becher et al. have fabricated Laponite-based nanohydrogel for drug
delivery in combination with polyacrylates and sodium phosphate salts. The unique
property of nanohydrogels is that they are not soluble or degradable in physiological
media. It was also mentioned that soft nanohydrogels can show easy deformation
that might facilitate their passage through crucial physiological barriers that results
in increase in their blood circulation lifetime improving their therapeutic efficiency.
These nanohydrogels very efficiently encapsulated cisplatin, cyclophosphamide, and
4-fluorouracil. It was demonstrated that IC50 of the drug loaded nanohydrogels was
much lesser as compared to that of the free drugs. In vivo experiments also revealed
that these nanohydrogels do not accumulate in any organ [49]. Supraphysiological
dose of therapeutic growth factors and other therapeutic proteins that are used today
results in a variety of adverse effects that could be serious. In a recent report by
Cross et al. [50] Laponite nanodiscs were used for loading and delivering physiolog-
ical dose of protein therapeutics using rhBMP2 and TGF-β3 as the model drugs. This
nano-system was used to differentiate human MSCs into osteoblasts (by rhBMP2)
and chondrocytes (by TGF-β3 ). Therefore, this LAP-based nanodisc formulation of
growth factor delivery revealed the usage of physiological effect of these drugs that
are being used in supraphysiological dose currently.
As mentioned in previous sections, HNTs are the natural alternative to CNTs on
the structural front, but the former is much cheaper and biocompatible than CNTs.
HNTs could also be seen as rolled kaolin sheets. Almost a decade ago, Vergaro
et al. presented a detailed report on cytotoxicity and biocompatibility of HNTs and
concluded that HNTs are highly biocompatible with very minimal cytotoxicity [51].
14 A. Kumar and A. Kumar

Lvov and team have probably done a considerable amount of work on HNTs. Three
beautiful reviews by his team cited as Lvov et al. [52], Santos et al. [53], and Lvov
et al. [54] concerning various detailed aspects of HNTs are worth reading.

6 Nanosilicate Composites in Wound Healing

and Hemostasis

Wound healing is a complex physiological cascade that includes various hierarchical

stages such as homeostasis stage (blood clotting and immune activation), inflam-
mation (recruitment of immune cells, production of related cytokine, and growth
factor), proliferation phase, tissue neoformation (reepithelialization, angiogenensis,
and granulation), and subsequent remodeling of the newly generated tissue. More-
over, non-healing wounds (diabetic foot ulcers, venous leg ulcers, and pressure ulcers)
and burns impose substantial morbidity and mortality, deeply affecting the quality
of life. Secondly, surgical wounds are an important class of wounds that need proper
care and any act of negligence could become medical emergency at times. Thirdly,
battlefield wounds are serious concern that, if not provided proper treatment, could be
detrimental in several ways. Severe cutaneous wounds are a major medical concern,
with approximately 300 million chronic and 100 million traumatic wound patients
affected worldwide. Moreover, approximately 37 million patients are affected glob-
ally by chronic wounds [55]. As mentioned in the previous section, clay minerals
have been traditionally used for wound healing since ages. In modern-day practice
too, clay minerals, primarily kaolin, has been an important constituent of dermatocos-
metic products. Since the traditional clay mineral forms have impurities in them and
because of the therapeutic effects of clay minerals over wound, they have been fabri-
cated into better wound healing constructs for better therapeutic results. Pacelli et al.
fabricated injectable hydrogel nanocomposites incorporating methacrylated gellan
gum and Laponite that could release suitable drug and antibiotics for wound healing
purpose [56]. Ambrogi et al. [57] have fabricated MMT-Chitosan-chlorhexidine films
to inhibit biofilm formation in chronic wounds. In another report Aguzzi et al. [58]
MMT-chitosan silver sulfadiazine loaded nanocomposites were fabricated for wound
healing purpose. In a recent report by Peng et al. [59], tetracycline loaded Laponite
nanodiscs were incorporated in PLLA solution to form a porous membrane that could
be effectively used as a wound dressing. The fabricated membrane was shown to be
highly effective against Staphylococcus aureus that was taken as the model organism.
Secondly, the membrane displayed good biocompatibility in vitro. Villen et al. [55]
fabricated MMT-Norfloxacin composite powder for application of various kinds of
acute and chronic wounds.
The blood clotting process, medically known as hemostasis, is a physiological
cascade that inhibits blood loss through the formation of a stable cellular plug known
as hemostatic clot at the site of injury/bleeding. It is a complex physiological mecha-
nism that involves the coordinated activation and action of platelets, a range of plasma
Biomedical Applications of Nanosilicate Composites 15

proteins, and coagulation factors. These coagulation factors reach the bloodstream
in an inactivated state and get activated as soon as the hemostatic cascade initiates.
Overall, the hemostatic action happens via the coordinated action of three physio-
logical mechanisms viz. vasoconstriction, platelet plug formation, and finally blood
clotting. Pourshahrestani et al. [60] have published a comprehensive review regarding
certain commercial hemostatic constructs that apply zeolite and nanoclays as the
major hemostatic constituents. Where QuikClot granular powders, Advanced Clot-
ting Sponge (ACS), and ACS+ are the Zeolite-based hemostatic dressings, QuikCLot
Combat Gauge (QCG), QCG XL, QCG Trauma Pad, and QC Interventional are the
kaolin-based commercial hemostatic products while WoundStat is a smectite-based
commercial hemostatic product available in certain countries.

7 Conclusions

While a lot has already been mentioned in the chapter, conclusively we can see that
natural as well as synthetic nanoclays/nanosilicates are readily available and are a
cheaper alternative to various other nanoparticles available for biomedical appli-
cations. While many biomaterials find restricted applications, nanoclays have been
used in perhaps all major biomedical applications ranging from tissue engineering,
drug delivery, dental restoration, wound healing, and hemostatic agents because of
their consistent biocompatibility, non-immunogenicity, and non-toxicity. Still, the
use of nanoclays for medical application, apart from the use in hemostatic dressings,
is in nascent stage. With FDA approval of various nanoclays for certain medical
applications, researchers can utilize the properties of these nanoclays to construct
useful products.

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Polysaccharide-Based Composites
for Biomedical Applications

Patrícia Alves, Filipa Gonçalves, and M. H. Gil

1 Introduction

Polysaccharides have been widely used in the most diverse applications, like pack-
aging, water treatment, bionsensors, and more extensively in biomedical applica-
tions such as drug delivery vehicles, scaffolds, wound dressing, and biosensors,
among others (Fig. 1). Natural polysaccharide-based composites offer biocompati-
bility, bioavailability [1], and biological recognitions, making them highly used in
biomedical applications. For that reason, in this chapter, the authors gathered the
most relevant and more recent advances in the development of polysaccharide-based
composites and their uses in biomedical fields.

2 Natural Polysaccharides

The potential of polysaccharides in biomedical fields is explained not only by their

natural sources but also due to their similarity with the extracellular matrix compo-
nents (EMC) [2]. Also, the existence of distinct functional groups promotes the
modification of polysaccharides and therefore their wide range of applicability in
biomedical areas. Polysaccharides can have different sources: animal, plant, microor-
ganisms, and algae. Starch, cellulose, pectins, and exudate gums are examples of the
most common polysaccharides obtained from plants. Alginates and carrageenan are
from algae sources while chitin, chitosan, and hyaluronic acid (HA) are derived from
animal sources. Dextran, pullulan, xantham gum, bacterial cellulose are examples of

P. Alves · F. Gonçalves · M. H. Gil (B)

CIEPQPF, Department of Chemical Engineering, University of Coimbra, 3030-790 Coimbra,
Portugal
e-mail: [email protected]
© Springer Nature Singapore Pte Ltd. 2021 19
A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_2
20 P. Alves et al.

Fig. 1 Main biomedical fields of polysaccharides

polysaccharides obtained from microorganisms (Fig. 2) [1]. Among these biopoly-

mers, cellulose, chitin, and chitosan are some of the most reported polysaccharides
in biomedical field in the last decades, not only due to their properties, but also due
to their high availability [3, 4].
Cellulose is the most abundant natural polysaccharide, presenting a linear-chain
polymer of poly-β-(1 → 4)-D-glucosamine units, with a flat ribbon-like conformation
(Fig. 3) [5]. This polysaccharide is characterized by a high amount of hydroxyl
groups, three per each glucose unit. These –OH groups are extremely important
since they are responsible for cellulose reactivity [2]. Although extensively used
due to its high toughness, biodegradability, renewability, and availability [4], this
biopolymer has some disadvantages, such as poor solubility, high hydrophilicity,
low thermoplasticity, and the lack of antimicrobial properties. These drawbacks can
be overcome by modifying the cellulose structure, i.e., introducing specific functional
groups can overcome these weaknesses [2].
Chitin, poly (β-(1-4)-N-acetyl-d-glucosamine), is found in the exoskeleton of
crustaceans (crabs, lobsters, and shrimps), insects, mollusk, and fungi (cell walls) and
is the second most abundant polysaccharide in the biosphere [6]. Chitosan is the most
important and used derivative of chitin and can be obtained by the deacetylation of
chitin under alkaline conditions or by enzymatic hydrolysis [3]. Both biopolymers are
non-toxic, biocompatible, biodegradable, and have antibacterial properties, however,
chitosan presents a higher hydrophilicity and water retention capacity [4]. Also,
the presence of reactive primary amines and hydroxyl groups in chitosan structure
Polysaccharide-Based Composites for Biomedical Applications 21

Fig. 2 Main sources of natural polysaccharides (algae, plants, animals, and microorganisms)

promotes its chemical modification [7]. The use of these natural polymers in tissue
engineering and pharmaceutical fields is largely explained by their specific biological
properties—antioxidant, analgesic, antitumor, and hemostatic properties—that have
been recently described by several authors [6, 8, 9].
Hyaluronic acid (HA) is also a very important biopolymer, since it is one of
the elements present in the extracellular matrix (ECM) with extensive usage in
cosmetics for skin rejuvenescence [10]. HA is a polymer formed by repeating units
of N-acetylglucosamine and D-glucuronic acid. Its large use in biomedical fields
is strongly connected with its pharmacological activity—anti-inflammatory, wound
healing, skin rejuvenescent, anti-aging, anticancer properties [11]—and can be used
in the form of hydrogel, injectable creams and foams, scaffolds, membranes, gels,
or even encapsulated in a nanocarrier. The broad range of applicability combined
with their unique properties makes this polysaccharide one of the main candidate for
extensive research and developments.
The previously mentioned biopolymers are highlighted and discussed in this
chapter, side by side with other polysaccharides—e.g., dextran, alginic acid, cellu-
lose derivatives—which are extensively explored for a wide range of biomedical
applications (Fig. 3).
22 P. Alves et al.

Fig. 3 Main natural polysaccharides used in biomedical fields and respective chemical structure
Polysaccharide-Based Composites for Biomedical Applications 23

3 Natural Polysaccharides-Based Composites Applications

Polysaccharide-based composite materials are subject of great interest in a variety

of fields, mainly in tissue engineering and drug delivery. The preparation of
different polysaccharide-based composites, with different types of reinforcements,
is explored in the next sections. Drug delivery, tissue engineering, hydrogels, scaf-
folds, bionsensors, wound dressings, are some of the most relevant applications of
polysaccharide-based composites.

3.1 Drug Delivery

Several developments have been recently made in the field of delivery systems.
These developments were made in order to provide therapeutic agents or natural-
based active compounds to their target location for treatment of several pathologies
[12, 13]. However, there are still some challenges that need to be addressed for a
successful delivery of drugs to their target sites. The way that a drug is delivered
highly affects the efficacy of the drug. Drug delivery systems (DDS) are pharma-
ceutical formulations or devices for the targeted delivery and/or controlled release
of therapeutic agents. Drug delivery systems can be used either to deliver small
molecules or large molecules (peptides, nucleic acids, or polymers). For this purpose,
different types of delivery systems have been successfully used such as liposomes,
particles, micelles, and niosomes [12]. In the last years, due to their ability to form
stimuli-responsive hydrogels, polysaccharides are gaining interest as drug delivery
systems. Polysaccharide-based drug delivery carriers usually present an initial burst
of the drug due to their lack of mechanical properties. In order to overcome this
problem, a filler is usually added into the polysaccharide matrix [1, 14], such as
Fe3 O4 [15], graphene oxide [16], or silica nanoparticles [17].
Some researchers [18] have developed a system to transport and deliver iron along
the gastrointestinal tract. Iron deficiency is still a worldwide public health problem
combined with the fact that the incorporation of this metal into food matrices does
not completely resolve the problem if iron is not properly transported, iron-pectin
beads were prepared by ionic gelation and showed to have a potential application in
the food and pharmaceutical industry.
Temperature-sensitive hydrogels can be obtained from polysaccharides and
applied in the drug delivery area. Almeida et al. [19] prepared thermoresponsive
hydrogels from crosslinked copolymers based on dextran and chitosan to prepare
OndansetronTM delivery systems, a drug used for the treatment of nausea and
vomiting, to be applied as sublingual formulations. Also, Schmitt et al. [20] produced
a temperature-sensitive nanocomposite by dispersing halloysite nanotubes into a
starch matrix.
One of the most common methods of drug administration is by using the oral route,
since it is easy, convenient, and there is no risk of infection. But they need to pass the
24 P. Alves et al.

gastrointestinal tract, which presents a wide range of pH medium. Changes in pH may

affect the efficiency of the drug or even degrade it. Therefore, pH-sensitive polysac-
charide composites can be prepared in order to protect the drugs from unwanted
environments especially in the stomach before reaching the intestine, where they can
be absorbed. Quintana et al. [21] developed a novel strategy for probiotics controlled
delivery by encapsulating bacteria using the layer-by-layer self-assembly technique
(LbL) with chitosan and poly(acrylic acid) obtaining a permeable high performance
drug and storage delivery system.
Another approach is to use polysaccharides to alter de drug delivery device in
order to modify the surface of the original material and tune the release of the encap-
sulated drug. Recently, some studies reported the use of the layer-by-layer technique
(LbL), using polysaccharides, such as chitosan [22, 23] and alginate [23] to control
ophthalmic drug release from lens materials.

3.2 Bionsensors

By definition, biosensors are analytical devices applied for detection of chemical

substances, by combining a biological component and a physicochemical detector
(a transductor). They can be categorized either according to the biological recog-
nition element (enzymes, DNA, whole cells and antibodies) or to the signal trans-
duction method (electrochemical, optical, thermal, and mass-based) (Fig. 4) [24].
They can be applied in many fields, mainly in biomedical applications. In general,

Fig. 4 Schematic representation of bionsensors actuation, divided by analyte detection and

respective receptor, signal transduction method, and signal output
Polysaccharide-Based Composites for Biomedical Applications 25

the sensing molecule is immobilized in a support whose properties must enhance

the stability and activity of the biological compound. This matrix should have the
right hydrophobicity, chemical composition, mechanical properties, and biocompat-
ibility, considering the biosensors application as biomaterials. Additionally, it should
be resistant to physiological pHs and ionic strengths. Besides, they should be able to
co-immobilize more than one biological compound.
Polysaccharides are polymeric materials with excellent properties to be applied
in the field of biosensors since they present the right chemical constitution, good
microenvironment for the biological compound, and the right hydrophilicity. For
instance, cellulose and cellulose derivatives composites have been widely used in
the area of biosensors.
Gil et al. immobilized unease on cellulose derivatives for urea detection [25].
Promising results were obtained by Edwards et al. [26] who developed a composite of
proteins and nano-crystalline cellulose to obtain a fluorescent biosensor for elastase.
Due to the excellent properties of cellulose, namely its biocompatibility, biodegrad-
ability, and low cost, a vast amount of research work has been made with this polysac-
charide and its derivatives. Ratajczaket al. [27] published a review concerning the
development of cellulose paper-based biosensors for biomedical application. Abdi
et al. [28] optimized the fabrication of a new cholesterol biosensor based on a
composite of nanocellulose and polyaniline (PANi), with very promising results.
Other researchers used a poly (4-vinylaniline) polyaniline-functionalized bacterial
cellulose to develop a new flexible electrochemical biosensor [29].
Due to their chemical properties, reactivity, hydrophilicity, biocompatibility, and
capacity of gel formation, chitosan and chitin are widely applied in the preparation
of composites for biosensors [30–33].
Other polysaccharides are reported to be used in the preparation of biosensors.
Fois et al. [34] used a starch-based hydrogel composite to develop a biosensor for
glucose detection. Gautam et al. [35] characterized a composite based on PANi,
starch, and a multi-wall carbon nanotube (MWCNTs) to be used in glucose and
hydrogen peroxide detection.

3.3 Tissue Engineering

The incidence of bone disorder and conditions is increasing as a result of aging with
obesity and few exercise. One of the most promising techniques in orthopedic surgery
and tissue engineering is to repair and reconstruct bone and cartilage by autogenous
tissue transplantation [36, 37]. The development of biomaterials for tissue regenera-
tion comprises not only to create a scaffold that is biocompatible and biodegradable,
but also with suitable mechanical properties and porosity that supports the differenti-
ation of cells [38]. Sometimes is not possible to prepare a biomaterial from only one
polymer with all the desired properties. Therefore, composite or hybrid materials
appear in order to combine them all. Scaffolds are commonly composites produced
from both synthetic and natural polymers. Among the synthetic polymers, aliphatic
26 P. Alves et al.

polyester like poly glycolic acid (PGA), poly lactic acid (PLA), poly lactic-co-
glycolic acid (PLGA), polycaprolactone (PCL), are widely used in tissue engineering
due to their biodegradable nature. The natural polymers commonly used are mainly
polysaccharides such as chitosan (CS), alginate, agarose, and hyaluronic acid (HA),
which choice is justified by their biocompatibility, renewability, and sustainability.
Along with protein-based materials collagen, gelatin, and fibrin are also popular
for engineering bioactive scaffolds because of their advantages in mimicking the
extracellular environment [39, 40].
Jithendra et al. [39] prepared a 3D scaffold consisting of fish collagen, chitosan,
and Aloe Vera by the freeze-drying technique. This 3D scaffold presented all the
desired physical and biological properties to attract, attach, and proliferate fibroblasts,
meaning that it could be used as a promising biomaterial for tissue engineering.
Electrospinning technique has been one of the most explored techniques for the
preparation of polymer-based scaffolds able to sustain and improve tissue or organs
regeneration. Coimbra et al. [41] prepared core-shell fibrous meshes by coaxial elec-
trospinning, with a polycaprolactone (PCL) core and a functionalized gelatin shell,
and then photocrosslinked the meshes under UV light, aiming to be used in vascular
tissue regeneration. Using the same approach and for the same application, fibrous
meshes were also prepared by electrospinning blends of PCL and photocrosslinkable
gelatin (GelMA) [42]. It is known that the inclusion of extracellular matrix-derived
materials as gelatin, in polymeric blends, increases biological activity of the scaf-
folds promoting cell adhesion, proliferation, and, consequently, tissue regeneration
[42, 43].
Moreover, another type of hydrogels have been gaining interest in the area of tissue
engineering. Injectable hydrogels have been explored as substrates into a defect,
presenting several advantages. This method appears as a non-invasive approach in
a way that may avoid surgery procedures and, at the same time, allows an easy and
homogenous drug or cell distribution for bone tissue engineering applications [37].
Injectable hydrogels can be produced by thermal gelation, ionic interaction, physical
self-assembly, photo-polymerization, and chemical crosslinking [37, 44].
Ren et al. [37] prepared a gel scaffold by the Schiff-base reaction using the
linkages between oxidized alginate and carboxymethyl chitosan, which allowed to
obtain a polysaccharide-based hydrogel. Hydroxyapatite nanoparticles and calcium
carbonate microspheres loading tetracycline hydrochloride were blended to form a
composite gel scaffold to enhance bioactive properties and mechanical strength.
Injectable beads of pullulan and dextran with hydroxyapatite were prepared by
Fricain et al. [45] and showed to be safe and efficient bone engineering in the field
of oral and maxillofacial surgeries.
Cheng et al. [46] prepared chitosan-cellulose nanofiber composite injectable
hydrogels with self-healing properties for neural regeneration.
Also, an injectable sodium alginate/bioglass composite hydrogel containing bone
marrow stem cells (BMSCs) was prepared by Zhu et al. [47] and used for subchon-
dral bone regeneration. These authors also prepared an injectable thermosensi-
tive alginate/agarose composite hydrogel with co-culture of BMSCs and articular
chondrocytes was applied for articular cartilage regeneration.
Polysaccharide-Based Composites for Biomedical Applications 27

Another interesting approach in the area of the injectable hydrogel compos-

ites was proposed by Hu et al. [48]. These authors prepared dual-structural pH-
sensitive nanocarrier for growth factor delivery with the pH-sensitive acetalated
β-cyclodextrin and heparin nanogel interpenetrating component for growth factor
protection, which was later incorporated into a hyaluronic acid hydrogel to form a
cell scaffold composite hydrogel, that maintained cell activity and supported cell
growth.

3.4 Wound Dressing

Wound healing is a very complex process occurring after damage and is divided into
four distinct steps: (a) coagulation and hemostasis; (b) inflammation and release of
pro-inflammatory cytokines, (c) proliferation, resulting new tissue formation, and
finally (d) remodeling where the new tissue is remodeled, defining the nature of the
final scar [49]. Mainly, a wound dressing must be able to increase epidermal migra-
tion, to connect the tissue production, protect against bacterial infection, stimulate
angiogenesis and connective tissue production, and also to keep a moist environment,
allowing the gas exchange. Non-adhering and easy to remove after healing, non-toxic,
sterile and non-allergic are also requirements for an ideal wound dressing [50]. The
traditional wound dressings comprise gauze, natural and synthetic bandages, lint, and
plaster. Overall these types of wound healing dressings require frequent change to
avoid macerating the new tissues and its removal is usually traumatic to the patience
[49]. The use of natural polysaccharides in wound dressings, besides their biocom-
patibility and biodegradability, is due to its similarity with biological ECM and
therefore the recognition by biological systems [51]. Considering the well-known
disadvantages of the traditional tissues, like the lack of a moist environment, their
replacement by modern wound dressing was inevitable.
Regarding the most common polysaccharide used in wound dressing applica-
tion, the use of chitosan, chitin, pullulan, starch, cellulose, and alginate can be
highlighted. In recent years, several works have reported the use of polysaccha-
rides, being chitosan one of the primary choice for wound healing—hemostasis
and bacterial activity are the main reasons [52, 53]. A vast research in the prepa-
ration of wound dressings based on polysaccharides (homo and hetero), inorganic
compounds, and antibiotics have been explored [54]. It is well-known that the combi-
nation of different types of polysaccharides can enhance the performance of the final
wound dressing, with improved mechanical properties or antimicrobial activity [55].
Shabunin et al. [56] described the preparation via electrospinning of a two-layered
wound dressing, based on a copolymer of poly(ε-caprolactam) nanofibers (CoPA)
and a composite comprising chitosan and chitin nanofibrils. They showed the impor-
tance of the combination of these two layers to promote the wound healing. The
preparation of cellulose-chitosan sponges, with controlled structure and morphology
was also reported [52] and presented promising results as possible wound dressings
for diabetic patients. Kaczmarek et al. [57] developed a polymeric blend based on
28 P. Alves et al.

chitosan and tannic acid, showing the improvement of the physiochemical proper-
ties of the final film due to the tannic acid incorporation. Other studies explored the
combination between chitosan and tannic acid to obtain an enriched wound dressing
[58–60]. There are several works exploring the use of polysaccharide composites
with different polysaccharides but also with inorganic materials, such as the use
of gold nanoparticles (AuNPs) [61–65]. These nanoparticles are characterized by
a high antimicrobial activity and for being an effective antiviral agent. Tran et al.
[66] prepared a composite based on AuNPs, cellulose, wool keratin, and chloroauric
acid with possible application for chronic ulcerous infected wounds, or even as a
controlled drug delivery. The use of metal nanoparticles, such as gold, silver, or zinc,
is becoming particularly common, once these particles proved to have an outstanding
antibacterial effect [67]. Along with their capability to prevent bacterial infections,
they also have the ability to accelerate wound healing, and guarantee a constant
moist environment. For instance, Shao et al. [68] developed a wound dressing
composite with chitosan and silver sulfadiazine (AgSD) and the final composite
revealed extremely good antibacterial properties.
The use of other polysaccharides, such as alginate, cellulose, and derivatives, and
derivatives of chitosan had also been explored. Gao et al. [69] prepared a wound
dressing based on a derivative of chitosan, carboxymethyl chitosan, and calcium
alginate fibers. While carboxymethyl cellulose is characterized by its high biocom-
patibility, high moisture retention ability, and high antimicrobial activity, the algi-
nate is responsible for enhancing the mechanical properties of the final wound
dressing. Minocycline, a second-generation tetracycline antibiotic, was loaded in
these structures to promote the antimicrobial activity of the composite. This combi-
nation resulted in a wound dressing with skin-like mechanical properties, long-term
antimicrobial activity, biocompatibility, and with drug sustained-release properties.
Bacterial cellulose is also one of the main addressed topics on the preparation
of improved wound dressings which is strongly related with its ability to retain
water, high porosity and crystallinity, and superior mechanical properties [70, 71].
Wound dressings based on bacterial cellulose, alginate, chitosan, and copper sulphate
were prepared by Wichai et al. [72], showing an enhanced antibacterial performance
against gram-negative Escherichia coli (E. coli) and gram-positive Methicillin-
resistant Staphylococcus aureus (MRSA). Recently, a composite of gelatin and
bacterial cellulose was prepared using glutaraldehyde as the crosslinker [73]. This
composite with a controlled morphology was loaded with ampicillin and the results
suggested high antibacterial activity and biocompatibility. The construction of a
wound dressing focused on a specific type of wound, a third-degree skin burn,
was made through the combination of bacterial cellulose with copolymers of 3-
hydroxybutyric and 4-hydroxybutyric acids [P(3HB/4HB] [74], which were also
loaded with wound healing drugs and cells.
Wound dressings based on alginate composite have been widely investigated since
this biopolymer presents also interesting properties for wound healing—biocompat-
ibility, non-immunogenicity, non-toxicity, and high absorption capacity [75]. Rezva-
nian et al. [75] prepared a simvastatin-loaded wound dressing composite, based on
alginate, pectin, and gelatin, using the solvent casting method and the obtained results
Polysaccharide-Based Composites for Biomedical Applications 29

proved to have the required properties to be used as wound dressings, with excel-
lent mechanical properties. The combination of alginate with nanomaterials such
as graphene oxide and polyvinyl alcohol (PVA) has also been reported by several
authors [76–78].
Side by side with chitosan and cellulose, the incorporation of HA in wound
dressing is also gaining attention lately. This polysaccharide is well-known for its
ability to promote the wound healing with remarkable properties, and extremely used
in wound dressing applications [79, 80]. Eskandarinia et al. [81] developed a wound
dressing based on HA, cornstarch, and ethanolic extract of propolis, with an enhanced
wound healing and good cell biocompatibility. Makvanbdi et al. [82] synthesized an
injectable antibacterial thermosensitive hydrogel comprising HA, corn silk extract,
and nanosilver. The use of HA with cellulose nanocrystals and gelatin is also reported
in the preparation of new wound dressing with biocompatible character [83]. Other
authors [84] revealed that loading an antimicrobial peptide to a composite made of
alginate, HA, and collagen, resulted in a high antimicrobial activity, promoting also
the re-epithelialization and angiogenesis.
The incorporation of antibacterial agents is also a procedure used to promote
the functionalization of the wound dressings [85], something notorious along this
section.

4 Conclusions

Polysaccharide-based composites have proved their value in the biomedical field over
the past few years. The combination of these biopolymers with other type of mate-
rials—carbon nanotubes, proteins, other polysaccharides, inorganic compounds—
usually results in an enhancement of the properties of the composites, in terms
of biocompatibility, biodegradability, mechanical performance, and antimicrobial
activity, among others. The growing use of this biopolymers in biomedical fields is
not only explained by their unique properties but also by their high bioavailability,
low cost, and non-toxicity.
Overall, this chapter covers the most recent developments of polysaccharide-
based composites in biomedical fields, showing they are a suitable alternative to the
petroleum-based polymers. By using renewable and sustainable sources, a positive
outcome is expected, both economically and environmentally. Therefore, chitosan,
chitin, cellulose, starch, alginate, hyaluronic acid, and several other polysaccha-
rides will be continually used in the preparation of biocomposites to overcome the
polysaccharide’s main weaknesses.
30 P. Alves et al.

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Biomedical Nanocomposites

Amit Kumar Nayak, Saad Alkahtani, and Md Saquib Hasnain

1 Introduction

A huge volume of research endeavors has been covered for the advancements in
material science engineering and technologies to develop several kinds of biomate-
rials for biomedical applications [1–11]. These advanced biomaterials for biomed-
ical applications cover the designing of gels, hydrogels, membranes, films, patches,
polymer-blends, composites and nanocomposites, scaffolds, etc. [12–23]. The latest
studies in the domain of nanotechnology have been recognized as the popular most
and flourishing areas for the preparation of a variety of nanomaterials for biomed-
ical applications [24–29]. Some of these key biomedical nanomaterials are polymeric
nanoparticles and nanocapsules [30–32], lipid nanoparticles [33], metallic nanoparti-
cles [27, 34], ceramic nanoparticles [35], nanogels [14, 36, 37], nanovesicles [38–40],
nanotubes [41–43], nanocomposites [44, 45], etc.
Amongst these nanomaterials, nanocomposites are consisting of a number of
nanoscale substances or nanoscale substances incorporated into the bulk substances
[46, 47]. Since past few decades, a number of nanocomposites (i.e., organic-organic,
inorganic-inorganic, and organic–inorganic nanocomposites) are being prepared for
biomedical applications (for example drug delivery, wound healing, gene delivery,
tissue regeneration, dentistry, antimicrobial, bioimaging, biosensors, etc. [45–54].

A. K. Nayak (B)
Department of Pharmaceutics, Seemanta Institute of Pharmaceutical Sciences, Mayurbhanj,
Odisha, India
e-mail: [email protected]
S. Alkahtani
Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451,
Saudi Arabia
M. S. Hasnain
Department of Pharmacy, Palamau Institute of Pharmacy, Chianki, Daltonganj, Jharkhand, India

© Springer Nature Singapore Pte Ltd. 2021 35

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_3
36 A. K. Nayak et al.

This chapter presents an inclusive review on the nanocomposites for biomedical

applications.

2 Nanocomposites

In general, nanocomposites belong to a category of nanoengineered materials

possessing the structures modulated in the nanosizing scales [46, 47]. In general,
nanocomposites differ from the conventional composites owing to the higher ratio
of surface to volume for the reinforcing-phase, augmented ductility without the
decreasing of material strength profile as well as scratching resistances [55]. During
the past few decades, numerous kinds of nanocomposites are being investigated
and developed. Most of these nanocomposites are made of natural, semi-synthetic
and synthetic polymers, metals and their oxides, ceramics, structured carbons,
and other inorganic components [47–54]. Various materials are usually reinforced
to improve physical, physico-chemical, mechanical, and biomedical properties of
the composite/nanocomposite matrices. The materials reinforced for the syntheses
of nanocomposites are polymeric nanoparticles, inorganic nanomaterials (e.g.,
nanostructured carbons, metallic nanoparticles, metal oxide nanoparticles, nanoce-
ramics, etc.), nanofibres (e.g., electrospun fibers, etc.), nanotubes (carbon nanotubes,
etc.), nanosheets (e.g., exfoliated clay stacks), etc. [56–59]. Various nanocompos-
ites can be classified on the basis of composition and matrix-reinforcement: (i)
organic-organic nanocomposites (e.g., polymeric nanocomposites, etc.), (ii) organic–
inorganic nanocomposites (e.g., polymeric-ceramic nanocomposites, polymeric-
metallic nanocomposites, etc.), (iii) and inorganic-inorganic nanocomposites (e.g.,
metallic nanocomposites, ceramic nanocomposites, metallic-ceramic nanocompos-
ites, carbon nanotube-ceramic nanocomposites, carbon nanotube-metallic nanocom-
posites, etc.).
A number of synthesis methodologies are being used to synthesize many kinds of
nanocomposites. The extensively used synthesis methodologies of nanocomposites
are: physically mixing, film-casting, dip-coating, in situ preparation, co-precipitation,
ionic-gelation, covalent coupling, electro-spinning, colloidal assembly, layer-by-
layer assembly, etc. [48, 58]. These synthesis methodologies facilitate the advan-
tages of hydrogen bonding, electrostatic interactions, coulombic interactions, ionic
interactions, covalent bonding, hydrophobic effects, assembly formation, etc. [48].
The challenges in synthesis of different nanocomposites involve the controlling of
synthesis methodologies, assurance of compatibility with the component materials,
achieving the product of unique as well as desirable material properties [56, 58].
Recent years, the applications of nanocomposites direct a variety of newer technolo-
gies along with the functional and classy viewpoints for numerous fields including
biomedical area [45–58].
Biomedical Nanocomposites 37

3 Nanocomposites for Biomedical Applications

Nanocomposites designed for the use in various biomedical applications are usually
well-known as ‘biomedical nanocomposites’. The most important applications of
biomedical nanocomposites include drug delivery, gene delivery, antimicrobial,
tissue regeneration, wound healing, dentistry, bioimaging, biosensors, etc. [45–58].
Various important applications of biomedical nanocomposites are illustrated in Fig. 1.

3.1 Drug Delivery

The area of drug delivery applications demands the designing and development of
efficient dosage forms capable of delivering different drugs to the body for manage-
ment as well as treatment of various diseases and ailments [60]. Since long, drug
delivery has been recognized as a most important healthcare area where the deliv-
ering of different kinds of drugs via the administration dosage forms is requisite to
accomplish the optimum therapeutics to treat various diseases and ailments [61, 62].
Many biomaterials like natural, synthetic and semi-synthetic biopolymers, bioce-
ramics, metal and metal oxides, etc., are being employed to formulate drug delivery
systems [61, 63–84]. Using these biomaterials, various dosage forms like tablets
[85–88], capsules [89, 90], topical gels [91], hydrogels [92–96], in situ gels [14],
beads [97–109], microparticles [110–114], nanoparticles or nanocapsules [25, 31,

Fig. 1 Important applications of biomedical nanocomposites

38 A. K. Nayak et al.

32, 115], buccal patches [21, 116], dental pastes [117, 118], emulsions [118–120],
implants [121–125], etc., are being formulated for a variety of drugs and used for
treatment or management of many diseases. In recent times, nanocomposite has
become a promising carrier system for drug delivery because of their improved
improve physical, physico-chemical, mechanical, and biomedical properties [47, 48,
58, 59, 126]. In addition, high drug loading ability and nanoscale reinforcement have
further unlocked new realms in the field of drug delivery.
In a research, alginate-montmorillonite (MMT) nanocomposite bead system
loaded with irinotecan was developed by Illiescu et al. [127]. These alginate-MMT
nanocomposite beads of irinotecan were synthesized via ionic gelation method. Both
air-drying and freeze-drying were used for drying these nanocomposite beads of
irinotecan. Irinotecan-loaded nanocomposite beads prepared by air-dried process
were found as smaller sized as compared to that of nanocomposite beads prepared by
freeze-dried process. The reinforcement of MMT within the alginate matrix exhibited
the sustained release of loaded irinotecan over a longer period. In another research,
the same research group synthesized MMT-alginate nanocomposite beads loaded
with carboplatin via ionic gelation method [128]. These carboplatin-loaded MMT-
alginate nanocomposite beads exhibited sustained drug release profile over a longer
period.
5-fluorouracil releasing alginate-chitosan-MMT nanocomposite system was
synthesized by Azhar and Olad [129]. The synthesized nanocomposite systems
containing 30% wt MMT demonstrated a sustained releasing profile of loaded 5-
fluorouracil, in vitro. The in vitro 5-fluorouracil releasing from the alginate-chitosan-
MMT nanocomposite system was found to follow Korsmeyer–Peppas kinetic model.
MMT-chitosan nanocomposite hydrogel was prepared for controlled release appli-
cation of vitamin B12 under electro-stimulation [130]. The results of in vitro study
demonstrated the pulsatile release of vitamin B12 and excellent anti-fatigue behavior.
Justin and Chen developed chitosan-reduced graphene oxide nanocomposite form
transdermal delivery of drug [131]. These nanocomposites were prepared via the
reduction of graphene oxide. The reinforcement of graphene oxide within the
chitosan-based matrix improved the electrical conductivity, which could help in
electroporation and iontophoresis for enhanced transdermal drug permeation.
Venkatesan et al. prepared hydroxyapatite (HAp)-chitosan nanocomposites for
colonic delivery of celecoxib in colon cancer therapeutics [132]. The prepared
HAp-chitosan nanocomposites demonstrated high celecoxib loading efficiencies and
sustained celecoxib releasing behavior. In vitro cell culture studies using HT 29 and
HCT 15 colon cancer cells revealed antiproliferation, apoptosis, and significant time-
dependent cytoplasmic uptake. Additionally, a substantial tumor growth inhibition
on the xenografted colon tumor in the nude mouse was indicated by the in vivo
studies. The order of decreasing tumor volume after treating with free celecoxib,
nanocomposites without celecoxib and celecoxib loaded nanocomposites demon-
strated the highest decreasing of tumor volume in comparison to free celecoxib and
nanocomposites without celecoxib.
Nanda et al. developed chitosan-polylactide nanocomposites using cloisite 30B
and investigated the releasing potential of paclitaxel (an anticancer drug) from these
Biomedical Nanocomposites 39

developed nanocomposites [133]. The results of in vitro drug release study demon-
strated that the paclitaxel releasing from these nanocomposites was found dependent
on the polymer matrix and pH of drug release media. In addition, in vitro drug
release study suggested that the excellent release could be achieved in the basic pH
milieu. For the development of controlled doxorubicin (an anticancer drug) releasing
in cancer therapeutics, Wu et al. fabricated chitosan-based mesoporous magnetic
nanocomposite using silica (SiO2 ) and magnetite [134]. In this chitosan-based meso-
porous magnetic nanocomposite, chitosan was employed as blocking excipient for
the prevention of early releasing of doxorubicin. The doxorubicin releasing from
these magnetic nanocomposite system was found pH dependent.
Like other organs, bones are also susceptible to many orthopaedic diseases
like osteoarthritis, osteoporosis, osteomyelitis, bone metastasis, osteosarcoma, etc.
[135]. Since past few decades, huge volume of research and development in the
orthopaedic drug delivery has been noted to treat the said orthopaedic diseases
[121–125]. As a result of this, a variety of nanocomposites have been reported
as orthopaedic drug delivery carrier matrices. In a research, a nanocomposite of
calcium sulfate and nanocrystalline apatite was synthesized by Hesaraki et al. for
orthopaedic release of indomethacin [136]. The synthesized nanocomposite exhib-
ited a slower sustained releasing profile of indomethacin. The setting time and of
injectability of this nanocomposite system was found relatively superior than those
of the pure calcium sulfate. In addition, the indomethacin loading within the calcium
sulfate-nanocrystalline apatite nanocomposite system slightly enhanced the setting
time and of injectability, which did not produce any influence on the compres-
sive strength. The results of in vitro biocompatibility evaluation of indomethacin-
loaded calcium sulfate-nanocrystalline apatite nanocomposites demonstrated good
cytocompatibility, when tested on mouse fibroblast L929 cell line.
Mesoporous silicate MCM-48/HAp nanocomposite loaded with ibuprofen was
synthesized by Aghaei et al. via the in situ synthesis method [137]. The in vitro
ibuprofen release from this nanocomposite was found slightly rapid when ibuprofen
loading was done using 25 mg/ml ibuprofen concentration. In vitro cytocompatibility
of the mesoporous silicate MCM-48/HAp nanocomposite was tested by MTT [3-(4,
5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] assay using osteosarcoma
cell line. The nanocomposite loaded with ibuprofen showed above 40% toxicity indi-
cating the cytocompatibility of the ibuprofen-loaded nanocomposites for orthopedic
drug delivery application.
In another research, ciprofloxacin-loaded HAp-polycaprolactone nanocomposite-
based film for orthopedic implantable drug delivery in treatment of osteomyelitis
was developed by Nithya et al. [138]. The nanocrystalline HAp was synthe-
sized via the precipitation process using eggshell and used to develop HAp-
polycaprolactone nanocomposite-based film. The ciprofloxacin-loaded HAp-
polycaprolactone nanocomposite implantable film was prepared via solvent evap-
oration procedure. In vitro drug releasing profile indicated its capacity for faster
ciprofloxacin releasing. In addition, the results of in vitro cytotoxicity evaluation
using fibroblast NIH-3T3 cell line and osteoblast MG 63 cell line indicated that this
40 A. K. Nayak et al.

ciprofloxacin-loaded HAp-polycaprolactone nanocomposite film possessed excel-

lent biocompatibility. Some recently reported nanocomposite systems used in drug
delivery applications are presented in Table 1.

3.2 Gene Delivery

At present, gene delivery has expansively been introduced and employed for the
treatment/management of various kinds of diseases, such as genetic diseases, cystic
fibrosis, cancers, autoimmune diseases, etc. [161]. In this context, the delivery option
of entrapped gene within the nanoscale carriers like nanoparticles and nanocompos-
ites can improve cellular uptake via the endocytosis and the prevention of the prema-
ture releasing of gene into the non-targeted organs [162]. In recent years, an extensive
volume of researches on the applications of nanocomposites has been performed for
the delivery of gene [163].
In a research, Kashkouli et al. developed and investigated aminotetrazole-
functionalized magnetic chitosan nanocomposite for targeted gene delivery [164].
This magnetic nanocomposite was fabricated using Fe3 O4 /chitosan grafted with
organosilane modified 5-amino-1H-tetrazole via the chemical modification process.
The magnetic nanocomposite demonstrated the capacities of high loading and
targeted delivery of plasmid as well. An improved releasing of plasmid from the
nanocomposite at pH 7.4 was found with the enhancement of gene expression, when
studied in HECK-293 T cell line.
In another report, Xie et al. developed anionic-charged hybrid nanocomposite for
delivery of siRNA [165]. This nanocomposite was synthesized using polyethylene
grafted carboxymethyl and calcium phosphate via the self-assembly procedure in an
aqueous environment. Zhu et al. reported doxorubicin loaded cholesterol siRNA/low-
density lipoprotein coupled N-succinyl chitosan nanocomposite for delivery of
siRNA [166]. Yan et al. developed Tat tagged and folate modified N-succinyl
chitosan nanocomposite for tumor targeted gene therapy [167]. This Tat tagged and
folate modified nanocomposite was prepared via the self-assembly procedure. Some
recently reported nanocomposites for gene delivery are presented in Table 2.

3.3 Antimicrobials

In recent times, many antimicrobial biomaterials are being developed to offer

promising microbial inactivation [175]. In general, antimicrobial agents are used
to develop these kinds of biomaterials. In most of the cases, the loading or
impregnating or reinforcing of antimicrobial agents with other materials has been
used to develop many effective antimicrobial biomaterials [176]. In recent years,
numerous antimicrobial nanocomposites have been developed by many researchers
and scientists.
Biomedical Nanocomposites 41

Table 1 Some recently reported nanocomposite systems used in drug delivery applications
Nanocomposite systems Drug released References
Carboxymethyl cellulose/starch/zinc oxide Doxorubicin Gholamali and
nanocomposite hydrogel beads Yadollahi [139]
pH-sensitive nanocomposite beads of folic acid Folic acid Mallakpour and
intercalated layered double hydroxide and chitosan Hatami [140]
Pectin-zinc oxide hybrid nanocomposite Donepezil Kodoth et al. [141]
Oral colon-specific drug delivery system based on the 5-Fluorouracil Wang et al. [142]
pectin/modified nano-carbon sphere nanocomposite
gel films
Magnetite/silica nanocomposites Doxorubicin Taufiq et al. [143]
Chitosan-based magnetic/fluorescent nanocomposites Doxorubicin Ding et al. [144]
Cassava starch acetate–PEG/gelatin nanocomposites Cisplatin Raj and Prabha
[145]
Alginate-polyvinyl pyrrolidone-nanoHAp composite Diclofenac sodium Hasnain et al.
matrices [146]
Sodium caseinate-magnesium aluminum silicate Acetaminophen Kajthunyakarn
nanocomposite films for modified-release tablets et al. [147]
Magnesium aluminium silicate-polyethylene oxide Diltiazem HCl Asare-Addo et al.
nanocomposite matrices [148]
HAp-alginate nanocomposite beads Ofloxacin Roul et al. [149]
Polycaprolactone-forsterite nanocomposite fibrous Dexamethasone Kharaziha et al.
membranes [150]
Chitosan-starch nanocomposite particles Bis-desmethoxy Bala Subramanian
curcumin analog et al. [151]
Dextran sulfate-modified pH-sensitive layered double Methotrexate Wang et al. [152]
hydroxide nanocomposites
Agarose encapsulated mesoporous carbonated HAp 5-fluorouracil and Kolanthai et al.
nanocomposites powder amoxcillin [153]
MMT-alginate nanocomposites Vit. B1 and B6 Kevadiya et al.
[154]
Chitosan-poly(aminopropyl/phenylsilsesquioxane) 5-fluorouracil Kummari et al.
hybrid nanocomposite membranes [155]
PEG-chitosan-iron oxide nanocomposites Methotraxate Lin et al. [156]
Chitosan/ZnO bio-nanocomposite hydrogel beads Ibuprofen Yadollahi et al.
[157]
Chitosan-fibrin nanocomposites Methotraxate Vedakumari et al.
[158]
Hollow chitosan nanocomposites Ramipril Basu et al. [159]
Sodium alginate/chitosan/HAp nanocomposite Doxorubicin Taleb et al. [160]
hydrogels
42 A. K. Nayak et al.

Table 2 Some recently reported nanocomposites for gene delivery

Nanocomposite systems Gene delivery References
Bioactive nanocomposite coatings Surface-mediated gene delivery Yao et al. [168]
based on collagen/gold
nanoparticles under visible light
illumination
Collagen-silica nanocomposites Modulating inflammation in a Wang et al. [169]
cutaneous chronic wound model by
IL-10 released
Reduced graphene Photothermally controlled gene Kim and Kim [170]
oxide-polyethylenimine delivery
nanocomposite
DNA-amorphous calcium Surface-mediated gene delivery Oyane et al. [171]
phosphate nanocomposite spheres
Mannose-conjugated layered Targeted siRNA delivery Li et al. [172]
double hydroxide nanocomposite
Stabilized calcium phosphate pH-responsive siRNA delivery Zhou et al. [173]
hybrid nanocomposite using a
benzoxaborole-containing polymer
Graphene oxide-HAp HSV-TK suicide gene delivery to Cheang et al. [174]
nanocomposites inhibit human breast cancer growth

Butchosa et al. developed bacterial cellulose-based nanocomposites possessing

strong antibacterial activity [177]. These bacterial cellulose-based antibacterial
nanocomposites were synthesized by post-synthetic modification via mixing aqueous
suspension of partially deacetylated chitosan nanocrystal with bacterial cellulose.
The antibacterial action of these bacterial cellulose-partially deacetylated chitosan
nanocrystal nanocomposites was found to be increased with the increment of partially
deacetylated chitosan nanocrystal concentrations.
Rinehart et al. developed antimicrobial nanocomposite loaded chitosan/polyvinyl
alcohol (PVA) hydrogel, which showed remarkable bactericidal action against
both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria
(Escherichia coli) [178]. In addition to improve the mechanical stability, this antimi-
crobial nanocomposite was found capable to prevent the formation of biofilm and
also, to enhance the growth of fibroblasts. In a research, Elbarbary and El-Sawy
developed antimicrobial nanocomposite membranes of chitosan, polyvinyl alcohol
and silver [179]. These antimicrobial nanocomposite membranes were synthesized
by γ-irradiation. These membranes were found to be antibacterial, nonthrombogenic,
and haemolytic in nature. Youssef et al. developed chitosan-gold and chitosan-silver
nanocomposite films for antimicrobial applications [180]. These nanocomposite
films exhibited considerable antimicrobial actions against Gram-positive bacteria
(Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aerugenosa).
Likewise, these chitosan-gold and chitosan-silver nanocomposite films also showed
their effectiveness against fungi (Aspergillus niger) and yeast (Candida albicans).
Mohamed and Sabaa reported antimicrobial nanocomposite of silver nanoparticles
Biomedical Nanocomposites 43

Table 3 Some recently reported antimicrobial nanocomposites

Antimicrobial nanocomposites References
Graphene oxide-chitosan and graphene oxide-ethylene Khalil et al. [183]
diamine tetraacetic acid nanocomposites
Silver nanoparticles-loaded chitosan nanocomposite Chen et al. [184]
Zinc-mineralized alginate nanocomposites Malagurski et al. [185]
Chitosan based silver nano-biocomposites Davoodbasha et al. [186]
Bacterial cellulose-lignin-cellulose nanocrystal Sá et al. [187]
nanocomposite films
N,N,N-trimethyl chitosan chloride/poly (acrylic acid)/silver Mahmoud et al. [188]
nanocomposites
Polyvinyl alcohol/silver nanocomposite films Mathew et al. [189]
Polystyrene-silver nanocomposite Jabbar et al. [190]
Silver-zinc oxide nanocomposites Noohpisheh et al. [191]
Multivalent and synergistic chitosan oligosaccharide-silver Mei et al. [192]
nanocomposites
Chitosan-titanium dioxide nanocomposite film Siripatrawan et al. [193]
Antibiotic-functionalized silver nanocomposites Guom et al. [194]
Grafted sugarcane bagasse/silver nanocomposites Abdelwahab and Shukry [195]
Streptomycin-loaded chitosan-coated magnetic El Zowalaty et al. [196]
nanocomposites
Oleo-polyurethane-carbon nanocomposites Ahmadi et al. [197]
Nanocellulose/carboxymethyl cellulose and nanochitosan/ Jannatyha et al. [198]
carboxymethyl cellulose composite films

with chitosan [181]. This kind of nanocomposite exhibited its effectiveness against
several bacteria, namely Staphylococcus aureus, Streptococcus faecalis, Pseu-
domonas aerugenosa, Escherichia coli, Bacillus subtilis, and Neisseria gonorrhoeae
and fungal species like Candida albicans. Farhoudian et al. developed antimicro-
bial nanocomposite composed of CuO nanoparticles within chitosan hydrogel beads
[182]. These chitosan hydrogel beads were prepared by ionic-gelation reaction using
sodium tripolyphosphate as ionic cross-linker. The nanocomposite showed signif-
icant antibacterial action against Gram-positive bacteria (Staphylococcus aureus)
and Gram-negative bacteria (Escherichia coli). Some recently reported antimicrobial
nanocomposites are presented in Table 3.

3.4 Tissue Regeneration

In the regenerative medicine, tissue regeneration is well recognized for aiming to

repair the diseased/damaged tissues of the body [199]. The tissue regeneration
strategies deal with the development of scaffold materials via the exploitation of
44 A. K. Nayak et al.

different biomaterials (such as natural, synthetic and semi-synthetic biopolymers,

bioceramics, metals, etc.) and these scaffold systems are being implanted in the
diseased/damaged sites for tissue repair through regeneration [9, 47, 48, 199]. Gener-
ally, scaffold materials are the fundamental constituents of the tissue regeneration
approaches as these scaffold materials present an architectural milieu, wherein extra-
cellular matrix, cell–cell interactions, and cell-growth factor interactions combine to
ease the regenerative niche at the implanted sites [9, 200]. Therefore, the tissue regen-
eration approaches mimic the extracellular matrix and also facilitates cell attachment
signaling, proliferations and differentiations of cells to meet up the requisite for the
tissue regeneration.
Meskinfam et al. reported the synthesis of nanohydroxyapatite (nHAp)-starch
composites for tissue regeneration [201]. In this work, the nanocomposites were
synthesized by a biomimetic process. The in vitro biocompatibility was tested via
MTT assay demonstrated that the nHAp reinforcement with starch influenced the
cell proliferation. These nanocomposites did not display any adverse outcome on the
cultured cell structure, cell viability, and cell proliferation, in vitro. Similar kinds
of starch-based nanocomposite scaffolds for bone tissue regeneration were reported
to be developed by Sadjadi et al. [202]. These starch-based nanocomposite scaf-
folds were also synthesized by in situ biomimetic methodology [202]. In another
research, Huang et al. developed an injectable nanocomposite hydrogel composed of
hyaluronic acid, glycol chitosan, and nHAp [203]. The injectable nanocomposite
hydrogel exhibited a good promise for its probable use in bone tissue regener-
ation. Chae et al. developed and tested alginate/HAp nanocomposite-based scaf-
folds for bone tissue regeneration [204]. These nanocomposite-based scaffolds were
prepared by electro-spinning and in situ synthesis of HAp mimicking bone miner-
alized collagen fibrils. The prepared alginate/HAp nanocomposite-based scaffolds
were fibrous in nature.
Liu et al. synthesized alginate/halloysite nanotubes composite scaffolds for
bone tissue regeneration [205]. These nanocomposite scaffolds were prepared by
employing solution-mixing and freeze-drying methodologies. The in vitro biocom-
patibility was tested via MTT assay using mouse fibroblast cells and the results of
these testing demonstrated active mitochondrial activities of the living cells, when
treated by these nanocomposite scaffolds. The overall results of in vitro biocom-
patibility testing suggested its use in bone tissue regeneration. In another research,
Kawaguchi et al. synthesized alginate-carbon nanotube nanocomposite scaffolds for
tissue regeneration [206].
Correia et al. fabricated chitosan-HAp nanocomposite for the use in cartilage
tissue repair [207]. This kind of chitosan-HAp nanocomposite possessed the network
configuration and was found to improve the production of extracellular matrix. Some
recently reported nanocomposite systems for tissue regeneration applications are
presented in Table 4.
Biomedical Nanocomposites 45

Table 4 Some recently reported nanocomposite systems for tissue regeneration applications
Nanocomposite systems Tissue regeneration References
types
Bioactive electrospun nanocomposite scaffolds Bone tissue Patel et al. [208]
of poly(lactic acid)/cellulose nanocrystals
Fibrin hydrogel incorporated with graphene Bone tissue Pathmanapan et al. [209]
oxide functionalized nanocomposite scaffolds
Biocompatible nanocomposite scaffolds based Bone tissue Saber-Samandari and
on copolymer-grafted chitosan Saber-Samandari [210]
Graphene and HAp self-assemble free Bone tissue Xie et al. [211]
standing nanocomposite hydrogels
Zirconium oxide nanoceramic modified Bone tissue Bhowmick et al. [212]
chitosan-based porous nanocomposites
Organically modified clay supported Bone tissue Bhowmick et al. [213]
chitosan/hydroxyapatite-zinc oxide
nanocomposites
3D fiber-deposited Bone tissue De Santis et al. [214]
poly(ε-caprolactone)/iron-doped
hydroxyapatite nanocomposite magnetic
scaffolds
Collagen-inspired mineral-hydrogel Bone tissue Patel et al. [215]
nanocomposites
Chitosan–gelatin-alginate-HAp Bone tissue Sharma et al. [216]
nanocomposite scaffold
Bioinspired collagen-apatite nanocomposites Bone tissue Liu et al. [217]
Nano-hydroxyapatite/β-cyclodextrin/chitosan Bone tissue Shakir et al. [218]
nanocomposite
Osteoblast-conditioned nHAp/gelatin Bone tissue Samadikuchaksaraei
composite scaffold et al. [219]
nHAp–pullulan/dextran composite Bone tissue Fricain et al. [220]
macroporous material
Intercalated chitosan/HAp nanocomposites Bone tissue Nazeer et al. [221]
Bioactive gum Bone tissue Mirza et al. [222]
Arabic/κ-carrageenan-incorporated nHAp
Nanocomposites
Argon plasma modified nanocomposite Cartilage tissue Griffin et al. [223]
polyurethane scaffolds
Photopolymerized maleilated Cartilage tissue Zhou et al. [224]
chitosan/methacrylated silk fibroin
micro/nanocomposite hydrogels
Poly 3-hydroxybutyrate-chitosan-multiwalled Cartilage tissue Mirmusavi et al. [225]
carbon nanotube/silk nano-micro composite
scaffold
(continued)
46 A. K. Nayak et al.

Table 4 (continued)
Nanocomposite systems Tissue regeneration References
types
Porous poly(d,l-lactic acid)/vertically aligned Osteochondral tissue Stocco et al. [226]
carbon nanotubes/nHAp Scaffolds
Nanocomposites complexed with gold Nerve Kim et al. [227]
nanoparticles on polyaniline
Electroactive alginate hydrogel nanocomposite Nerve Homaeigohar et al.
reinforced by functionalized graphite [228]
nanofilaments
Chitosan-selenium biodegradable Nerve Dolkhani et al. [229]
nanocomposite
Carbon nanotube-polyurethane nanocomposite Cardiac tissue Shokraei et al. [230]

3.5 Prosthesis

In prosthetic applications, a variety of nanomaterials are being used since past

few decades. Amongst these prosthetic nanomaterials, nanocomposites are recently
being used in cardiac prosthetics, orthopaedic prosthetics, dental and maxillofacial
prosthetics, etc. [231, 232]. In recent years, many polymeric nanocomposites are
being developed via the reinforcement of fibers [50]. These fiber-reinforced poly-
meric nanocomposites are being used in a variety of orthopaedic prosthetic devices
because of the capability of their higher mechanical strength [231]. In a work, Ghan-
bari et al. investigated novel kinds of cardiac prosthesis made of modified polyhe-
dral oligomeric silsesquioxane-nanocomposite [232]. This nanocomposite material
showed self-endothelialization potential for the use in heart valve prosthesis. Some
recently reported biomedical nanocomposites used in prosthetics are presented in
Table 5.

3.6 Dentistry

During past few years, numerous kinds of nanomaterials are being developed and
used for dentistry applications like dental drug delivery, dental tissue regener-
ation, dental pulp capping, pulpotomy, etc. [51–53]. Amongst these nanomate-
rials, dental nanocomposites are the widely used nanocandidate category, which
are designed and investigated for periodontal drug delivery, dental restoration,
dentin-pulp regeneration, enamel substitution, etc. [51–54].
In a research, nanocomposite microspheres for periodontal drug delivery were
synthesized by Pataquiva Mateus et al. using nHAp using sodium alginate [245].
Within these nHAp-alginate nanocomposite microspheres, two different kinds of
antibiotics namely, erythromycin and amoxicillin were loaded. These antibiotics-
loaded nanocomposite microspheres showed a promise for their sustained drug
Biomedical Nanocomposites 47

Table 5 Some recently reported nanocomposites used in prosthetic devices

Biomedical uses in prosthetics Nanocomposites References
Orthopedic prosthetics Nano-alumina/multi-walled Dabees et al. [233]
carbon nanotubes/high-density
polyethylene hybrid
nanocomposites for hip joint
replacement
Bioresorbable β-TCP-FeAg Swain et al. [234]
nanocomposites for load
bearing bone implants
Pullulan/dextran/nHAp Schlaubitz et al. [235]
macroporous composite beads
for repairing of a femoral
condyle defect in rats
Biomimetic nanocomposites of Garai and Sinha [236]
carboxymethyl cellulose-HAp
as three dimensional load
bearing bone grafts
Osteoblast-seeded Johari et al. [237]
bioglass/gelatin nanocomposite
for critical-size calvarial defect
repair in rat
Carbon nanotube reinforced Medupin et al. [238]
natural rubber nanocomposite
for anthropomorphic prosthetic
foot purpose
Dental prosthetics Nanocomposite fibrous scaffold Manju et al. [239]
of silica coated
nanoHAp-gelatin reinforced
with electrospun poly(L-lactic
acid) to promote new bone
formation and osseointegration
in mandibular defect
Chemically cross-linked Liu et al. [240]
poly(ε-caprolactone)-HAp
nanocomposite scaffold for
repairing of a mandibular bone
defect
Ceria-stabilized Hagiwara and Nakajima [241]
zirconia/alumina
nanocomposite for fabricating
the frameworks of removable
dental prostheses
Cardiac prosthetics Nanocrystalline cellulose-fibrin Brown et al. [242]
nanocomposites for artificial
vascular graft applications
Poly(vinyl alcohol)-bacterial Mohammadi et al. [243]
cellulose nanocomposite for
mechanical aortic heart valve
prosthesis
(continued)
48 A. K. Nayak et al.

Table 5 (continued)
Biomedical uses in prosthetics Nanocomposites References
Barium Ang et al. [244]
sulfate/poly-L-lactide-based
nanocomposites for coronary
stents

releasing prospects in the treatment of periodontitis. In another work, similar kinds of

erythromycin and amoxicillin-loaded nanocomposite microspheres were developed
Ferraz et al. [246]. The in vitro drug releasing from these nanocomposite micro-
spheres, antimicrobial activities, and in vitro cytocompatibility with osteoblasts
were assessed. The outcome of this research suggested the use of erythromycin
and amoxicillin-loaded nanocomposite microspheres for periodontal drug delivery
carriers. Madhumathil et al. developed antibiotic-loaded gelatin-alginate/apatite
nanocomposite films [247]. These antibiotic-loaded nanocomposite films were found
bioactive in nature and can be used in the treatment of periodontal infrabony defects.
In recent years, different dental nanocomposites are being developed and investi-
gated for the fortification of multifaceted vital dentin pulp [52]. Dental nanocompos-
ites composed of biopolymers and nanoHAp and/or inorganic metallic components
have been developed and investigated to fill the tooth perforations [248–250]. Dental
pulp capping nanocomposites made of polyglutamic acid (PGA) and ∞-melanocyte
stimulating hormone (α-MSH, an anti inflammatory hormone), which demonstrated
the acceleration of the regeneration of pulp connective tissue resulting in adhesion
as well as growth of pulp fibroblasts [251]. Lipopolysaccharide was used to moti-
vate the growth of pulp fibroblasts via incubating the PGA-α-MSH nanocomposites.
These PGA-α-MSH nanocomposites have been proved to stimulate the adhesion of
pulp fibroblast along with improved proliferation for fibroblast cell. The outcome of
this research indicated that these nanocomposites are capable to lessen the inflam-
matory condition of lipopolysaccharide restorative dental pulp fibroblasts, which
commonly occurred during infections by Gram-negative bacteria. These PGA-α-
MSH nanocomposites can be used for the treatment of endodontic injury as well as
lesions.
Panahi et al. prepared tricalcium silicate-based nanocomposites for root-end
dental application [252]. Besides tricalcium silicate, chitosan and dicalcium phos-
phate were used for the fabrication of these dental nanocomposites. The researchers
noticed the occurrence of synergic influence of chitosan and dicalcium phosphate
on these tricalcium silicate-based dental nanocomposites. Some recently reported
dental nanocomposites are presented in Table 6.
Biomedical Nanocomposites 49

Table 6 Some recently reported dental nanocomposites

Dental nanocomposite systems Purpose References
Bioactive upconversion nanocomposites Treatment of periodontal Zhang et al. [253]
containing chlorin e6 diseases
Amoxicillin-loaded electrospun Fibrous-based antibiotic Furtos et al. [254]
nanocomposite membranes based on carrier system for dental
poly(ε-caprolactone) and nHAp and tissue engineering
applications
Porous tri-layered nanocomposite hydrogel Concurrent regeneration Sowmya et al. [255]
scaffold composed of of cementum, periodontal
chitin-poly(lactic-co-glycolic ligament, and alveolar
acid)/nanobioactive glass ceramic /cementum bone
protein 1
Piroxicam loaded biodegradable Periodontal regeneration Farooq et al. [256]
chitosan/poly(vinyl alcohol)/HAp
electrospun nanocomposite scaffolds
2-methacryloyloxyethyl For inhibition of Wang et al. [257]
phosphorylcholine-dimethylaminohexadecyl periodontal pathogens,
methacrylate Class V nanocomposite combat periodontitis and
protect the periodontium
Alginate/HAp-based nanocomposite Dental pulp Sancilio et al. [258]
biomineralization and
differentiation

3.7 Wound Healing

During past few years, a considerable amount of interest has been paid in the research
and development for the advancements of wound healing therapeutics [8]. Many
groups of researchers and scientists are trying to develop improved and efficient
wound healing biomaterials, which should possess biocompatibility, ability to stop
bleeding, nonallergic and noninfective properties, permeability for gases, ability to
retain moisture and to absorb exudates, capability of promoting skin regeneration, and
fastening wound healing process [8, 259, 260]. In recent years, many nanocomposite
biomaterials are being developed and employed for the use in wound healing.
Shah et al. developed moxifloxacin (a fluoroquinolone derivative antibiotic)
loaded chitosan-silver-sericin nanocomposite films for wound healing applications
[261]. These moxifloxacin loaded nanocomposite films were prepared by solvent
casting technique. These moxifloxacin loaded nanocomposite films demonstrated a
potential antibacterial action against both Gram-positive bacteria (Staphylococcus
aureus and Staphylococcus epidermidis) and Gram-negative bacteria (Acineto-
bacter baumannii and Pseudomonas aeruginosa). In addition, moxifloxacin loaded
films showed their antibacterial efficacy against methicillin-resistant Staphylococcus
aureus (MRSA) mediated hospital-acquired skin infections. In the in vivo testing
on the burn-injured wound model developed Sprague Dawley rates, these moxi-
floxacin loaded chitosan-silver-sericin nanocomposite films displayed significantly
50 A. K. Nayak et al.

faster wound healing results. In another research, Kumar et al. developed chitosan
hydrogel-zinc oxide nanocomposite bandages for wound healing applications [262].
These nanocomposite bandages showed significant antibacterial efficacy against
both Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria
(Escherichia coli). Sandri et al. fabricated nanocomposite composed of chitosan and
halloysite [263]. This kind of chitosan-based nanocomposite showed remarkable
wound healing results in cases of burns and skin lesions types of wounds. Anisha
et al. prepared nanocomposite sponge composed of chitosan-hyaluronan/chondroitin
sulfate nanoparticles for wound healing uses [264]. In addition, the nanocomposite
sponge demonstrated enhanced blood clotting and platelet activation abilities with
a controlled swelling and biodegradation. The results of in vitro cytotoxicity testing
on human dermal fibroblast cells indicated the biocompatible (nontoxic) nature of
this nanocomposite sponge.
Figueiredo et al. developed bacterial cellulose-based nanocomposite films for
wound dressings in combination with poly(2-hydroxyethyl methacrylate) [265].
These nanocomposite films were synthesized via an in situ radical polymeriza-
tion of 2-hydroxyethyl methacrylate and in this synthesis, poly(ethylene glycol)
diacrylate was employed as cross-linking agent. The bacterial cellulose-poly(2-
hydroxyethyl methacrylate) nanocomposite films exhibited desirable biocompati-
bility with improved cell adhesion and cell proliferation for human adipose-derived
mesenchymal stem cells. Some recently reported nanocomposites for wound healing
applications are presented in Table 7.

3.8 Bioimaging and Biosensor

In recent years, nanocomposites are broadly used in many bioimaging and biosensor
applications [284]. Lin et al. prepared polyethylene glycol (PEG)-chitosan-iron
oxide nanocomposite containing fluorescent cyanin dye for bioimaging applica-
tions. The prepared PEG-chitosan-iron oxide nanocomposite was found potential
for fluorescence imaging and magnetic imaging (MRI) [285].
Fu et al. developed gold-embedded chitosan nanocomposite for the use as surface-
enhanced Raman scattering sensor (SERS) [286]. The 3D architecture of gold-
embedded chitosan nanocomposite was noticed to serve as an outstanding SERS
substrate for detecting 4-mercaptobenzoic acid. In addition, because of the pH-
sensitive characteristics, the chitosan nanocomposite-based SERS was found capable
of detecting the charge of dye molecules. In another research, Liu et al. reported
the fabrication of chitosan-carbon nanotubes composite for biosensing applications
[287]. This nanocomposite was found efficient as glucose electrochemical sensor by
immobilizing the glucose oxidase. Some recently reported nanocomposites used in
bioimaging and biosensors are presented in Table 8.
Biomedical Nanocomposites 51

Table 7 Some recently reported nanocomposites for wound healing applications

Nanocomposites for wound healing References
applications
Chitosan-laponite nanocomposite scaffolds Gonzaga et al. [266]
Basil seed (Ocimum basilicum L.) Tantiwatcharothai and Prachayawarakorn [267]
mucilage-zinc oxide nanocomposite
Curcumin-loaded electrospun Sadeghianmaryan et al. [268]
polycaprolactone/MMT nanocomposite
Bacterial cellulose-zinc oxide nanocomposites Khalid et al. [269]
Chitosan/banana peel powder nanocomposites Kamel et al. [270]
MMT-norfloxacin nanocomposite García-Villén et al. [271]
A novel bilayer zein/MMT nanocomposite Gunes et al. [272]
Titanium dioxide nanotubes incorporated Razali et al. [273]
gellan gum bio-nanocomposite film
Alginate/acacia based nanocomposites of zinc Manuja et al. [274]
oxide nanoparticles
Citric acid cross-linked carboxymethyl guar Prabhakar and Matta [275]
gum nanocomposite films loaded with
ciprofloxacin
Bacterial Cellulose/methylglyoxal Yang et al. [276]
nanocomposite
Edaravone-loaded alginate-based Fan et al. [277]
nanocomposite hydrogel
Alginate/silver/nicotinamide nanocomposites Montaser et al. [278]
for treating diabetic wounds
Cellulose based nanocomposite hydrogel films Koneru et al. [279]
consisting of sodium
carboxymethylcellulose-grapefruit seed extract
nanoparticles
Incorporated plant extract fabricated Renu et al. [280]
silver/poly-D,l-lactide-co-glycolide
nanocomposites
Keratin-chitosan/zinc oxide nanocomposite Zhai et al. [281]
hydrogel
Bioactive antibacterial silica-based Li et al. [282]
nanocomposites hydrogel scaffolds
Polyurethane nanocomposite impregnated with Najafabadi et al. [283]
chitosan-modified graphene oxide

4 Conclusion

In recent years, there has been a growing interest for the development of different
nanomaterials for various biomedical applications. Amongst these nanomate-
rials, different nanocomposites, such as organic-organic, inorganic-inorganic, and
52 A. K. Nayak et al.

Table 8 Some recently reported nanocomposites used in bioimaging and biosensors

Nanocomposites Applications (bioimaging References
and/or biosensors)
Bi/phthalocyanine manganese Trimodal imaging directed Wang et al. [288]
nanocomposite photodynamic and
photothermal therapy
Fluorescently-labeled magnetic Optical and magnetic Zhu et al. [289]
nanocomposites resonance imaging
Fluorescent gold nanocrystals-silica hybrid Bioimaging Kim et al. [290]
nanocomposite
MnO2 -DNAzyme-photosensitizer Cell imaging Wang et al. [291]
nanocomposite
Magneto-fluorescent perovskite Directed cell motion and Tan et al. [292]
nanocomposites imaging
Graphene/polyvinylpyrrolidone/polyaniline Novel paper-based Ruecha et al. [293]
nanocomposite cholesterol biosensor
Graphene–polyaniline nanocomposite Biosensor for detection of Radhapyari et al.
antimalarial drug artesunate [294]
in pharmaceutical
formulation and biological
fluids
Multifunctional zirconia-reduced graphene Ultrasensitive Chen et al. [295]
oxide-thionine nanocomposite electrochemical DNA
biosensor
Polyaniline–bismuth oxide nanocomposite Biosensor for quantification Jain et al. [296]
of anti-parkinson drug
pramipexole in solubilized
system
Mushroom-like polyaniline and gold Detection of silver ions and Yang et al. [297]
nanoparticle nanocomposite DNA biosensor
Polyaniline capped Bi2 S3 nanocomposite Impedimetric DNA Zhu et al. [298]
biosensor
Polyaniline@nickel metal–organic Electrochemical biosensor Sheta et al. [299]
framework nanocomposite
Cysteine-silver nanoparticles/graphene oxide Biosensor Wang et al. [300]
nanocomposite

organic–inorganic nanocomposites are being developed for biomedical applica-

tions. From this chapter, we can conclude that these biomedical nanocomposites
possess several unique properties, such as physical, physico-chemical, mechan-
ical and biomedical properties for the inclusive range of applications, such as drug
delivery, gene delivery, antimicrobial, tissue regeneration, wound healing, dentistry,
bioimaging, biosensors, etc.
Biomedical Nanocomposites 53

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Antimicrobial Nanocomposites

El-Refaie Kenawy, Mohamed M. Azaam, Syed Anees Ahmed,

and Md Saquib Hasnain

1 Introduction

Since last few decades, a huge leap of material researches in particular biomaterial
development and applications have been experienced [1–19]. In recent years, a wide
variety of advanced materials for biomedical applications have been engineered using
biopolymers (such as synthetic, semi-synthetic, and natural biopolymers) [19–36],
metals and metallic oxides [37–38], bioceramics [39–40], bioglasses [39], struc-
tured carbons [41–50], etc. These engineered biomaterials includes polymer-blends
[51–57], gels [2, 58], hydrogels [59–69], particulates (nanoparticles, microparticles,
beads, etc.) [70–82], composites and nanocomposites [83–89],scaffolds [90], fibers
[91], membranes and films [92–93], pastes [15, 94], emulsions [95–96], suspensions
[97–98], nanotubes [99–100], etc. These advanced biomaterials are being used in
many important biomedical uses like drug delivery [101–113], cancer therapy [114],
protein encapsulation [115], gene delivery [116, 117], antimicrobial [118], tissue
engineering [119–120], wound healing [121], dentistry [122], bioimaging [116],
biosensor [123], etc. With the fast progress of science, the nanotechnology has been
proved as the prosperous field for biomedical applications [124–125]. The signif-
icant biomedical nanomaterials include polymeric nanoparticles [126–127], lipid
nanoparticles [128], metallic nanoparticles [129], ceramic nanoparticles [130–131],

E.-R. Kenawy (B) · M. M. Azaam

Polymer Research Group, Department of Chemistry, Faculty of Science, Tanta University, Tanta,
Egypt
e-mail: [email protected]
S. A. Ahmed
Department of Pharmacology, Hygia Institute of Pharmaceutical Education and Research,
Lucknow, UP, India
M. S. Hasnain
Department of Pharmacy, Palamau Institute of Pharmacy, Chianki, Daltonganj, Jharkhand, India

© Springer Nature Singapore Pte Ltd. 2021 71

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_4
72 E.-R. Kenawy et al.

nanogels [132–133], nanofibers [91], nanovesicles [134–136], nanotubes [137–138],

nanocomposites [139–145], etc.
Recently, most of the nanocomposites are made of biopolymers (such as synthetic,
semi-synthetic, and natural biopolymers), metals and metallic oxides, bioceramics,
bioglasses, structured carbons, etc. [83–88, 139–145]. In biomedical field, these
nanocomposites are being used for drug delivery [83–87, 141], cancer therapy [146],
gene delivery [147], antimicrobial [148], tissue engineering [139, 141], wound
healing [149], prosthesis [140], dentistry [142–144], bioimaging [146], biosensor
[123, 150], etc. In recent years, numerous antimicrobial biomaterials are being devel-
oped for microbial inactivation and characterized by many researchers and scientists
[118]. The present chapter presents a widespread assessment of the applications of
antimicrobial nanocomposites.

2 Application of Nanocomposites as Antimicrobial Agents

Devi et al. (2019) used a waste material such as jute stick for the sustainable applica-
tion [151]. Silver loaded activated carbon (Ag-AC) nanocomposite exhibited remark-
able antimicrobial activity against gram (−) and (+) bacteria due to the enhancement
of the silver nanoparticles’ as antibacterial. The antimicrobial activities of (Ag-AC)
were better than silver nanoparticles. The nanocomposite also showed an excellent
photocatalytic activity [151].
Another manuscript describes an efficient synthesis of Ag nanoparticles immo-
bilized on the magnetic polymeric nanocarriers for applications as antibiotics [152].
Polyethylene glycol coated Fe3 O4 nanoparticles (Fe3 O4 @PEG) were synthesized,
and Ag nanoparticles were supported. The Ag magnetic polymer nanocomposites
showed a high antibacterial effect. Besides, magnetic nanocomposites could be
readily separated from the solution after the disinfection process by applying an
external magnetic field [152].
Hosseini et al. [153] reported the preparation of nanocomposite based on MnS2 ,
MnS2 /Chitosan-calcium alginate (MnS2 /CS-CaAlg), and MnS2 /Chitosan sodium
alginate (MnS2 /CS-NaAlg) [153]. The antibacterial and antifungal activities of the
nanocomposites showed good activities as compared to MnS2 . Also, the MnS2 ,
MnS2 /CS-NaAlg, and MnS2 /CS-CaAlg showed outstanding mechanical and antiox-
idant properties. The antibacterial effect was observed after 60 min. The reduction
of the surviving bacteria under visible light irradiation was observed in the presence
of a catalyst in comparison to materials from MnS2. The size of the fungal colonies
was reduced by MnS2 /CS-NaAlg and MnS2 /CS-CaAlg nanocomposites that may be
due to degradation of the cell wall and structure of the cell membrane [153].
Corn silk as anagricultural waste was introduced on polyester to augment the cell
viability of zinc oxide on polyester fabric [154]. Good antimicrobial properties were
obtained on the material containing corn silk/zinc oxide nanocomposites close to
100 and 87%, respectively. The advantage of corn silk and ZnO nanoparticles as a
polyester fabric is a low cytotoxicity antimicrobial agents [154].
Antimicrobial Nanocomposites 73

Nanocomposites based on pectin, polyethyleneimine, and Cu nanoparticles were

synthesized [155]. The antimicrobial investigations of the prepared nanocomposites
revealed they have high antimicrobial activity against S. aureus and E. coli strains.
The oxidized nanofibers of cellulose were formulated with cadmium oxide
nanostructure from bagasse pulp, and their antibacterial activity was studied [156].
The Minimum Concentration of Inhibition (MIC) highlighted that the formulated
nanocomposite has potent antimicrobial activity. MIC values were 1.56 μg/mL for
gram (−) bacteria, less than 1.56 for gram (+) bacteria and 0.19 μg/mL for Candida
albicans [156].
The antimicrobial activity of pure Ag2 S, CdS, and CdS-Ag2 S composites was
evaluated [157]. The CdS to Ag2 S ratio was varied to increase antibacterial activity
at an optimum level between two compounds. The increase in CdS ratio in CdS-Ag2S
nanocomposites improved antibacterial activity.
Alginate-mediated biomineralization with the Zn-mineral phase was prepared as
a simple and cost-effective procedure [158]. Both forms of Zn-mineralized nanocom-
posites had a clear antimicrobial effect on Candida albicans, Escherichia coli, and
Staphylococcus aureus. Mineralized samples had higher Zn(II) content and more
stable in a biological environment. Also, they released Zn(II) in a controllable and
sustained fashion when compared to a non-mineralized sample. Alginate-mediated
biomineralization represents promising antimicrobial biomaterials.
Chitosan (CH)/polyvinyl alcohol (PVA) hydrogel and CH/PVA/ZnO nanocom-
posite was prepared [159]. The antimicrobial activities of the prepared composites
were higher than Erythromycin and Metronidazole. Also, the activity was highly
improved after ZnO nanocomposites formation. The increase of the ZnO ratio in the
nanocomposite increases their antimicrobial activities.
Modified HDPE nanocomposite monofilaments based on MMT were synthesized
using nanoparticles made from copper, silver, and zinc oxide [160]. HDPE/modified
MMT nanocomposite monofilaments displayed the enhanced activity in bacterial
colonies against HDPE/Ag-MMT monofilaments and gram (−) and (+) bacteria. The
kinetics of metal ions/NPs dissolution from modified MMT nanostructure reflected
its potential as having antimicrobial action. The antimicrobial HDPE monofilaments
produced aim to minimize occurrences of these undesired abnormalities. The level
of antimicrobial activity for each load (1, 3, and 5 weight percent) of modified MMT
is HDPE/Ag-MMT > HDPE/Cu-MMT > HDPE/Zn-MMT [160].
Ghosh et al. (2019) designed a nanohybrid by simultaneous reduction of copper(II)
chloride dehydrate and graphene oxide (GO) in the presence of octadecylamine
(ODA) [161]. The nanohybrid was then included in the interpenetrating polymer
network (IPN) of bio-based PU and polystyrene (PS). The nanocomposites exhib-
ited high antibacterial properties. Consequently, the studied nanocomposites repre-
sent a new track in the surface-active advanced thin-film field. Fascinatingly, high
hydrophobicity through intrinsic water repellent behavior and the strong antimicro-
bial property is achieved for the nanocomposites. As a result, the studied nanocom-
posites are used as protective functional surface materials for biomedical and
electrical devices [161].
74 E.-R. Kenawy et al.

The graphite (Gr), graphene oxide (GO), and reduced graphene oxide (RGO) reac-
tion formed cross-linked antimicrobial oleo-polyurethane nanocomposites (PUCs)
with high performance[162]. The composites prepared showed the biocidal effects
of GO and RGO PUCs against all those bacterial classes. Surface-active cross-linked
LO-based PUCs utilizing 0.5 wt% of pristine Gr, GO, and RGO was formulated as
nano-fillers. GO and RGO PUCs exhibited efficient antimicrobial properties against
S. aureus and E.coli. The present study may provide new methods and materials
for the preparation and designing of advanced contact active nanocomposite films
required in various areas, such as the development of medical equipment, functional
films, and active packaging [162] (Fig. 1).
Hydroxyapatite nanoparticles (nHAP) with zinc oxide (ZnO) nanoparticle
nanocomposite were synthesized [163]. Nanocomposite showed antimicrobial and
antibiofilm activity in a ratio of 90:10, 75:25, and 60:40 nHAP and ZnO NPs. For
both pathogens, the MIC was found to be 0.2 mg/mL in a ratio of 90:10 nanocom-
posites. For S. aureus and E. coli, the minimum bactericidal concentration (MBC)
was 0.2 mg/mL of 75:25 and 60:40 nanocomposites. The maximum biofilm inhi-
bition percentage was found at ratio 60 (nHAP): 40 (ZnO NPs). It was against S.

Fig. 1 The permanent antimicrobial effect of GO-PUC against; a E. coli and b S. aureus. c The
three-step antimicrobial action mechanism of Gr-based PUCs [162] (with permission @ 2019
Elsevier Ltd.)
Antimicrobial Nanocomposites 75

aureus as well as E.coli bacteria having inhibition of 52 percent as well as 54 percent

and for green synthesized ZnO NPs have inhibition of 51 percent and 52 percent
against E.coli and S. aureus, respectively. Biomaterials that involve multiple ratios
of nHAP-ZnO NPs could be used in bone regenerative medicine as antibiofilm mate-
rials. Increasing the proportion of ZnO NPs in the nanocomposite prevented bacterial
infection in orthopedic implants [163].
Silver nanocomposites (AgNPs@Tob&PAGA) was prepared by reaction of
AgNPs with tobramycin (Tob) and poly(2-(acrylamide) glucopyranose) (PAGA)
[164]. The antibacterial activities of nanocomposites were determined. The introduc-
tion of PAGA onto silver nanocomposites enhanced cytocompatibility and antibac-
terial activity. AgNPs@Tob&PAGA showed more attractive antimicrobial conse-
quence than Tob against E. coli and S. aureus. The introduction of (PAGA) into
nanocomposites improved the compatibility and the antibacterial effect [164].
The antibacterial activity of different silver sulfide (Ag2 S) nanoparticles cross-
linked with chitosan was evaluated [165]. The cross-linked nanopolymers prevent
the production of gram (−) antibiotic-resistant bacteria and gram (+) bacteria from
64 up to 32 μg/mL resulting in a positive charged surface. Using glucose as a green
capping agent generates nanostructures with acceptable size distribution [165].
Chitosan/silica nanocomposite membrane doped with Al2 O3 nanoparticles was
synthesized [166] and their antimicrobial activities were determined. The modified
membranes with a lower concentration of Al2 O3 ions display better antimicrobial
properties due to the high cross-linking between the chitosan matrix and silica-based
nanoparticles [166].
Nezami et al. (2019) formulated composites AgNPs, St-A-E/M, and St-A-E/M-Ag
nanocomposite beads, and then used as controlled-release drug delivery applications
for methyl prednisolone [167]. The release of the nanocomposite drug was signifi-
cantly high than the composite of free-NPs, as well as the release increased up to 3.3%
(V/V) in the polymer matrix by the increase in AgNPs number. The nanocomposite
demonstrated more significant antibacterial activity compared to the corresponding
composite of free-NPs. The results showed that the amount of drug released from the
nanocomposite was around 92 percent compared to free AgNPs composite (68%)
for release media having pH 7.4 for 9 h.The nanocomposite with 3.3 (V/V) AgNPs
exhibited the highest antibacterial activity against E. coli (ZOI = 10.48 mm) and
S. aureus (ZOI = 8.37 mm) compared to the free AgNPs composite. Therefore, all
these observations unanimously illustrated the high potential of the nanocomposite
as an oral drug delivery system [167].
An ethnonanocomposite was formulated employing Curcuma zedoaria and a ZnO
nanoparticle [168] that was immobilized for effective delivery on the microspheres
of poly (ÿ-caprolactone) (PCL). The antimicrobial activity confirmed the notable
effectiveness of microspheres loaded with nanocomposite. This study is significant
due to the biocompatibility, biodegradability and stability of PCL. The prepared
microspheres can be used as efficient antimicrobial agents in applications that require
the instant and sustained release of effective antimicrobial agents [168].
Fe3 O4 /Ag3 PO4 @WO3 nanocomposites were prepared by in-situ industrial
production and electrospinning to enhance photocatalyst efficiency for methylene
76 E.-R. Kenawy et al.

blue (MB) deterioration and antibacterial activity [169]. The nanocomposites showed
high photocatalytic and antibacterial activity. The Ag3 PO4 /Fe3 O4 @WO3 composite
has high antibacterial activities than the other prepared composites. The oleic acid
functionalized composite Fe3 O4 /Ag3 PO4 @WO3 also exhibited a high inhibition
zone [169].
An oxidation redox coating approach was used to prepare a hybrid Ag@CeO2
nanostructure composite [170]. The synthesized hybrid showed excellent antibacte-
rial activity against gram (−) and (+) bacteria. The hybrid nanostructure bind with
the bacterial cell membrane, followed by cell rupturing and finally cell death. The
prepared nanocomposites Ag@CeO2 showed more potent bacteriostatic properties
compared to other kinds of nanoparticles [170].

3 Application of Antimicrobial Nanocomposites in Food

Packaging

Siripatrawan et al. developed antimicrobial packaging nanocomposite based on

chitosan and nanosized titanium dioxide (TiO2 ) [171]. They reported that 1% TiO2
was optimal for antimicrobial properties against gram (−) and (+) bacteria and fungi
[171] (Fig. 2).

Fig. 2 A schematic diagram of the possible mechanism of photocatalytic degradation of ethylene

and antimicrobial activity of the chitosan-TiO2 nanocomposite film [171] (with permission @ 2018
Elsevier Ltd.)
Antimicrobial Nanocomposites 77

Certain antimicrobial nanocomposites were synthesized with the root extract of

Salvadorapersica L. (SPE) of carboxymethyl cellulose (CMC), cellulose nanofiber
(CNF), and miswak [172]. The pure nanocomposites activated by SPE and SPE exhib-
ited strong antibacterial activity. Nanocomposite films based on pullulan/AgNPs and
pullulan/pectin/AgNPs films were prepared by Lee et al. (2019) [173]. They showed
high antimicrobial activity against food-borne pathogens to maintain food safety
[174].
Yu et al. (2019) prepared cellulose nanofibril/silver nanoparticle (CNF/AgNP)
as antimicrobial nanocomposite and evaluate their toxicity against human colon
cells [175]. The prepared nanocomposite exhibited antimicrobial activities against
two important food-borne pathogens, including Escherichia coli and Staphylococcus
aureus.
The nanocomposite CuO/hc-pCUR consisting of copper (II) oxide nanoparti-
cles and strongly cross-linking poly(curcumin) nanospheres (hc-pCUR-NS) was
prepared [176]. Relative antimicrobial assays against gram (−) and (+) bacteria
were investigated. Hc-pCUR-NS demonstrated more antimicrobial activity than free
CUR. Copper nanoparticles added improved the hc-pCUR-NS inhibitory effect.
Polyvinyl alcohol (PVA) reinforced with silver nanoparticles (AgNPs) film was
prepared [177]. The presence of AgNPs in the film improved UV and light barrier
properties in addition to antibacterial activity against the food-borne pathogen
Salmonella Typhimurium and Staphylococcus aureus.
The in-situ precipitation and a casting method in combination [178] devel-
oped nanocomposite films (Zn-carbonate and Zn-phosphate/agar) Reinforcement
of the Zn-mineral process enhanced the mechanical properties of the carbonate-
mineralized films. Still, they had a marginal effect on the phosphate-mineralized
samples. Both nanocomposites exhibited improved optical, thermal and antimi-
crobial properties. Zn-mineralized nanocomposite agar films have been used as
accessible, environmentally safe and active food packaging materials [178].
CuO/BiVO4 (BVO) nanocomposite photocatalyst was immobilized on cotton
fabrics through polydopamine (PDA) templating. These nanocomposites showed
efficient visible-light-driven photocatalysis, antimicrobial activity and UV protec-
tion [179]. The CuO/BVO@ cotton was an antimicrobial agent with a 99.2% reduc-
tion in bacterial load against Escherichia coli. Also, it was UV protective with the
UPF value of 237.17 and UVA and UVB transmittance value of 0.47% and 0.39%,
respectively [179].
The dual sputtering method for the mid-range frequency was used to prepare thin
films of Ag-plasma polymer fluorocarbon (PPFC) nanocomposite [180]. The thin
films of Ag-PPFC nanocomposite, coated on a substratum of polyethylene tereph-
thalate (PET), display higher visible light transmittance than the bare substratum
and excellent water repellent. The Ag nanoparticles have had no observed effect
on the thin film’s optical and surface properties. It has been found that the Ag-
PPFC nanocomposite thin films have more potent antimicrobial properties because
it reduces bacterial growth by up 92.2% compared to uncoated substrates.This antimi-
crobial activity is attributable to the decreased humid atmosphere and Ag nanopar-
ticle’s antimicrobial characteristics. These coatings have been used in many areas,
78 E.-R. Kenawy et al.

such as outdoor products, usable fibers, sportswear, medical equipment, touch screen
displays, and flexible solar cells [180].
These were manufactured the gelatin-based nanocomposite consisting of chitosan
nanofiber (CHINF) and ZnO nanoparticles (ZnONPs) [181] which displayed potent
antibacterial activity against pathogenic bacteria raised in food. Chicken fillet and
cheese were chosen as food prototypes to evaluate the efficacy of this film for
food packaging. Results revealed that coating with nanocomposite film reduced
(p < 0.05) the growth of inoculation bacteria in chicken fillet and cheese samples
significantly.The weight loss of the coated chicken fillet and cheese specimens with
nanocomposite at the end of 12-day storage was 18.91 ± 1.96 and 36.11 ± 3.74%,
respectively. This study was significant due to: (1) ZnONPs were antimicrobial
agents and improved physical properties and barrier properties. (2) They enhance
the synergistic effects on antimicrobial properties. (3) biocompatibility of gelatin.
G/CHINF/ZnO NPs antibacterial nanocomposite film is suitable for the packaging
of poultry meat and cheese, representing a great promise for improving the quality
and shelf life of these food products [181].
Nano-sized Sr1−xAgxTiO3 system was prepared using modified Pechini method
(x = 0, 0.02, 0.05, and 0.07) [182]. Antimicrobial properties have been observed for
undoped and Ag-doped nano-sized SrTiO3, while the parent SrTiO3 has no specific
region. Different silver concentrations were added to polymer matrices to be used
on paper sheets as energetic coating materials. The mechanical and short period
behaviors were improved by active nanocomposite coating. Antimicrobial behavior
was added to the SrTiO3 parent by doping with silver metal as successfully active
packaging coating. Besides, the mechanical properties of Kraft paper were enhanced
in tensile, roughness, short span, and water absorption characters [182].

4 Application of Antimicrobial Nanocomposites in Water

Treatment

Nanocomposites were prepared, consisting of chitosan and silver nanoparticles

(AgNPs), copper nanoparticles (CuNPs), and carbon nanotubes (CNTs) [183]. They
have higher antimicrobial activity against gram (−) and (+) bacteria and antifungal
activity isolated from the local wastewater sample. The prepared multifunctional
nanocomposites act as promising materials in water treatment such as heavy metals
removal and water disinfection against different microbes [183] (Figs. 3 and 4).
Nanocomposite based on polyaniline (PANI) and copper nanoparticles solved the
problems of easy oxidation and aggregation of copper nanoparticles in addition to
their antimicrobial activities [184]. They can control microorganisms in contaminated
water before further processing.
A nanocomposite (PPP-TiO2 ) was prepared by the reaction of pristine
pomegranate peel extract (PPP) and titanium dioxide nanoparticles (TiO2 NPs) [185].
Antimicrobial Nanocomposites 79

Fig. 3 TEM image and Schematic representations for the suggested structure of chitosan/Ag NPs
nanocomposite [183] (with permission @ 2018 Elsevier B.V.)

Fig. 4 TEM image and Schematic representations for the suggested structure of chitosan/CNTs
nanocomposite [183] (with permission @ 2018 Elsevier B.V.)

PPP-TiO2 antimicrobial activity was 1.5 times that of PPP and TiO2 NPs. The Diam-
eter Inhibition Zone (DIZ) had the highest inhibition of S. aureus as compared to
E-coli and P. aeruginosa. The findings investigated lower BOD5 values for PPP-
TiO2-containing samples as compared to TiO2 NPs.The approach is simple, feasible,
cost-effective, and minimize environmental impact. The samples containing PPP-
TiO2 showed lower BOD5 values and consequently indicated that the sample has
lower organic matter and the composites can disinfect water sources without any side
effects [185].

5 Antimicrobial Nanocomposites Are Used as Anticancer

Agents

Abu Elella et al. synthesized water-soluble polyelectrolyte complex (PEC) with a

reaction of N, N, N-trimethyl chitosan chloride (TMC), and poly (acrylic acid) (PAA)
80 E.-R. Kenawy et al.

in an acidic environment to synthesize AgNPs producing water-soluble nanocompos-

ites (TMC/PAA/Ag) [186]. The formulated nanocomposites had good antimicrobial
activity, which increased with an increase of Ag percent. In contrast, the most cyto-
toxic impact against colon cancer was TMC/PAA/Ag (3%). The nanocomposites
have no major harmful effects on the VERO cell lines.
Pristine polyaniline (PANI), PANI-Au nanocomposites, and PANI-Au–Pt
nanocomposites were prepared [187]. They demonstrated antibacterial activity
against bacterial pathogens with gram (−) and (+) bacteria. They are in vitro
anticancer investigations against HepG2 liver cancer cells revealed the highest
cytotoxicity for PANI-Au–Pt nanocomposite followed by PANI-Au nanocomposite
(32 mg/mL) and pristine PANI (49 mg/mL) [187].
Aminoclays/poly(vinyl alcohol) (AC/PVA) hydrogel films were prepared after
three cycles of the freeze-thawing process [188]. Both MgAC/PVA and ZnAC/PVA
hydrogel films exhibited antimicrobial effects. The silver nanopaerticles enhanced
the antibacterial activity, but it did not decrease the gel fraction or swelling ratio
of the MgAC/PVA hydrogel films. The presence of AgNW did not change the gel
fraction or swelling ratio but did increase the antibacterial activity of MgAC/PVA
hydrogel films [188].

6 Application of Antimicrobial Nanocomposites in Dental

Application

Alamgir et al. [189] synthesized poly (methyl methacrylate) and TiO2 nanocompos-
ites using a twin-screw extruder by melt mixing the compounds and used in dental
applications [189]. TiO2 nanoparticles with different percentages (1 and 2 wt%) were
mixed with PMMA through a melt compounding process. The PMMA/2wt%TiO2
sample exhibited the best mechanical and physical properties [189].

7 Conclusion

Production of various nanocomposites for different biomedical applications has

received considerable attention. Numerous antimicrobial biomaterials for micro-
bial inactivation are being developed in recent years and are described by many
researchers and scientists. This chapter is focused on the various applications of
nanocomposites as antimicrobial agents, in food packaging, wound dressings, water
treatment, anticancer and dental applications.
Antimicrobial Nanocomposites 81

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Potential Application of Silver
Nanocomposites for Antimicrobial
Activity

Shagufta Haque, Mamatha Julappagari, and Chitta Ranjan Patra

Abbreviations

AFM Atomic force microscopy

ATP Adenosine triphosphate
BSA Bovine serum albumin
CAGR Compound annual growth rate
DLS Dynamic light scattering
DSC Differential scanning calorimetry
EDAX Energy dispersive X-ray analysis
EPS Extracellular polymeric substance
FESEM Field emission scanning electron microscope
FTIR Fourier transformation infrared spectroscopy
GSH Glutathione peroxidase
HPLC High performance liquid chromatography
ICP-OES Inductively coupled plasma optical emission spectroscopy
MBC Minimum bacterial concentration
MDR Multidrug resistant
MIC Minimum inhibitory concentration
PVA Poly (vinyl alcohol)
PVP Poly(N-vinyl-2-pyrrolidone)
ROS Reactive oxygen species
RT-PCR Reverse transcription polymerase chain reaction
TEM Transmission electron microscope

S. Haque · M. Julappagari · C. R. Patra (B)

Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, UP, India
e-mail: [email protected]; [email protected]
M. Julappagari · C. R. Patra
Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road,
Tarnaka, Hyderabad 500007, Telangana, India

© Springer Nature Singapore Pte Ltd. 2021 93

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_5
94 S. Haque et al.

TGA Thermogravimetric analysis

XRD X-ray diffraction analysis
XPS X-ray photoelectron spectroscopy
WHO World Health Organization

1 Introduction

The microbial infections have resulted as one of the foremost reasons of increased
morbidity and mortality worldwide. The infections make the immune system of the
individuals weak resulting in a comorbid situation [1–3]. Sometimes, these infec-
tions make the individual susceptible to cancer and other highly fatal diseases. The
most common pathogens responsible for the infections include Gram-positive and
Gram-negative bacteria, fungi, protozoans and viruses [4–7]. The global statistics
indicate the loss of huge sum of monetary expenditure, time and health behind the
microbial infections cases which affects millions of lives worldwide [8–10]. The
global market value of the antibiotic treatments is accountable to billions in terms of
USD [11]. The conventional therapies available for the treatment of microbial infec-
tions target them through different routes resulting in either killing or inhibiting the
growth of the microbes [12, 13]. But there exist certain limitations of the conventional
therapies including the antibiotic resistance, side effects arising from the antibiotics,
the cost of production, etc. [14]. Thereafter, scientists have been exploiting various
technologies for antimicrobial therapies. In this context, nanotechnology is in every
field of biomedical applications that is helping mankind to lead a better life [15–17].
The nanomedicines are being used for the therapeutic and diagnostic purposes of
several diseases including cancer, diabetes, antimicrobial agents, etc. [18]. Among
the several applications, nanostructured composites are increasingly preferred as a
long-term substitute for additives and antibiotics in different yield to confer antimi-
crobial effect [19–22]. Furthermore, nanotechnology plays a significant role in the
development for the treatment of microbial infections.
In nanotechnology diverse nanocomposites are used to overcome micro-
bial resistance by exhibiting a potential antimicrobial agent. The nanoparti-
cles/nanocomposites (both organic and inorganic) act well against a broad range
of bacterial strains. Among the different types of nanocomposites, scientists are
focusing on inorganic silver metal nanocomposites for modern-day antimicrobial
applications [22–25]. Silver nanocomposites have emerged as a potential candidate
as antimicrobial agent owing to its versatile applications, efficiency and effective-
ness to augment the outcome of conventional antibacterial agents toward multidrug
resistance bacteria [19–21]. Therefore, the upcoming sections of the book chapter
dwell into the microorganisms and their ways of antimicrobial activity along with
the effect of silver nanocomposites in curbing them.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 95

1.1 Pathogenic Strains for Infection

Microorganisms though are invisible to our naked eyes, but are one of the major
reasons of woes and worries all over the world. Some microorganisms are also
within us. Some are beneficial to us while a majority of them lead to morbidity and
mortality [1–3]. The infections are generally caused by viruses, fungi, bacteria and
parasites. The pathogens can be further classified as intracellular and extracellular
depending on the immunopathological point of view [26]. Generally, Gram-negative
bacteria are found to be pathogenic, harmful and hence some of the examples are
described herewith. Among the Gram-negative bacteria of family enterobacteriaceae
Escherichia coli and Klebsiella pneumoniae are responsible for causing pneumonia,
infections of wounds, bloodstream and urinary tract [6]. Neisseria gonorrhoeae is
another pathogen which causes sexually transmitted disease called gonorrhea. The
symptoms of the disease include abdominal pain, pelvic inflammatory disease as
well as infertility [27].
Again multidrug resistant (MDR) Acinetobacter baumannii causes infection
through wound and bloodstream [28]. Another MDR bacterium includes Pseu-
domonas aeruginosa that induces skin rashes and skin infections. Shigella bacteria
are found in the stool of the infected human beings and cause abdominal pain, diar-
rhea and fever. Infection can occur by consuming raw uncooked poultry or unpasteur-
ized milk [29]. Another most common pathogen is the tuberculosis-causing bacteria
Mycobacterium tuberculosis that mostly affects the lungs [30]. Other disease-causing
pathogens include Candida which is a yeast belongs to fungus family. Candida leads
to infections of mouth, throat oesophagus along with vagina [7]. Even there are reports
of different other pathogenic strains of microbes responsible for microbial infections
as published in some previously reported literatures. The readers can consult with
the published reports to know more about microbial infections in detail [2, 4, 5].

1.2 Global Statistics and Market Value of Bacterial Infections

To evade microorganisms for leading a healthy life, a large amount of economy, labor
and time is being spent for decades. In view of a report, infection and contamination
caused by microorganisms have lead to mortality of nearly 3 million people in devel-
oping countries [22]. If one goes by the statistical analysis, antibiotic resistance is
the reason for 25,000 deaths per year in European Union [8–10]. India is helmed to
carry the largest trouble regarding drug resistant pathogens worldwide including the
high cases of MDR tuberculosis [31, 32]. In India, with passing of bacterial resistant
infection from mothers has lead to death of 58,000 babies [8–10]. It is estimated that
nearly two million deaths are estimated to happen in India due to antimicrobial resis-
tance by 2050 [32, 33]. The antibiotic resistance global market value was estimated
at USD 7.81 billion in 2017. It is being anticipated to attain a CAGR of 5.6% from
2018 to 2025.
96 S. Haque et al.

The increased resistance to antibiotic along with the rise of the multidrug resis-
tance pathogens has led to the augmenting of the market. Various companies such
as Melinta Therapeutics, Nabriva Therapeutics and Achaogen are in the process of
developing new therapeutics for antibiotic resistance. The Gram-positive antibiotic-
resistant bacteria is responsible for a vast amount of infections but the availability
of large amount of treatment therapies has decreased the overall cost of treatment
in comparison with Gram-negative bacteria [12]. The drug market includes lipogly-
copeptides, oxazolidinones, tetracyclines, combination therapies and others. Among
them it is estimated that lipoglycopeptides will occupy 17% of the overall revenue
by 2025. On the other hand, the combination therapies comprising of fixed doses of
combination of beta-lactamase and beta-lactams inhibitors are anticipated to have
maximum growth within forecast period [34]. The revenue for the antibiotic resis-
tance market has been received maximum from North America in 2017. The U.S.
will continue to be a major contributor for the revenue during the forecast period.
After U.S., Europe is the second largest contributor of revenue in this market by
2017, the reason being the increased number of antibiotic resistance infections [11].

1.3 Conventional Treatment Strategies for Bacterial

Infections

The conventional treatment strategies available for the therapeutic usage of microbial
infections are antibiotics that work against microbial infections. They work either
through killing the bacteria or inhibiting the growth of bacteria [35]. Antibiotics
are either produced naturally by a certain organism in response to another organism
(such as penicillin) or synthetically (such as antiseptics and sulphonamides). There
are varieties of antibiotics targeting the microbe through various pathways. Even the
antibiotics are classified on the type of bacteria they are attacking such as targeting
a specific group including the Gram-negative and Gram-positive or targeting a wide
range of bacteria classified as broad-spectrum group [36]. Some of them target the
bacterial cell wall namely cephalosporins and penicillin, other hinders the bacte-
rial enzymes from working properly such as quinolones, sulphonamides, rifamycins
[12, 13]. Also some antibiotics help in the inhibition of the protein synthesis such
as lincosamides, macrolides and tetracyclines. These protein synthesis inhibitors are
usually bacteriostatic in nature [37]. Along with these, there are certain antibiotics
that are semisynthetic in origin such as beta-lactam antibiotics which are a combina-
tion of penicillin, carbapenems and cephalosporins. Although the antibiotics hold a
major portion of the antimicrobial treatment, there lie major disadvantages in using
them. From grave side effects to costly treatments antibiotics are helmed respon-
sible. But the main disadvantage faced by the antibiotics is the growing resistance
by the bacterial strains [14]. Thereafter to combat the limitations of the conventional
treatment new alternatives are being checked out.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 97

1.4 Limitations to Current Treatment Strategies

The bacteria is attaining resistance to antibiotics which pose as a major threat and
initiate the emergence of multidrug resistance (MDR). These bacteria and super-
bacteria are resistance to all antibiotics owing to the unobstructed uses of antibi-
otics leading to genetic and morphological changes in bacteria overtime [38]. The
main causes of resistance owe to the expression of efflux pumps, expression of
some enzymes such as aminoglycosides and modification of cell components. There
are even reports of a gene regarded as supergene NMD-1 (New Delhi metallo-β-
lactamase) in superbacteria responsible for broad-spectrum antibiotic resistance [39].
Some bacterial species are reported to show microbial aggregation on solid surface
by interaction with other bacteria and also secretes extracellular polymeric substance
(EPS) on its outside. The bacterial aggregates on the EPS are regarded as biofilm. The
bacteria within the mature biofilm produces the superantigens that attack the immune
system and pose a serious threat to healthcare and biomedical areas [40–42]. Also
depending upon the market, majority of the pharmaceutical companies are less active
in the development of antibiotics. The reason for this is that the antibiotic resistance
market offers slow growth in marketed drugs revenues which in turn account for
low return on investments [43]. Hence a new technology is urgently required to
overcome all these limitations. To this end nanotechnology may play significant
role in combating bacterial infections and overcome the existing limitations of the
conventional therapies.

1.5 Nanotechnology and Its Applications

With the growing backdrops of the conventional therapies toward the treatment of
microbial infections, scientists are exploring nanotechnology as an alternative means
to combat limitations. Nanoparticles/nanocomposites have the capability to enter into
the bacterial cell causing interference in the molecular mechanistic pathways thus
exhibiting antimicrobial actions [17, 22–25]. They have the tendency to overcome
the commonly faced limitations from antibiotic resistance mainly inactivation of
enzymes, changes in the target site followed by more efflux of the efflux pumps [22].
The characteristic features of the nanocomposites also play a major role in imparting
the antimicrobial activity such as increased surface to volume ratio.
In nanotechnology different types of nanoparticles are exploited for treating bacte-
rial infections. Among various nanoparticles, metal-based nanocomposites involving
gold, silver, zinc copper, magnesium, nickel, selenium, aluminium, palladium and
iron are reported to exhibit antibacterial activities as per published literatures [23, 25,
44–46]. For example, gold platinum bimetallic nanocomposites exhibit antibacterial
actions against E. coli which is a multidrug resistant bacteria. The nanoparticles cause
bacterial membrane potential dissipation along with increase in adenosine triphos-
phate (ATP) levels [23]. In another report, iron oxide nanoparticles have been found
98 S. Haque et al.

to cause damage to the bacterial cell by increasing oxidative stress [22]. Even there
are reports of copper nanoparticles exhibiting bactericidal and fungicidal activity
through either penetrating the bacterial cell wall and damaging the nucleic acid or
directly binding with helical structure of DNA and destroying it [46, 47]. Table 1
depicts the list of various metal nanocomposites and their role as antibacterial agents.
TiO2 nanoparticles also exhibit antimicrobial activity through ROS generation
mainly through OH free radicals formation as shown in Table 1 [22]. Among all these
metals, silver nanoparticles as well as nanocomposites have drawn much attention
because of several advantages such as easy synthesis procedures, unique properties,
characterization and biological property such as antimicrobial activity [16, 19–21].
Silver nanocomposites have been found to be highly toxic toward microbes hence
provides efficient antimicrobial activity. The upcoming sections focus on various
aspects of silver toward treating microbial infections.

2 Historical Background of Silver

If one traces back to the old time, before antibiotics, silver is utilized in various
applications. Silver has been used for making vessels, liquid container for water
storage, silver coins, sutures, shaving and lastly medicinal requirements that was
first documented in 750 AD [48]. Silver was being used for treating certain diseases
and ailments even before its discoveries having antibacterial properties. It has been
recorded that several civilizations of Phoenicians, primordial Romans, Egyptians
and Greeks used silver for food preservation and in water [49]. It was reported that
Macedonians and Hippocrates were the first to use silver as wound healing agent.
Gabor in 702–705 AD used silver as silver nitrate. There are reports of Avicenna in
980 AD and Angelo Sala in 1614 utilized silver for the purpose of blood purification,
wound healing and other infections [50, 51]. Silver was earlier used in the forms of
silver zeolite and silver sulfadiazine in clinical applications such as wound healing
[52, 53].
Later the silver was converted to silver nanoparticles and silver nanocomposites
with the advent of nanotechnology for several biomedical applications. With the
formation of the nanoform of silver, it exhibits different properties like increased
surface area to volume ratio leading to elevated bioavailability allowing efficient
antimicrobial activity with respect to bulk material [54]. The nanosilver at same
molar concentration shows less toxicity in comparison to its ionic form. The antimi-
crobial action of the silver nanocomposites largely depends on their physicochemical
features including shape, size, charge and stability. The features in turn are formed,
based upon the process of synthesis conditions such as temperature, nature of solvent,
concentration and strength of reducing agent [55, 56].
If one goes by the commercialization of the nanoparticles, then silver in the struc-
ture of nanoforms occupies 50% of the worldwide nanomaterial end user products.
The annual manufacture market estimation of silver nanoforms accounts for 320 tons
of manufacture that is being utilized for healthcare along with food products [57].
Potential Application of Silver Nanocomposites for Antimicrobial Activity 99

Table 1 Role of different metal nanocomposites as antibacterial agents

Nanoparticle Conjugation Targeted microbes Mode of action References
Gold Cefotaxime Klebsiella Damage to the cell [123]
pneumoniae, wall of bacteria
Escherichia coli followed by DNA
disruption
Carbapenems Klebsiella Changing the [124]
(meropenem and pneumoniae, osmotic balance
imipenem) Acinteobacter leading to the
baumannii and damage of the cell
Proteus mirabilis integrity as well as
cell wall
Aluminium Oxide – Staphyloccocus Generation of ROS [125]
(Al2 O3 ) aureus that damages the
bacterial cell
membrane
– Escherichia coli Deposition and [126]
penetration within
the cell wall of
bacteria
Titanium Oxide – Escherichia coli Peroxidation and [127]
accumulation of the
fatty acids of the
bacterial cell
membrane
– Escherichia coli Production of ROS [128]
that disrupts the
bacterial cell
membrane
Iron Oxide – Escherichia coli Damaging the cell [129]
membrane of
bacteria with the
help of magnetic
core shell
nanoparticles
coupled with
radiofrequency
– Staphyloccocus Pentration through [130]
aureus, the bacterial
Escherichia coli membrane and
and Pseudomonas interference with
aeruginosa the electron transfer
Zinc Oxide PVA Escherichia coli Generation of ROS [45]
and damage to
bacterial cell
membrane
(continued)
100 S. Haque et al.

Table 1 (continued)
Nanoparticle Conjugation Targeted microbes Mode of action References
– Bacillus subtilis Inhibition of [131]
bacterial cell
growth and biofilm
formation
Copper – Pseudomonas Penetration through [46]
aeruginosa the cell wall
followed by
destruction of
nucleic acid
– Staphyloccocus Release of Cu ions [132]
aureus that binds with the
helical structure of
DNA and disrupts
them
Silver – Escherichia coli, Bacterial cell [24, 25]
Bacillus subtilis, membrane damage
Klebsiella
aerogenes,
Pseudomonas
aeruginosa

It has been even assumed that the global market will exceed 3 billion by the year
2020. The demand for the silver nanocomposites is rising in a variety of commercial
markets of textile, paint industry, food packaging and water treatment all owing to
its antibacterial and antifungal properties [13, 58, 59].

3 Synthesis and Characterization of Silver Nanocomposites

The designing, synthesis and assembly of silver nanocomposites occurs through

different processes but is majorly classified into two categories: bottom-up and top-
down. In this the bottom-up approach involves silver nanocomposites production
using photochemical, chemical and biological reactions. The top-down approach
on the other hand employs breakdown of solid materials into nanocomposites [60].
Among the different synthesis techniques employed for silver nanocomposites, there
are mainly three methods that are: chemical, physical and biological synthesis [25,
61–63]. The synthesis procedures play major role behind imparting antibacterial
property to the silver nanocomposites [64]. Although the main objective of this book
chapter is to demonstrate the potential antimicrobial activity of the silver nanocom-
posites hence it is beyond the scope of the chapter in indulging about the synthesis
and characterization. Thereafter, we very briefly describe about the synthesis and
characterization of silver nanocomposites in this section.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 101

The fabrication of silver nanocomposites by chemical methods involves the reduc-

tion process, thermal reduction, microwave assisted synthesis and polyol process.
Among the other types include electrochemical synthetic method, microemulsion,
photochemical reduction methods and irradiation method [65–68]. Various scientists
from all over the world are involved in the process of synthesis of silver nanocompos-
ites by different chemical methods. To this context, our group synthesized various
silver nanocomposites including silver nitroprusside (AgNNPs) and silver Prus-
sian blue analogue nanocomplex using chemical methods (SPBANPs) [24, 25]. The
formed silver nanocomposites were characterized with the help of several character-
ization techniques such as DLS, XRD, TEM and FESEM. The overall characteristics
exhibit the crystalline nature of the nanocomposites [24, 25]. The significance of the
chemical methods is that it needs decreased reaction time, increased yield of product
with lesser energy consumption.
Another method of productions of silver nanocomposites are physical methods
[61]. The various physical methods include evaporation and condensation, laser abla-
tion, ac power, thermal, arc discharge, etc. [62, 69, 70]. These methods are used
because they prevent any sort of contamination occurring from the solvent. The
physical methods help in formation of size of silver nanocomposites in the narrow
range [64].
Apart from the chemical and physical synthesis methods, biosynthesis is another
procedure for silver nanoparticles/nanocomposites formation. The biosynthesis
process is an economic, simple and non-toxic method. The extracts of plant, micro-
bial (such as bacteria, fungi, algae) agents are attributed to the biosynthesis process
[71–73]. The chemical and physical methods have certain disadvantages such as
presence of toxic precursors or use of large amount of energy, which can be easily
overcome by biosynthesis measures. There are several reports on biosynthesis of
silver nanocomposites using plant and microbial extracts as bioresource that acts as
reducing agent [19]. Many scientists all over the world have biosynthesized silver
nanocomposites. The phytochemicals present within the leaf extract helped in the
reduction of the silver into its nanoforms. Our group synthesized silver nanocom-
posites with the help of Olax scandens plant extracts by the reduction of silver
nitrate [74]. Similarly, Tripathi et al. [63] biosynthesized poly (vinyl alcohol)-silver
nanocomposites by using Ficus benghalensis leaf extract [63].

4 Role of Shape, Size and Charge of Silver Nanocomposites

on Antimicrobial Activity

The silver in its nanoform exhibits unique features. There exists a strong connection
between the physical characteristics and the morphological properties (shape, size,
charge and surface chemistry) of the silver nanocomposites [18]. The morphological
features of the silver nanocomposites are responsible for most of the biological
activities of the silver nanocomposites including cellular activation, cellular uptake,
102 S. Haque et al.

intracellular distribution as well as antibacterial activity [21]. The morphological

features in turn are dependent upon certain criteria including synthesis procedures,
reducing and stabilizing agents as described in the above Sect. 3. The production of
nanoforms takes place through three defining steps namely, nucleation, coalescence
and growth [75]. The following section briefly describes the different morphological
features involved in imparting antimicrobial features to the silver nanocomposites.
The shape of the nanoparticles and nanocomposites (for example nanocubes,
triangular, spherical, pyramidal, nanorods, nanoprism, nanobars, flower shaped,
nanowires, octahedral as well as tetrahedral) plays a major role in determining
their antibacterial property [76, 77]. In this context Logaranjan et al. [78] designed
silver nanocomposites that were octahedral in shape [78]. The nanocomplex was
synthesized using the extracts of aloe vera plant that acted as reducing agent in
both conditions of microwave irradiation and room temperature. The range of the
particles was from 5 to 50 nm and exhibited excellent antimicrobial effect against
various strains of bacteria. The results revealed that the octahedral-shaped parti-
cles exhibited more antibacterial activity in comparison with other nanocomposites
[78]. In another report Chakraborty et al. [79] synthesized silver nanocomposites
with different shapes by employing various proteins [79]. The protein used during
synthesis acts as capping agent. The different types of proteins used are ovalbumin,
BSA, catalase, hemoglobin, avidin, apo-transferrin, lysozyme and β-lactoglobulin.
The silver was reduced to silver cluster with the help of citric acid followed by
mixing with various proteins. Also, the silver nanocomposites were synthesized
with individual amino acids in order to check the contribution of each amino acid
in determining the shape of the silver nanocomposites. The results depicted that the
cystine and lysine formed spherical-shaped nanocomposites and the other amino
acids glycine, alanine, valine, threonine, isoleucine and serine produced anisotropic
nanocomposites. The results also exhibited that only lysine helped in formation of
stable particles [79].
The size of the silver nanocomposites plays a major role in imparting antibacte-
rial property. The smaller the size of the nanocomposites the greater is the surface to
volume ratio resulting in increased interaction with the microorganisms exhibiting
more antibacterial activity. To exhibit the size-dependent antimicrobial activity of
silver nanoparticles Dogru et al. [80] synthesized silver nanocomposites (sizes
ranging from 70 ± 5, 52 ± 5, and 37 ± 5 nm) with the extracts of Matricaria
chamomilla [80]. The silver nanocomposites exhibited efficient antibacterial activity
against E. coli and S. aureus. All the three size range particles exhibited antibacterial
effect with 37 ± 5 nm particle having the maximum one. This corroborates with the
fact that the small size of the nanocomposites has greater surface volume ratio and
hence interacts more efficiently with the bacteria [80].
Similar to shape and size of the nanoparticles, charge helps in inferring certain
characteristic features to the nanocomposites. The charge of the silver nanocom-
posites is usually dependent upon the process of synthesis. The capping and the
stabilizing agents used during synthesis affects the surface charge of the nanocom-
posites which in turn confers stability and bioactivity to the nanoparticles. In this
context Salvioni et al. [81] synthesized silver nanocomposites using tannic and citric
Potential Application of Silver Nanocomposites for Antimicrobial Activity 103

acid as reducing agents [81]. The formed particle was characterized by several char-
acterization techniques which revealed 20 nm size along with negative charge of
the formed nanocomposites. The silver nanocomposites exhibited good antibacte-
rial activity against Gram-negative and Gram-positive bacteria in comparison with
commercially available silver nanocomposites [81].

5 Silver Nanocomposites and Their Antimicrobial Activity

The silver nanocomposites are produced by the combination of various sources and
precursors. They either act as matrix bed or reducing agents for silver nanocompos-
ites formation and some compounds enhance the antimicrobial property of silver
nanoforms through synergistic effect [20, 82].

5.1 Graphene Silver Nanocomposites

There are many reports of graphene being used as an antibacterial agent. There-
after this property of graphene was used in synthesizing graphene associated silver
nanocomposites to impart a synergistic antibacterial effect. In one such report Beiran-
vand et al. [20] produced graphene oxide/hydroxyapatite/silver (rGO/HAP/Ag)
nanocomposite with the help of graphene oxide nanosheets, (NH4 )2 HPO4 , Ca(NO3 )2
and AgNO3 using a hydrothermal method [20]. The nanocomposites were character-
ized by various analytical tools. The results revealed the formation of graphene-based
silver nanocomposite. The nanocomposite was checked for its biological activity
which clearly exhibited antibacterial activity through disk diffusion method against
Gram-positive and Gram-negative bacteria. The results were affirmative toward
Gram-positive bacteria [20]. In another report Song et al. [83] synthesized graphene
oxide-based silver nanocomposites [83]. The nanocomposites with size of 5–15 nm
were characterized by several characterization techniques. The graphene oxide-silver
nanocomposites were evaluated for bactericidal activity in water through different
assays such as SEM analysis, bactericidal dosage, effect of pH and lipid peroxida-
tion assay using strains of Staphylococcus aureus and Escherichia coli. The bacte-
rial dosage study depicted that the nanocomposites exhibited dose-dependent and
time-dependent antibacterial activity against both the strains. Time of 20–30 min is
required for 99.99% killing activity of both the strains. Also the silver could permeate
easily in Gram-negative E. coli in comparison to Gram-positive S. aureus due to
the presence of thicker cell wall of the latter. The nanocomposites worked better in
showing antibacterial activity in acidic pH as compared to basic one. The SEM results
showed the damage of the cell wall membrane of both the bacterial strains with E.
coli being more. The antioxidant enzyme measurement study revealed the increased
oxidative stress followed by cell membrane damage is the mechanism behind the
antibacterial activity of the graphene silver nanocomposites. Figure 1 shows the
mitochondria through its electron transport chain generates ROS causing damage to
104 S. Haque et al.

Fig. 1 Schematic mechanisms for antibacterial behaviors of GO-Ag. Blue dots represent silver
nanoparticles loaded on GO sheets, and red dots are silver ions. The roman numerals (I, II, III and
IV) refer to different stages of the bactericidal process [92]. Reprinted with permission from Song
et al. [83]. Copyright © 2016 Elsevier

lipids, proteins and DNA. This in turn leads to damage of cellular membrane with the
release of cell contents. The oxidation of the biomolecules paves way for the destruc-
tion of the bacterial cell [83]. Similarly Jaworski et al. [84] coated graphene oxide
with silver nanoparticles leading to the formation of silver nanocomposites [84]. The
nanocomposites were checked for the biological activity through presto blue assay,
ROS analysis and lactate dehydrogenase assay against four bacterial strains as well
as pathogenic yeast. The results exhibited excellent antimicrobial activity against
both bacterial and fungal strains [84].
Potential Application of Silver Nanocomposites for Antimicrobial Activity 105

5.2 Chitosan-Based Silver Nanocomposites

Chitosan is another such component used for the production of silver nanocompos-
ites acting as a reducing agent for the reduction of silver metal into its nanoform
[82]. Vimala et al. [85] designed curcumin encapsulated chitosan-polyvinyl alcohol
(PVA)-silver nanocomposites through their reduction in acidic medium [85]. The
formation of the silver ions into its respective nanocomposites in the acidic solu-
tion was due to the reducing ability of polyvinyl alcohol (PVA) and chitosan (C)
which contained functional groups such as –OH, –NH2 and –COOH. The formed
nanocomposites were characterized with the help of several characterization tech-
niques. The visualization of the SEM images indicated the presence of silver
nanocomposites embedded within the chitosan and polyvinyl alcohol (PVA) matrix.
The size of the nanocomposites was around 16.5 nm as indicated by TEM. The
effect of the nanocomposites on the bacterial strains (Escherichia coli, Staphylo-
coccus, Pseudomonas, Micrococcus, Pseudomonas aeruginosa) as well on the fungal
strain (Candida albicans) was checked with the help of various assays. The results
clearly depicted the antibacterial and antifungal activity of the chitosan–curcumin-
based silver nancomposites being more than the individual components [85]. In
another work, Kaur et al. [86] synthesized silver nanocomposites with the help of
chitosan [86]. The nanocomposites were established to have characteristic features
through the characterization techniques such as TEM, FTIR, DSC and XRD. The
formed nanocomposites showed excellent antibacterial activity against Staphylo-
coccus aureus MTCC 1809, Salmonella entrica MTCC 1253 and Pseudomonas
aeruginosa MTCC 424 through disk diffusion methods [86].

5.3 Nitroprusside-Based Silver Nanocomposites

Sodium nitroprusside is the class of drug used for the treatment of high blood pres-
sure. Being FDA approved the sodium nitroprusside is used as precursor molecules
for the synthesis of several nanocomposites. Our group synthesized silver nanocom-
posites with the help of sodium nitroprusside. The nanocomposite produced is silver
nitroprusside nanocomplex using silver nitrate and sodium nitroprusside [24]. The
nitroprusside helps in providing extra stability to the silver and results in its slow
release from the complex. The formed nanocomposites were characterized by the
help of DLS, XRD, TGA, FTIR, HRTEM, Raman, ICP-OES and FESEM. The char-
acterization results revealed the optimized features of the nanocomposites such as
20–80 nm of size in HRTEM and XRD showing the characteristic peaks of silver
as crystalline nature. The nanocomposites were initially evaluated for its biocom-
patibility in normal cell lines (EA.hy 926 cells, NIH 3T3) and also through chick
embryonic assay. Both the results confirmed the biocompatible nature of the formed
silver nanocomposites. They were now checked for the antibacterial activity with
the help of different methods including zone of inhibition assay, colony formation
106 S. Haque et al.

assay, bacterial cell damage assay and protein expression assay on Escherichia coli,
Staphylococcus epidermidis, Bacillus subtilis, Klebsiella aerogenes, Pseudomonas
aeruginosa bacteria. The results showed excellent antibacterial activity through all
the assays. Figure 2 illustrated that on treating the bacteria at log phase with the silver
nanocomposites at a concentration of 0.5–2 μg/mL for a period of 24 h showed excel-
lent antibacterial activity in comparison to untreated and positive control (commer-
cially available streptomycin). The bacterial colony was inhibited by the treatment
of silver nanocomposites as compared to other groups. Figure 3 shows that when
the bacterial cells are damaged they have a tendency to join with one another. The
confocal images clearly exhibit that the cell membrane adheres to one another. The
SEM images depict the damage of the silver nanocomposites treated bacterial cells.
The silver nanocomposites so formed were further reported to have shown accelerated
wound healing activity in mice due to macrophage conversion [24].

Fig. 2 a–d Calculation of MIC of AgNNPs (0.5–2 μg/mL) on both Gram-positive and Gram-
negative bacteria. The bacteria at various time points by measuring OD at 600 nm. Here streptomycin
(2 μg/mL) used as positive control [54]. Reprinted with permission from Rao et al. [24]. Copyright
© 2018 American Chemical Society
Potential Application of Silver Nanocomposites for Antimicrobial Activity 107

Fig. 3 Confocal microscopic study of bacterial membrane damage. E. coli and B. subtilis treated
with AgNNPs (1 μg/mL), streptomycin (1 μg/mL) for 6 h. AgNNPs treated E. coli and B. subtilis
displayed enhanced membrane damage causing enhanced green fluorescence (FITC:100 μg/mL).
Nuclear staining was performed with DAPI (100 μg/mL). First row: untreated control, second row:
streptomycin (1 μg/mL) treated bacteria, third row: AgNNPs (1 μg/mL) treated bacteria. Cells were
observed under 100X objective, scale bar: 5 μm. The arrows in the corresponding figures represent
membrane damage of bacteria [54]. Reprinted with permission from Rao et al. [24]. Copyright ©
2018 American Chemical Society

5.4 Prussian Blue-Based Silver Nanocomposites

Prussian blue (ferric hexacyanoferrate) is generally used as an oral antidote, also

known as Radiogardase for the treatment of poisoning due to radioactive or non-
radioactive thallium and radioactive cesium [87]. Being FDA approved it has been
utilized for the synthesis of various compounds. Several reports have shown that the
Prussian blue and its analogues have been used for the anticancer activity [88]. The
Prussian blue prevents the leakage as well as helps in better circulation of silver and
forms a stable complex. Therefore, our group used Prussian blue for the synthesis of
silver Prussian blue nanocomposites that exhibited excellent antibacterial activity.
The silver nanocomposites were designed with silver nitrate and Prussian blue in
the presence of PVP [poly(N-vinyl-2-pyrrolidone)] [25]. The process of synthesis
is simple, economical and environment friendly. The formed nanocomposites were
characterized by different techniques such as UV–visible spectroscopy, TEM,
DLS and XRD. The results revealed 2–40 nm size of the nanocomposites through
TEM followed by the XRD peaks matching with the earlier published silver
nanoparticles. The nanocomposites revealed antibacterial property through various
assays such as growth inhibition, zone of inhibition and colony forming assay on
both Gram-positive and Gram-negative strains of bacteria. Figure 4a, b depicts the
more inhibition of growth of the bacterial strains upon treatment with the 30 μM
108 S. Haque et al.

Fig. 4 a–e: a, b Growth inhibition of E. coli and B. subtilis in presence of SPBANPs. c Zone of
inhibition of various bacteria treated with SPB(1,1)ANPs-40 and standard antibiotics (1 mg/mL)
(P: penicillin; K: kanamycin; S: streptomycin and Z: gentamicin: 0.5 mg/mL. B: blank) in agar
plate. d, e Optical images of bacterial colony formed by E. coli and B. subtilis after 24 h treatment
with SPBANPs [42]. Reprinted with permission from Mukherjee et al. [25]. Copyright © 2020
American Chemical Society

concentration of nanocomposites with respect to the control group. Figure 4c

shows the results of the zone of inhibition assay where the nanocomposites helped
in maximum zone inhibition in comparison with the untreated and four positive
controls (treated with kanamycin, gentamycin, streptomycin, penicillin). The colony
formation is visible in Fig. 4d, e where the nanocomposites treated bacterial colony
of both the strains exhibited maximum inhibition in comparison with untreated and
positive control. Figure 5 shows the TEM images of the bacterial cell which upon
treatment with the silver nanocomposites shows extensive membrane damage as
compared to the untreated ones. Thus the mechanism underlying the antibacterial
activity of the silver nanocomposites includes interaction of the bacterial cell with
the silver nanocomposites leading to damage of their membrane and leaking of
the cellular contents. Also the silver nanocomposites increased the ROS generation
resulting in the suppression of the antioxidant enzymes. Oxidative stress generated
by the silver Prussian blue analogue complex causes lipid peroxidation leading to
plasma membrane damage [25].
Potential Application of Silver Nanocomposites for Antimicrobial Activity 109

Fig. 5 a–d: TEM images of a, b untreated E. coli and c, d E. coli treated with SPBANPs [42].
Reprinted with permission from Mukherjee et al. [25]. Copyright © 2020 American Chemical
Society

5.5 Cellulose-Based Silver Nanocomposites

It has been well established that cellulose polymers are used for the synthesis of
silver nanocomposites [61]. The cellulose usually acts in the form of matrix for the
formation of silver nanocomposites [89]. To this regards, Li et al. [90] synthesized
silver nanocomposites with the help of AgNO3 and cellulose fibers by heating at a
temperature of 80 °C for 24 h [90]. The formed nanocomposites were checked for its
characteristics features through several characterization tools. Later the antibacterial
studies were performed on Staphylococcus aureus strain. Figure 6 clearly depicted
the enhanced antibacterial property of the silver nanocomposites as compared to the
untreated ones. The antibacterial activity augmented with the increase in the concen-
tration of the silver nanocomposites and size of the fibers. The mechanism behind the
antibacterial activity of the silver nanocomposites is damage to the bacterial cell wall
[90]. In another work Pinto et al. [91] synthesized silver nanocomposites by using
different cellulosic substrates (bacterial and vegetables) [91]. The silver nanocompos-
ites exhibited excellent antibacterial activity against three different strains of bacteria.
110 S. Haque et al.

Fig. 6 Antibacterial test of cellulose/silver composite fibers. a, e-B1, b, f-B3, c, g-B4, d, h-B5.
a–d: Staphyoccocus aureus; e–h: Echerichia coli. The scale bar is 1 cm [100]. Reprinted with
permission from Li et al. [90]. Copyright © 2014 Elsevier

The nanocomposites were effective in showing the antibacterial activity at a concen-

tration even low as 5.0 × 10−4 wt.%. Thus this cellulosic silver nanocomposites
exhibits an effective potential antibacterial agent [91].

5.6 Synthetic Polymer-Based Silver Nanocomposites

The synthetic polymers are used for wide range of biomedical applications including
synthesis of nanocomposites. Many reports suggest silver nanocomposites are
produced with the help of synthetic polymers that either acted as reducing agents or
matrix bed [89]. To this Palomba et al. [92] used synthetic polymer polystyrene to
produce silver polystyrene nanocomposites [92]. The synthesis took place by mixing
silver 1,5-cyclooctadiene-hexafluoroacetylacetonate and amorphous polystyrene
resulting in a solid solution. During the reaction the silver atoms are decomposed
from its initial reactants followed by accumulation in the polymer. The polymer-based
silver nanocomposites were checked for its antibacterial activity through several
assays such as BacLight fluorescence-based test, turbidity assay on Gram-negative
Escherichia coli bacteria. The nanocomposites were used in two concentrations (10
and 30% wt) in correspondence with the pure form of silver. Figure 7 depicts the
results of BacLight fluorescence-based test where in Fig. 7a shows the green fluores-
cence of the entire bacterial culture indicating live cell population after 16 h of incu-
bation in liquid medium for the growth of bacteria. Figure 7c, d show the increased
red fluorescence indicating dead bacteria upon treatment with silver nanocomposites
of 30% wt with mild and strong annealing temperature, respectively. The red fluo-
rescence is due to labeling of propidium iodide with the membrane compromised
Potential Application of Silver Nanocomposites for Antimicrobial Activity 111

Fig. 7 Live-dead cell staining assay: microscopic analysis and quantitative evaluation. a High
number of green-labeled living bacteria after 16 h incubation in LB, without Ag/PS. c, d: high
number of red-labeled dead bacteria after 16 h incubation +30 wt.% Ag/PS film (1 cm × 1 cm—
0.0177 mg) obtained by using a mild thermal annealing treatment (c); similar results were observed
in bacteria culture after 16 h of incubation with Ag powder (d). Scale bar represents 20 μm.
b Quantitative analysis of the E. coli viability (%) after 16 h incubation at each condition reported
in Table 1 (a–e) [101]. Reprinted with permission from Palomba et al. [92]. Copyright © 2012
Palomba et al.

dead bacteria. The results were then quantitatively analyzed as shown in Fig. 7b. The
quantitative results revealed decrease in the percentage viability of the Escherichia
coli is more with the treated samples. The results revealed that the 30% wt of the
nanocomposites exhibited the maximum antibacterial activity in comparison with the
pure silver [92]. Another polymer-based silver nanocomposite was synthesized by
Jabbar et al. [93] where he used silver nanoparticles biosynthesized from Parthenium
leaf extract doped over polystyrene dissolved in Tetrahydrofuran [93]. The formed
nanocomposites hold potential as an antibacterial agent since silver itself imparts
antibacterial properties [93].
112 S. Haque et al.

5.7 Biosynthesized Silver Nanocomposites

Apart from chemical means, silver nanocomposites are also synthesized with the
help of plants and microorganisms that impart antimicrobial properties. Our group
biosynthesized silver nanocomposites with the help of Olax scandens plant extracts
[74]. The reduction of the silver ions into its nanoform is achieved with the help of
the plants extracts. The formed silver nanocomposites were characterized with the
help of several characterization techniques. The results revealed the 2–40 nm size
of the silver nanocomposites through TEM images and the XRD shows the crys-
talline nature of the nanocomposites. Several antibacterial assays such as the growth
kinetics assay, colony forming assay and SEM analysis were done on Escherichia
coli bacterial samples. Figure 8 shows the results of antibacterial studies performed
with the help of the biosynthesized silver nanocomposites. Figure 8a shows the inhi-
bition kinetics in graphical manner where the inhibition increased with the dose of the
nanocomposites as compared to control and positive control ampicillin. The colony
forming assay has been depicted in Fig. 8b–e with control, ampicillin and biosynthe-
sized silver nanocomposites at concentrations of 18 μM and 30 μM, respectively. The
30 μM concentration of the nanocomposites exhibited highest antibacterial activity
as compared to the other groups. The SEM images of the bacterial cell are depicted
in Fig. 8f–h as control, Olax and silver nanocomposites, respectively. Even the SEM
images clearly depict damage of the biosynthesized silver nanocomposites treated
Escherichia coli cell wall in comparison with the other groups. Figure 9 illustrates
the overall working antibacterial mechanism of silver nanocomposites. The silver
ions released from the nanocomposites result in oxidation of the glutathione which
in turn leads to generation of ROS causing toxicity and inhibiting bacterial growth.
The glutathione oxidation by silver nanocomposites causes lipid peroxidation that
results in membrane damage followed by leakage of cellular components. The silver
nanocomposites also disrupt the DNA (both plasmid and chromosomal) by the inter-
action with the phosphorus groups. Overall the biosynthesized silver nanocomposites
act as an excellent antibacterial agent [74].
In another report, Tripathi et al. [63] designed and biosynthesized poly (vinyl
alcohol)-silver nanocomposites with the help of Ficus benghalensis leaf extract [63].
Initially the silver nanoparticles were made from the plant extracts then the nanocom-
posites were formed by incorporating nanoparticles into poly (vinyl alcohol) (PVA).
The formed nanocomposites were characterized with the help of AFM to exhibit
the incorporation of nanoparticles in the film. The silver nanocomposites exhibited
good antimicrobial activity against Salmonella typhimurium [63]. Scientists are now
extensively working upon hydrogel-based silver nanocomposites for several appli-
cations. In this context Novientri et al. [94] synthesized sodium alginate-polyvinyl
alcohol-g-acrylamide (NaAIg-PVA-g-AAm) nanocomposite with the help of brown
algae [94]. The silver ions get trapped in the hydrogel matrix. The various characteri-
zation studies revealed the characteristic features of the nanocomposites. The results
revealed the size of the silver nanocomposites were of 20 nm that helped them to
easily embed in the matrix of the hydrogel. The silver nanocomposites exhibited
Potential Application of Silver Nanocomposites for Antimicrobial Activity 113

Fig. 8 Study of antibacterial activities: a liquid growth inhibition kinetics of E. coli using different
concentrations of b-AgNPs. b-AgNP-30 (at 30 μM) shows almost 100% growth inhibition. Ampi-
cillin has been used as a positive control (PC) & NC: negative control or untreated E. coli. The
numerical number indicates the concentration of b-AgNPs in μM, b–e optical images of bacterial
colonies formed by E. coli cells, i.e. colony counting assay (after 24 h): b: Control, c: Ampicillin
(100 μg/ml), d: b-AgNPs (18 μM), e: b-AgNPs (30 μM) and f–h SEM images of E. coli cells
f without being treated (control), g treated with Olax for 1 h, h treated with b-AgNPs (30 μM) for
1 h. The SEM images show the silver nanoparticles damages the bacterial cell membrane (marked
by blue arrow), whereas, the bacterial membranes of untreated and treated E. coli with Olax is
intact [81]. Reprinted with permission from Mukherjee et al. [74]. Copyright © 2020 Ivyspring
International Publisher

antibacterial activity against Staphylococcus aureus and Escherichia coli. The ratio
of minimum bacterial concentration (MBC) to minimum inhibitory concentration
(MIC) was four when both the bacterial strains were treated with the silver nanocom-
posite hydrogels. The nanocomposite exhibited prominent results of antibacterial
activity against both the Gram-negative and Gram-positive bacteria [94]. Table 2
shows the silver nanocomposites produced by different components as well as their
mechanism of antibacterial activity.
114 S. Haque et al.

Fig. 9 Schematic presentation of antibacterial activity of b-AgNPs toward E. coli using biosynthe-
sized silver nanoparticles [81]. Reprinted with permission from Mukherjee et al. [74]. Copyright
© 2020 Ivyspring International Publisher

6 Antimicrobial Activity of Silver Nanocomposites:

Mechanistic Approach

Silver nanocomposites exhibit excellent activities such as antiviral, antifungal,

antibacterial, antiangiogenic and anti-inflammatory because of the characteristic
properties. The exact mechanism of action is under research and hence needs further
examination for the proof of its antimicrobial activity. There are several reports that
demonstrate the probable mechanism of the antibacterial activity of silver nanocom-
posites. The reports suggest the ability of silver nanocomposites to damage the bacte-
rial membrane leads to release of cellular components and killing the bacterial cell
[95]. The effect of silver nanoparticles and nanocomposites depends upon the pepti-
doglycan layer of the Gram-positive and Gram-negative bacteria. Some reports high-
light that the positive charge of the silver nanocomposites helps in easy interaction
with the bacterial membrane which is negatively charged. The size, shape and charge
of the silver nanocomposites play major role in their internalization into the bacte-
rial cells [96]. Also these properties are responsible for the interaction of the silver
nanocomposites with the intracellular components such as nucleic acids, proteins,
lipid leading to the inactivation and subsequently death of the bacterial cells. Another
mechanism that is being employed by the silver nanocomposites for killing the bacte-
rial cells involves generation of reactive oxygen species (ROS) involving the release
of hydrogen peroxide, singlet oxygen and superoxide anion [97]. The augmented
Potential Application of Silver Nanocomposites for Antimicrobial Activity 115

Table 2 Silver nanocomposites from different substances

Precursors Nanocomposites Bacterial strain Mechanism References
of action
Graphene Graphene Staphylococcus Changing [20]
Oxide/Hydroxyapatite/silver aureus, Bacillus properties of
cereus, Escherichia bacterial
coli Klebsiella membrane
pneumoniae and cell wall
and
disturbing
basic
metabolic
processes
Graphene oxide-based silver Staphylococcus Generation [83]
nanocomposites aureus, Escherichia of ROS
coli leading to
bacterial cell
damage
Chitosan curcumin encapsulated Escherichia coli, Biocidal [85]
chitosan-poly vinyl alcohol Staphylococcus, activity
(PVA)-silver Pseudomonas,
nanocomposites Micrococcus,
Candida albicans,
Pseudomonas
aeruginosa
Chitosan-silver Staphylococcus Inhibition of [86]
nanocomposites aureus, Salmonella bacterial
entrica, growth
Pseudomonas
aeruginosa
Sodium Silver nitroprusside Escherichia coli, Damaging [24]
nitroprusside (AgNNPs) Staphylococcus cell
epidermidis, membrane of
Bacillus subtilis, the bacteria
Klebsiella
aerogenes,
Pseudomonas
aeruginosa
(continued)

ROS function results in the decrease of the enzymes such as superoxide dismutase,
catalase and glutathione peroxidase (GSH) and also causes DNA damage as well as
lipid peroxidation [98]. The toxicity toward the bacterial cell is due to the release of
silver ions. But there are also reports that silver ions have less antibacterial activity
in comparison with silver nanocomposites, which is an indicative of the fact that the
silver in its nanocomposites has different silver ion discharge and different binding
pathways [55].
116 S. Haque et al.

Table 2 (continued)
Precursors Nanocomposites Bacterial strain Mechanism References
of action
Prussian blue Silver Prussian blue Bacillus subtilis, Generation [25]
analogue nanoparticles Escherichia coli, of ROS
Pseudomonas suppresses
aeruginosa, the
Klebsiella antioxidant
pneumoniae activity and
oxidative
stress results
in lipid
peroxidation
causing cell
damage
Polymers Silver nanocomposites with Staphylococcus Bacterial cell [90]
cellulosic fibres aureus wall damage
Silver polystyrene Escherichia coli Decrease in [92]
nanocomposites viability of
bacterial cell
along with
cell wall
damage
Biosynthesized Olax scandens synthesized Escherichia coli Generation [74]
silver nanocomposites of ROS
causing
toxicity to
bacterial cell,
glutathione
oxidation
causes lipid
peroxidation
and damages
cell
membrane
Sodium alginate-polyvinyl Staphylococcus Exhibit [94]
alcohol-g-acrylamide aureus, Escherichia bacteriostatic
(NaAIg-PVA-g-AAm) coli and
hydrogel with silver bacteriocidal
nanocomposite activity

The resistance to antibiotics has emerged as one of the leading causes of increased
and stable microbial infections. In context to this, Surwade et al. [99] used a combina-
tion therapy by combining antibiotic ampicillin with silver nanocomposites [99]. The
combination therapy was used against methicillin-resistant Staphylococcus aureus
(MRSA) bacteria. The combination was effective at a lower dose of ampicillin and
sublethal dose of silver nanoparticles. When combined both acted synergistically
[99]. The second most drawbacks to microbial infection after antibiotic resistance
Potential Application of Silver Nanocomposites for Antimicrobial Activity 117

are the biofilm formation. The extracellular matrix produced by the bacteria on
solid surfaces such as medical devices, pacemakers, catheters, etc., are referred to
as biofilms [3]. This is done by the bacteria in order to prevent the action of the
antibiotics on their community. To prevent these, Siddique et al. [100] exhibited the
role of silver nanocomposites in the inhibition of biofilm formation [100]. The silver
nanocomposites were synthesized with AgNO3 , polyvinyl pyrrolidone (PVP) and
sodium borohydride (NaBH4 ) where PVP was used for the stabilization of the silver
nanocomposites. The silver nanocomposites showed antibacterial activity through
broth microdilution and agar diffusion method. When the formed nanocomposites
were used upon K. pneumoniae strains through tube method and microtiter plate
assay, it exhibited decreased biofilm formation. Thus the silver nanocomposites
exhibited both antibacterial and antibiofilm formation [100]. In another study Yu
et al. [101] exhibited the antibacterial and antibiofilm role of silver nanocompos-
ites on E.coli strain obtained from dairy cows affected with mastitis [101]. The
silver nanocomposites were formed by the combination of silver nanoparticles with
quercitin which is a plant derived drug. The nanocomposites were used to perform
experiments such as antibacterial assays, scanning electron microscopy (SEM) exper-
iments, biofilm formation assays as well as real-time reverse transcription PCR (RT-
PCR) assays. The results revealed increased antibacterial and antibiofilm activity of
the nanocomposites in comparison with its single counterparts. Figure 10 depicts
the SEM experiment which clearly shows that the biofilm of the controlled group is
normal in comparison with the treatment groups. Moreover the biofilms treated with
the silver nanocomposites showed intense damage with respect to the other groups
treated with only quercitin and silver nanocomposites. The molecular studies done
with the RT-PCR even showed inhibition of transcription biofilm associated genes
(bcsA, fliC, csgA, motA, fimA, and wcaF) [101]. Thereafter Fig. 11 illustrates the
overall probable mechanism for the antibacterial activity of the silver nanoparticles
and nanocomposites that involves adhering to the bacterial cellular membrane, pene-
trating through the bacterial nucleus, generation of ROS and cell signal modulation
leading to the death of the bacterial cells [102].

7 Commercial Applications of Antibacterial Activity

of Silver Nanocomposites

Silver nanocomposites due to their antibacterial activity are finding utility in

several biomedical applications [16]. From wound healing to medical devices,
silver nanocomposites are being used as an antibacterial agent that has been briefly
highlighted in the following sections.
118 S. Haque et al.

Fig. 10 Effect of Qe, Ag NPs, and QA NPs on biofilm integrity of E. coli strain ECDCM1 were
monitored by SEM. a The control group with no antimicrobial agents; b E. coli strain ECDCM1
with exposure to Qe; c E. coli strain ECDCM1 with exposure to Ag NPs; d E. coli strain ECDCM1
with exposure to QA NPs [109]. Reprinted with permission from Yu et al. [101]. Copyright © 2018
Yu et al.

7.1 Wound Healing

Wound healing is one such important aspect of medical field that involves a series of
processes. It is a known fact that wound causes an exposure over the skin leading to
bacterial infections which in turn causes complications in its healing process [103].
The bacterial and fungal infections cause a delay in wound healing followed by
sepsis that sometimes proves fatal to the patient. Since silver is known to be used
as a wound healing agent from ancient times, several reports suggest accelerated
wound healing. Several groups impregnated wound dressing material with silver
nanocomposites for improved wound healing through acting as an antibacterial agent
[104]. In this regards, Kim et al. [105] impregnated silver nanocomposites over cotton
wool by dipping the cotton in a solution containing alcoholic silver-2 ethylhexyl-
carbamates along with thermal reduction at a temperature of 120 °C [105]. The
formed cotton fabrics exhibited excellent antibacterial activity against Escherichia
coli and Staphyloccocus aureus along with good anticoagulant action. The silver
nanoform impregnated cotton dressings even blocked UV rays in the range of UV-R.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 119

Fig. 11 The four most prominent routes of antimicrobial action of AgNPs. 1, AgNPs adhere to
microbial cell surface and results in membrane damage and altered transport activity; 2, AgNPs
penetrate inside the microbial cells and interact with cellular organelles and biomolecules, and
thereby, affect respective cellular machinery; 3, AgNPs cause increase in ROS inside the microbial
cells leading to cell damage and; 4, AgNPs modulate cellular signal system ultimately causing cell
death. Reprinted with permission from Dakal et al. [102]. Copyright © 2016 Dakal et al.

These properties helped in increased wound healing [105]. Similarly, Adibhesami

et al. [58] reported that the antibacterial activity of silver nanocomposites that helped
in healing of wounds [58]. For the experiment, firstly the wound was created on the
back of mouse then the Staphyloccocus aureus solution was applied. The results
revealed that the silver nanoforms at a concentration of 0.08 mg/kg of body weight
of mice proved to be the most effective one in decreasing the bacterial load along
with reduction in wound as compared to untreated and antibiotic gentamicin [58].

7.2 Medical Devices

Apart from medicines, medical devices are considered as an inevitable part for the
therapeutic and diagnostic purposes. But there remains a major issue regarding infec-
tion from the medical devices due to bacterial growth as well as biofilm forma-
tion on the exterior of the medical devices [3]. In this regards, application of silver
nanocomposites is considered for its antibiomicrobial activity for medical devices.
Dybowska-Sarapuk et al. [106] synthesized graphene-based silver nanocomposites
for coating of catheters since there exists a serious issue of biofilm formation in the
120 S. Haque et al.

Fig. 12 Photographic images of an AgNP-grown PDMS film at 36 h after inoculation of S. aureus on

the surface. a Control PDMS was prepared without the AgNP growing reaction. From b to d, AgNP-
grown PDMS films subjected to one, two or three rounds of reactions, respectively. Inset in each
figure is an enlarged image of the figure. Reprinted with permission from Kim et al. [107]. Copyright
© 2017 Kim et al.

catheters of bloodstream and urinary tract infection [106]. The antibiofilm activity
was checked in S. epidermidis. The results revealed inhibition in the biofilm forma-
tion on the catheter surface and the methods were even simple and economical [106].
The other major problem faced by the medical implants is that they are invaded by the
bacteria and cause infections. Kim et al. [107] used silver nanocomposites embedded
on poly-dimethyl-siloxane (PDMS) for the fabrication of silicon films [107]. The
silver nanocomposites formed were permitted to bind electrostatically to the surface
of the film through the hydroxyl groups formed on the latter through air plasma
treatment. The film when evaluated exhibited excellent antibacterial effect against
Bacillus subtilis and Escherichia coli in comparison with the commercially available
silver nanocomposites. Figure 12 clearly shows that the antibacterial activity of the
PDMS with silver nanocomposites increased in a concentration-dependent manner
in comparison with the naked PDMS without silver nanocomposites. The highest
antibacterial activity was shown with three rounds of reaction of silver nanocompos-
ites on PDMS. Thus these silicon films can be effectively used as implanting devices
as offering biosafety toward infections caused medical devices [107].

7.3 Tissue Engineering

Silver nanocomposites are even being utilized for biomedical engineering designing
works. This owes to their unique characteristic features such as antiangiogenic, anti-
inflammatory and most importantly antibiofilm as well as antibacterial activity [16].
Silver nanocomposites are extensively used for bone and dental tissue engineering.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 121

Regarding bone tissue implants, Aurore et al. [108] worked to prevent osteoclast
infection by Escherichia coli and Staphyloccocus aureus by synthesizing silver
nanoforms [108]. The results clearly depicted that the silver nanocomposites reduced
the bacterial load and prevent the infection. The mechanism behind its activity owes to
ROS generation followed by damaging of the bacterial cell. These silver nanocom-
posites can be effectively used for orthopedic and bone implants which can even
prevent bacterial infections [108]. For dental care silver nanocomposites are used
from centuries as dental amalgams. Due to their antimicrobial property they are
highly preferred for dental products. In this context, Inbakandan et al. [109] biosyn-
thesized silver nanocomposites with the extracts of Haliclona exigua which is a
marine sponge [109]. The shape of the silver nanocomposites was flower like. The
silver nanocomplex exhibited excellent oral antibiofilm activity against S. salivarius,
S. orulis and S. mitis. The results also revealed that at the lowest concentration (10 μg)
the silver nanocomposites inhibit the growth of these bacteria [109].

7.4 Other applications

Apart from the biomedical applications, the silver nanocomplexes and nanocompos-
ites are utilized for textiles, food processing, cosmetics, paints and water management
because of its antibacterial activity [110]. The silver nanocomposites are employed
for the production of cosmetics as they are effective in removing pathogens from the
skin surface without affecting the epidermal lipid layer of the skin [111]. Regarding
this Bansod et al. [112] synthesized silver nanocomposites functionalized oil for
the production of shampoo, soaps and ointments. The formed compound proved
to be antibacterial when topically applied over wounds and other microbial infec-
tions [112]. Silver is being widely preferred for the purpose of food packaging since
it imparts prevention of the food particles from being destroyed by the microbes.
Pulit-Prociak et al. [113] reported to have used silver nanocomposites associated dye
for the purpose of dyeing cotton fabrics and imparting antimicrobial protection to
the fabric [113]. The silver nanocomposites are being included in paints in order to
make them antimicrobial resistance. The silver nanocomposites help the paint from
weathering by the microbial particles [114]. The microbes form biofilm over the paint
resulting in the retention of spores and dirt. The silver nanocomposites generate ROS
and destroy the cell membrane of the bacteria and also interfere with the antioxidant
activity of the bacterial cell [115]. All these results exhibit the potential of the silver
nanocomposites of being as an efficient antibacterial agent.
122 S. Haque et al.

8 Toxicity of Silver Nanocomposites

The silver nanocomposites have gained lot of interest among the scientists due to their
promising outcomes toward antibacterial activity. The commercialization of the silver
nanocomposites requires certain criteria’s to be fulfilled where toxicity occupies a
major portion. Certain experimental reports exhibit work regarding the toxicity of
the silver nanocomposites [57]. In one toxicity report Sujarania et al. [116] studied
the toxicity of silver nanocomposites anchored with [2-(2,2-diphenylethylimino)
methyl)phenols] [116]. The nanocomposites were characterized using the techniques
NMR, IR and UV–Visible. Following that rhodamine B marker was attached to the
material. The in vitro and in vivo studies were performed to analyze the toxic effects of
the silver nanoparticles anchored 2-[(2,2-dipenylethylimino)methyl] phenols which
clearly represented no toxicity. The study demonstrated that the silver nanocompos-
ites along with the Schiff bases is biocompatible in nature [116]. In another work
Saha et al. [117] checked for the toxicity of the biosynthesized silver nanocomposites.
The silver nanocomposites were produced by coupling with Euphorbia thymifolia
[117]. For the toxicity studies to be conducted, Swiss albino mice were procured
of 6–8 weeks old after in compliance with the rules of Organization for Economic
Cooperation Development (OECD). The silver nanocomposites were administered
intraperitoneally with a single dose of 2000–5000 mg/kg of body weight to the mice.
Even the compounds silver oxides as well as Euphorbia thymifolia were provided
at low doses to the mice that exhibited biocompatible results. The experiment was
performed for 14 days keeping up a constant check on the body and behavioral
changes. The end results displayed that the mice were healthy even after the single
dose of the silver nanocomposites and showed no toxicity [117].
In a further study, Yu et al. [118] synthesized silver nanocomposites with the
help of cellulose fibril [118]. The formed nanocomposites were characterized with
different techniques and also exhibited antibacterial property against Staphylococcus
aureus and Escherichia coli. Then the toxicity study was assessed through the effect
of the silver nanocomposites on human colon cells. The formed nanocomposites at
low concentration exerted no toxicity to the human colon cells but decreased cell
viability at higher doses [118]. But some results highlighted the toxic effects of the
silver nanocomposites. In one report Ema et al. [119] evaluated the effect of silver
nanocomposites on the developmental and reproductive processes of female and
male rats [119]. The results revealed that the silver nanocomposites accumulated in
the testis of male. In the female rats the formed nanocomposites were found to be
accumulated in the visceral yolk sac at time of early gestation followed by deposition
in placenta, pre- and postnatal offspring as well as in breast milk in late gestation
period. The developmental defects caused by the silver nanoforms include defective
cognitive behavior and physical disabilities [119]. The silver nanocomposites when
released in the environment can cause some amount of dysfunction. Hence, they
must be properly treated before being discharged in nature.
Potential Application of Silver Nanocomposites for Antimicrobial Activity 123

9 Future Perspectives and Challenges

Nanotechnology is paving its way into the everyday life of living beings along with its
advancement and improvement. Various diseases including antibacterial disease are
diagnosed and cured with the help of nanotechnology [18]. The increased amount
of antibiotic resistance toward conventional drugs has led to commercial markets
dwell into nanotechnology to formulate new diagnostics and therapeutics. In this
aspect silver nanocomposites have emerged as one of the most eligible candidates
for antibacterial therapeutics owing to its unique characteristics features [69]. A good
number of silver nanocomposites related products are under experimental conditions
for clinical trials and FDA approvals.
Although the silver nanocomposites have the potential to establish itself as a
strong antibacterial agent, but for enhancement of its activity certain things are to
be considered. The primary one is the proper surface functionalization of the silver
nanocomposites. This can be done by attaching certain antibiotics, gene, protein and
target moieties that will help in the augmentation of the antibacterial activity of the
nanocomposites [23]. The second is increase in the yield and proper stability of the
silver nanocomposites on a large scale in order to reach a wider population. Usually
chemical stability of the silver nanocomposites is preferred for the slow release
of silver ions from the complex allowing a longer retention time. Even positively
charged silver nanocomposites are preferred for better interaction with the cellular
membranes. The shapes of the silver nanocomposites should be given more pref-
erences for proper contact with the bacteria [60]. Since silver is metal, the toxicity
experiments should be conducted in a vigorous amount to eliminate any possibility
of toxicity through the silver nanocomposites. Also the right amount of dosage as
well as accumulation study of the silver nanocomposites possesses a challenge that
needs to be met [120]. More amounts of mechanistic studies regarding the antibac-
terial activity of silver nanocomposites should be conducted. This is due to the fact
that the silver nanocomposites is available in various sizes, shapes and charge and
each have its own separate mechanism of action [102]. This can even act as a tool to
combat antibiotic resistance exhibited by the microbes.
But there remain certain challenges that act as backdrop regarding the usage of
silver nanocomposites. The first and the foremost is the resistance of the bacteria
toward silver. Certain reports exhibit ineffectiveness of the silver nanocomposites
toward some specific bacterial strains [38]. Another challenge involves consumption
of the silver nanocomposites on a regular basis does pose a threat of its accumulation
in the skin tissues. This even leads to argyrosis following sunlight exposure [121].
Other than these the general difficulties regarding nanocomposites includes cost of
production, environmental hazards, immunological effects as well as the pharma-
cokinetics and pharmacodynamics that needs to be addressed [122]. Thereafter, if
all these future perspective and challenges are timely met then it will result in the
development of excellent treatments for antibacterial therapy.
124 S. Haque et al.

10 Conclusions

Since microbial infections are increasing on an alarming rate in day-to-day life even
with the availability of commercial products, nanotechnology has paved the way to
curb it in its own ways. The book chapter overall focuses on the different pathogenic
strains causing the microbial infections, their global statistics in terms of mortality
and morbidity followed by the conventional treatment strategies available. But the
limitations and backdrops of the commercially available products have led to the
rise of nanotechnology especially silver nanocomposites as an alternative treatment
for microbial infections. The silver nanocomposites have exhibited excellent antimi-
crobial activity produced in combination with several precursors and agents through
various mechanisms. The silver nanocomposites exhibit its antimicrobial strategy
through cell membrane breakage resulting in the leakage of cellular components,
damage to DNA, interference in the synthesis of nucleic acids and proteins, genera-
tion of oxidative stress, etc. The silver nanocomposites are even utilized for further
commercial products. The book chapter further highlights the toxicity related issues
of the silver nanocomposites. Finally the future perspective and challenges section
discusses the different backdrops of silver nanocomposites such as cost of produc-
tion, toxicity which when met can prove to produce wonders in the treatment of
microbial infections.

Acknowledgements Financial support from Nano Mission-DST (SR/NM/NS-1252/2013; GAP

570), New Delhi, to C.R.P. is duly acknowledged. S.H is thankful to CSIR, New Delhi for supporting
Senior Research Fellowship. We thank Director, CSIR-IICT (Ms. No. IICT/Pubs./2020/184 dated
July 3, 2020) for providing all the required facilities to carry out the work.

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Cellulose and Chitin Nanofibers:
Potential Applications on Wound Healing

Athira Johnson, M.S. Neelakandan, Jiya Jose, Sabu Thomas,

and Nandakumar Kalarikkal

1 Introduction

“Wound” is defined as an injury to the living tissue caused by a scratch, cut, shock,
burn, or another impact in which the skin is cut or broken. Wound healing is a step by
step process that includes four major stages, hemostasis, inflammation, proliferation,
and maturation. Blood factors have a vital role in this biological process [1]. Human
skin is composed of three layers, namely, epidermis, dermis, and hypodermis. The
outer most layer epidermis is hydrophobic in nature. Connective tissues, hair follicles,
and sweat glands are found in dermis layer beneath the epidermis. Inner layer hypo-
dermis consists of fat and connective tissues. The epidermis is rich in keratinocyte and
dermis associated with the presence of collagen. Generally, the wound is arisen with
swelling, redness, pain, bleeding, and weakening in function. There are two types of
wound called acute and chronic. During the past, plant extract and honey are used for
healing. Now, these methods are changed by the invention of advanced technologies
like nanotechnology and biotechnology. Bionanotechnology knew to be a trend in
research field help to plays role in biomedical research. Concentrating onto wound
healing, attraction toward biopolymers in nano form has largely increased, which is
able to promote cell proliferation, reduce inflammation, and facilitate cell migration.
Cellulose and chitin are widely used biopolymers for wound healing due to their
availability in nature and attractive properties. Formulation of cellulose nanofibers

A. Johnson · M.S. Neelakandan · J. Jose · S. Thomas (B)

International and Inter University Centre for Nanoscience and Nanotechnology, Mahatma Gandhi
University, Kottayam 686560, Kerala, India
e-mail: [email protected]
N. Kalarikkal
School of Chemical Sciences, Mahatma Gandhi University, Kottayam 686560, Kerala, India
S. Thomas
School of Pure and Applied Physics, Mahatma Gandhi University, Kottayam 686560, Kerala, India

© Springer Nature Singapore Pte Ltd. 2021 133

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_6
134 A. Johnson et al.

and chitin nanofibers helps to overcome the problems associated with ordinary poly-
mers. Glucose monomers connected by a β 1–4 linkage to form cellulose and chitin
are derived by elongation of N-acetyl glucosamine units. Cellulose nanofibers and
chitin nanofibers were fabricated by electrospinning method with good antimicrobial,
anti-inflammatory, and healing properties.

2 Process of Wound Healing and Trends in Wound Dressing

Wound formation by an injurydestroys the integrity of the skin layers. If a wound

is formed, the healing process initiates with the formation of a clot. Generally, the
healing process is a four-stage process. Removal of microorganisms and facilitation
of the activity of growth factors and immune cells like neutrophils, macrophages,
endothelial cells, keratinocytes, and fibroblast occurs in the initial stage called
hemostasis. New blood vessels were formed in the second stage, angiogenesis [2]. In
a wound part, various intracellular and extracellular path way is activated to restore
normal structure and functions of the skin. During proliferation, the wound is rebuilt
and new tissues are formed to restore the normal structure of the skin. The wound
is completely recovered its functions and structure at remodeling phase. Most of the
case, the extracellular matrix disorganized and form scar. Depends on the time to heal,
the wound is categorized in to acute and chronic. An acute wound is an injury occurs
suddenly and heals at an expected rate. A chronic wound develops when any acute
wound fails to heal in the expected time, it last for a long term. The process of wound
healing is directed by the activity of growth factor called cytokines. Not only the
cell growth, migration, matrix formation, differentiation, and enzyme expression but
also wound healing activity was controlled by growth factors called cytokines. The
process of healing starts when the platelets come in contact with collagen results in
the formation of fibrin clot. During this time, platelet produces two important signals
called platelet derived growth factor (PDGF), that initiate chemotaxis together with
mitogenesis process and transforming growth factor-β (TGF-β) responsible for initi-
ating healing cascade. As a result, connective tissue is deposited at the site of injury
and inflammatory stage starts. Within 24 h neutrophils arrives at the wounded area and
remove damaged matrix, bacteria, and foreign material. The inflammatory process
is accompanied by the signs of redness, heat, swelling, and pain. Macrophage cell is
known to be the most important inflammatory cell in wound healing process. Inflam-
matory phase is followed by proliferative stage. Epithelialisation and angiogenesis
were taken place during this stage. Fibroblast begins to produce collagen. Finally,
the remodeling of collagen happens and the skin is regenerated [3].
The mechanical distraction of tissue and inflammation are two main issues that
arise during an injury formation. Ancient times, human exploits the benefit of natural
resources such as honey, animal fat, and plant extract for healing the wound. Honey
provides a moist healing environment, rapidly clears the infection, and reduces
inflammation, edema, and exudation; promote angiogenesis, granulation, and epithe-
lialization. Some better materials like bandages, cotton wool, lint, and gauze were
Cellulose and Chitin Nanofibers: Potential Applications on Wound Healing 135

Fig. 1 a Hydrocolloid and b hydrogel (Boateng et al. 2007)

used for covering the wounded area and thereby preventing inflammation, keeping
the wound dry, and allow evaporation in later times [4]. The use of green tea, mush-
room, bronze instrument, and other plant powder are used in Chinese medicine which
promotes tissue granulation and reduces infection. There is strong evidence for using
honey as a wound healing agent from Mesopotamian times. Before dressing, they
wash the wound with milk and water. In Greece, the wounded area is washed with
vinegar, wine [5]. Adherence is the major problem associated with wound healing
process in ancient technique. The first non-adherent dressing material was a double-
layered material prepared by using cotton, balsam, and paraffin [6]. In 1947, Blaine
developed Calcium alginate extracted from brown seaweed which can be made up of
fibers, films, and forms for wound curing [7]. Another type of wound dressing mate-
rial is called hydrocolloids developed in 1982. It is made up of sodium carboxymethyl
cellulose, gelatin, polyisobutylene, and pectin [8]. Hydrogels are another type formed
from polymers; they can provide a moisture environment for the dry wounded area.
They are able to possess antimicrobial activity. The picture of hydrocolloid and
hydrogel are illustrated in Fig. 1.
Polyurethrane foams are also used as dressing materials. The scaffold is the group
of dressing material that have rigid nature and promote the infiltration of cells during
the healing process [9]. A wound dressing material should possess some proper-
ties such as water vapor permeability, sterilization, anti-microbial activity, oxygen
permeability, non-toxicity, and biodegradability.

3 Cellulose and Cellulose Nanofibers (CNFs)

Cellulose is known to be the most abundant biopolymer in the world.Anselme Payen,

a French Chemist discovered and isolated cellulose from green plants in 1938. It is
136 A. Johnson et al.

Fig. 2 Illustration of the structure of cellulose from plant cell wall (https://public.ornl.gov/site/gal
lery/detail.cfm?id=181)

the main constituent of the plant and is also presents in bacteria, fungi, animals,
and algae [10]. Cellulose provides protection together with a structural framework
for plants and helps to maintain their rigidity. It is insoluble in water and present in
almost all part of the plant such as stem, leaves, and stalks. Cellulose (C6 H10 O5 )n is
a polysaccharide composed of a linear chain of several hundred to many thousands
of β(1 → 4) linked D-glucose units. Figure 2 illustrates the distribution of cellulose
in plants.
Cellulose present in the form of microfibrils with asize between 2 to 50 nm.
The crystallize microfibrils may exist in the form of cellulose Iα and Iβ and will be
organized further to form the layers, cell walls, and fiber. The number of glucose unit
is depended on the location where they found, which means, about 8000 glucose
unit per chain (DP = 8000) is present on the primary cell wall, while in the case
of the secondary cell wall is seems to be around 15,000 units per chain [11]. Apart
from that, cellulose nanofibers (CNFs) have high surface area to volume ratio, high
tensile strength, and low-coefficient of thermal expansion. CNFs has size <100 nm
in one dimension with a length in micrometer and width in the nanometer range.
Nano cellulose fibrils can be divided into two broad categories: amorphous form
(cellulose nanofibers) and crystalline form (cellulose nanocrystals). Currently, green
nanotechnology is widely spread in almost all part of the world and the attraction
toward this area has largely increased especially for fabricating nanocomposite, gas
barrier films, and biomedical applications [12]. Commonly CNFs are produced by
high-pressure homogenization, refining, and grinding [13].
Cellulose and Chitin Nanofibers: Potential Applications on Wound Healing 137

Quality and standard of dressing materials are important for measuring the rate of
healing process. Mechanical parameters like flexibility, tensile strength, and elastic
property of the material is considered during dressing material preparation. Stretchy
and soft membrane is easy to handle and readily detached from skin. Air perme-
ability of the wound dressing material prevents the activity of bacteria by the open
pores present over it. As compared with hydrophobic material, hydrophilic materials
are more efficient because they can absorb wound exudates. Controlling of moisture
retention is significant to prevent dehydration and exudates formation. Wound infec-
tion is reduced by the fluid uptake ability of wound dressing material [15]. Due to
the similarities with extra cellular matrix (ECM), biocompatible and biodegradable
nature of CNFs is suitable for wound healing.
Ancient methods are replaced by the advancement in technology; the situation is
turned into an immense world of research and studies regarding wound healing. Flex-
ibility and porosity of CNFsmake them very attractive material for wound healing.
There are certain criteria required for choosing a material as “wound healing mate-
rial” such as maintaining sterile condition and moisture balance, allow gas exchange,
ability to absorb cell exudates, nontoxicity, and non-adherent to wound [14]. Liu
et al., 2012 shows that nanofibrous membranes prepared from cellulose acetate (CA)
and polyester urethane (PEU) perform good wound healing activity. CA fibers can
improve the hydrophilicity and permeability to air and moisture. They prepared by
using the blend electrospinning method. Polyhexamethylene biguanide (PHMB) was
added to this fibers to prevent clinical infections.
One of the main issue relatedto wound healing material is inflammation, arises
due to the activity of microbes which results in the delay of the healing process. The
bandage is prepared by the immobilization of cell lytic enzyme called lysostaphin
on to the biocompatible fibers {obtained from the solution of cellulose, cellulose-
chitosan, and cellulose-poly (methyl methacrylate) (PMMA)} by electrospinning
method. Results show that it has an antimicrobial activity against S. aureus with low
toxicity toward keratinocytes [15]. The fibers prepared by electrospinning are asso-
ciated with the high surface to volume ratio and length to diameter ratios. Because
of these advantages, ultrafine nanofibers exhibit stronger antimicrobial activity than
natural fibers. [16] prepared a solution of cellulose acetate ultrafine fibers with silver
nanoparticle by an electrospinning method and evaluate the antimicrobial property.
The results show that this combination has good activity against microbes [16]. Bacte-
rial cellulose associated in bacterial cell wall, incorporated with silver nanoparticle
demonstrated that it performs significant antimicrobial activity against Staphylo-
coccus aureus. From this experiment, it is clearly evident that this combination can
reduce inflammation and promote wound healing [17]. Centella asiatica extracts with
cellulose acetate nanofibers are used to heal the wound. In this case, the percentage
of water uptake and diameter of the fibers are increased but the contact angle between
fiber and water droplet is decreased with increase in solution content. Considering
the percentage of water uptake is a remarkable factor for wound healing process. The
combination of nanofibers and Centella asiatica extract helps to provide a suitable
condition for the healing process [18]. Hydrogels are polymer network extensively
swollen in water with a high rate of absorption. Currently, there are lots of research
138 A. Johnson et al.

focused on the preparation and modification of hydrogel for wound healing appli-
cation. Acetobacter xylinum is a bacteria modified to form 2, 3 dialdehyde cellulose
(DABC) by an oxidation process using sodium metaperiodate as an oxidizing agent
and this DABC loaded with chloramphenicol (CAP) for preparing a hydrogel. Studies
display that, this hydrogel has good biocompatibility, biodegradability, and antimi-
crobial activity [19]. Nanofibrous membrane mimics extra cellular matrix (ECM)
because of its high porosity and high surface area makes them proper material for
good wound healing. The ECM consists of proteins and polysaccharides. Electrospun
cellulose acetate and gelatin membranes promote proliferation during skin regener-
ation and perform as a scaffold for wound healing applications [20]. BC is excellent
for cleaning and penetration of active substance to the wounded area. Nanocom-
posite films prepared by BC and montmorillonite (MMT) have good antibacterial
activity against Escherichia coli and Staphylococcus aureus with high tissue regen-
eration capacity. The BC-MMT composite indicates the attachment and penetration
of MMT particles in BC matrix; this penetration provides strength to the composite
[21].
Aerogels are formed by removing theliquid part from a gel without damaging the
network through freeze drying (lyophilization) or critical point drying [22]. CNFs
obtained from wood powder are oxidized by sodium periodate to form dialdehyde
CNFs and introduction of collagen into this CNFs forms a matrix. Collagen grows in
dialdehyde CNFs and forms composite aerogels. They have a high porosity between
90 and 95% and strong water absorption up to 4000 times. They also exhibit biocom-
patibility and active proliferation. It support wound healing process [23]. In conclu-
sion, Cellulose nanofibers wound materials have good porosity, surface to volume
ratio, biocompatibility, biodegradability, low density, and unique morphology. Due
to these characteristics, it is widely employed in wound healing applications [24].
Jin et al. [25] prepared cellulose nanofibrous aerogel by the dissolution of cellulose
in calcium thiocyanate followed by drying. Aerogels prepared by solvent exchange
drying are good because it has high porosity and specific surface area [25] (Fig. 3).

Fig. 3 SEM of CF11 cellulose aerogels by solvent exchange drying a 0.5%; b 1.0%; c 2.0% and
d 3.0%
Cellulose and Chitin Nanofibers: Potential Applications on Wound Healing 139

4 Chitin and Chitin Nanofibers (CNFs)

Chitin (C3 H13 O5 N) n is a biopolymer formed by the monomer N-acetylglucosamine

linked through β 1–4 linkage, which is a primary component of the cell wall in
animals, fungi, the exoskeleton of crustaceans, insects, and the scales of fish and
lissamphibians. It is a white, hard, nitrogenous polysaccharide with a high percentage
of nitrogen (6.89%) [26].
Min et al. studied theformation of chitin nanofibers by an electrospinning method
with an average diameter of 110 nm and concluded that they can form a matrix for
wound healing in 2004. The solubility of chitin was improved by depolymerizing
with gamma irradiation. The chitin matrix gets converted into chitosan matrix by
deacetylation [27]. Chitin is known to be the second most abundant biopolymer after
cellulose. Compared to cellulose, chitin has low attention from the research field due
to its poor solubility and deacetylation of chitin into chitosan. Major investigations
associated with chitin in biomedical fields related to wound healing applications.
Noh et al. illustrated that these fibers with extracellular matrix proteins like type
1 collagen can promote cell attachment, distribution of keratinocyte and fibrob-
last during wound healing [28]. The scaffold prepared from chitin has high oxygen
permeability, porosity, the morphological similarity with ECM, and promote cell
adhesion and cell proliferation. Chitin prepared from shrimp is treated with butyric
anhydride results in a water-soluble chitin derivative called Dibutyrylchitin (DBC).
This compound elevates glycosaminoglycans (GAG) level and promotes granula-
tion. Charernsriwilaiwat N et al. and his co-workers prepare hydrogels from chitin
nanofibers and are also applicable for wound healing process. The wound made
on the back of the rat was completely re-epithelialized while using hydrogels [29].
The wound healing activity is evaluated by using lysozyme loaded mixture of chitin
fiber-ethylenediaminetetraacetic acid show potential activity during healing [30].
Kang et al. 2012 show that heat-treated polyvinyl alcohol (H-PVA)—chitosan
nanofibrous matrix is suitable for wound dressing. Compared to control PVA,
chitosan loaded H-PVA shows good collagen regeneration and thick epidermis forma-
tion [31]. Chitin nanofibers are prepared by melt blown, self-assembly, spinning,
and phase separation [32]. Chen et al. prepared a composite nanofibrous membrane
(NFM) with the use of chitosan, type 1 collagen, and polyethylene oxide in 2008,
with a fiber diameter of 134 ± 42 nm. It had been reported that Young’s modulus
and diameter can be increased by adding cross-linker and, water absorption capacity,
tensile strength, and strain are decreased. [33] show that 3T3 fibroblast cells are used
for measuring cytotoxicity. From the experiment, it was understood that there is no
cytotoxicity and good biocompatibility. Animal studies show that NFM is better than
normal gauze [33]. The composite composed by using chitosan and silk fibroin show
good anti-microbial activity against Escherichia Coli and Staphylococcus aureus
and have good proliferation capacity during wound healing [34]. The composite
prepared from β-Chitin, and zinc oxide nanoparticle is used as bandages during
wound healing process. In vivo evaluation in Sprague Dawley rat show faster healing
and high collagen deposition [35]. During inflammation, the attack of microorganism
140 A. Johnson et al.

is increased. The scaffold prepared from chitin and nanosilver is a good candidate
for biocompatibility, antimicrobial activity, and blood clotting [36].
By radiation-induced cross-linking, hydrogels are prepared from carboxymethyl
chitosan (CM-Chitosan) and gelatin solution. When chitosan content is increased,
the swelling ability is also increased but the compressive modulus decreased. After
lyophilisation, there is a homogenous pore structure seen. It promotes cell attachment
and fibroblast formation [37].
Park et al. [38] formulatedPolyglycolic acid/Chitin blend nanofibers for wound
healing applications. They have an average diameter of 140 nm. As can be seen from
Fig. 4 their study concluded that these nanofibers coated with bovine serum albumin

Fig. 4 Cell attachment of proliferating NHEF plated onto PGA/chitin blend nanofibrous matrixes.
Microphotographs and level of cell attachment of cultured cells onto PGA/chitin blend nanofibrous
matrixes without a or with b BSA coating. Data are expressed as mean (SD (n) 4). ANOVA: P <
0.05. Pairwise comparisons: *P < 0.05 versus pure PGA nanofibers. a polystyrene surface, b pure
PGA, c PGA/chitin (75/25), d PGA/chitin (50/50), e PGA/chitin (25/75), and f pure chitin. Bar 100
ím
Cellulose and Chitin Nanofibers: Potential Applications on Wound Healing 141

promote cell attachment and migration [38]. Requirements for wound dressing are
increased the time to time according to the development of new technologies. Preven-
tion of infection and rapid curing are the two major conditions for every dressing
material. The materials which provide growth factors and antimicrobial agents get a
good impact on the clinical area. Plant and animal derived product are also employed
for this purpose.

5 Conclusion

Cellulose and chitin are known to be the most abundant polymers in nature. Cellulose
is obtained from plant, fungi, algae, and animal-like tunicates. Chitin is obtained
primarily from the exoskeleton of crustaceans and insects. Cellulose and chitin
nanofibers are capable of the wound healing process. They are manipulated mainly
by electrospinning method. Studies show that when transformed into nano range the
properties of fibers are changed and possess advantages than the ordinary one. This
fiber can be framed into composite, hydrogel, aerogel, and thin films, etc. They are
non-toxic and biocompatible in nature. They are able to promote cell migration, cell
proliferation, and prevent infection by the activity microorganism.

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Bionanocomposites for In Situ Drug
Delivery in Cancer Therapy: Early
and Late Evaluations

Luiza Steffens Reinhardt, Pablo Ricardo Arantes, Jeferson Gustavo Henn,

and Dinara Jaqueline Moura

Abbreviations

α-CD A-cyclodextrin
BBB Blood brain barrier
CT Chemotherapy
CTC Circulation tumor cells
DCA Sodium dichloroacetate
DOX Doxorubicin
DPPE Dipalmitoyl phosphatidylethanoiamine
MWCNT Multi-walled carbon nanotubes
NF Nanofiber
NP Nanoparticle
PCL Polycaprolactone
PDLLA Polylactide
PDT Photodynamic therapy
PEG Poly (ethylene glycol)
PEI Poly(ethylene imine)
PEO Polyethylene glycol
PLA Polylactic acid
PLGA Poly (lactic-co-glycolic acid)
PLLA Poly-l-lactic acid
pNIPAM Poly(N-isopropylacrylamide)
PS Photosensitizer
PTX Paclitaxel
PVA Polyvinyl alcohol

L. S. Reinhardt (B) · P. R. Arantes · J. G. Henn · D. J. Moura

Laboratory of Genetic Toxicology, Federal University of Health Sciences of Porto Alegre
– UFCSPA, Sarmento Leite 245, Lab.714, Porto Alegre, Rio Grande do Sul, Brazil
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2021 145

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_7
146 L. S. Reinhardt et al.

ROS Reactive oxygen species

RT Radiotherapy
SPIONs Superparamagnetic iron oxide NPs
T1 Two-photon absorption compound
TMZ Temozolomide
VEGF Vascular endothelial growth factor

1 Cancer Outline

Cancer is a general word for a complex category of diseases described by unregulated

cellular processes such as growth and death [1]. Theoretically, all cancers develop as
an outcome of mutations in the DNA sequence, which may be hereditary, provoked
by environmental dynamics or a consequence of replication errors of DNA [2].
These explanations are elucidated by the carcinogenesis multistep model (Fig. 1)
comprising initiation, promotion, and progression stages [3, 4]. Initiation begins
when healthy cells are damaged by contact with physical, biological, or chemical
carcinogens resulting in mutations in the DNA [5]. Promotion is described by the
clonal growth of initiated cells, inhibiting apoptosis [6]. Lastly, tumor progression
is described by a malignant phenotype expression, including characteristics such as
uncontrolled growth, genomic instability, metastases development, and modifications
in the morphological and biochemical features of cells [6, 7].
Conventional cancer management embraces surgical procedure, systemic
chemotherapy (CT), and radiotherapy (RT). The CT therapeutics frequently damage
normal cells developing severe side effects [8, 9]. An alternative to the treatment
is a nanotechnological intervention since it has transformed the cancer therapy by
overcoming existing restrictions of conventional CT, and offering better targets
with sustained and controlled drug release also improving both distribution and
pharmacokinetics [9–11].

2 Drug Delivery for Cancer: General Reflections

Targeted drug delivery for cancer therapy refers, quantitatively and selectively, to
drug accumulation within tumor tissue [12, 13]. Subsequently, targeted medicine or
targeted therapy means precise drug efficiency combine with minor side effects and
interaction, at the molecular level, between a biomolecule and a drug. As a matter
of fact, the concentration of drugs and biomolecules must be high at the tumor site,
while its concentration in healthy non-target tissue should be almost undetectable
preventing systematic negative reactions [12, 13].
Bionanocomposites for In Situ Drug Delivery … 147

Fig. 1 Carcinogenesis multistep model comprising initiation, promotion, and progression stages
148 L. S. Reinhardt et al.

Preferably, for formulations to be efficient in cancer therapy, they should be

biofunctionalized including simple features as size and surface charge and multi-
functional features as biomolecules combination. Furthermore, they need to fulfill
the requirement to:
a. Enhance drug pharmacodynamics and pharmacokinetics profiles.
b. Selectively eradicate tumor cells without affecting healthy cells.
c. Prolong and control the release of the active form of drugs.
d. Improve the cellular uptake of delivered drugs.
e. Diminish dose-limiting toxicities [14, 15].
In all assurance, to reach a successful CT without harming healthy tissues has been
the key effort of targeting approaches. It is estimated the enhancement of drug delivery
to cancer cells with an improved well-developed nanoproduct [12, 13], however,
even with intense research developing in the bionanocomposites field, no significant
results with meaningful changes in the life of patients have been accomplished.
It is a challenge to bypass drug delivery issues such as early drug degradation,
bloodstream circulation time, tissue membranes, and systemic toxicity, therefore,
for some cancers, the main promising approach to achieve suitable drug delivery is
an in situ platform. In fact, several nanoproducts present an initial burst release that
can cause acute toxicity or release the total amount of a therapeutic compound before
reaching the tumor cells, fortunately, both of these issues can be addressed by using
local drug delivery.

3 Bionanocomposites

It is known that biopolymers possess some limitations in contrast to synthetic poly-

mers in terms of stability, transparency, and mechanical properties nevertheless, these
drawbacks can be overcome by combining nanosized materials while producing
bionanocomposites. Among all advantages of bionanocomposites, this approach can
avoid nanoparticle (NP) agglomeration during nanocomposite preparation by using
a polymeric matrix where the NP can be dispersed [16], moreover, nanocomposite
biodegradability increases after producing a composite with nanosized materials
[17].
By rule, bionanocomposite is a two-phase system, where, at least one part had
a nanosized dimension (lower than 100 nm) and the word “bio” indicates the use
of biodegradable material [18]. The main feature of bionanocomposites is a large
surface area owing to nanosized material resulting in enhanced interaction between
its components with the composite matrix (Fig. 2) [19].
Bionanocomposites for In Situ Drug Delivery … 149

Fig. 2 Features of bionanocomposites

3.1 How to Choose the Right Polymer

In the last decade, the use of synthetic biodegradable polymers as drug delivery
systems increased significantly [20], mainly because synthetic polymers made of
petrochemicals are toxic and take a long time to degrade. Biodegradable polymers
(Table 1) can be synthetic such as polyglycolic acid (PGA), polylactic acid (PLA),
polycaprolactone (PCL) and poly-l-lactic acid (PLLA), or natural such as collagen,
chitosan, and silk [35, 36].
Features of biocomposite materials such as optical and mechanical properties
and conductivity usually are improved from bulk material although matrix compo-
sition is the same [37]. Polymers as PCL, poly (ethylene glycol) (PEG), PLA,
and poly (lactic-co-glycolic acid) (PLGA) that are synthetic and biodegradable are
commonly used as substrates for the production of scaffolds. Moreover, during
the past years, other polymers with stimuli-responsive behavior such as poly(N-
isopropylacrylamide) (pNIPAM) and poly(4-vinylpyridine) have been investigated
as thermo and pH-responsive [38].
It is also essential, during the development of biocomposites as drug delivery
devices, the solvent selection, which needs to offer adequate solubility for both drug
and polymer. The compatibility of polymeric mixture components and their physic-
ochemical properties such as surface tension, conductivity, and viscosity have key
effects on the resulting morphology of the carrier [39, 40]. The state of both compo-
nents, their crystallinity, and their distribution within the composite have significant
influences on the drug release kinetics. Furthermore, the establishment of both phases
can be influenced by the addition of amphiphilic molecules, which can facilitate the
release of hydrophobic drugs to the aqueous environment [41, 42].
 150

Table 1 Features of biodegradable polymers

Polymer Biodegradation Mechanical properties Physicochemical properties Key-points References
Synthetic polymers PLGA Hydrolysis Strong Amphiphilic Good solubility, weak toxicity, [21, 22]
application in several
biomedical devices and fast
biofilm formation
PLA Hydrolysis Strong Hydrophilic Weak toxicity and application [23, 24]
in several biomedical devices
PCL Hydrolysis Strong Hydrophobic Application in long-term [25]
implantable devices
PVP Hydrolysis Strong Hydrophilic Good solubility and application [26, 27]
as antimicrobial nanofibers
PVA Enzymatic Strong Hydrophilic Good solubility, nontoxic, and [28, 29]
non-carcinogenic
PEG Enzymatic Weak Hydrophilic Good solubility, high range of [30]
viscosity, contains toxic
impurities, and is nephrotoxic
if applied to damaged skin
Natural polymers Collagen Enzymatic Weak Hydrophilic Weak toxicity [31]
Chitosan Enzymatic Weak Hydrophilic Molecular weight dependent [32, 33]
solubility, weak toxicity, and
several applications in
pharmaceutical industry
Silk Enzymatic Strong Hydrophilic Moderate toxicity [34]
PLGA poly (lactic-co-glycolic acid), PLA poly (lactic acid), PCL poly (1-caprolactone), PVP polyvinylpyrrolidone, PVA polyvinyl alcohol, PEG polyethylene
glycol
L. S. Reinhardt et al.
Bionanocomposites for In Situ Drug Delivery … 151

3.2 How to Investigate Polymer-Drug Interactions

Polymers present particular characteristics which range from the angstrom level of
an individual bond between atoms to nanometers of the polymer chain, microme-
ters, millimeters, larger in solutions, and polymeric nanofibers. Due to its particular
characteristics, the best way to investigate the polymer-drug interactions is through
in silico studies [43–47]. The different time scales for each material properties may
range from femtoseconds to seconds or even hours. On the literature, there are many
examples of the multiscale nature of polymer systems [48–55]. Because of this,
several computational methods were developed in order to address these issues [56–
64]. These novel methods introduce new possibilities to design, optimize, and predict
the structures and properties of polymers. Not only that, these methods allowed the
study of these materials with other molecules [43–47]. In the case of this chapter, is
the possibility of studying the polymer-drug interactions. One of the main methods
to study polymer-drug interactions, nowadays, is the atomistic calculations where all
atoms are explicitly represented and treated by a single sphere. The force field, typical
interactions in the system, is responsible for the potential energy of the system. These
interactions include the bonded interactions that are the bond length, the bond angle,
and the dihedral angle potentials between atoms. In addition to the bonded inter-
actions, force fields also contain non-bonded interactions. Non-bonded interactions
act between atoms in the same molecule and those in other molecules. Force fields
usually divide non-bonded interactions into two: electrostatic interactions and Van
der Waals interactions.
Molecular dynamics (MD) simulations are one example used to model molecular
processes involving a larger group of atoms, such as drugs, biological compounds
[65], carbohydrates [66–69], proteins, membranes [68, 69], nucleic acids, and poly-
mers [43–45]. On the MD simulations studies, usually, to analyze the polymer-drug
interactions the root means square deviation (RMSD) [43], solvent accessible surface
area (SASA) [43], interaction energy [44, 45], number of contacts [46], and number
of hydrogen bonds [46, 47] are the main choices. With these analyses, it is possible to
study the polymer behavior in the presence and absence of the drug, verify the drug
exposition and interaction to the solvent, verify the drug affinity for the polymer, and
calculate the interaction number of contacts between the polymer and drug. The study
of polymer-drug interactions requires a comprehensive knowledge of the phenomena.
The computational methods, mainly MD simulations allow the study of these systems
at the atomistic scale. With this method it is possible to verify some important features
on the polymer-drug interaction, solving problems that experimental studies are not
able to.
152 L. S. Reinhardt et al.

3.3 Current Achievements in Bionanocomposites for In Situ

Drug Delivery

Recently, several researchers are developing bionanocomposites for in situ drug

delivery aiming to address issues caused by systemic CT (Table 2). A significant
drawback in cancer therapy is that 90% of patients with cancer die from cancer
metastasis [75, 76]. Metastasis is a complex multistep process comprising tumor cells
release and their proliferation at a secondary site [77, 78]. Nowadays, nanotechnology
has been addressing this issue with controllable and functionalized structures able
to recognize circulating tumor cells (CTCs) [79, 80], nevertheless few products are
able to induce in situ CT in blood circulation. Chunmiao Liu and colleagues (2019)
developed a multifunctional nanocomposite by conjugating FePt NPs onto the surface
of Fe3 O4 @SiO2 NPs, which, in tumor pH, was able to release Fe2+ catalyzing reactive
oxygen species (ROS) formation and inducing ferroptosis in tLyP-1 receptor-positive
CTCs such as MCF-7, HeLa, HepG2, and 4T1. Thus, this system could effectively
separate and capture CTCs suggesting it as a promising approach for in situ cancer
treatment [73].
Angiogenesis displays a critical role in tumor proliferation and development in
which, vascular endothelial growth factors regulate the development of new blood
vessels and act binding tyrosine kinase receptors including vascular endothelial
growth factor (VEGF) receptor 1 and 2 [81, 82]. As expected, these receptors are
overexpressed in several tumors and have been studied as a target for treatments
[83, 84]. Recently, Carenza et al. developed PLGA nanocapsules containing super-
paramagnetic iron oxide NPs (SPIONS) and VEGF165 in a polymeric shell able to

Table 2 Bionanocomposites for in situ drug delivery

System Drug Outcome References
Silica-calcium phosphate Cisplatin Lower tumor growth in the rats [70]
nanocomposite (SCPC75) discs treated with SCPC75-cisplatin
discs when compared to
systemic cisplatin
CuS nanoparticles-graphene DOX 75% cancerous cells death after [71]
oxide with polyethylene glycol irradiation at NIR laser
(PEO-GO/CuS)
Albumin, PLGA and magnetic 5-Fluorouracil Reduction of tumor size [72]
nanoparticles with
diphenylhexatriane
FePt nanoparticles – Ferroptosis induction in tLyP-1 [73]
receptor-positive CTCs
PLGA nanocapsules with VEGF165 Improved accumulation in the [74]
SPIONs tumor cells
CuS copper sulfide; DOX dooxorubicin, PLGA poly (lactic-co-glycolic acid), FePt Iron–platinum,
CTCs circulation tumor cells, SPIONs superparamagnetic iron oxide nanoparticles
Bionanocomposites for In Situ Drug Delivery … 153

accumulate in the tumor cells [74], showing that magnetic bionanocomposites could
be used in targeted and controlled drug delivery.
Multifunctional nanocomposites can be produced as biofunctionalized drug
delivery systems that combine the drug transport with biomolecules such as proteins,
enzymes, antibodies, nucleic acids, and membrane receptors [85]. Biomolecules are
not able to penetrate some body barriers such as the brain-blood barrier (BBB), which
creates the necessity of novel drug delivery routes for solid tumors treatment.

3.3.1 Injectable Hydrogels

Photodynamic therapy (PDT) stands for a therapeutic that sensitizes cells to a partic-
ular wavelength of light. The radiation and the photosensitizer (PS) are inoffensive
when used separately however when combined, they can form several ROS and
trigger local inflammation, cell apoptosis, and localized ischemia. Therefore, PDT
is very effective as cancer in situ therapy [86]. Recently, Luo and coworkers (2019)
established a micellar thermosensitive nanocomposite gel for intra-tumoral two-
photon PDT by developing methoxy PEG-polylactide copolymer (mPEG-PDLLA)
micelles loaded with PS and two-photon absorption compound (T1). The liquid char-
acteristic of the micelles enabled an in situ gelification after injection maintaining
the composite into the breast cancer tumor model and it was shown that the radiation
promoted deep tissue penetration and enhanced tumor inhibition in around 50% [87].
Another study aiming PDT developed an injectable nanocomposite hydrogel with
chitosan micelles loaded with paclitaxel (PTX) and PEGylated gold nanorods to treat
local hepatocellular carcinoma by using xenograft tumor model [88]. After laser
irradiation, the photothermal-induced damage was restrained to the tumor without
damaging the adjacent healthy tissue and the sustained release of PTX enhanced
the antitumor effect of treatment prolonging mice survival compared to PDT alone
[88]. These studies indicate that photothermal-CT associated with nanocomposites
present improved antitumor effects by suppressing tumor recurrence and prolonging
survival.
Glioma is a common central nervous system tumor that presents no significant
improvement in therapy in the past years, therefore, the average of patients’ survival
sits still around 14 months [89]. The challenges in drug delivery for this type of
cancer include the existence of the BBB that provides an impediment to standard
CT routes of delivery [90]. It was confirmed by Bodell et al. [91] that in situ drug
delivery enhances by 113 times the drug concentration when compared to intra-
venous injection. By exploiting this approach, Ding and colleagues (2017) devel-
oped a nanocomposite thermoresponsive hydrogel using mPEG-dipalmitoyl phos-
phatidylethanoiamine (mPEG-DPPE) calcium phosphate NP and the double emul-
sion method for PTX and temozolomide (TMZ) delivery. Their results suggest that
the autophagy induced by the combination of the drugs regulated tumor cell apoptosis
and that the local delivery of the gel on C6 tumor-bearing rats improved significantly
the treatment antitumor efficacy [92].
154 L. S. Reinhardt et al.

An injectable hydrogel system based on the polypseudorotaxane development

between cationic mPEG-b-PCL-b-poly(ethylene imine) (PEI) and α-cyclodextrin (α-
CD) was developed for the delivery of plasmid DNA, more specific the Nur77 gene.
Given the addition of cyclic α-CD, this biocomposite could form solid hydrogels
in situ, owing to the poly pseudorotaxane establishment between α-CDs and PEG.
The authors showed that the controlled drug release was able to improve the tumor
resistance in vivo [93].
Even that hydrogel studies seem promising for local therapy of cancer, there
are some drawbacks to their application. If solid, the samples are usually very
stiff and this makes the administration challenging in soft tissues and if liquid, the
variable release and distribution could trigger side effects in healthy cells. Thus,
different approaches are required for improving biocomposite hydrogels or intro-
ducing malleable materials with enhanced release properties such as nanofibrous
drug delivery systems.

3.3.2 Electrospinning

The electrospinning method can be used for fibrous bionanocomposites as nanofibers

(NF) combined with nanocarriers. The equipment consists of a conductive collector
plate, a pump, a high voltage power apparatus, a tip needle, and a syringe containing
the polymeric solution [37]. When the solution is loaded in the equipment, the high
voltage is applied and the drop in the edge of the needle is transformed into a
Taylor cone producing nanofibrous material jetted in the collector plate. By using
this method, some features of the nanoproduct can be modulated such as diameter
and drug release rate by choosing the adequate properties of the polymeric solution
composition as polymeric concentration, viscosity, elasticity, conductivity, and the
distance between the collector and the needle [38].
Generally, electrospinning is used for producing NF blends of drugs and polymers,
however, it can be used to fabricate fibrous nanocomposites. These systems are
constructed by the incorporation of other nanocarriers including nanoparticles (NP),
vesicles, and micelles into the NF (Fig. 3). Table 3 shows several recent studies with
promising NF for cancer applications.
It is known that the simple incorporation of drugs into nanocarriers commonly
leads to burst drug release and to overcome this disadvantage the introduction of
these nanocarriers into NF can expand the applicability of nanocarriers as cancer
drug delivery, improving their release rate, stability, and acute toxicity. Lately, NF
systems aiming at local drug delivery for both tumor therapy and prevention have
been significantly studied by researchers worldwide. Table 4 presents an overview
of studies focused on the design and evaluation of fibrous bionanocomposite in vivo.
Within these studies, the treatment was focused on solid tumors therapy. These NF
can increase the specificity of drugs associated with the tumor microenvironment
and given their release properties tumor recurrence can be diminished.
One of the most frequent forms of gynecological tumors is cervical cancer,
presenting an incidence rate of 25 years in young adults [103]. This type of tumor is
Bionanocomposites for In Situ Drug Delivery … 155

Fig. 3 Fibrous nanocomposites scheme constructed by the incorporation of nanocarriers into

nanofibers

developed as a consequence of oncogenic types of human papillomavirus infec-

tion [104] and regardless of the mortality and incidence rates are considerably
decreasing over the last few years in developed countries, patients with stage IV
tumor have a survival rate of only 5 years occasioning in more than 270,000 deaths
each year around the world [105]. The cervical cancer therapy depends on the cancer
stage varying from surgical resection and RT at stages I to III and CT as a pallia-
tive treatment of stage IV (advanced stage or recurrent tumor). Amid the drugs
used for the therapy the most common drugs are cisplatin (Pt), ifosfamide, doxoru-
bicin (DOX), and sodium dichloroacetate (DCA) [106]. Albeit platinum compounds
present high efficacy against cervical carcinoma, their toxicity on healthy tissues
and their systemic toxicity is a drastic therapy problem. To overcome this issue,
Zhang and colleagues [107] produced a system by using polyvinyl alcohol (PVA)
NF loaded with DCA and Pt(IV)-backboned micelles [107]. Firstly, DCA release
induced cell death by apoptosis through the inhibition of pyruvate dehydrogenase
kinase and the micelles could highly accumulate in tumor cells via enhanced perme-
ation and retention effect. In tumor pH, Pt(IV) is reduced to Pt(II) that is its active
form which can cause DNA crosslinks and consequently inhibition of DNA replica-
tion. The nanoproduct presented improved in vitro cytotoxicity against HeLa cells
when the two drugs were combined showing their synergistic effect. The in vivo
evaluation was performed by using Kumming mice injected with the cervical cancer
cell line U14 in the armpit, and the results showed an enhanced drug accumula-
tion on the tumor of mice treated locally with NF when compared to intravenously
treated mice. The tumor volume analyses after 16 days of treatment revealed that
micelles-DCA-NF reduced tumor volume when compared to single-drug NF and
intravenous injection. This study showed the importance of local treatment with
synergic drug effects implying that this implantable bionanocomposite could be
useful for successful therapy reducing system risks [107].
156 L. S. Reinhardt et al.

Table 3 Fibrous bionanocomposites for in situ drug delivery

System Drug Cancer Outcome References
Core-shell NF – SKOV-3 After the [94]
Core: iron oxide nanoparticles ovarian cancer application of
Shell: PS and collagen NF cell line an AMF during
10 min to the
mats, the cancer
cells deposited
on the
Fe3 O4 -in-fibers
were eliminated
Core-shell NF DOX B16F10 Higher cell [95]
Core: cobalt ferrite and melanoma cell death was
titanium oxide nanoparticles line observed when
Shell: DOX-loaded chitosan hyperthermia
NF and
chemotherapy
were combined,
achieving a
synergistic
effect, which
enhances the
cytotoxic effect
and allows a
reduction of
side effects
Core-shell NF 5-Fluorouracil and – The release [96]
Core: vesicles of CTAB and paenolum investigations
SDBS with 5-Fluorouracil concluded that
and paenolum the hydrophilic
Shell: PEO drug was
released in an
increased
manner when
the molar ratio
of CTAB/SDBS
was higher
Core-shell NF 5-Fluorouracil – The different [96]
Core: three different vesicles drug delivery
composed of systems showed
didodecyldimethylammonium different release
bromide, CTAB/SDBS (7/3) rates and
and CTAB/SDBS (3/7) pH-responsive
introduced into chitosan and behaviors
sodium alginate nanocapsules
Shell: PEO
Core-shell NF Rhodamine-B L929 murine Reduction in the [97]
Core: pluronic (F127) fibroblast cell release rate and
vesicles line increased cell
Shell: PCL NF viability
(continued)
Bionanocomposites for In Situ Drug Delivery … 157

Table 3 (continued)
System Drug Cancer Outcome References
Electrospun of a mesh using SN-38 HT-29 Decreased cell [98]
10% of a hydrophobic colorectal viability
PGC-C18 and 90% of PCL cancer cell
loaded with the SN-38 line
Core-shell NF 5-Fluorouracil and HepG-2human Decreased cell [99]
Core: colloidal structures cefradine liver cancer viability
formed by a block copolymer cell line
composed by mPEG-PLA
with 5-Fluorouracil and
cefradine
Shell: chitosan and PEO
Core-shell NF DOX and HeLa cell line Increased [100]
Core: silica and hydroxycamptothecin thermal stability
hydroxyapatite nanoparticles (topoisomerase and inhibited
containing DOX and inhibitor) tumor cells
hydroxycamptothecin growth
Shell: PLGA
Core-shell NF DOX HeLa cell line Sustained and [101]
Core: silica nanoparticles prolonged
with DOX release and
Shell: PLLA higher antitumor
efficacy
TMZ and polycaprolactone TMZ U-87 MG The [102]
diol combined with gold glioblastoma nanoformulation
nanoparticles cell line achieves a
greater cell
death overtime
in contrast with
free TMZ (25%
more)
NF nanofiber, PS polystyrene, AMF alternating magnetic field, DOX dooxorubicin, CTAB
cetyl trimethylammonium bromide, SDBS sodium dodecylbenenesulfonate, PEO polyethylene
glycol, PCL polycaprolactone, PGC poly(glycerol monostearate-co-ε-caprolactone, mPEG-PLGA
methoxypoly(ethyleneglycol)-block-poly(l-lactide), PLGA poly (lactic-co-glycolic acid), PLLA poly-
l-lactic acid, TMZ temozolomide

Another study with cervical cancer focused on CT in combination with hyper-

thermia by producing PLLA NF loaded with DOX and multi-walled carbon
nanotubes (MWCNT) [108]. During the in vitro drug release studies it was found
that when near-infrared irradiation was applied, DOX was released in a burst rate in
both acid and physiologic pH, however, when the radiation was stopped DOX release
returned to slow mode, this behavior was explained by the relation between polymer
mobility and the diffusion rate of DOX. By using Kunming mice implanted with
U14 cells, this effect was also observed in vivo after 48 h of NF implantation into the
tumor and irradiation treatment, moreover, it was detected that DOX accumulated
in the tumor tissue more than in other vital organs. Finally, the in vivo antitumor
effect of the nanocomposite was assessed by placing the material directly on the
158 L. S. Reinhardt et al.

Table 4 Overview of studies focused on the design and evaluation of fibrous bionanocomposite
in vivo
System Drug Cancer Outcome References
Core-shell DCA, Pt(IV) Cervical In vivo release showed 58% [107]
Core: DCA and of Pt released during
Pt(IV)-micelles 30 min, 90% of Pt released
Shell: PVA NF during 24 h and the tumor
volume on day 15 was
found to be half of the
untreated control group
tumor size
Core-shell DOX Cervical Enhanced DOX release [108]
Core: DOX and MWCNTs after irradiation and
Shell: PLLA NF preferable accumulation of
DOX in the tumor than in
vital organs
Core-shell DOX Breast Drug concentration [111]
Core: PVA micelles loaded increased in the tumor site
with DOX and activefolate after 48 h (18 μg/g) when
groups on the surface compared to free DOX
cross-linked with gelatin (2 μg/g) and other organs;
Shell: PCL/PEO NF-treated animals
presented a significate
increased survival rate
Thixotropic silk NF DOX Breast The system was capable of [112]
hydrogels loaded with solidifying at a specific site
DOX and releasing to certain pH
conditions. The in vivo
studies: at the fifth week,
showed significant
differences in the volume
and weight of the tumor
treated with DOX-loaded
hydrogels compared to free
DOX
DCA sodium dichloroacetate, Pt(IV) platinum(IV), NF nanofiber, DOX dooxorubicin, MWCNTs
multi-walled carbon nanotubes, PLLA poly-l-lactic acid, PVA polyvinyl acid, PEO polyethylene
glycol, PCL polycaprolactone

tumor site and after 24 h of implantation the site was irradiated and as expected the
MWCNT-DOX NF were effective against cancer cells, nevertheless, cells detected
profound inside the tumor presented a certain resistance. Analyzing this study, it was
possible to conclude that CT and PDT using MWCNT-DOX-loaded PLLA fibrous
composite could have a synergic effect on antitumor activity [108].
A critical type of cancer in relation to mortality worldwide is breast cancer. This
type of disease is the leading cause of women’s deaths related to cancer, with more
than half a million deaths [105] and nearly 2.5 million new cases in 2015 [109]. One
important characteristic of this cancer is that the recurrence rate within 10 years can
Bionanocomposites for In Situ Drug Delivery … 159

reach 15% even after resection [110]. The standard therapy consists of mastectomy,
RT, systemic CT, or hormonal blockade. However, systemic treatment is associated
with severe cardiotoxic effects.
Aiming to diminish DOX side effects and reduce the risk of recurrence, Yang and
colleagues [111] produced a complex active-targeting system. Firstly, DOX micelles
were developed by using PCL-PEO copolymer with active folate groups on their
surface. Then, core-shell NF were produced by coaxial electrospinning where the
micelles were mixed with PVA to form the core phase and a cross-linked gelatin
solution was used as the shell. After NF implantation, the micelles were released
from the NF as a consequence of polymeric matrix degradation, then they were able
to penetrate into the tumor cells and undergo endocytosis throughout binding to the
folate receptors on the cells’ membranes. To perform in vivo biodistribution and
antitumor activity, 4T1 tumor-bearing Balbc nude mice were used. The distribution
results showed a strong DOX accumulation at the tumor when compared to other
organs such as heart, lung, kidney, and liver, suggesting that this bionanocomposite
is an effective and safe system for DOX delivery. The antitumor activity evaluation
compared two different deliveries: (a) intravenous injection of free DOX or micelles
and (b) implantation of NF near the tumor. The NF exhibited a pronounced tumor
volume reduction after 21 of implantation when compared to the other groups, in
addition, the NF-treated mice presented increased body weights and survival rates,
and therefore this system presents significant improvements to the breast cancer
therapy [111].
It is known that most frequent actions for in vitro release studies embrace:
(a) cumulative release that occurs when a compound is released into the same
amount of media volume and;
(b) non-cumulative release that occurs when there is a continuous replacement of
the media mimicking a living organism where the drug concentration drops.
Related approaches are used in in vitro biological analysis focus on cytotoxic
experiments on different cell lines. Though experimental conditions of in vitro eval-
uations simulate those of living organisms, for a system as bionanocomposites to be
acknowledged as suitable and safe for local antitumor treatment, in vivo investiga-
tions are still essential. Unfortunately, only a few studies tested its bionanocomposites
products in vivo, therefore the local application is still a challenge given the invasive
procedures and the lack of in vivo evaluations.

4 Final Considerations

Bionanocomposites show great properties related to effective therapy of cancer such

as increased thermal stability, enhanced tensile strength, reduced side effects and
improved targeting. Drug delivery obtained by in situ approaches has the prospective
to overcome the CT limitations by their capacity to increase drug concentration in the
tumor and in the same instance decreasing the damage to the healthy tissue. Though
160 L. S. Reinhardt et al.

bionanocomposites may enable the efficacy of individualized drug delivery, issues

such as the difficulty of testing the scaffolds and to develop surfaces that are able to
maintain an appropriate environment may affect the drug distribution. It is necessary,
in the future of bionanocomposites research, for a collaboration among researchers,
regulatory agencies, and industry to ensure that novel approaches are safe and effec-
tive and will be provided to the society. Further development in bionanocomposite
technology may prove potent, safe, controlled, and targeted drug delivery.

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Tumor Microenvironment
and Intracellular Signal-Activated
Nanocomposites for Anticancer Drug
Delivery

Yilan Huang, Yiheng Huang, Yuefei Zhu, Xiaowen Zhu, and Zhiqing Pang

1 Introduction

Malignant tumors have become a major disease threatening human health worldwide.
The World Health Organization’s statistical survey indicates that about 8.8 million
people die of cancer each year worldwide. In 2010, the total cost of cancer on the
global issue was about 1.16 trillion U.S. dollars, and this figure continues to grow.
Research on cancer treatment has gradually attracted people’s attention. The current
clinical therapeutic methods of tumors mainly include surgery, chemotherapy, radi-
ation therapy, and biological therapy, among which chemotherapy is very common
and important [1]. However, as most chemotherapeutic drugs lack selectivity for
normal cells and tumors, they not only affect the growth and proliferation of normal
cells when killing tumor cells, but also are hardly accumulated in tumor tissues to
achieve effective drug concentrations.

Y. Huang · Y. Huang · X. Zhu · Z. Pang (B)

Key Laboratory of Smart Drug Delivery, School of Pharmacy, Ministry of Education, Fudan
University, 826 Zhangheng Road, Shanghai 201203, China
e-mail: [email protected]
Y. Huang
e-mail: [email protected]
Y. Huang
e-mail: [email protected]
X. Zhu
e-mail: [email protected]
Y. Zhu
Department of Biomedical Engineering, Columbia University Medical Center, 3960 Broadway
Ave, New York, NY 10032, USA
e-mail: [email protected]

© Springer Nature Singapore Pte Ltd. 2021 167

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_8
168 Y. Huang et al.

In recent years, with the rapid development of nanotechnology, the unique advan-
tages of nanocomposites in the field of targeted tumor therapy have become increas-
ingly prominent. Due to their special size effect (1–100 nm), quantum effect and
surface effect, nanocomposites could enhance aqueous solubility, stability, bioac-
tivity, and bioavailability of drugs, and reduce their adverse toxicities [2]. Diverse
classes of nanocomposites have been developed for improved anticancer drug
delivery, including polymeric nanoparticles, liposomes, micelles, polymersomes,
mesoporous silica nanoparticles, dendrimers, and inorganic nanoparticles made of
iron oxide, quantum dots, gold, or metal oxide frameworks. Although much progress
has been achieved in preclinical animal or human models, the majority of nanocom-
posites for anticancer drug delivery still suffers from their limitations, such as fast
blood clearance, premature drug release in circulation, nontargeted accumulation
in healthy tissues, poor tumor penetration capability, and insufficient drug release
in tumor cells [3]. In order to resolve these limitations, maximizing the thera-
peutic efficacy and minimizing the side effects of loaded drugs, smart nanocom-
posites responding to environmental stimuli have been developed. These stimuli-
activated nanocomposites are equipped with environmental sensitive modalities
within their structures and can make responses to specific environmental stimuli
(such as tumor microenvironment/intracellular signals) to change their chemical
or physical properties, exhibiting greater potential in anticancer drug delivery than
traditional nanocomposites [4].
The tumor microenvironment (TM) refers to the normal cells, molecules, and
blood vessels that surround and feed tumor cells, including surrounding blood vessels,
immune cells, fibroblastic cells, bone marrow-derived inflammatory cells, lympho-
cytes, signaling molecules and the extracellular matrix (ECM) composed of collagen
and proteoglycans [5]. It is an integral part of cancer since it supplies a nurturing
environment for the malignant process and contributes to cancer development and
progression by affecting the complex interplay of genetic and epigenetic changes
within the cells themselves [6]. Compared with normal tissues or cells, abnormal
physiologic conditions exist in the TM such as acidic extracellular pH, high concen-
trations of specific enzymes (matrix metalloproteinases, hyaluronidase, cathepsin
B, legumain, etc.), hypoxia, extra- and intracellular glutathione (GSH) and adeno-
sine triphosphate (ATP) gradients, and excessive amounts of reactive oxygen species
(ROS). Based on a well understanding of the TM, the prominent distinctions between
tumor sites and normal tissues have been utilized as stimuli to trigger specific changes
in stimuli-activated nanocomposites, thus enhancing the therapeutic efficacies and
reducing the side effects of chemotherapeutics.
In this chapter, we will provide a comprehensive outline of the tumor microenvi-
ronment (Table 1) and intracellular signal-activated nanocomposites for anticancer
drug delivery (Table 2) and try to provide an in-depth look at the design of these drug
delivery systems to maximize their benefits for cancer treatment.
Tumor Microenvironment and Intracellular Signal-Activated … 169

Table 1 A summary of tumor microenvironment and intracellular signals

Signals Details References
Low pH Inflammation-associated acidic pH: 7.2–6.5 for [7]
extracellular pH in tumor
Over-expressed enzyme Matrix metalloproteinases, hyaluronidase, cathepsin B, [8]
urokinase plasminogen activator, β-galactosidase,
legumain, secretory phospholipase A2
Glutathione Intracellular environment: 0.5–10 mM; extracellular [3]
environment: 2–20 μM
Hypoxia Hypoxia (low oxygen levels in tissues): regions with [9]
low oxygen concentration often exist in solid tumors
H2 O2 Excessive amounts of H2 O2 have been observed in [10]
various cancer cells
ATP Intracellular environment: 1–10 mM; extracellular [11]
environment: <0.4 mM

2 Tumor Microenvironment and Intracellular Signal

2.1 The Acidic pH

It is well-known that pH values vary significantly in different tissues and cellular

compartments, and in disease states, such as ischemia, infection, inflammation,
and tumorigenesis [12] (Table 1). Studies over the last few decades have demon-
strated that the pH value of the tumor extracellular environment is maintained with
a range from 6.5 to 7.2 [13], whereas that in blood circulation and normal tissues is
around 7.4 [7]. The pH decrease is mainly a consequence of enhanced glycolysis and
plasma membrane proton-pump activity of tumor cells, both in aerobic and anaer-
obic conditions, making tumor cells produce more lactic acid than normal cells and
then leach out the acid to the surrounding environment [14]. The high production of
acidic metabolites, termed as the Warburg effect, provides a growth advantage for
tumor cells in vivo [13]. Besides, much lower pH also exists in tumor intracellular
microenvironment: the pH values decrease to the range 5–6, 4–5, and around 6.4 in
endosomes, lysosomes, and Golgi apparatus, respectively [15].

2.2 The Over-Expressed Enzyme

Enzymes participate in a variety of physiological and pathophysiological processes

and accelerate biochemical reactions by lowering their activation energy. Compared
to normal tissues, a great number of enzymes are overexpressed in pathological condi-
tions, such as cancer and inflammation (Table 1). The altered expression of specific
170 Y. Huang et al.

enzymes in tumors is linked to several key events, including tumor progression,

growth, neovascularization, intravasation, extravasation, and metastasis [16].
Matrix metalloproteinases (MMPs), especially MMP2 and MMP9, have been
observed in almost every type of human tumors. The MMPs family is comprised
of at least 23 structurally related, zinc-dependent extracellular matrix remodeling
proteases [8a]. These endopeptidases play essential roles in a number of pathologic
processes, including proteolytic degradation of ECM, alteration of the cell-cell and
cell-ECM interactions, cell migration, and tumor angiogenesis [17]. Elevated cellular
level of hyaluronidase (HAase), a kind of enzyme that can catalyze the degradation
of hyaluronic acid through preferential cleavage of glycosidic linkages, has been
particularly documented in the prostate, urinary bladder, breast, as well as laryn-
geal cancers [8b]. The effect of its overexpression is still not clear now, and a few
reports suggest that it may be involved in the metastatic process [18]. Cathepsin B
is a lysosomal cysteine protease that is involved in various pathologies and onco-
genic processes in humans. Upregulation of cathepsin B is found in a wide variety
of human cancers and often induces secretion into the ECM [8c]. Urokinase plas-
minogen activator (uPA), another important protease to degrade fibrin, is closely
associated with tumor growth, invasion, and metastasis in a wide range of tumors,
including glioma [19]. It has been demonstrated to be highly expressed in tumors
(for instance, glioma neo-vascular cells and glioma cells, especially in the leading
edge of glioma [20]), about 4–10 folds higher than that in normal tissues [21]. Other
enzymes overexpressed in human malignant tumors include urokinase plasminogen
activator, β-galactosidase, legumain, secretory phospholipase A2 and so on [8d−g].

2.3 The Reductive Environment

Glutathione (γ-glutamyl-cysteinyl-glycine; GSH) is the most abundant low-

molecular-weight biological thiol [22]. As an important antioxidant, GSH is involved
in protecting cellular components from the noxious effect of reactive oxygen species,
such as free radicals, peroxides, lipid peroxides, and heavy metals. It is also capable
of forming disulfide bonds with cysteine residues of proteins, and participating in
nonenzymatic conjugation with some chemicals [23]. Many evidence has shown
that there is a large GSH concentration gradient between the intracellular environ-
ment and the external matrix of tumor tissues (Table 1). Generally, inside cells,
the GSH concentration could be up to 0.5–10 mM, while only around 2–20 μM
in extracellular environments [3]. In addition, studies have shown that the intracel-
lular concentration of GSH in tumor tissues is at least four fold higher than that in
the normal cells [24], and it is often elevated in multidrug resistance (MDR) cancer
cells. GSH/glutathione disulfide (GSSG) is the major redox couple in human cells and
determines the anti-oxidative capacity of human cells. GSH/GSSG is kept reduced
by NADPH and glutathione reductase. The intracellular concentration of GSH is
also dependent on other redox couples such as NADH/NAD+ , NADPH/NADP+ ,
Tumor Microenvironment and Intracellular Signal-Activated … 171

and thioredoxinred/thioredoxinox [25]. Moreover, the endo/lysosome is also redox-

active. It also possesses a high reducing potential controlled by a specific reducing
enzyme gamma interferon-inducible lysosomal thiol reductase (GILT) in the co-
presence of L-cysteine, not GSH [26]. The redox-active lysosome also contains
low-mass iron. The iron is kept in a reduced state (Fe2+ ) by the concentration of
thiols.

2.4 The Hypoxia

Hypoxia, defined as a state of reduced tissue oxygen availability or decreased oxygen

partial pressure, is a common characteristic of the tumor microenvironment [27]
(Table 1). To grow beyond a diameter of approximately 1 mm, newly developing
tumors must form their own vascular network and blood supply, which they accom-
plish either by incorporating preexisting host vessels or by forming new microvessels
through the influence of tumor angiogenesis factors. However, the newly formed
blood vessels in tumors are often highly abnormal, with dilations, incomplete or
absent endothelial linings and basement membranes, leakiness, irregular and tortuous
architecture [9]. Tumor hypoxia is generated when a tumor rapidly outgrows its blood
supply, leaving portions of the tumor with regions where the oxygen concentration
is significantly lower than in healthy tissues [28]. Emerging evidence has indicated
that the hypoxic response is a series of hypoxia-induced proteomic and genomic
changes, and primarily driven by the transcription factor hypoxia-inducible factor 1
(HIF-1) [29].
Many clinical studies have demonstrated that tissue oxygen partial pressures,
which are measured from those cancer patients, are near zero mm Hg, which is
significantly lower than that in normal tissue (about 30 mm Hg) [27]. Hypoxia is
a negative factor for anticancer therapy. It has been shown that hypoxia is strongly
associated with tumor propagation by enhancing MDR in cancer as well as malignant
invasiveness and metastasis. Under hypoxia conditions, tumor cells divide more
slowly than that in oxygen-rich environment, which makes these tumor cells less
susceptible to traditional antiproliferative drugs that target rapidly dividing cells.
In addition, tumor cells undergo genetic alterations that allow them to survive and
grow in the hypoxic environment, such as local invasive growth, perifocal tumor
cell spreading, and distant tumor cell spreading. However, hypoxia is rarely seen in
normal tissue, indicating it is a primary target in the development of anticancer drug
delivery.

2.5 Reactive Oxygen Species

Studies have shown that compared with normal cells, tumor cells can constantly
produce high levels of reactive oxygen species (ROS), such as hydrogen peroxide
172 Y. Huang et al.

(H2 O2 ), hydroxyl radicals (•OH), and superoxide anion (O•2− ), which are derived
from by-products of aerobic metabolism due to oncogenic transformation (Table 1).
Many evidence shows that tumor cells suffer from a higher level of ROS than normal
cells due to the fast proliferation and enhanced metabolic rate of tumor cells. In
addition, MDR cells show even higher ROS levels than their wild-type cells. It is
because MDR cells need more energy to elevate mitochondrial activity for the over-
expression of membrane transporters [30]. ROS is a double-edged sword. It can
stimulate the growth and proliferation of tumor cells, and have genetic instability
and senescence evasion [10]. ROS also play an important role in maintaining tumor
phenotype. However, producing excessive ROS will cause cellular damage, such
as lipid peroxidation, protein oxidation, DNA adduct formation, and so on. These
damages can finally cause cell death by apoptosis or necrosis [31]. H2 O2 is a major
component of ROS and is a common marker of oxidative stress, which can induce
apoptosis of many tumor cells. Hydroxyl radical is far more reactive than H2 O2 and
O•2− , and can also mediate intracellular cytotoxicity [31].

2.6 The ATP Gradient

It is well-known that ATP, referred to as the “molecular unit of currency” of intracel-

lular energy transfer, participates in various kinds of metabolic processes. Recently,
researches revealed that ATP has a very high concentration (approximately 1–10 mM)
in intracellular cytosol, much greater than that in the extracellular environment (below
0.4 mM) (Table 1) [11]. Such a prominent difference inspires researchers to exploit
ATP as a new trigger for the controlled release of anticancer drugs both in vitro and
in vivo [32].

3 Tumor Microenvironment and Intracellular

Signal-Activated Nanocomposites for Anticancer Drug
Delivery

3.1 pH-Activated Nanocomposites for Anticancer Drug

Delivery

The acidic pH of the extracellular and intracellular environment of tumors is an

appropriate internal trigger to control the release of anticancer drugs from pH-
activated nanocomposites in tumor tissues and/or tumor cells (Table 2). Generally,
pH-activated nanocomposites can be designed by incorporating pH-labile chem-
ical bonds into the polymer components or the polymer-drug conjugates, such as
acetal, hydrazone, cis-acotinyl, orthoester, b-carboxylic acid amide, and glycerol
ester groups [3]. These nanocomposites are quite stable under neutral conditions,
Tumor Microenvironment and Intracellular Signal-Activated … 173

Table 2 A summary of representative TM and intracellular signal-activated nanocomposites for

anticancer drug delivery
Stimulus Nanocomposites Disease/Application References
pH Polydopamine-coated mesoporous silica Colorectal cancer [33]
nanoparticles
Nanoparticle composed of Prostate cancer [34]
pH-responsive
PEGylated polymer and cell-targeting
and -penetrating peptide-amphiphile
Nanoparticle constructed by assembly of Pancreatic cancer [35]
platinum-prodrug conjugated
polyamidoamine dendrimers
Micelles composed of poly Non-small cell lung [36]
(lysine)-b-polycaprolactone and poly cancer
(glutamic acid)-g- methoxyl poly
(ethylene glycol)
Nanoparticle with poly(ethylene Breast cancer [37]
glycol)-b-poly(2-(hexamethyleneimino)
ethyl methacrylate) diblock copolymer
Enzyme PbS/CdS/ZnS core/shell/shell quantum Colon cancer [38]
dots
Nanoparticle comprising Breast cancer [39]
MMP-cleavable octapeptide
(Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln)
Nanoparticle developed from MMP2 Melanoma [40]
and MMP9-sensitive copolymers and
folate receptor targeted copolymers
Hyaluronic acid-conjugated mesoporous Colon cancer [41]
silica nanoparticle
Polycation/DNA complexes coated with Liver cancer [42]
PEGylated hyaluronic acid
Rotaxane-modified MSNs based on Cervical cancer [43]
alkoxysilane tether, α-cyclodextrin and
multifunctional peptides
Nanoparticle comprising human serum Pancreatic cancer [44]
albumin and cathepsin B cleavable
peptide
Reduction/GSH Thiolated silica nanoparticle Hepatocellular [45]
carcinoma
Poly(amido amines) nanoconjugate Liver cancer [46]
H2 O2 Nanoparticle comprised of Photodynamic [47]
semiconducting polymer core and therapy
manganese dioxide (MnO2 ) shell
Thioether-bridged dendritic mesoporous Breast cancer [48]
organosilica nanoparticle
(continued)
174 Y. Huang et al.

Table 2 (continued)
Stimulus Nanocomposites Disease/Application References
ATP Nanocomplex based on ATP-responsive Lung [49]
aptamer duplex adenocarcinoma
Polypeptide-wrapped Real-time [50]
mesoporous-silica-coated nanoparticle monitoring
pH and GSH PEG-SS-PTMBPEC micelles Cervical cancer [51]
Amphiphilic PCL-PDEA and Cervical cancer [52]
MPEG-SS-PCL mixed micelles

but they can rupture at acidic pH to release their payload rapidly because of the
hydrolysis of these bonds. Zheng et al. designed mussel-inspired polydopamine
(PDA)-coated mesoporous silica nanoparticles (MSNs) as pH-responsive nanoparti-
cles (MSNs@PDA) [53]. MSNs with a large pore diameter were synthesized by using
1,3,5-trimethylbenzene as a pore-expanding agent and were modified with a PDA
coating by virtue of oxidative self-polymerization of dopamine in neutral pH. The
tailored mesoporous structure, high surface area, and tunable pore size of MSNs gave
them significant advantages in stimuli-activated controlled drug delivery [54]. Mean-
while, PDA coating worked as the “gatekeepers” to block the drugs inside MSNs at
neutral pH and trigger their release at lower pH. In order to evaluate the drug release
profiles, doxorubicin (DOX) was loaded in MSNs@PDA (MSNs-DOX@PDA). It
was shown negligible release of DOX from MSNs-DOX@PDA was found in pH
7.4 PBS. Decreasing the pH value of the release medium could increase the release
rate of DOX from MSNs-DOX@PDA. In addition, the color of the supernatant solu-
tion turned brown in acidic PBS but not in neutral one, which agreed with the PDA
coating’s peeling off from the surface of MSNs in an acidic environment.
RNA-based therapeutics, such as small-interfering RNAs (siRNAs), microRNAs
(miRNAs), and antisense oligonucleotides (ASOs), have shown great potential in
targeting a large part of the currently undruggable genes or gene products in
cancer therapy [55]. However, existing challenges lie in the delivery of RNAi
molecules such as siRNAs to diseased sites in vivo [56]. Recent years have
witnessed developments in RNAi molecule delivery systems in nanooncology,
especially nanoparticles encapsulating siRNA [57]. Xu et al. fabricated a pH-
responsive multistage nanoparticle platform for effective and systemic targeted
siRNA delivery to tumor cells [34]. This nanoparticle platform consisted of a PEG
surface shell, a hydrophobic polymer poly(2-((hexamethyleneimino)ethyl methacry-
late) (PHMEMA) core which was sensitive to tumor microenvironment extracellular
(TME) low pH, and charge-mediated complexes of siRNA and cell-targeting and
-penetrating peptide-amphiphile (TCPA) encapsulated in the nanoparticle core. As
shown in Fig. 1, after intravenous injection, the pH-responsive multistage nanoparti-
cles exhibited a long circulatory half-life because of PEG outer shell on these nanopar-
ticles and thus enhanced their accumulation in the tumor tissue due to the enhanced
permeability and retention (EPR) effect. When these nanoparticles entered tumors,
Tumor Microenvironment and Intracellular Signal-Activated … 175

Fig. 1 Schematic illustration of the TME pH-responsive multistage nanoparticle platform for
systemic targeted siRNA delivery and effective cancer therapy. Reprinted from Ref. [34] with
permission, Copyright American Chemical Society, 2017

extracellular low pH-activated rapid nanoparticle disassembly led to the exposure of

siRNA-TCPA complexes, which could then target and penetrate tumor cells via the
RGD (Arg-Gly-Asp) ligand segment of TCPA. Finally, the cell-penetrating ability of
TCPA attributable to its cationic polyarginine segment enhanced the cytosolic siRNA
transport to achieve efficient gene silencing. In order to evaluate the pH-responsive
behavior of these nanoparticles, transmission electron microscopy (TEM) imaging
was employed. TEM images exhibited a spherical morphology of the nanoparticles
at pH 7.4 PBS. After incubation at pH 6.8 PBS for 3 min, some large amorphous
aggregates and small size particles were observed, which were confirmed as the
ionized polymers and exposed siRNA-TCPA complexes. In addition, more than 80%
of siRNA and less than 20% of siRNA was released from these nanoparticles when
nanoparticles were incubated in pH 6.8 PBS and 7.4 PBS for 4 h, respectively. In vitro
cellular uptake showed that HeLa cells took in more siRNA at pH 6.8, compared to
cells incubated at pH 7.4. More importantly, the internalized siRNA molecules at pH
6.8 exhibited a much higher distribution in the cytoplasm than that of the internalized
siRNA molecules at pH 7.4. Further flow cytometry demonstrated the siRNA uptake
by cells incubated at pH 6.8 is >5-fold more than that by cells incubated at pH 7.4.
Moreover, ex and in vivo results suggested the multistage siRNA delivery platform
could silence the expression of bromodomain 4, a conserved member of the BET
family of chromatin readers regulating signaling network in prostate cancer (PCa),
and significantly inhibit PCa tumor growth [34].
Apart from low delivery efficiency to cancer, another challenge faced by cancer
nanomedicine is poor tumor penetration. It is well-known that nanoscale drug
delivery systems preferentially accumulate in solid tumors through the EPR effect
due to their inherent size advantages [58]. However, the large size also limits
176 Y. Huang et al.

the deep penetration of nanomedicine into the tumor parenchyma. Several liter-
ature reported that after nanosystems reached tumors, drug transport and distri-
bution would be hindered by unique architectural features of solid tumors such
as elevated interstitial fluid pressure (IFP) and dense extracellular matrix (ECM)
[59]. To solve this paradox, Li et al. developed a class of ultra-pH-sensitive
cluster nanobombs (SCNs) with instantaneous size switching in the acidic tumor
microenvironment [35]. The platinum (Pt)-prodrug conjugated SCNs (SCNs/Pt) were
self-assembled from poly(ethylene glycol)-b-poly(2-azepane ethyl methacrylate)-
modified PAMAM dendrimers (PEG-b-PAEMA-PAMAM/Pt), in which the PAEMA
block contains ionizable tertiary amine groups for pH-responsiveness. At neutral pH,
PAEMA was hydrophobic and directed the assembly of PEG-b-PAEMA-PAMAM/Pt
into SCNs/Pt, which had an initial size of ~80 nm for prolonged blood circulation
and selective extravasation into tumor tissue. While in the acidic tumor microen-
vironment, PAEMA was rapidly protonated, leading to a hydrophobic-hydrophilic
transformation and subsequently instantaneous disintegration of SCNs/Pt into small
nanoparticles for deep penetration into tumor parenchyma. The Pt-prodrug was
covalently attached to the polymer and specifically activated by intracellular redox,
resulting in the release of active cisplatin [60]. The tumor-pH-triggered size transi-
tion of SCNs/Pt was testified by using dynamic light scattering (DLS) and TEM. At
neutral pH, the size was 80 nm in diameter. Then the size showed a dramatic transi-
tion from ~80 nm at pH 6.8 to ~10 nm at pH 6.7, which was superfast and completed
within seconds, indicating the rapid dissolution of the superstructure into small parti-
cles. The penetration capability of SCNs/Pt was investigated by employing BxPC-3
human pancreatic cancer cell-derived tumor spheres as an in vitro model. To monitor
the penetration activity, SCNs and pH-insensitive cluster nanostructure (ICNs) were
labeled with Cy5 (denoted as SCNs/Cy5 and ICNs/Cy5). Confocal laser scanning
microscopy (CLSM) showed limited penetration of both SCNs/Cy5 and ICNs/Cy5
into tumor spheres when incubated at pH 7.4. However, a significant improvement
of SCNs/Cy5 in penetration capability (at a depth of 85 μm into tumor spheres) was
found at tumor extracellular pH (pH 6.7). Next, tumor growth inhibition study in nude
mice bearing poorly permeable BxPC-3 tumor xenografts revealed tumor suppres-
sion rate of 22%, 24%, 41%, and 82%for PAMAM/Pt, free cisplatin, ICNs/Pt and
SCNs/Pt (at a Pt dose of 2 mg/kg), respectively. All these results demonstrated that
the pH-triggered size switching is a viable strategy for improving drug penetration
and therapeutic efficacy of nanomedicine [35].
Surface charge also plays a determining role in the performance of nanoparticles.
The high affinity of positively charged nanoparticles to biological interfaces makes
them easily taken up by tumor cells via electrostatic attraction mediated targeting
[61], but this strong interaction also limits their tumor permeation due to shortened
blood circulation [62]. Thus, a class of novel delayed charge reversal nanoparticles
were fabricated to investigate the influence of the charge reversal profile of nanopar-
ticles on tumor penetration [36]. These nanoparticles were constructed by an anionic
coating with poly (glutamic acid)-g-methoxyl poly (ethylene glycol) (PGlu-g-mPEG)
and a drug-loading cationic inner core composed of poly (lysine)-b-polycaprolactone
(PLys-b-PCL). Manipulation of charge reversal profile under lower pH was achieved
Tumor Microenvironment and Intracellular Signal-Activated … 177

by varying the shell cross-linking degree of the cationic core, prior to electrostatic
interaction in the polyanion complex layer. The changes in ξ-potential of these
nanoparticles with different cross-linking degrees (CTCL0, CTCL30, and CTCL70)
incubated in pH 6.5 PBS revealed a relation between the charge reversal profile of
nanoparticles and cross-linking degree. After 6-h incubation, no changes in the ξ-
potential of CTCL0 nanoparticles were observed, indicating that it was non-charge
reversible; CTCL70 nanoparticles accomplished charge reversal after 1-h incuba-
tion, while it took 2 h for the ξ-potential of CTCL30 to turn positive. However,
the ξ-potential of the three nanoparticles remained constant after 24-h incubation
in pH 7.4 PBS. The amount of remained PGlu-g-mPEG on nanoparticles during
incubation was determined by gel permeation chromatography to study the charge
reversal caused by decoating of PGlu-g-mPEG. Results showed the coating material
remained on CTCL30 and CTCL70 nanoparticles were 34.7% ± 0.77% and 8.57%
± 6.1%, respectively, while the percentage was over 70% for CTCL0 nanoparticles.
These results indicated that the lowered environmental pH was responsible for charge
reversal, and controllable charge reversal could be achieved by changing the degree
of cross-linking. The in vivo intratumoral penetration and cellular uptake of CTCL30
and CTCL70 nanoparticles were compared, and it was found that CTCL30 nanopar-
ticles with a delayed charge reversal profile increased the number of cells exposed
to the nanoparticles by the wider intratumoral nanoparticle distribution as well as
improved nanoparticle–cell interaction due to tumor acidity induced time-dependent
charge reversal.
PEGylation of nanoparticles is a good way to extend their blood circulation
time, but researchers also found it could dramatically shield the targeting effect and
suppress the cellular internalization. Thus, novel nanoparticles, including stimuli-
activated nanoparticles, have been developed to address the above problem and
improve tumor distribution of PEGylated nanoparticles. Wang et al. designed a
tumor extracellular acidity-triggered ligand-presenting (ATLP) nanoparticle for
tumor-specific penetration and cellular uptake [37]. The ATLP nanoparticles
were composed of a Ce6-modified acid-responsive poly(ethylene glycol)-b-poly(2-
(hexamethyleneimino) ethyl methacrylate) (PEG-b-PHMA, denoted as PHMA)
diblock copolymer as a shield matrix and an iRGD-modified polymeric prodrug
of doxorubicin (iPDOX) as an amphiphilic core. In the tumoral acidic microenviron-
ment, PHMA diblock copolymer underwent amphiphilic to hydrophilic transition
and dissociated from ATLP to expose the iRGD ligand for cellular uptake of the
PDOX prodrug and restore the fluorescence imaging of Ce6 for real-time moni-
toring. The acid-sensitivity of the ATLP nanoparticles was evaluated by monitoring
their size change by dynamic light scattering after incubation in PBS with different
pH values. The average particle size and surface charge of ATLP at pH 7.4 were
34 ± 2.0 nm and 1.1 ± 0.4 mV, respectively. When pH was decreased to 6.6, the
nanoparticles swelled dramatically to 155 ± 12 nm, with a positive surface charge
of 4.6 ± 0.2 mV, presumably due to protonation-induced dissociation of the PHMA
matrix and formation of iPDOX aggregates with iRGD presented on the surface.
At the same time, no changes in particle size and surface charge of acid-insensitive
nanoparticles at both pH values were observed. Next, the tumor penetration ability
178 Y. Huang et al.

of ATLP was investigated using MCF-7/ADR-derived multicellular spheroid, which

could mimic the acidic microenvironment of solid tumors. The DOX fluorescence
of ATLP perfused throughout the tumor spheroid at pH 6.6, while the fluorescence
distribution at pH 7.4 was limited, verifying acid-triggered exposure of iRGD ligand
dramatically promoted tumor penetration of iPDOX.

3.2 Enzyme-Activated Nanocomposites for Anticancer Drug

Delivery

3.2.1 Matrix Metalloproteinases

Fluorescence (FL) imaging into the second near-infrared region (NIR-II) at 1,000–
1,700 nm have attracted much interest due to its high spatial resolution through deep
tissues. However, bright and compact fluorophores are rare in this region, and sophis-
ticated control over NIR-II probes has not been fully achieved yet. Thus, Jeong et al.
fabricated a protease-activated NIR-II quantum dot probe (PA-NIRQD), which could
exhibit FL upon matrix metalloprotease activity in the tumor microenvironment [38].
Bright and stable PbS/CdS/ZnS (core/shell/shell) NIRQDs that emitted at 1200 nm
were synthesized, and activatable modulators, containing an MMP-cleavable peptide
sequence and methylene blue (MB) which could quench FL via photoexcited electron
transfer (PET), were attached to the surface ligands. The NIRQDs were quenched
by PET from the MB when the QDs were in close proximity to MB, and FL was
activated upon modulator cleavage. The peptide sequence was optimized to impart
flexibility, facilitating enzyme access, and guaranteeing effective quenching upon
activation. To evaluate the ability of PA-NIRQD to respond to the tumor microen-
vironment, fresh colon tissues were prepared from azoxymethane/dextran sulfate
sodium-treated colorectal tumor-bearing mice due to the high-level expression of
MMPs. The colon tissues were excited by a time-modulated 910 nm pulse laser,
and then the FL intensities were recorded over time. A rapid increase in FL ranging
from 150% to 300% was observed with 10 min at tumor site, while no changes
occurred in the area of normal mucosa. Time-dependent contrast-to-noise ratio values
at tumor site were also plotted, indicating the PA-NIRQD probe could distinguish
tumors from the normal tissues. In contrast, administered PA-NIRQD in normal
colon tissues showed minimal activation, and the FL intensity remained constant in
another experiment using NIRQD instead of PA-NIRQD, attributed the low MMP
activity and lack of activation event, respectively. In conclusion, PA-NIRQD enabled
the simple and rapid visualization of MMP enzyme activity and successfully distin-
guished tumor sites in the colorectal tumor mouse model. Thus, it has the potential
for the image-guided biopsy of cancers.
Zhu et al. designed a novel multifunctional liposomal nanocarrier, which could
respond to the up-regulated extracellular MMP2 in tumors, resulting in the enhanced
Tumor Microenvironment and Intracellular Signal-Activated … 179

tumor targetability and internalization [39]. An MMP-cleavable octapeptide (Gly-

Pro-Leu-Gly-Ile-Ala-Gly-Gln) was used as a sensitive linker between a liposomal
lipid and its long-chain PEG block to serve as a steric shield for the nanoparticle and
surface-attached cell-penetrating peptide TAT in the blood. The multifunctional lipo-
somes could be retained in the tumor site due to the EPR effect and the active targeting
effect by tumor cell-specific antinucleosome monoclonal antibody (mAb 2C5). Then
the high expressed extracellular MMP2 cleaved the octapeptide linker and removed
the protective long-chain PEG, providing the exposure of TATp for the enhanced
intracellular uptake. Results showed that the MMP2-cleavable peptide did not lose
its “cleavability” after the conjugation with both PEG and DOPE at its two ends.
The MMP2-responsive liposomes were prepared using the film hydration method
followed by extrusion and surface modification with mAb 2C5. The particle size and
zeta-potential of the liposomes were 207.5 ± 75.5 nm and 10.23 ± 1.21 mV, respec-
tively. To demonstrate the in vitro cellular uptake of the MMP2-responsive multifunc-
tional liposomes, mouse breast cancer cells (4T1), and normal rat cardiomyocytes
(H9C2) were treated with fluorescence-labeled liposomes at different conditions.
Fluorescence-activated cell sorting suggested enhanced targetability and internal-
ization of the MMP2-responsive multifunctional liposomes in 4T1 cancer cells, as
expected. Besides, it was also shown that the MMP2-sensitive liposomes minimized
nonspecific uptake in normal cells and maximized their penetration into cancer cells.
Folate receptor has been well documented to be over-expressed in some malig-
nant tissues, which provides an effective target site for drug delivery systems [63]. To
enhance programmable long circulation and tumor targeting ability, MMP-sensitive
nanoparticles (SNPs) were constructed by mixing a MMP2/9-sensitive copolymers
(mPEG-Pep-PCL) and folate receptor-targeted copolymers (FA-PEG-PCL) [40]. The
SNPs exhibited a sandwich structure composed of a detachable PEG outmost layer,
a targeting middle layer of folate ligands, and a hydrophobic innermost core loaded
with camptothecin (CPT). In the tumor microenvironment, the MMP2/9-sensitive
linker was expected to be cleaved, followed by the detachment of the PEG layer and
then the exposure of folate, via which the selective binding to the tumor cells and the
cellular uptake could be strengthened. The size of CPT-loaded SNPs was determined
to be around 160 nm by DLS, in line with previous literature which claimed that
the effective size for the drug delivery systems should be below 200 nm since the
threshold vesicle size for the extravasation into a tumor’s extracellular space was
around 400 nm [64]. To demonstrate changes in SNPs in response to MMP, SNPs
were incubated with gelatinases for 24 h. Serious aggregation and precipitation were
observed in nanoparticle suspension, attributing to the detachment of the PEG layer
induced by the breakup of mPEG-Pep-PCL. In contrast, MMP-insensitive nanopar-
ticles (INPs) composed of mPEG-PCL and FA-PEG-PCL exhibited no significant
size variation due to the absence of peptide. In vitro uptake by B16 melanoma cells
was investigated using a fluorescence microscope to compare the cytotoxicity of
CPT-loaded SNPs and INPs. After 4-h incubation, no fluorescence was found in the
group of INPs, indicating no or negligible amount of CPT was inside the B16 cells.
However, fluorescence was observed at the initial 2 h in the SNPs-treated group, and
more fluorescence appeared when the incubation time was extended to 4 h, indicating
180 Y. Huang et al.

that CPT was delivered into the B16 cells more efficiently by SNPs. In vivo antitumor
studies in B16 melanoma xenograft models demonstrated that a significantly higher
tumor group inhibition was achieved by CPT-loaded SNPs treatment compared with
other treatments with saline, empty NPs, CPT, CPT-loaded INPs.

3.2.2 Hyaluronidase

Fluorouracil (5-FU) is a pyrimidine analog used to treat cancer of the breast, glioblas-
toma, colon, rectum, stomach, and pancreas [65]. It interferes with DNA and RNA
synthesis by mimicking the building blocks necessary for synthesis. However, the
growth of normal cells may also be affected by 5-FU. Side effects will also occur, such
as fever, unusual bleeding, and trouble breathing. Thus, effective delivery strategies
for 5-FU are needed. Jiang et al. prepared hyaluronic acid (HA)-conjugated 5-FU-
loaded mesoporous silica nanoparticles (HA/FMSN) to improve its therapeutic effi-
cacy in colon cancer [41]. The surface-conjugated HA with large molecular weight
could prevent the drug release in normal conditions and target the CD44 receptors,
which are overexpressed in tumor tissues. After entering the tumor site, HA would be
degraded into lower molecules with the assistance of hyaluronidase (HAase), an over-
expressed enzyme at the tumor microenvironment, leading to the release of the drug.
HA/FMSN were perfectly spherical and well-ordered in appearance with particle size
and zeta potential of around 190 nm and −18.5 mV, respectively. When incubated
in colon cancer cells for 2 h, HA/FMSN exhibited superior targeting efficiency with
a stronger fluorescence over FMSN, indicating a typical receptor-mediated endo-
cytosis uptake of the former particles. In MTT assay, the IC50 value of free 5-FU,
FMSN and HA/FMSN were 6.89 μg/ml, 4.52 μg/ml, and 1.08 μg/ml, respectively,
which suggested a significantly higher anticancer effect of HA/FMSN compared
with other formulations. As expected, higher apoptosis of cancer cells induced by
HA/FMSN was also observed in colon cancer cell-bearing mice. Altogether, these
results suggested that the hyaluronidase-responsive targeted nanoparticles could hold
enormous potential in colon cancer targeting.
PEGylation can not only improve the biocompatibility and long-circulation of
nanoparticles in vivo, but also inhibits cellular uptake and endosomal escape and leads
to low transfection efficiency [66]. To overcome the “PEG dilemma,” PEGylated
HA, i.e., HA-g-PEG (HgP), was synthesized to coat the binary complexes of core-
shell cationic polycaprolactone-graft-poly (N, N-dimethylaminoethyl methacry-
late) nanoparticles/DNA (NP-D) for constructing a novel enzyme-activated poly-
cation/DNA complexes (NP-D-HgP) for gene therapy [42]. The coating of HgP on
NP-D could inhibit the release of the inner layer of nucleic acid due to strong elec-
trostatic interaction between the polyelectrolytes. DNA release could be achieved by
the high level of HAase in the tumor microenvironment, which promoted HA degra-
dation, resulting in enhanced endosome escape and transfection efficacy. To confirm
the detachment of HgP coating, zeta potential and size of NP-D-HgP were measured
in the presence of HAase at different times. Results showed a dramatic increase in
the diameter of NP-D-HgP from 60 to 8591 nm within the prior 1 h and a subsequent
Tumor Microenvironment and Intracellular Signal-Activated … 181

decrease to 200 nm. The zeta potential of NP-D-HgP went through a charge reverse
from −10 mV to +5 mV, which could be attributed to the detachment of the HA
layer. Compared with the control nanoparticles composed of PCL-g-PDMAEMA
NPs/DNA/PGA-g-PEG (NP-D-PGgP), the nanoparticle uptake by HepG2 cells was
enhanced by HgP coating, partly caused by the CD44-mediated endocytosis. The
effect of HgP and PGgP coating on cellular uptake was further evaluated using
confocal microscopy. After incubation for 9 h, NP-D nanoparticles were found to
aggregate outside the cell and stay with the surface of cells, while NP-D-HgP were
stable and the cell uptake efficiency was remarkably improved. In vivo RFP gene
transfection of NP-D-HgP was performed at a dose of 1.5 mg/kg of RFP plasmid
DNA via intravenous injection. It was shown that mice treated with NP-D died in
several hours after injection, while no death occurred in NP-D-HgP and NP-D-PGgP
groups. At 24 h after injection, higher RFP expression was observed in mice admin-
istered with NP-D-HgP than other groups. Taken together, NP-D-HgP showed high
potential in gene delivery to cancer cells.

3.2.3 Cathepsin B

Among various kinds of nanocarriers, functional mesoporous silica nanoparticles

(MSNs) have exhibited unique advantageous properties, such as chemically inert,
easy functionalization, and large loading capacity, attracting research interest for
drug storage and delivery. In order to achieve efficient drug delivery and reduce
adverse effects, functionalized MSNs are usually constructed by covering the surface
of MSNs with nanovalves, including rotaxanes, polypseudorotaxanes, cucurbiturils,
and so on, which serve as a “gatekeeper” to retain drugs until external stimuli. Cheng
et al. designed a drug delivery system based on rotaxane-modified MSNs [43]. The
rotaxane structure was formed with alkoxysilane tether, α-cyclodextrin, and multi-
functional peptides, which were composed of a cell-penetrating peptide of seven argi-
nine (R7) sequence, a cathepsin B-cleavable peptide of GFLG, and a tumor-targeting
peptide of RGDS. When incubated with tumor and normal cells, the DOX-loaded
MSNs could target tumor cells via the specific interaction between RGDS and inte-
grins receptor overexpressed on tumor cells, followed by penetrating cell membrane
with the aid of R7 sequence. After endocytosis into tumor cells, drug release was
triggered due to the breakage of GFLG peptide induced by cathepsin B, resulting in
enhanced antitumor activity. In vitro drug release behavior of the DOX-loaded MSNs
was investigated in PBS buffer at pH 7.4 or 5.0, simulating physiological environ-
ment and lysosome environment, respectively. In the presence of cathepsin B, about
60% of DOX has been released from the nanoparticles at pH 7.4 PBS for 24 h. While
in the absence of cathepsin B, less than 10% DOX has been released within the same
time scale, attributed to the protection of the gatekeeper on the surface of the MSNs.
The DOX release behavior at pH 5.0 also confirmed that cathepsin B could cleave
the GFLG sequence and thus remove the gatekeeper from the surface of the MSNs.
These results showed MSNs had great potential in controlling drug release in cancer
cells.
182 Y. Huang et al.

Gemcitabine (GEM) is a chemotherapy medication used in a number of types

of carcinomas, including breast cancer, ovarian cancer, non-small cell lung cancer,
pancreatic cancer, and bladder cancer. However, the therapeutic efficacy of GEM is
limited because it could be rapidly delaminated in the blood to the inactive metabo-
lite 2 ,2 -difluorodeoxyuridine, causing a short plasma half-life [67]. To increase the
stability and bioavailability of GEM in vivo, new GEM delivery strategy should be
designed. Han et al. developed enzyme-sensitive gemcitabine-conjugated albumin
nanoparticles as a versatile theranostic nano platform for pancreatic cancer treat-
ment [44]. The novel nano platform was developed by covalent conjugation of
GEM to human serum albumin (HSA) via cathepsin B cleavable peptide GFLG and
then bounding with near-infrared (NIR) cyanine dye IR780 (HAS-GEM/IR780).
It was shown the release of GEM from HAS-GEM/IR780 was inhibited until the
breakage of GFLG triggered by cathepsin B, which is overexpressed in pancreatic
cancer cells, leading to superior tumor inhibition activity with minimal side effects.
As expected, improved cellular proliferation inhibition of HSA-GEM/IR780 was
observed in MTT assays, which suggested efficient drug release and cytotoxicity
toward pancreatic cancer cells. Furthermore, the in vivo antitumor efficacy revealed
that HSA-GEM/IR780 exhibited significantly stronger tumor inhibition compared
with other GEM formulations, indicating a distinct improvement of the therapeutic
effect of GEM.

3.3 Reduction (or GSH)-Activated Nanocomposites

for Anticancer Drug Delivery

Reduction-sensitive nanocomposites can be applied for the development of anti-

cancer drug delivery systems (Table 2). These nanocomposites usually contain
characteristic disulfide bonds (S-S) in the main chain, in the side chain, or in
the cross-linker [68]. The disulfide bonds are sufficiently stable in the extracel-
lular environment, but under reductive conditions in the cytoplasm, they tend to
be rapidly cleaved after minutes or hours through thiol–disulfide exchange reactions
[69]. Thiol-disulfide exchange reactions are rapid and readily reversible. GSH is the
most abundant thiol in mammalian cells [23], and the significant difference in GSH
concentration between tumor cells and extracellular fluid renders GSH-responsive
nanocomposites most appealing for targeted anticancer drug delivery.
Due to the reducing potential in tumor cells, reduction-responsive nanocomposites
targeting tumor cells can significantly enhance the release of intracellular drugs and
achieve the rapid accumulation of drugs (Fig. 2). In addition, compared with the pH-
sensitive nanocomposites, reduction-sensitive nanocomposites have several unique
advantages, such as high stability against hydrolytic degradation, rapid response to
the intracellular reducing environment, and the selective and rapid release of anti-
cancer drugs, at the same time, reducing side effects at normal tissues. Reduction-
responsive nanocomposites (also called redox-responsive nanocomposites) have
Tumor Microenvironment and Intracellular Signal-Activated … 183

Fig. 2 The illustration of the intracellular trafficking pathway of GSH-responsive nanocomposites

including steps of cellular internalization, endosomal escape, reduction-triggered vehicle degrada-
tion, and drug release. The redox potential of the cytosol is mainly determined by GSH/GSSG,
while that of the endo/lysosome is controlled by GILT and co-factor cysteine. GSH-responsive
nanocomposites may also be partially degraded in the endo/lysosomal compartments. Reprinted
from Ref. [25] with permission, Copyright Elsevier, 2011

been used to confer sensitivity to the intracellular reducing potential for the release
of anticancer drugs into tumor cells, such as liposomes [70], polymersomes, poly-
meric nanogels [71], micelles [72], dendrimers, and protein nanocapsules [73]. The
disulfide bond has been widely used as the cleavable/reversible linker in nanocom-
posites to render redox potential sensitivity to nanocomposites [26]. In addition,
disulfide bonds can be introduced in the repeated units of the polymers, and cause
the rapid degradation of the polymer nanocomposites under reductive conditions. In
general, reduction-activated nanocomposites contain characteristic disulfide bonds
which can be rapidly cut in highly reducing intracellular environments by a GSH-
mediated thiol-disulfide exchange reaction to release anticancer drugs [3], showing
great potential in targeted anticancer drug delivery.
Ong et al. developed reduction-activated liposomes comprised of quinone-
dioleoyl phosphatidylethanolamine (Q-DOPE) lipids for site-specific release [70].
In these liposomes, a “trimethyl-locked” quinone redox switch was attached to
the N-terminus of DOPE lipids, and underwent a cleavage (liposome opening) by
two-electron reduction to liberate the payload. Ryu et al. synthesized highly stable
polymeric nanogels with lots of advantages, such as high encapsulation capability,
tunable release kinetics, and no loss of drugs before entering tumor cells [71]. It
was a self-cross-linked polymeric nanogel composed of pyridyl disulfide-grafted
184 Y. Huang et al.

oligoethyleneglycol-based polymers by intra-/intermolecular disulfide cross-linking.

The kinetics of drug release could be fine-tuned by control over cross-linking density
in nanogels. It was shown that the encapsulated dye molecules could be released
in response to a redox trigger (GSH) via the degrading of cross-linking disul-
fide bonds in the reducing environment. In addition, these nanogels could be used
to sequester hydrophobic chemotherapeutics and released them intracellularly to
achieve drug-induced cytotoxicity [71].
Shi et al. developed a multifunctional star-shaped micellar system based on
a four-arm amphiphilic copolymer, which had both active targeting ability and
redox-responsive behavior [72]. In this copolymer, the hydrophobic and hydrophilic
segments were connected by disulfide bonds, which provided the redox-sensitivity
to the multifunctional star-shaped micellar system and then triggered the release of
drugs. The terminal groups of the hydrophilic segments were decorated with folate
ligands, which provided a highly active targeting to tumor cells. Compared with
linear polymeric micelles, these star-shaped polymeric micelles exhibited unique
features such as better stability, smaller size distribution, and higher drug loading
capacity. Moreover, under stimulated normal physiological conditions, the redox-
responsive star-shaped micelles could maintain great stability to avoid the premature
drug release, while under reducing and acid conditions, drugs would be rapidly
released. In vivo fluorescence imaging showed that these folic acid-functionalized
star-shaped polymeric micelles had much better specificity to target solid tumors.
In vivo antitumor efficacy study demonstrated that the active targeting and redox-
responsive star-shaped micelles had better therapeutic effects to solid tumors and
higher safety to the normal tissues in contrast to the redox-insensitive micelles [72].
Direct delivery of proteins to the cytosol of cells has tremendous potential in
biomedical applications, and biodegradable polymeric nanocapsules have attracted
much attention for anticancer therapy. Zhao et al. prepared redox-responsive single-
protein nanocapsules for intracellular protein delivery. The target protein could be
encapsulated into a positively charged polymeric shell interconnected by disulfide
cross-linking. Under reducing conditions, the polymeric shell would dissociate, and
the target protein would be released. Studies showed that the active caspase 3 could
be efficiently internalized and induced apoptosis in human tumor cells (such as HeLa,
MCF-7, and U-87 MG) by the nanocapsules [73a].
Drug resistance in tumor cells and unexpected cytotoxicity in healthy cells are
major problems in tumor chemotherapy. To overcome these obstacles, Chang et al.
constructed a redox-responsive DOX/siRNA (small interfering RNA) co-delivery
system (cationic vesicle) consisting of a novel ferrocenium capped amphiphilic
pillar[5]-arene (FCAP) [74]. Due to the high GSH level in tumor cells, this cationic
vesicle could disassemble after entering cancer cells, leading to a rapid release of
encapsulated siRNA and drug. SiRNA could knock down drug resistance genes and
restore the drug sensitivity of cells to enable effective chemotherapy. Meanwhile,
due to the absence of the specific stimulus, drug release did not occur or occurred
slowly in healthy cells, thus efficiently avoiding severe side effects. It was shown that
the co-delivery of siRNA and DOX into cancer cells by cationic vesicles resulted in
a clear synergistic inhibition of SKOV-3 cells.
Tumor Microenvironment and Intracellular Signal-Activated … 185

In addition to the disulfide bonds, more reactive diselenide (Se–Se) bonds have
been integrated into nanocomposites to achieve redox responsiveness. Ma et al.
synthesized a redox-sensitive micelle that was self-assembled by an amphiphilic
diselenide-containing triblock copolymer (PEG–PUSeSe–PEG) [75]. The results
revealed that the redox responsive micelles were quite stable and showed a high
sensitivity to the external redox stimulus.
In conclusion, the reduction-sensitive nanocomposites meet the requirements of
an ideal anticancer drug delivery system, such as high stability in circulation, rapid
degradation in tumor cells, enhanced therapeutic effect, and minimal carrier-related
in vitro and in vivo cytotoxicity. Reduction-sensitive nanocomposites are in particular
suited for the efficient delivery of active nucleic acid drugs like siRNA to the cytosol
of tumor cells. However, the reduction-sensitive nanocomposites still need further
improvement, especially in aspects such as reduction-sensitivity, biocompatibility,
long circulation time, and cellular or intracellular targeting ability for better clinical
applications.

3.4 Hypoxia-Activated Nanocomposites for Anticancer Drug

Delivery

The tumor microenvironment is commonly hypoxic. It is different from normal

tissues due to the imbalance between the supply and the consumption of oxygen. The
vascular networks within solid tumors are very irregular and are unable to deliver
sufficient oxygen and nutrients to all regions. That situation then results in the lack
of oxygen in tumor cells [76]. Hypoxia in tumors is mainly a pathophysiologic
consequence of structurally and functionally disturbed microcirculation.
Studies have reported that low oxygen pressure can cause conformational changes
in hypoxia-responsive nanocomposites via the cleavage or reduction of hypoxia-
responsive functional groups (Table 2) [77]. There are three representative classes
of hypoxia-responsive moieties, including nitroimidazoles, nitrobenzyl alcohols,
and azo linkers, which are useful for the design of nanocomposites for cancer
therapy. These hypoxia-responsive moieties contain reducible functional groups
(such as nitro, azo, and quinoa), which can accept electrons under hypoxic condi-
tions. When the hypoxia-responsive nanocomposites are exposed to a hypoxia envi-
ronment, the hydrophilicity of the nanocomposites can be altered as the reduction of
hypoxia-responsive moieties produces hydrophilic functional groups [77].
Thambi et al. reported self-assembled nanoparticles (HR-NPs) that could selec-
tively release the hydrophobic agents under hypoxic conditions (Fig. 3) [78]. The
HR-NPs were composed of carboxymethyl dextran integrated with a hydrophobic
2-nitroimidazole derivative for the hypoxia-triggered drug release. HR-NPs were
stable under physiological conditions and were capable of selectively releasing
hydrophobic drugs under hypoxic conditions. Studies showed that DOX-loaded
HR-NPs had higher cytotoxicity to hypoxic tumor cells than to normal cells, and
186 Y. Huang et al.

Fig. 3 Schematic illustration of the formation of drug-loaded HR-NPs and in vivo tumor-targeting
pathways. The HR-NPs can reach the tumor site via the EPR effect, followed by intracellular drug
release at hypoxic tissues. Reprinted from Ref. [78] with permission, Copyright Elsevier, 2014

HR-NPs could effectively accumulate at the tumor site, resulting in significantly

higher anticancer efficacy than free DOX. To enhance anticancer therapy, Thambi
et al. also developed a novel type of hypoxia-sensitive polymeric micelles (HS-
PMs) [79] exhibiting significant physicochemical changes at the early stage of
tumor progression and responsiveness to tumor cells [80]. It was shown that HS-
PMs composed of an amphiphilic block copolymer poly(ethylene glycol)-poly(e-(4-
nitro)benzyloxycarbonyl-L-lysine) had higher cytotoxicity to SCC7 (squamous cell
carcinoma) cells under hypoxic conditions than under normal conditions.
Since hypoxic regions are distant from blood vessels and have increased efflux
transporters for siRNA delivery to tumor cells with nanocomposites, it is necessary to
protect siRNA from degradation and to promote its cellular internalization and endo-
somal escape. Perche et al. developed hypoxia-activated nanoparticles composed of
polyethyleneglycol 2000, azobenzene, polyethyleneimine (PEI), and 1,2-dioleyl-sn-
glycero-3-phosphoethanolamine (DOPE) units for siRNA delivery to tumor cells
[81]. It was shown that these nanoparticles exhibit hypoxia-activated gene-silencing
capacity both in vitro and in vivo.
Qian et al. reported on light-activated hypoxia-responsive nanocomposites for
enhanced anticancer therapy [82]. The nanoparticle was composed of three parts:
ROS-generating and hypoxia-sensitive 2-nitroimidazole-grafted conjugated polymer
(CP-NI), polyvinyl alcohol (PVA)-based surface coatings, and the encapsulated
DOX. It was a novel drug delivery system for the first time using a light-activated
hypoxia-responsive pathway, which was able to produce reactive oxygen species and
Tumor Microenvironment and Intracellular Signal-Activated … 187

induce hypoxia to release drugs into tumor cells. This drug delivery system combined
both photodynamic therapy and chemotherapy and could enhance the efficacy of
anticancer therapy. Photodynamic therapy depended on the ability of photosensi-
tizers to transfer energy from light to the surrounding molecular oxygen (3 O2 ) to
produce cytotoxic ROS (such as singlet oxygen 1 O2 ). During the conversion from
molecular oxygen (3 O2 ) to singlet oxygen (1 O2 ), oxygen was consumed and blood
vessels constricted, which produced a hypoxic condition. Therefore, these nanopar-
ticles combining a photodynamic system with a hypoxia-responsive drug delivery
system efficiently enhanced the anticancer therapy.
In conclusion, tumors are complex and exhibit molecular and cellular hetero-
geneity. Hypoxia is the hallmark of solid tumors. The properties of the hypoxic tumor
environment can be used in diagnostic and therapeutic strategies [77]. Targeting the
tumor hypoxia microenvironment provides an important strategy to overcome the
problem of tumor heterogeneity, and the development of hypoxia-activated nanocom-
posites is important for enhancing anticancer therapy. However, there still are some
challenges in hypoxia-activated nanocomposites for anticancer drug delivery. For
example, the ability of nanocomposites to deliver anticancer drugs to tumors depends
on their physicochemical and biological properties, such as the circulation time,
targeting ability, and the uptake of tumor cells. A small variation in these parameters
may lead to a dramatic alteration in their circulation and tumor targeting efficiency.
Although there are many limitations in hypoxia-activated nanocomposites due to the
lack of biodegradability and biocompatibility, which limit the clinical application
of nanocomposites, they are still promising in anticancer drug delivery for targeted
tumor therapy.

3.5 ROS-Activated Nanocomposites for Anticancer Drug

Delivery

The increased cellular levels of ROS are the biochemical property of tumor cells.
ROS-responsive drug delivery systems targeting oxidative microenvironment in
tumor cells can selectively deliver anticancer drugs to tumor cells and can effec-
tively reverse their drug resistance (Table 2) [31]. de Gracia Lux et al. developed new
biocompatible, self-degradable, and H2 O2 -triggered nanoparticles that could release
drugs upon exposure to the low concentrations of H2 O2 [83]. These nanoparticles
were prepared from a bio-responsive polyester bearing aryl boronic ester groups that
had a high sensitivity to H2 O2 . Every cleavage of the boronic ester led to polymer
backbone degradation and could be degraded easily. At the inflamed or cancerous
cells, the boronic ester cap was removed to produce phenols and led to a quinoneme-
thide rearrangement, thus disrupting the nanoparticles and releasing the drugs [3].
The nanoparticles could be degraded into small molecules, which were easily cleared
by human body. Since cytokines and chemokines produced by neutrophils could
188 Y. Huang et al.

affect the recruitment and activation of inflammatory cells in the tumor microenvi-
ronment, and provide an immunosuppressive microenvironment for tumor growth,
metastasis, and angiogenesis, using these nanoparticles to deliver drugs that inhib-
ited neutrophils recruitment in tumor tissues could limit tissue damage and improve
anticancer therapy.
Shim and Xia synthesized a cationic polymer poly(amino thioketal) (PATK) with
ROS-cleavable thioketal linkages for mediating intracellular gene delivery [84]. In the
study, PATK and DNA could self-assemble into nanoparticles, and these nanoparti-
cles could be easily cleaved under ROS-abundant conditions due to the biodegradable
thioketal linkages. It was the probable mechanism that the ROS-cleavable thioketal-
based nanoparticles could enhance the efficiency of the intracellular gene delivery
in tumor cells by facilitating the intracellular release of the encapsulated nucleic
acids in response to the high levels of intracellular ROS. The results showed that
the DNA/PATK nanoparticles exhibited efficient gene transfection in prostate cancer
cells.
Liu et al. developed H2 O2 -responsive nanoparticles based on an amphiphilic
hyperbranched polymer with hydrophobic selenide groups and hydrophilic phos-
phate segments in the dendritic backbone framework [85]. The nanoparticles could
be disassembled under an exclusive oxidative tumor microenvironment that contained
a high level of H2 O2 (the level of H2 O2 in tumor cells was increased at the speed
of 0.5 nmol/104 cells/h [86], compared with normal cells). Because hydrophobic
selenide groups could be easily oxidized into hydrophilic selenone groups, the
amphiphilic hyperbranched polymers became hydrophilic hyperbranched polymers
after oxidation and then nanoparticles could rapidly and selectively release drugs in
tumor cells. In this study, the DOX-loaded H2 O2 -responsive nanoparticles could be
rapidly disassembled in the target tumor cells under intracellular oxidative condi-
tions, thus rapidly releasing DOX for inducing cell apoptosis. In addition, selenium
compounds enhanced the immune response, produced anticancer metabolites that
could efficiently disturb the metabolism of tumor cells, inhibited angiogenesis and
finally induced the apoptosis of tumor cells [85]. Therefore, these nanoparticles that
contained selenium compounds showed potential in cancer therapy.
High ROS level in tumor cells is essential for ROS-activated anticancer drug
release in tumor cells. Su et al. developed novel ROS-activated and positive feed-
back micelles that could replenish the ROS upon their disassembly to maintain high
ROS levels [87]. These micelles not only responded to original ROS signals in tumor
cells for drug release, but also responded to self-regenerating ROS signals in the cyto-
plasm by amplifying ROS signal. These micelles were prepared by a new amphiphilic
polymer (TBH) that was composed of D-α-tocopherol polyethylene glycol 1000
succinate (TPGS), hyaluronic acid (HA), and arylboronic ester. TPGS was an effec-
tive P-gp efflux inhibitor, selective tumor cell killing agent and, most importantly, a
powerful ROS inducer upon its interaction with mitochondrial respiratory complex
II [88]. The arylboronic ester was as a ROS sensing linker for imaging and targeting
of tumor cells. TBH was synthesized by linking HA as hydrophilic chains to the
hydrophobic TPGS with arylboronic esters [87]. Therefore, TBH micelles could
efficiently degrade in response to ROS in tumor cells and disassembled to release
Tumor Microenvironment and Intracellular Signal-Activated … 189

TPGS rapidly. Because of the ROS-induced activity of TPGS, ROS regenerated

and increased. These results could be observed both in MCF-7/ADR cells and MCF-
7/ADR tumor xenografts after TBH micelles treatment. In addition, HA was an active
target ligand for CD44 positive malignant cells and it could increase the uptake of
the TBH nanoparticles [89]. DOX, as a model anticancer agent, was loaded in TBH
micelles (DOX-TBH). The results showed that compared with free DOX, DOX-TBH
could significantly reduce the tumor volume, increase the percentage of apoptotic
or necrotic tumor cells, and prolong the survival time of tumor-bearing nude mice.
In addition, after DOX-TBH treatment, no obvious pathological abnormalities were
observed in normal tissues such as heart, lung, spleen, liver, and kidney.
In conclusion, many studies have observed an increased generation of ROS and
altered redox status in tumor cells. Targeting ROS signal in tumor cells provides
the possibility for selectively eliminating tumor cells and some studies have achieved
ROS-activated anticancer drug delivery by using ROS-sensitive nanocomposites.
ROS has a mixed effect on tumor cells, both good and bad. Evidence suggests that
transformed cells use ROS signals to promote proliferation. However, this confers a
state of increased basal oxidative stress for tumors that can make them more vulner-
able to chemotherapeutic agents and can further augment the generation of ROS or
weaken antioxidant defenses of cells [90]. Developing ROS-activated nanocompos-
ites to amplify ROS level in tumors, keeping the intracellular ROS at a high level,
increasing the oxidant stress and sensitivity to chemotherapeutic agents, and finally
causing cell death represents a new way for tumor treatment.

3.6 ATP-Activated Nanocomposites for Anticancer Drug

Delivery

ATP is the basic energy currency in the physiology environment. It has a sharp
concentration contrast between extracellular (<0.4 mM) and intracellular (1–10 mM)
environments, especially in the tumor sites, which makes ATP a feasible cue for regu-
lating drug release (Table 2) [91]. ATP-activated drug release is always achieved by
competitive binding of ATP with other DNA sequences to ATP aptamer. Generally,
drugs (for example, DOX) are loaded inside the double-chain structure composed of
ATP aptamer (DNA sequence) and its corresponding cDNA. When it enters tumor
cells that are full of ATP, ATP will compete with cDNA to bind with the ATP
aptamer. The binding of ATP and ATP aptamer is much stronger than the inter-
action between ATP aptamer and cDNA [92] and thus the previous double-chain
structure is destroyed, which results in drug release.
Yang et al. firstly demonstrated that ATP aptamer-ATP interaction could be used as
a stimuli-responsive model for controlling drug release. They prepared fluorescein
isothiocyanate (FITC) dye-loaded mesoporous silica nanoparticles (MSN), which
were capped with ATP aptamer-based molecular gates to control the drug release
from MSN [93]. First, they covered MSN with a derivative of ATP through amidation
190 Y. Huang et al.

reaction. Then ATP aptamer-modified Au nanoparticles (AuNPs) acted as molecular

gates to close the pores of MSN through adenosine-aptamer interaction (MSA).
When MSA met ATP in tumor cells, ATP would compete to bind the ATP aptamer,
which replaced the previous interaction of aptamer and adenosine, causing the pores
to open and drug releasing from MSA. It was shown that AuNPs could cap MSN and
dissociate from MSA once exposed to ATP, confirmed by TEM microscopy. In vitro
experiments showed that the drug release of MSA was nearly 0% without ATP and
the drug release of uncapped MSN was very rapid. In the presence of 8 mM ATP,
drug release from MSA reached around 40% after 10 h. The results showed MSA
could effectively control drug release in response to ATP.
As AuNPs still have some cytotoxicity and may accumulate in the liver, Ozalp
et al. simplified these molecular gates by extending 7 more bases to 3 end of ATP
aptamer (5 -CACCTGGGGGAGTATTGCGGAGGAAGGTTCCAGGTG-NH2-3 )
where the first 7 bases in 5 -end could be hybridized with the extended 7 bases
to close the pores of MSN [94]. It was demonstrated that the release of drug from
MSN increased dramatically in the presence of 1 mM ATP and it reached around
80% release after 1-h incubation. To further confirm the specificity of ATP aptamer
to ATP, 1 mM GTP was added to aptamer-capped MSN and it was found that drug
release from MSN was very slow. In addition, drug release from MSN increased
with increasing concentration of ATP, indicating that drug release from MSN could
be controlled in response to ATP.
Changing ATP aptamer sequence may impact its affinity with ATP. To avoid
this problem, He et al. designed a “sandwich structure” composed of ATP aptamer,
ssDNA1 and ssDNA2 to serve as molecular gates of MSN (Fig. 4) [95]. The sandwich
structure would close the pores of MSN by the interaction of ssDNA1 and ssDNA2
with ATP aptamer until it met ATP, which could bind with ATP aptamer, resulting in
the opening of pores. To test the ATP-responsive drug release system, Ru(bipy)2+ 3 , as
the model drug, was loaded in MSN. It was shown that Ru(bipy)2+ 3 release from MSN
after 48-h incubation correlated with the increasing ATP concentration and it reached
a platform when ATP concentration was above 10 mM. These results indicated that
the more ATP existed in cells, the more drugs were released from MSN. To test the
stability of the aptamer-MSN system, He et al. added it into mouse serum to test
their precipitation at 37 °C. After incubating mouse serum with MSN for about 3 h,
the supernatant was still transparent, which indicated the nanoparticles were quite
stable. Moreover, to demonstrate the ATP-response mechanism, the drug release of
MSN was tested in Ramos cells as a large amount of ATP appeared to be in Ramo
cells. Once MSN was incubated with broken Ramo cells, drug release from MSN
increased dramatically, suggesting great potential in cancer drug delivery.
Mo et al. used ATP aptamer as the connecting arm, which joined DNA-graphene
oxide (GO) complex pieces together to develop ATP-activated graphene nanosheets
(Fig. 5) [96]. In this system, DNA1 and DNA2 had sequences of “head and tail,”
which would bind to aptamer (with head) and GO (with tail). ATP aptamer was
added to the DNA-GO complex to cause the DNA-GO complex to self-assemble into
ATP-activated graphene nanosheets. DOX, as a model drug, was loaded into DNA-
GO nanosheets with high loading efficiency because of π–π stacking interaction
Tumor Microenvironment and Intracellular Signal-Activated … 191

Fig. 4 Schematic illustration of aptamer-based ATP-responsive MSN system. ATP aptamer

combined with ssDNA1 and ssDNA2 forming sandwich-type DNA which could block the drug
release in the absence of ATP. When it was exposed to ATP, the sandwich structure was destroyed and
triggering drug release. Reprinted from Ref. [95] with permission, Copyright American Chemical
Society, 2012

between DOX and GO. Once ATP appeared, ATP aptamer would bind with ATP
and thus dissociated from the DNA-GO nanosheets causing the release of DOX.
The results demonstrated that in the presence of 3 mM ATP, the particle size of
DNA-GO nanosheets reduced from 550 to 270 nm. To test the intracellular ATP-
responsive DOX release, the uptake of DNA-GA nanosheets on HeLa cells was
tested by measuring the fluorescence intensity of tumor cells. It was shown the
fluorescence intensity of DOX in tumor cells increased significantly after incubating
DNA-GA nanosheets with HeLa cells at 37 °C. To confirm the triggering of drug
release was caused by ATP, the temperature was reduced to 4 °C to stop ATP synthesis
of tumor cells and almost no increase in DOX release after incubating HeLa cells
with DNA-GA nanosheets, indicating DNA-GA nanosheets had a good response to
ATP.
192 Y. Huang et al.

Fig. 5 Schematic illustration of ATP-responsive DNA-GA nanosheets for controlled drug delivery.
Reprinted from Ref. [96] with permission, Copyright Elsevier, 2015

3.7 Multi-stimuli Activated Nanocomposites for Anticancer

Drug Delivery

Single stimuli-responsive nanocomposites have achieved some progress in anti-

cancer drug delivery. Smart nanocomposites responding to multiple (two or more
than two) stimulus can take advantages of different responsive mechanisms and
make up their disadvantages, which may fulfill the different requirements in anti-
cancer drug delivery and has become an important direction in tumor drug delivery
(Table 2). Notably, these combined stimulus activations take place either simultane-
ously at the same pathological site or in a sequential manner in different settings.
These multi-stimuli activated nanocomposites with programmed site-specific drug
delivery features have shown superior in vitro and/or in vivo anticancer efficacy.
DOX is an important anticancer drug, but it has high cytotoxicity, such as
cardiotoxicity. Zhang et al. designed enzyme/pH-responsive nanoparticles to control
the DOX release in tumor cells and thus reduce the cytotoxicity (Fig. 6) [97]. In
these nanoparticles, DOX was inserted into the negative double strand of DNA,
which was complexed with cationic gelatin to form compact nanoscale complexes
(CPX1). Another pH-sensitive material histamine (His)-modified alginate (pKa 6.9)
was used to cover CPX1 to form enzyme/pH-responsive nanoparticles (CPX2). Under
a physiological environment (pH 7.2), these nanoparticles were stable. Once these
nanoparticles went into the tumor sites (pH 6.2–6.7), His-alginate would become
Tumor Microenvironment and Intracellular Signal-Activated … 193

Fig. 6 Fabrication of CXP1 and CPX2 and the mechanism of enzyme/pH-triggered drug release.
First, polyGC-DOX and C-gelatin formed into CPX1, and then CPX1 was covered by His-alginate-
PEG to form CPX2. When CPX2 was under physiological condition (pH > 7.0), it was stable. When
it was exposed to lower pH and enzyme in tumor tissues, it would be broken and release DOX.
Reprinted from Ref. [97] with permission, Copyright Elsevier, 2010

cationic and dissociate from CPX2. Then, the cationic gelatin could be digested by
gelatinase, which was highly expressed in tumors, and DOX was released after DNA
was digested with DNase. It was shown that CPX2 and CPX1 both had a high DOX
release (nearly 80%) within 5 h after incubation with the tumor tissue homogenate
supernatant, indicating that these enzyme/pH-responsive nanoparticles might work
well in the tumor environment. In addition, as CPX2 was covered by His-alginate,
which responded to low pH, it showed low DOX release (<30%) in liver tissue
homogenate supernatant (LHS) while CPX1 has a relatively high DOX release in
LHS. In vivo bio-distribution results revealed that CPX2 has the highest DOX release
in tumor cells and the lowest release in the liver at 48 h after an intravenous injec-
tion of CPX2 at the dose of 20 mg DOX/kg body weight. Anticancer experiments
in sarcoma tumor-bearing mice demonstrated that only 30% of the mice survived
after free DOX treatment, while 90% of the mice survived more than 4 weeks after
CPX2 treatment. The results indicated that CPX2 could effectively increase the drug
194 Y. Huang et al.

release in tumors and reduce drug release in livers, thus improving the anticancer
efficacy and reducing cytotoxicity of chemotherapeutics.
Generally, the anticancer efficacy of nanomedicines is typically limited by poor
tumor cell uptake and low intracellular drug release. To achieve noninvasive and
pinpointed intracellular drug release, Wang et al. reported multi-stimuli-activated
nanohybrids by coating DOX-loaded gold nanocages with hyaluronic acid (AuNCs-
HA) [98]. These well-prepared nanohybrids could be efficiently internalized into
cancer cells by the receptor-mediated process via HA-CD44 interactions. After
cell internalization, the coated HA molecules on AuNCs could be degraded by
hyaluronidase in lysosomes, resulting in the accelerated release of DOX from AuNCs
in acid lysosomes. In addition, by taking advantage of the excellent photothermal
properties of AuNCs, near-infrared (NIR) irradiation could enhance DOX release
and induce a higher therapeutic efficacy. In vitro results confirmed that in the acetate
buffer (pH 4.5) and in the presence of hyaluronidase, there was subsequent DOX
releasing from AuNCs-HA. While in pH 7.4 PBS and in the absence of hyaluronidase,
only little DOX was released from AuNCs-HA within 12 h. These results indicated
the encapsulated drug could be pinpointedly released in intracellular environments.
Biodistribution results in breast cancer-bearing mice revealed that much more Au
was accumulated in tumors of AuNCs-HA-treated mice than those of AuNCs-treated
mice. More importantly, a complete inhibition of tumor growth treated with AuNCs-
HA with the help of NIR irradiation was observed in vivo. In a word, this study
provided new insights into developing multi-stimuli responsive intracellular drug
release systems for cancer therapy.
Breast cancer appears to have higher ATP concentration in tumor cells (1–10 mM)
than that in tumor extracellular space (<0.4 mM) and pH inside tumor cells is 5.0–5.5.
According to these pathology properties, Yu Zhang et al. designed ATP/pH-activated
nanoparticles for breast cancer therapy, in order to improve tumor cell uptake and
intracellular drug release of nanomedicine [99]. First, DOX was conjugated to PEG-
poly(aspartic acid) to form PA-DOX, which could be hydrolyzed in low pH. Then
PA-DOX was connected with ATP aptamer by intercalating DOX into the double-
strand DNA to form nanoparticles. In this case, positive-charged polymers were not
needed in this nanoparticle structure, which implied lower cytotoxicity to the body.
Moreover, Des-Arg analog D-[des-Arg10] kallidin was conjugated to nanoparticles
(D-PA-DOX) for active targeting to bradykinin receptor1 upregulated in the breast
cancer cells. When the nanoparticles came to the tumor tissue, [des-Arg10] kallidin
facilitated the endocytosis by tumor cells quickly. In the tumor cells, the nanoparticles
could be destroyed by the binding of ATP to the aptamer and the breakdown of
the acid liable bond, and release DOX dramatically to improve the killing ability
of DOX. It was shown that nearly 70% of DOX was released from D-PA-DOX at
pH 5.0 in the presence of 4 mM ATP within 24 h. At a lower ATP concentration
(0.4 mM) and pH 5.0, DOX release from D-PA-DOX reached only about 40%. In
addition, at pH 7.4 and 4 mM ATP, DOX release from D-PA-DOX was even lower
than 30%. These results proved D-PA-DOX had a dual response to ATP and pH
in vitro. Orthodox tumor-bearing nude mice were used as the animal model to test
the in vivo biodistribution and tumor accumulation of D-PA-DOX. It was shown that
Tumor Microenvironment and Intracellular Signal-Activated … 195

DOX concentration in tumors for the D-PA-DOX group was approximately 100-fold
that for the free DOX group, suggesting good accumulation of D-PA-DOX in tumors.
After 3 cycles of treatment at the DOX dose of 5 mg/kg, breast cancer cell-bearing
mice in the D-PA-DOX group exhibited significantly smaller tumor volume than
any other group, indicating these ATP/pH-activated nanoparticles could effectively
control the drug release and treat tumors.
Redox and low pH are the two most appealing stimuli because they exist naturally
in all cancer cells, and they have been well identified as ideal stimuli for triggering
rapid destabilization of nanoparticle drug delivery systems inside cancer cells. To
provide effective therapeutic and diagnostic strategies against ovarian cancers, Lin
et al. developed a redox and pH-sensitive self-assembling hyaluronic acid nanopar-
ticle with active targeting peptide LHRH [100]. Anticancer drug DOX is grafted
onto hyaluronic acid via cis-aconityl linkage and disulfide bond to possess pH sensi-
tivity and redox property, respectively. With the amphiphilic property, this conjugate
could self assemble to nanoparticle and load NIR for in vivo tracking. The results
showed that the nanoparticles have a drug loading amount of about 6.2% and their
particle size was approximately 229.0 nm. Synergistic controlled DOX release from
these nanoparticles was triggered by acidic and reductive environment, indicating
these nanoparticles could release DOX rapidly under the trigger of reductive reagents
in the cytoplasm and low pH value in lysosomes. In orthotopic ovarian tumor models,
these nanoparticles possessed a preferable tumor imaging capability and an excellent
antitumor ability to almost 30% of original size in 20 days.
Apart from smart nanocomposites described above, which were all activated
simultaneously in tumor cells, nanocomposites experiencing distinct two-step
delivery kinetics under the stimuli of the tumor microenvironment and intracellular
microenvironments, have been developed for deep tumor tissue penetration, cellular
uptake, and controlled drug release. Ruan et al. developed a kind of novel nanocarrier,
G-AuNPs-DOX-PEG, which was constructed with shrinkable gelatin nanoparticles,
DOX tethered gold nanoparticles, and long-chain polyethylene glycol (PEG) [101].
GAuNPs-DOX-PEG could be degraded by MMP-2 proteins, an overexpressed extra-
cellular enzyme, with a size shrink from 186.5 to 59.3 nm. The release of DOX from
G-AuNPs-DOX-PEG was in a pH-dependent manner. Tumor spheroid penetration
and collagen gel diffusion showed G-AuNPs-DOX-PEG with pre-incubation with
MMP-2 could significantly enhance its penetrating efficiency. In vivo and ex vivo
imaging revealed that G-AuNPs-DOX-PEG could distribute into 4T1 and B16F10
tumors at the highest intensity. Correspondingly, 4T1 and B16F10 tumor-bearing
mice treated with G-AuNPs-DOX-PEG displayed the lowest tumor growth rate.
These results suggested that G-AuNPs-DOX-PEG could enter the deep tumor region
and then release DOX, resulting in a powerful antitumor effect.
196 Y. Huang et al.

4 Conclusion and Perspectives

With the up-to-date advancements in nanotechnology, biopharmaceutics, materials

science, and chemistry, the design and application of stimuli-triggered nanodevices
for drug delivery have drawn our increasing attention and sprung up one after
another. Based upon the in-depth study of pathophysiological characteristics of the
endogenous tumor microenvironment and the discrepancies in the physiological and
biological characteristics between the tumor and healthy tissue, cancer-associated
stimuli-responsive nanosystems have been increasingly considered for the delivery
of anticancer drugs. This review has demonstrated various applications for which
nanoparticles are being employed in the fight against cancer. In accordance with the
deeper understanding of the realistic conditions and interactions involved in the tumor
microenvironment, all kinds of strategies have been carried out to treat and control
the evolution and neoplastic nature of cancer. Nanoparticles drug delivery systems
with elaborately designed synthetic nanocomposites have attested to be a promising
alternative for the targeted delivery of anticancer drugs. The unique attributes of
nanocomposites like pH-activated, enzyme-activated, GSH-activated, and hypoxia-
activated peculiarities may have allowed clinicians to furnish them either as new
treatments (monotherapy) or as adjuncts to existing treatments (combined therapy) to
enhance therapeutic efficacy.
Regardless of the current remarkable progress, only a minority of these nanopar-
ticles have been examined in preclinical animal models in vivo, and few have been
applied clinically. For example, nanovehicles with multi-stimuli-activated features
or thermosensitive liposomes are still in their infancy. The efficacy of those stimulus-
responsive nanosystems depends mainly on their high specificity and sensitivity of
tumor-targeting so as to reduce systemic toxic side effects at normal tissues and
increase drug accumulation at the tumor site or at intracellular cytosolic compart-
ments in tumor cells. However, this remains many challenges, which incorporate the
off-tumor targeting caused by the exaggerated EPR effect in humans, non-specific
stimuli, a lack of understanding of the distribution and activity of some internal
stimuli, the low levels of some triggering signals that cannot meet the signal response
needs and so on. With regard to these, those newly developed nanocarriers need to
be thoroughly characterized pharmacologically, physiochemically, and immunolog-
ically before they can be approved for use in humans. Clinicians may also need to
tightly regulate the distribution of the nanoparticle size, consistency, and uniformity
between batches. Additionally, owing to the lack of adequate evidence and in-depth
researches on the safety, biocompatibility, and degradability of these novel materials,
transition of their applications from animal to human models has been considerably
hindered. In view of that, even though the potential of intracellular signal-activated
nanocomposites for anticancer drug delivery to enhance drug delivery cannot be
denied, their transition from bench to bedside still strongly rests upon the emerging
region of nanotoxicology to assess the toxic effects and elucidation of the mech-
anism of reversing chemoresistance. Nevertheless, with the deeper study of tumor
acidity, enzymes, endo/lysosomal pH, GSH, ROS, etc., we firmly believe that these
Tumor Microenvironment and Intracellular Signal-Activated … 197

tumor microenvironment-targeting strategies will offer a good chance for the prac-
tical translation of nanoparticle formulations into the clinic. Moreover, these further
well-established nanosystems may control the drug-releasing pattern spatiotempo-
rally and will improve the accumulation of chemotherapeutic agents in MDR tumors
or cells.
In general, this review discussed the exploitation of the tumor microenviron-
ment signal-activated nanocomposites for cancer therapy. The utility of tumor
microenvironment-sensitive nanoparticles holds promise in oncotherapy. We are
convinced that with rational design and continuing researches, a rosy future of these
smart nanocomposites can be foreseen for the effective treatment of multifarious
cancers.

Acknowledgements This work was supported by the National Natural Science Foundation of
China (81773283, 81472757, 81361140344).

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Nanocomposites for Cancer Targeted
Drug Delivery Therapeutics

Francisco N. Figueroa, Dariana Aristizabal Bedoya, Miriam C. Strumia,

and Micaela A. Macchione

1 Introduction

Cancer is a major public health problem and is the second leading cause of death
worldwide [1–3]. Globally, about 1 in 6 deaths is due to cancer. The 5-year relative
survival rate for all cancers combined has increased substantially since the early
1960s, but there are still many improvements to make advances in treatment, as well
as in earlier diagnosis [4].
Chemotherapy is the most common cancer treatment and plays a vital role in
treating undetectable cancer microfocuses and free cancer cells [2]. This treat-
ment uses highly potent drugs causing toxicity and subsequently, cell death [5].
Fast-growing cells are the main targets of chemotherapeutics. However, traditional
chemotherapeutic agents do not discriminate between normal cells that divide rapidly
and tumor cells causing severe side effects [2]. This lack of specificity of anticancer
drugs is the main drawback associated with conventional chemotherapy which makes
indispensable the development of novel formulations that significantly increase the
survival of these patients. In this sense, the superior anticancer ability of nanosized
carriers, usually called “nanocarriers,” has attracted the attention of scientists [6].
Nanocarriers can enhance the intracellular concentration of drugs in cancer cells
by using passive or/and active targeting strategies. “Passive targeting” is given by
the property of nanoparticles (NPs) of suitable sizes to accumulate in tumor tissues

F. N. Figueroa · D. Aristizabal Bedoya · M. C. Strumia · M. A. Macchione (B)

Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad Nacional de
Córdoba, Av. Haya de la Torre & Av. Medina Allende, Córdoba X5000HUA, Argentina
e-mail: [email protected]; [email protected]
CONICET, Instituto de Investigación y Desarrollo en Ingeniería de Procesos y Química Aplicada
(IPQA), Av. Velez Sárfield 1611, Córdoba X5000HUA, Argentina
M. A. Macchione
Instituto Académico Pedagógico de Ciencias Humanas, Universidad Nacional de Villa María,
Arturo Jauretche 1555, Villa María, Córdoba X5900KBJ, Argentina

© Springer Nature Singapore Pte Ltd. 2021 201

A. K. Nayak et al. (eds.), Biomedical Composites, Materials Horizons: From Nature
to Nanomaterials, https://doi.org/10.1007/978-981-33-4753-3_9
202 F. N. Figueroa et al.

much more than in normal tissues [7]. This effect is called the enhanced permeability
and retention (EPR) effect [8]. The reason for this phenomenon is that the abnormal
tumor blood vessels have larger fenestrations and consequently, promote vascular
permeability. On the contrary, free drugs can freely pass through the vasculature of
any tissue. On the other hand, “active targeting” is used to describe drug accumula-
tion within a target zone by, for example, specific interactions between drug/carrier
and the target cells, usually through specific ligand–receptor interactions [9].
In this context, pharmaceutical industry is evolving toward the development of
targeted drug delivery nanocarriers with the ability to transport a high amount of the
therapeutic agent specifically to cancer cells, decreasing damage to healthy tissues.
Furthermore, drug delivery systems should allow the administration of a drug to the
body with an improvement of its pharmacokinetic and biodistribution profiles [10].
Despite the fact that significant advances have been made in this research area,
substantial improvements are still needed to reach the next level of chemothera-
peutic formulations. Accordingly, it is imperative to understand the processes which
involve transport of drug to a target site after intravenous administration as well as
relevant issues for specific target diseases and the response of the body towards a drug
delivery system [9]. Rational planning of the nanocarrier structure by choosing the
components and combining them into an appropriate design should be done consid-
ering structure/properties relationships. Finally, identifying a successful targeted
drug delivery strategy provides insights to enhance the development of the next
nanocarriers.
Among nanocarriers, nanocomposites are systems integrated by more than one
material which have at least one physical dimension in the nanometer range [6]. This
multicomponent system should have different phase domains in which at least one
type of the phase domains is a continuous phase [11]. In particular, nanostructures
that incorporate nanosized particles into a matrix of another material are usually
called nanocomposites [12].
Nanocomposites for cancer targeted drug delivery should be able to facilitate
that the therapeutic cargo reaches the site of action from the administration site with
minimal nonspecific accumulations, release the therapeutic payload in/around the
target site achieving effective therapeutic levels at the site of action, protect the
therapeutic molecules from the detrimental effects of environmental factors and,
finally, avoid toxicity or adverse reactions of the chemotherapeutic molecule on
healthy tissues [10]. Furthermore, it is also important to mention that the interaction
of the drug with the nanocarrier should not alter its therapeutic action.
The interest in applying nanocomposites as drug delivery carriers is based on
their outstanding properties. Firstly, the multicomponent nature of nanocomposites
makes the resulting physicochemical properties at least an additive combination of
the properties of the individual constituents but, but even a synergistic effect can
take place giving unique properties to the composite. Additionally, nanocomposites
can be functionalized to selectively deliver drugs to specific cancer cells, thereby
improving therapeutic efficacy. Moreover, the wide variety of synthetic strategies and
nanomaterials which can be handle by the research community opens the possibility
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 203

of designing nanocomposites with multiresponsiveness to achieve better targeting

on a specific site of action.
Among nanocomposites, those which combine hard and soft nanomaterials which
are usually described as organic/inorganic nanohybrids have attracted intense atten-
tion for biomedical application due to their ability to respond to external stimuli
such as light, electric or magnetic field, etc., and their biological properties resulting
from the synergy of combining the advantageous physicochemical properties of
both the inorganic and organic component. In general, this kind of nanocomposites
is formed by inorganic NPs covered by an organic brush or shell. There are many
strategies for the construction of organic/inorganic nanohybrids which can be catego-
rized into three main kinds of methods, including surface functionalization, one-pot
synthesis, and wrapping (Fig. 1) [13].

Fig. 1 Construction of organic/inorganic nanohybrids through surface functionalization, wrapping,

and one-pot synthesis. Reprinted with permission from [13]. Copyright (2019) American Chemical
Society
204 F. N. Figueroa et al.

Surface functionalization is a powerful tool to provide nanohybrids with biocom-

patible hydrophilic surfaces and functional groups capable of interacting with
biomolecules for targeting or delivery. Otherwise, one-pot synthesis offers a simple
and efficient approach in which one of the components is formed directly during a
one-step reaction in the presence of the other component. Wrapping means the encap-
sulation of one of the components with the other through noncovalent interactions
[13].
Furthermore, those nanocomposites formed by polymers are a particularly inter-
esting type of nanocarriers. Polymers exhibit therapeutic and diagnostic advantages
over conventional medicines which lead them to be prevalent materials within those
approved by the FDA [14]. Polymeric materials modulate the toxicity and improve
cellular interactions, stability, and drug-release kinetics [15]. The polymer-drug
conjugates have an increased drug half-life and bioavailability [14]. The composition
of the protein corona layer can be modified by the addition of low-fouling polymer
coatings on the particle surface [14]. Beyond this, their facile synthesis proce-
dures which allow the obtention of stimuli-responsiveness or degradable polymer
architectures make them versatile materials for cancer targeted drug delivery.
In our description, we will take into account that the interactions that allow the
assembly of the different components of nanocomposites can be either physical or
chemical. However, it is worth highlighting that covalent binding seems to be safer
for medical applications because it does not exhibit the risk of disassembling in the
physiological environment [16].
There is a huge variety of nanocomposites reported in the literature for which
different materials have been used combined into different architectures. In this
chapter, the most important combinations of materials that can be merged to form
nanocomposites will be summarized by making a classification based on the nature
of one of the constituent materials. The emphasis will be placed on polymer-
based nanocomposites, clay-based nanocomposites, metal-based nanocomposites,
silica-based nanocomposites, magnetic-based nanocomposites, and carbon-based
nanocomposites. Moreover, we will discuss which are the features that make each
system interesting for cancer targeted drug delivery therapeutics. Subsequently, we
will highlight some examples of recent research that illustrate their potential in cancer
therapeutics.

2 Passive and Active Targeting

In general terms, the aim of targeted drug delivery is to obtain predominant drug
accumulation within a target organ or tissue selectively and quantitatively with low
systemic exposure [17].
Passive targeting means the extravasation of nanosized particles through leaky
tumor vasculature and a dysfunctional lymphatic drainage system which is charac-
teristic for solid tumors and inflamed tissues, leading to preferential accumulation
through the EPR effect [17, 18]. In the ideal case, the drug release occurs when NPs
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 205

translocate across the capillary endothelium and enter the interstitial space of the
tumor, producing an accumulation of pharmaceutical agents in the tumor intersti-
tium. In order to attain effective passive targeting, it is necessary to have enough
plasma concentrations of the drug/carrier conjugate for prolonged blood circulation
time. However, the majority (>95%) of administered NPs are known to accumulate
in other organs, in particular the liver, spleen, and lungs [9]. Consequently, nowadays
the focus is placed on developing drug delivery systems that can show passive and
active targeting simultaneously.
As it was mentioned in the introduction, active targeting means the functionaliza-
tion of drug carriers with the purpose of giving them affinity toward specific recep-
tors/markers on cells, tissues, or organs. The more quantity of available targetable
components on the target organ/cell (e.g., transferrin receptors in tumor) than in
normal cells is the basic principle of active targeting [10]. In addition, the employ-
ment of external stimuli, such as magnetic fields or ultrasound, acting on nanocarriers
to target and release drugs in the target tissue is also another kind of active targeting
[10].

3 Polymer-Based Nanocomposites

In polymer-based nanocomposites, the filler or the matrix are polymeric compounds,

the most common systems of this type are, generally, made up of a polymeric nanopar-
ticle core and a lipid or another polymer around. One of the major advantages of
these type of nanocomposites is the capability to employ natural polymers or poly-
mers like aliphatic polyesters such as polylactide, polyglycolides (PGA), and poly
(ε-caprolactone) (PCL) that are biodegradable and biocompatible with the human
body [19, 20].
Although natural polymers have good biocompatibility and biodegradability for
biomedical applications, they present low solubility and poor mechanical and thermal
properties [20]. An efficient alternative to overcome these disadvantages lies in
the possibility of combining natural and synthetic polymers, to achieve new mate-
rials with a desired combination of properties which are inherent to the polymers
employed [21]. Polymeric-based nanocomposites employed as drug delivery systems
show stable and controlled release, as well as, enhanced encapsulation efficiency and
loading capacity [22].
These features make polymer-based nanocomposites suitable for cancer therapy,
involving drug delivery, photothermal therapy, and photo-dynamic therapy, among
others. A large variety of properties can be found in polymer-based nanocompos-
ites, such as their nontoxic nature, the capability to target nucleus or another subcel-
lular compartment, passive targeting, on demand drug release and stimuli-responsive
systems, etc., which convert these systems into potential cancer treatment devices.
One particular characteristic that has to be controlled over the synthesis of
nanocomposites, in particular polymeric ones, is their size and polydispersity. The
strict control over these qualities remains a major challenge to solve in upcoming
206 F. N. Figueroa et al.

years. Different properties of the final material are related to the size, such as kinetic
stability, cellular uptake, biodistribution, and drug release kinetics.
Nanocomposites present several advantages over conventional cancer treatments,
including targeted drug delivery, strict control over the release rate of the drug,
enhanced drug absorption into tumors, and reduced side effects of the free drug by
avoiding healthy cells interactions [23, 24].
One of the most widely used polymers in the formulation of a drug delivery
nanocomposite is chitosan [25]. Chitosan-based nanocomposites have a very impor-
tant role in targeted drug delivery providing a nontoxic, biocompatible, stable, target
specific, and biodegradable delivery device. Chitosan possesses unique properties
including low cytotoxicity and inmunogenicity, biodegradability, and biocompati-
bility, making this polymer a good material for cancer therapy and other biomedical
applications [26]. Luo et al. prepared a chitosan-based nanocomposite using charge-
driven self-assembly in aqueous buffer, preventing the side effects of employing
organic solvent in synthetic methodologies. Doxorubicin (DOX) was covalently
attached to chitosan, which interacted with methoxyl poly (ethylene glycol)-block-
poly (glutamic acid) copolymer (PEG-PGA), to form nanocomposites with loaded
anticancer drug and legumain substrate peptide to confer targeted drug delivery.
These systems show high cytotoxicity against choroidal melanoma cell line (Mum-
2C) and reduced cytotoxicity on normal human corneal epithelial cells (HCEC). Drug
release remains a major problem in these systems, reaching a maximum release of
42% of encapsulated drugs [27].
Xu et al. have described a monomethoxy (polyethylene glycol)-poly(D, L-lactide-
co-glycolide) (mPEGPLGA) nanocomposite, for paclitaxel (PTX) and temozolo-
mide (TMZ) drug delivery. The thermo-sensitive gels exhibit a passive targeting
through an average size of 110 nm. This system has the potential to become a drug
delivery device for the interstitial chemotherapy of glioblastoma [28].

4 Inorganic-Based Nanocomposites

Inorganic nanomaterials have unique physicochemical properties which make them

extremely interesting for biomedical uses. Besides, synthesis and modification of
inorganic nanomaterials are usually easier procedures compared with those involved
in organic nanostructures. The available synthetic methods allow one access to the
control of the nanoparticle size, shape, and surface functionalization [29]. Inorganic
NPs can be made of robust materials that allow encapsulation and protection of
sufficient amounts of cargos for cancer treatment [30]. Furthermore, certain inorganic
nanomaterials have intrinsic functionalities that are not normally seen in organic
materials such as the ability to respond to external stimuli such as magnetic fields or
near-infrared (NIR) light [31]. They also exhibit thermal stability which is often an
advantage over organic nanocarriers.
Some inorganic nanocomposite can display targeting mediated by external stimuli
by themselves, but organic functionalization is commonly used to achieve active
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 207

targeting. Hydrophilic groups are also used to increase the stability of the nanocom-
posite dispersion in aqueous solution and consequently, prevent aggregation [32]
and improve compatibility [33]. Organic coatings are also used to give the chance to
encapsulate therapeutic drugs in hard NPs.
Furthermore, there are inorganic NPs which can load therapeutic agents inside
their pores, mesoporous inorganic NPs. However, in pure inorganic mesoporous
nanosystems the drug would be released immediately after administration because
it is simply physically adsorbed in the channels [34]. Once more, smart polymeric
frameworks are used as gatekeeper to ensure a controlled drug release in these cases.
The strategy of functionalization of the surface of inorganic nanocomposites with
organic molecules is very often used to low systemic toxicity [35]. It is well known
that pure inorganic nanocomposites become surrounded by a nonspecific protein coat
after being exposed to serum or extracellular environments, a process described as
the protein corona effect. This kind of biological coating masks the chemical nature
of this nanomaterials and provides them with biological properties [36]. Functional-
ization with polyethylene glycol (PEG), a strategy also known as PEGylation, is the
most widely used coating to shield the surface of NPs from aggregation, opsonization,
and phagocytosis, thereby prolonging circulation time [37].
In the next sections, different types of inorganic-based nanocomposites will be
described.

4.1 Clay-Based Nanocomposites

These nanocomposites are multicomponent systems where one of its components

consists of clay NPs, whether the clay NPs could be dispersed/covered by a polymer
matrix or clay NPs could be decorated with NPs of different nature. As it has
been mentioned throughout this chapter, nanocomposites have unique properties
that cannot be obtained by the materials separately [38]. The combination of these
two materials causes a synergistic effect in the improvement of physical properties,
including mechanical strength, thermal stability, among others [39]. But it is note-
worthy that clay-based nanocomposite has unique properties given by this kind of
inorganic nanoparticle, that make this nanocomposite has been reported as the most
used in various fields [40]. For example, most clays present notably biocompatibility
and biodegradation, they have lower toxicity compared to another kind of inorganic
NPs as those based on metal, oxides, silica, or carbon nanotubes [41]. Also, they can
be obtained in nature as clay minerals or by an easy and low-cost synthetic route
[6]. Besides, their two-dimensional (2D) structure gives them a high ions reserva-
tion capacity, they can incorporate ions both between the sheets and on their surface
[42]. And in the same way, clays can carry out ion exchange with ions from the
surrounding environment [41].
In general, clay structure is characterized by layered platelets stacked through
electrostatic interactions. Depending on the type of clay, it presents different kinds
208 F. N. Figueroa et al.

of isomorphic cation substitution in their structure that result in excess charge (posi-
tive or negative) on layers, this charge is balanced with the presence of water and ions
between the layered [43]. Clays can be classified into two large groups as natural
(clay minerals) or synthetic. On the one hand, clay minerals are composed of alter-
nating negatively charged aluminosilicate layers, with exchangeable interlaminar
cations [42]. In this group are kaolinite, illite, smectite (montmorillonite), chlorite,
halloysite, and vermiculite. The most used clays for biomedical applications are
kaolinite (Si2 Al2 O5 (OH)4 ) and montmorillonite (AlSi2 O5 (OH).xH2 O) [44]. On the
other hand, among the synthetic clays, the lamellar double hydroxides (LDHs), also
known as anionic clays, stand out. They present stacks of brucite (Mg(OH)2 )-like
layers that are positively charged, as a result of the isomorphic substitution of diva-
lent by trivalent cations. In this case, there are anions and water between interlaminar
spaces to balance the charge [45].
Features exposed above have allowed clays to be widely used as excipient or
active substances in pharmaceutical technology and dermopharmacy; for example
LDHs can be found in commercial antacid formulations (Almax© and Talcid©)
[46]. Currently, the possibility of reducing its size to nanoscale and incorporating
ionic drugs or biomolecules between clay sheets stabilized through electrostatic
interaction, makes them very attractive for the development of nanocarriers with
controlled release ability, by exchanging the drug with other ions present in biological
environment [42]. Though, clay-based nanocarriers have the drawback that under
physiological conditions, clay particles could have cytotoxic properties linked to
their dispersion pattern, they could form agglomerates due to the high ionic strength
and presence of different electrolytes in biological fluid [42]. However, the formation
of nanocomposites with clay and organic materials, like polymer or proteins, offers
an improvement for this inconvenience. For example, hydrophilic polymers offer
colloidal stability for inorganic NPs, that result in prolonged blood circulation and
low uptake by the reticuloendothelial system (RES) organs [13]. Likewise, another
advantage of this nanocomposite is that drug can be linked to organic material as
well as in the clay, increasing its storage drug capacity [6].
Depending on the organic material and clay employed, nanocomposites could
have three different morphologies: (a) tactoid, (b) intercalated, or (c) exfoliated
[42], as illustrated in Fig. 2.
Tactoid structures result when the polymer closes around to clay agglomerates.
Their X-ray diffraction (XRD) shows that the basal spacing of the clay does not
expand, due to poor affinity of the polymer for the clay [39].
Intercalated: in this case, the polymer presents moderate affinity for clay sheets
and their chains penetrate between of them. This causes an increase in basal space
of clay but the shape of the layered stack remains [47].
Exfoliated or delaminated structures are obtained when the polymer presents
high affinity for clay sheets, their chains penetrate between the sheets widening the
interlayer space until stacks of clay layers become disordered. Thereby, X-ray cannot
detect any regular structure and the diffractogram does not show any peaks [48].
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 209

Fig. 2 Possible morphologies obtained for clay-polymer nanocomposites

Specifically, the use of both natural and synthetic clay-based nanocomposites has
been reported for cancer targeted drug delivery therapeutics, some examples are
mentioned below.
Yang et al. have developed a nanocomposite based on Halloysite nanotubes
(HNTs) with chitosan oligosaccharide-grafted as a DOX carrier for treating breast
cancer. Through the grafting of these two materials (DOX@HNTs-g-COS), it was
possible to increase the biocompatibility of HNTs, as well as, the cellular uptake
ability of DOX. The studies in vitro and in vivo conditions showed in both cases
an improved chemotherapy effect of nanocomposite compared with the free drug.
In vitro study in MCF-7 cells showed that IC50 value was 1.17 μg/mL, while it
was 2.43 μg/for free DOX. Similarly, in vivo study in 4T1-bearing mice, the tumor-
inhibition ratio of DOX@HNTs-g-COS was 83.5%, while it was 46.1% for free DOX
[49].
Another kind of natural layered clay used in nanocomposites is montmorillonite
(MMT), Phan et al. synthesized a nanocomposite hydrogel for controlled release
of gemcitabine (GEM). They first formed a complex MMT-GEM through inter-
calation and adsorption of the drug in clay NPs, then this complex was dispersed
in a temperature-sensitive poly(-caprolactone-co-lactide)-b-poly(ethylene glycol)-
b-poly(-caprolactone-co-lactide) hydrogel. In vitro GEM release studies were
performed between pristine hydrogel and the nanocomposite, the latter exhibited
210 F. N. Figueroa et al.

longer GEM release time and a much lower initial burst. Likewise, nanocomposite
produced a significant inhibition of tumor growth on pancreatic tumor-bearing mice
[50].
On the other hand, Ray et al. designed a synthetic clay-based nanocom-
posite for treatment of osteosarcoma. An LDHs matrix was used for intercala-
tion of methotrexate (MTX), at the same time this matrix was encapsulated using
a nontoxic and biodegradable polymer, poly (D,L-lactide-co-glycolide) (PLGA).
In this way, they achieved to arrest the initial release and dose-dumping-related
toxicity. The authors conducted comparative studies between the nanocomposite
(PLGA-LDH-MTX), and its counterparts LDH, PLGA-MTX, and free MTX. Phar-
macokinetics, tissue distribution, survival rate, and in vivo antitumor efficacy in
osteosarcoma induced Balb/c nude mice were evaluated. The results showed that the
delivery efficiency of MTX using the nanocomposite was significantly higher than
bare MTX. In addition, this nanocomposite presented remarkably more effective
in vivo antitumor activity than free MTX and PLGA-MTX [51].

4.2 Metal-Based Nanocomposites

Metal NPs, especially gold and silver NPs, have been exploited as a potential platform
in controlled drug delivery for cancer therapy.
Gold nanoparticles (AuNPs) have promising applications in the fields of drug
delivery and photothermal therapy because of their unique optical and photothermal
properties. In addition, the size and shape of the particles can be easily tuned by
the appropriate choice of synthetic method, which allows tunable optical absorption
properties which make them especially suitable candidates for drug delivery appli-
cations. It is also worth highlighting that colloidal dispersions of AuNPs are one of
the most stable among metal NPs [18]. Moreover, their surface can be modified in
order to conjugate them with organic molecules.
The combination of AuNPs with other materials such as biomolecules, polymers,
and proteins can result in nanoplatforms with improved therapeutic properties. As an
example, it is worth mentioning that naked gold nanostructures are extremely toxic,
however, surface modification can decrease their toxicity [52]. PEGylation renders
the nanoparticle perfectly soluble in water and minimizes any protein adsorption
which can postpone or prevent the rapid clearance by the RES [52]. By PEGylation,
it is also possible to increase the tumor accumulation of gold-based nanocompos-
ites through the EPR effect. In particular, thiolated poly(ethylene) glycol is very
often used to functionalize the surface of AuNPs. Recently, Halas and collaborators
have reported the initial results of a clinical trial in which laser-excited gold-silica
nanoshells functionalized with thiolated poly(ethylene) glycol were used for local-
ized photothermal ablation of prostate tumors [53]. Although it is not a drug delivery
itself, cell death and tumor remission can be achieved in this case by photothermal
cancer therapy taking advantage of the ability of nanosized gold to convert absorbed
NIR light into heat and induce highly localized hyperthermia. At this point, it is
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 211

important to note that light penetration depth in tissue dramatically increases with
the increasing wavelength.
Furthermore, PEGylated magneto-plasmonic NPs with a hollow or semi-hollow
interior have been successfully synthesized and evaluated as drug carrier using
Rhodamine B isothiocyanate as a model compound [54].
Targeted DOX-loaded rattle-type gold nanorods/porous-SiO2 nanocomposites
were prepared as NIR-activated drug delivery systems for combined cancer chemo–
photothermal therapy. PEG molecules and cyclic Arg-Gly-Asp peptides were cova-
lently conjugated to the surface of the nanocomposites, providing them with biocom-
patibility and targeting capabilities. The multicomponent nanostructures exhibited
combined cancer chemo–photothermal therapy, which was much more efficient
in inhibiting cancer cell proliferation than chemotherapy or photothermal therapy
alone. They also exhibited higher drug loading capacity than conventional core–shell
nanostructures because of their hollow interior [55].
AuNPs coated with serum albumin have also improved properties such as greater
compatibility, bioavailability, longer circulation times, lower toxicity, and selective
bioaccumulation [56].
Many cancer cell types have abundant expression of transferrin receptors, conse-
quently, by decorating AuNPs with transferrin, the intracellular delivery of these
carriers is significantly increased compared with their non-targeted counterparts [57].
Polymers are also fascinating materials for the combination with gold in nanocom-
posites with defined architecture. Recently, soft poly(N-vinylcaprolactam) nanogels
surface-decorated with AuNPs were prepared and tested as potential functional
carriers. Small AuNPs on the surface of poly(N-vinylcaprolactam) nanogels are
heated via light or radiofrequency radiation triggering the nanogels to shrink [58].
On the other hand, it is well known that silver-based nanocomposites exhibit
remarkable antimicrobial effects but conversely to gold, they are not extensively
applied in cancer drug delivery applications. However, they are a promising tool
as anticancer agents in diagnostics and probing with strong effects against different
cancer cell lines. Similar to gold, silver can be synthesized by reproducible routes, its
surface can be easily functionalized and its optical properties can be tuned to have an
absorption at specific wavelengths that is useful for imaging and photothermal appli-
cations in tissue [59]. AgNPs are reported to have anticancer property themselves
because they can damage structures of cells, which finally induce cytotoxicity, geno-
toxicity, immunological responses, and even cell death. Additionally, silver-based
nanocomposites loaded with anticancer drugs exhibit synergistic antitumor effect of
both drug and AgNPs. For example, DOX loaded nanocomposites based on AgNPs
with a polyvinylpyrrolidone (PVP) coating, in combination with iodine-125 isotope
to track them in vivo after intravenous injection in normal and solid tumor bearing
mice, provided new radiolabeled Ag-based nanocomposites as tumor-specific agents
for both diagnostic and therapeutic applications [60].
212 F. N. Figueroa et al.

4.3 Silica-Based Nanocomposites

Silica-based materials have unique properties that justify their use in cancer targeted
drug delivery nanocomposites as for example hydrophilic surface, versatility for
surface functionalization by simple exploitation of the well-established silicate chem-
istry, ability to easily obtain different shapes and sizes, easy synthetic protocols on a
large scale with low production costs, lack of toxicity, biocompatibility, biodegrad-
ability, and controllable superficial charge [61, 62]. Due to their exceptional features,
these materials have been recognized as one of the most promising biomedical plat-
forms for therapeutic, diagnostic, prognostic, and combined applications [5]. In addi-
tion, silica-based materials have been approved by the FDA for use in diagnostic
imaging and silica NPs have been approved for human clinical trials. Therefore, it is
expected that in a short time they can be approved as a drug [63].
Silica/stimuli responsive polymer nanocomposites have interesting properties for
drug/siRNA delivery. For example, nanostructured silica cores wrapped within a
tuneable pH-responsive coating of poly(2-diethylaminoethyl methacrylate) hydrogel
have been synthesized in a one-pot step obtaining uniformly dispersed nanocarriers
in the aqueous phase [64]. In this case, thermogravimetric analysis shows that the
formation of covalent chemical bonds between the silica and the polymer increases
the stability of the organic phase around the inorganic core. These nanocomposites
were loaded with small interfering RNA (siRNA) which was efficiently delivered into
the cytoplasm of MDA-MB-231 breast cancer cells and inhibited the protein expres-
sion of CXCR4 with an efficacy comparable to that of a commercial transfection
reagent. In vivo studies show that these nanocomposites preferentially accumulated
in the tumors of human breast cancer-bearing mice and mediated CXCR4 silencing
in these tumors.
Among silica-based nanomaterials, ordered mesoporous silica materials (OMMs)
have received increasing popularity in drug delivery. These materials have a highly
mesoporous structure which provides a large surface area and high pore volume
which allow large quantities of drug molecules to be loaded into their structure,
which is one of the major challenges in the development of carriers for anticancer
drugs [5]. The size of the mesoporous framework can be tuned during synthesis,
making these structures adaptable hosts for load agents of various sizes [65]. Pores
and the surface of this kind of NPs can be modified with a huge number of molecules
including drugs and fluorescent dyes (Fig. 3).
Their unique architecture of mesoporous silica allows the development of
prolonged release systems for hydrophobic drugs. As an example, nanocompos-
ites made of mesoporous silica NPs within a hyperbranched polymer network made
from poly(vinyl alcohol) (PVA) and poly(acrylic acid) (PAA) were employed as ideal
host materials with high adsorption capacity for a model hydrophobic drug, anti-
inflammatory agent dexamethasone, exhibiting prolonged, sustained release profiles
for as long as two months [66]. Biological characterization shows that these nanocom-
posites can, thus, be highly appealing biomaterials for sustained and prolonged drug
release, such as wound dressing systems.
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 213

Fig. 3 Mesoporous silica nanoparticles and their functionalization alternatives

As it was mentioned in the introduction, drug delivery vehicles without side

effects to normal physiological tissues are the actual challenge for safety and effec-
tive nanomedicine. Recently, a multifunctional drug delivery vehicle containing an
ordered mesoporous resin as a polymer core and homogeneous Fe nanodots-doped
silica as the biodegradable shell was developed [67]. The Fe-doped silica shell acts
as a compact inorganic cap to seal DOX into the mesoporous polymer cores, but
also serves as a superparamagnetic agent for magnetic targeting and magnetic reso-
nance imaging (MRI). Via Fe extraction-induced degradation, the loaded drug can be
slowly released under the acidic tumor environment, achieving ultralow drug leakage
under normal in vivo blood circulation (physiological environment).
214 F. N. Figueroa et al.

4.4 Magnetic-Based Nanocomposites

This multicomponent material presents magnetic NPs that induce the response to a
noninvasive stimulus, the external magnetic field [68].
Currently, there has been a notable increase in the interest of developing magnetic
nanocomposites, mainly in drug delivery field, because they have three features that
highlight them among over of other nanocomposites. First of all, the magnetic NPs
present in these nanocomposites mean that they can be used in MRI [13]. Secondly,
they have magnetic guidance toward the target tumor or organ [40]. Finally, this
kind of nanocomposites can be used as theragnostic systems by themselves, namely,
they have both applications in diagnostic and therapy [69]. Therapy applications of
magnetic nanocomposites involve a hyperthermia process which consists in inducing
local heat when the magnetic nanoparticle is subjected to an oscillating magnetic
field [70]. Nowadays, this is an approved treatment as a therapy against cancer, but
it is necessary to combine it with chemotherapy or another different treatment [71].
Furthermore, the hyperthermia process can be used as a mechanism to release the drug
when the magnetic NPs are conjugated to a thermosensitive polymer. The magnetic
heating can induce conformational changes (swelling/collapse) in the polymer that
produce drug release [72].
Iron oxide NPs, magnetite (Fe3 O4 ) and maghemite (Fe2 O3 ), are commonly used to
develop magnetic nanocomposites. In addition to their magnetic properties, these are
easily obtained through a coprecipitation synthesis [13]. Moreover, they have good
chemical stability and low toxicity. Thus, the combination of these NPs with different
materials including polymers, clays, silica, carbon, or metal organic frameworks has
been extensively studied [68]. Some of these nanocomposites, whether they are
magnetic NPs as part of the core or as a decoration of the mentioned materials, are
described below.
Delavari et al. designed a theranostic nanocomposite for DOX release using Fe3 O4
NPs as a MRI contrast agent and a coating of α-lactalbumin which was precipi-
tated and cross-linked with poly(ethylene glycol) and glutaraldehyde. In addition,
the resulting nanocomposite was pH-sensitive due to the formation of imine bonds
between glutaraldehyde and amine groups on α-lactalbumin and polyethylenimine
that resulted in the higher release of DOX at acidic pHs. The studies both in vitro
and in vivo reflected greater action by the DOX-loaded nanocomposite compared to
the free drug. Likewise, nanocomposite was less immunogenic than free DOX [73].
Another example of magnetic nanocomposite with dual capacity was developed
by Meenach et al. Their system had the ability to deliver paclitaxel (PTX), as an
anticancer drug, and produce heat by an alternating magnetic force (AMF) for the
potential treatment of cancer. This consists of a hydrogel of poly(ethylene glycol)
loaded physically with iron oxide NPs entrapped and PTX as chemotherapeutic
agent. Their swelling, heating, and mechanical characteristics were studied according
to the cross-linking density. Also, cytotoxicity assays on three different cell lines
including the M059K (glioblastoma), MDA-MB-231 (breast carcinoma), and A549
(lung denocarcinoma) were conducted by exposing them to PTX only, hyperthermia
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 215

only, and both PTX and hyperthermia. The drug release experiments showed that the
lower cross-linked hydrogels released PTX more quickly, and in vitro cell studies
showed that combined PTX and hyperthermia treatment increases the antitumor
efficacy for A549 lung carcinoma cells [74].

4.5 Carbon-Based Nanocomposites

Since the discovery of graphene, the studies of carbon allotropes have gained signif-
icant attention because of their versatile applications. Graphene and its derivatives,
and single or multiple walled carbon nanotubes (SWCNTs or MWCNTs) are the
most employed systems in carbon allotropes family [10, 75]. They possess unique
properties that make them potential anticancer drug delivery devices, such as good
chemical and thermal stability, flexibility, large surface area with a high density of
easily modifiable functional groups, good biocompatibility, and high capability of
drug loading by π-π physical interaction [76–78].
Graphene oxide (GO) and reduced graphene oxide (rGO) nanocomposites have
drawn extensive attention in biomedical applications like drug delivery, bioimaging,
and sensing, owing to their biocompatibility, aqueous solubility, and ease of function-
alization [79]. GO and rGO toxicity has been widely studied, due to its physicochem-
ical characteristics like purity and 2-dimensional size may influence nanocompos-
ites cellular uptake [75]. It has been proved that GO exhibits cell toxicity depending
on time and dose of exposure [80]. GO can also destroy the cell membrane, their
unique structure enables it to develop strong electrostatic interactions with cellular
membrane, given by π-π stacking with biomolecules, proteins, and/or nucleic acids
[81]. Interactions with cellular membrane may be also given by negatively charged
oxygen in GO and the positively charged lipids in the membrane [82, 83].
Li et al. studied the interaction of graphene with three different cell type by
molecular dynamics simulations and fluorescence and microscopic imaging. They
discovered that the edge of graphene could penetrate through cellular membrane in
human skin and immune cells [84]. Taking this into consideration, greater efforts are
being made to further modify graphene in order to cause less toxicity [85].
Graphene-based nanocomposites are being widely used in biomedical applica-
tions, for all the previously mentioned qualities. This kind of nanocomposites can
be classified into different groups for cancer drug delivery, graphene-based metallic
nanocomposites, graphene-based quantum dots, graphene-based iron oxide magnetic
NPs and graphene-based polymeric nanocomposites.
Graphene-based metallic composites are synthesized by incorporation of metallic
NPs into graphene sheets. As stated previously, graphene sheets present strong Van
der Wall forces causing restacking issues in synthesis methods, which limits GO
and rGO applications. This problem is solved by the incorporation of metallic NPs
(metal and metal oxides NPs) into graphene; metallic NPs reduces significantly the
π-π stacking interactions. Gold (Au), silver (Ag), platinum (Pt), and cadmium (Cd)
NPs are the most employed in the formation of graphene-metal nanocomposites [75].
216 F. N. Figueroa et al.

The combination of carbon allotropes with metals or metallic oxides has become an
attractive alternative for developing novel nanocomposites with unique properties to
achieve efficient drug delivery.
Despite the inherent toxicity of GO, which can generate reactive oxygen species
(ROS) or destroy de cell membrane through strong electrostatic interaction, different
graphene-based nanocomposites with reduced adverse effects were synthesized and
employed as drug carriers. Kakran et al. have covalently functionalized GO with
hydrophilic and biocompatible Pluronic F38, Tween 80, and maltodextrin for loading
and delivery of a poorly water-soluble antioxidant and anticancer drug, ellagic acid
(EA). Studies have shown that loaded EA presents higher cytotoxicity rather than
free EA. Therefore, all three functionalized GO composites are suitable nanocarriers
for drug delivery because of their nontoxicity and high drug loading capacity [86].
Shi et al. synthesized a GO@Ag nanocomposite by chemical deposition of AgNPs
onto GO. DOX, one of the most effective drugs against a wide range of cancers, was
employed as drug with a high drug loading efficiency. GO@Ag-DOX nanocomposite
was further functionalized with PEG2000-co-(Asn-Gly-Arg) peptide, to achieve
active tumor-targeting capacity and excellent stability in physiological solutions.
Compared with free DOX, these nanocomposites afforded much higher antitumor
efficacy without obvious toxicity in a tumor model in vivo. This system can also
be employed as an agent for photothermal ablation of tumor. The ability of the
GO-NPs to combine chemotherapy with external photothermal therapy significantly
improved the therapeutic efficacy [87].
A new class of nanomaterial emerged when 2D graphene folds on itself, to form
a triangular 0D structure. Graphene quantum dots (GQD) exhibit strong quantum
confinement and edge effects with a size smaller than 10 nm [88], making these
systems potential drug delivery and theragnostic devices. In addition, they present
great stability and can be obtained by easy synthetic methods. GQD possess some
interesting properties, such as, great spin, optical and electrical properties. They can
be easily modified by carboxylic acid making them more hydrophilic resulting less
cytotoxic and more biocompatible [89].
GQD can be employed as targeted drug delivery systems because they can facil-
itate the transportation of the loaded molecule to the cell membrane and nucleus.
Moreover, QGD can efficiently improve the nuclear accumulation rate of drugs, also
enhanced the activity and cytotoxicity of these drugs against cancer cells [90]. Javan-
bakht et al. designed a hydrogel nanocomposite film by incorporation of GQD as
NPs into a carboxymethyl cellulose hydrogel. DOX was efficiently loaded into the
nanocomposite, as a broad-spectrum anticancer drug. GQD-carboxymethyl cellulose
nanocomposites showed an improvement in degradation, pH-sensitive drug delivery,
and significant toxicity against blood cancer cells [91].
An interesting alternative of graphene materials relies on combining GO with iron
oxide NPs. Fe3 O4 is less toxic, superparamagnetic, biodegradable, and biocompatible
than other magnetic NPs resulting in a promising graphene-based composite [75].
GO acts as an oxidizing agent converting Fe2 into Fe3 , generating Fe3 O4 and turning
itself into rGO, thus in situ deposition of the iron oxide on the rGO surfaces is
achieved. The final material shows properties from both components [92].
Nanocomposites for Cancer Targeted Drug Delivery Therapeutics 217

Carbon-based polymer nanocomposites are considered unique candidates to be

employed as drug delivery carriers. Inorganic carbon provides excellent thermal,
mechanical, and optical properties [93], and polymers are capable of encapsulating
and releasing cargo molecules with the ability to achieve active and passive targeting.
The physicochemical properties of the nanocomposites will be determined by the
dispersion of the graphene sheets inside the polymer matrix. Graphene sheets modi-
fications with polymers result in increased biocompatibility and higher drug loading
efficiency. Goncalves et al. grafted poly(methyl metacrylate) from the GO surface
via atom transfer radical polymerization (ATRP) to generate a novel nanocomposite
with a homogenous dispersion of GO in the polymer matrix [22].
The most employed polymer in carbon-based composites is chitosan, its biocom-
patibility and cationic nature enables to target cancer cell based receptors and nega-
tively charged surfaces of the cell [94]. Wang et al. investigated a novel drug delivery
system, composed of NPs of galactosylated chitosan/graphene oxide/DOX for cancer
treatment. The synthesized nanocomposite exhibited a high drug loading capacity and
presented a pH-responsive behavior to release the loaded molecule in a low pH envi-
ronment. Additionally, nanocomposite showed higher cytotoxicity compared to the
non-glycosylated nanocomposite (chitosan/graphene oxide/doxorubicin). Further-
more, Wang et al. synthesized a novel drug delivery system by encapsulation of
superparamagnetic GO and DOX with folic acid conjugated chitosan. Nanocompos-
ites exhibited a high loading efficiency and a prolonged release rate, which can be
controlled by varying the pH. This system could be applied as dual-targeted drug
nanocarrier, biological and magnetic targeting capabilities [81, 95]. These results
showed that different components, such as polymers and magnetic NPs, can be
combined with GO/rGO to obtain an improved material with the properties of all
components.
Carbon nanotubes (CNTs) are a carbon allotrope which is cylindrical. Carbon
nanotubes are hollow cylinders formed by graphene sheets rolled up on itself. These
systems can be single walled carbon nanotubes (SWCNT), in which a single graphene
sheet is rolled, or multiwalled carbon nanotubes (MWCNT), consisting of several
SWCNT of different diameter inside each other. Their aromatic structures enable
them to absorb several anticancer drugs, as well as, polymeric anticancer molecules
by π-π stacking interactions [10]. Taking this into account, CNTs have emerged as
a potential carrier for anticancer drug delivery and diagnosis. Chen et al. developed
a CdTe quantum dots with Fe3 O4 -filled CNTs. This specific nanoplatform achieved
magnetically guided anticancer drug delivery. The combination of iron oxide inside
CNTs not only provides the magnetic property but also protects the CNTs from
agglomeration, enhancing their stability and drug loading capacity. By coating the
CNTs surfaces with transferrin, dual responsiveness is achieved. These properties
make CNTs interesting nanocarriers for drug delivery [96].
218 F. N. Figueroa et al.

5 Conclusions and Perspectives

Becoming quite popular in the last years, nanocomposites have gained a lot of atten-
tion in the field of biomedicine due to their improved physicochemical properties.
The use of targeted nanocomposites in drug delivery ensures a controlled release
rate in the desired site avoiding the side effects of highly specifics drugs. The possi-
bility of combining two or more components into a single material enables them to
possess unique properties, such as high loading capacity, enhanced biocompatibility
and biodegradability, decreased toxicity, improved mechanical properties, and the
capacity to image guidance.
In this chapter, we have summarized the most important concepts and recent
advances in the use of nanocomposites in targeted cancer drug delivery. From the
discussion made, it is possible to conclude that the multifunctional characteris-
tics of nanocomposites make them highly suitable for this biomedical application.
Nonetheless, overcoming toxicity drawbacks related to the used of nanocomposites
in medicine remains a challenge to be solved in the upcoming years. Several issues
as possible side effects, the long-term effects of biodistribution, the accumulation
in nontarget tissues, as well as, the toxicity effects are imperative to be addressed
in depth by the research community in order to move along to clinical trials and
increase the number of FDA-approved nanomedicines. The need of interdisciplinary
points of view in order to achieved better understanding of the structure/property
relationships, biological performance and pharmaceutical effects of nanocomposites
should not be overlooked. Therefore, both the development of novel nanocomposites
and the in vivo evaluation of the safety and efficacy of those already reported ones
are necessary for continuous progress.
Furthermore, multicomponent systems can offer the opportunity of developing
image-guided cancer treatments or improving the antitumoral effect. Finally, all the
considerations made let us to conclude that inorganic materials will display better
targeted drug delivery properties when they are interacting with soft organic systems
as polymeric materials.
All in all, much work is still needed for the development of improved nanocom-
posites which can become the next generation of efficient chemotherapeutic
formulations.

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