Accepted Manuscript

Pharmaceutical applications of chitosan

Zahra Shariatinia

PII: S0001-8686(18)30277-X
DOI: https://doi.org/10.1016/j.cis.2018.11.008
Reference: CIS 1927
To appear in: Advances in Colloid and Interface Science

Please cite this article as: Zahra Shariatinia , Pharmaceutical applications of chitosan. Cis
(2018), https://doi.org/10.1016/j.cis.2018.11.008

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Pharmaceutical applications of chitosan

Zahra Shariatinia shariati@aut.ac.ir

Department of Chemistry, Amirkabir University of Technology (Tehran Polytechnic),

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P.O.Box:15875-4413, Tehran, Iran

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
Corresponding author.
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Abstract
Chitosan (CS) is a linear polysaccharide which is achieved by deacetylation of chitin,
which is the second most plentiful compound in nature, after cellulose. It is a linear
copolymer of β-(1→4)-linked 2-acetamido-2-deoxy-β-D-glucopyranose and 2-amino-2-
deoxy-β-D-glucopyranose. It has appreciated properties such as biocompatibility,
biodegradability, hydrophilicity, nontoxicity, high bioavailability, simplicity of modification,
favorable permselectivity of water, outstanding chemical resistance, capability to form films,
gels, nanoparticles, microparticles and beads as well as affinity to metals, proteins and dyes.

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Also, the biodegradable CS is broken down in the human body to safe compounds (amino
sugars) which are easily absorbed. At present, CS and its derivatives are broadly investigated

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in numerous pharmaceutical and medical applications including drug/gene delivery, wound

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dressings, implants, contact lenses, tissue engineering and cell encapsulation. Besides, CS has
several OH and NH2 functional groups which allow protein binding. CS with a deacetylation
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degree of ~50% is soluble in aqueous acidic environment. While CS is dissolved in acidic
medium, its amino groups in the polymeric chains are protonated and it becomes cationic
which allows its strong interaction with different kinds of molecules. It is believed that this
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positive charge is responsible for the antimicrobial activity of CS through the interaction with
the negatively charged cell membranes of microorganisms. This review presents properties
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and numerous applications of chitosan-based compounds in drug delivery, gene delivery, cell
encapsulation, protein binding, tissue engineering, preparation of implants and contact lenses,
wound healing, bioimaging, antimicrobial food additives, antibacterial food packaging
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materials and antibacterial textiles. Moreover, some recent molecular dynamics simulations
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accomplished on the pharmaceutical applications of chitosan were presented.

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Keywords: Chitosan; Pharmaceutical applications; Drug delivery systems; Tissue

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engineering; Wound healing; Antimicrobial activity

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1. Introduction
Chitosan (CS) is the most important derivative of chitin which is prepared by the
alkaline deacetylation of chitin [1]. Chitosan structure is comprised of β-1,4-linked 2-amino-
2-deoxy-β-D-glucose (deacetylated D-glucosamine) and N-acetyl-D-glucosamine units [2].
According to United States Food and Drug Administration (USFDA), it is a GRAS (Generally
Recognized as Safe) material and therefore it has found wide pharmaceutical and biomedical
applications [3]. It is known as a bioactive compound that has shown numerous biological
properties such as antitumor, immunoenhancing, antifungal, antimicrobial, antioxidant and

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wound healing activities. These features plus some exceptional properties such as non-
toxicity, biodegradability, biocompatibility, non-antigenic and low-cost have led to its

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extensive pharmaceutical applications including biomedicine with the possibility of clinical

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use [4], drug delivery systems [5-8], tissue engineering [9], food technology [10,11],
bioimaging [12], implants [13], contact lenses [14], gene delivery [15], protein binding [16],
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wound healing [17] and textile industry [18].
In this review, the key research findings on the most recent pharmaceutical applications
of CS in diverse biomedical fields such as wound healing, tissue engineering, drug/gene
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delivery, protein binding, cell encapsulation, preparation of implants and contact lenses,
bioimaging, food additives, food packaging and antibacterial textiles (Scheme 1) will be
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presented. The molecular dynamics simulations on the pharmaceutical applications of

chitosan were also afforded.
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2. Pharmaceutical application of chitosan in medicine

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2.1. Application of chitosan in pharmaceutics/drug delivery

Recently, pharmaceutical carriers such as polymers, micelles, liposomes and
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nanoparticles have received increased attention [19-22]. These systems reveal numerous
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advantages principally in enhanced efficacy and safety of the drugs. These systems can
incorporate both hydrophobic and hydrophilic active compounds, which depends on the
carrier nature. As well, they can provide higher stability for the therapeutics against chemical
and enzymatic degradation, longer drug influence in the target tissue, superior bioavailability
and drug targeting by inclusion of specific ligands [23]. These systems are used to carry small
active molecules, proteins, peptides, vaccines, genes and oligonucleotides which are
adsorbed, encapsulated, covalently and/or electrostatically attached to their surface or within
their matrix [24]. Drug delivery systems based on polysaccharides have revolutionized
medical treatments due to their efficient and appropriate drug delivery. Among various
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polymers, the CS-based drug delivery systems are attracting substantial interest as the
vehicles that are able to release their contents at the desired rate and location in the body [25].
In a recent study, aceclofenac-loaded nanocomposites were prepared using two natural
polysaccharides including CS and locust bean gum (LBG) and glutaraldehyde as cross-linker
[28]. The infrared spectra established the formation of composites and the chemical
compatibility of polymers and drug. The influence of polymeric components on the particle
size and drug entrapment efficiency (DEE) of the composites was investigated. It was shown
that increasing LBG amount really lessened the DEE from 72% to 40% and created larger

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particles (372-485 nm). Nonetheless, a reverse trend was observed when the CS concentration
was enhanced. Among these composites, the greatest DEE=78.92% and the smallest

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composites size (318 nm) was gained when LBG:CS mass ratio was 1:5. Also, the CS:LBG

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(1:5) exhibited the slowest drug release rate in phosphate buffer solution (pH=6.8) up to 8 h.
The drug release properties excellently supported the swelling results of the nanocomposites
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so that the composites proficiently suppressed the burst release of drug in acidic solution
(pH=1.2). The in vitro drug delivery by the nanocomposites was happened through anomalous
transport mechanism. Hence, these CS and LBG nanocomposite systems could decrease the
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gastrointestinal side effects of the drug through slow sustained release of the medication.
CS nanoparticles (NPs) were utilized for the encapsulation of levofloxacin antibacterial
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agent to treat ocular infection [29]. The CS NPs were obtained through ionic gelation process
using CS and sodium tripolyphosphate. The formulations displayed the particle size in
nanometer range with high loading and encapsulation efficiency. The optimized formulation
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was changed to a sol-gel system in order to increase the corneal residence time. The
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histopathology of cornea established that the optimized formulation was non-irritant and
benign for topical ophthalmic usage. Hen's egg test-chorioallantoic membrane (HET-CAM)
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test is a rapid, inexpensive and sensitive method used to check the irritation ability of
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ophthalmic formulations. The chorioallantoic membrane of the chick embryo contains

complete tissue which includes arteries, veins and capillaries that technically responds to
injuries with a complete inflammatory process. The LFX-CS-NPs, sodium chloride solution
(0.9%, negative control) and 0.1 N NaOH (positive control) were examined for their irritation
potentials so that the irritation scores were obtained in 5 min (Fig. 1). It was shown that both
of the LFX-CS-NPs and sodium chloride solution were almost non-irritating (score 1) but
0.1N NaOH solution exhibited coagulation which confirmed this solution caused irritating
effects (score 13.43). Thus, the non-irritant LFX-CS-NPs in situ gel system could be well
tolerated for ocular administration [29].
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The antimicrobial assay proved that the formulation had high antibacterial effect against
S. aureus and P. aeruginosa microorganisms. The pharmacoscintigraphic test showed the
decreased corneal clearance, nasolachrymal drainage and greater LFX retention compared to
LFX solution. It was determined that the levofloxacin loaded CS NPs in situ gel system was a
proficient vehicle for ocular levofloxacin delivery. Histopathology of goat eye cornea did not
illustrate any variations in the eye tissue after the optimized LFX-CS-NPs in situ gel
formulation was applied that verifies the LFX-CS-NPs was safe for ocular application without
any ocular irritation influence. It did not display any histopathological changes so that the

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cells in the corneal membrane were remained intact in the same position without any rupture
in the corneal section in comparison to the control (Figs. 2A and 2B) [29].

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Confocal laser scanning microscopy (CLSM) of the formulations on excised goat

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cornea showed the permeation extent using the fluorescent intensity. The dye loaded LFX-
CS-NPs in situ gel penetration depth into the cornea was 91.73 µm having a high fluorescence
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intensity compared to that of the LFX solution (47.65 µm depth), see Figs. 3A and 3B. High
penetration of LFX-CS-NPs can be associated to the bioadhesive nature and high viscosity of
CS which caused extended residence time on the cornea, hence intracellular and intercellular
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penetration was occurred [29].

In another research, negatively charged carboxylic curdlan (Cc) including a β-1,3-
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polyglucuronic acid structure was used to produce nanosized polyelectrolyte complexes

(PECs) with positively charged CS in aqueous solution as effective delivery carriers for
anticancer drug 5-fluorouracil (5Fu) [30]. Nanosized CS/Cc PECs were prepared by adding
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0.5 mg/mL solutions of CS and Cc in 1:1 (w/w) ratio at pH=3.0. Under the optimized
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conditions, the CS-Cc PECs revealed an average size of around 180 nm, spherical
morphology and a zeta potential of about 41 mV. The 5Fu drug was loaded to the nanosized
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CS/Cc PECs and exhibited outstanding loading content (10.81%) and encapsulation efficacy
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(86.47%). The in vitro drug release data specified that the nanosized CS-Cc PECs were
potential platforms for the sustained release of 5Fu having an anomalous transport mechanism
which followed the Ritger–Peppas model. Moreover, the CS-Cc PECs demonstrated low in
vitro cytotoxic activity against HeLa and SPCA-1 cell lines. Thus, it was suggested that the
developed nanosized CS-Cc polyelectrolyte complexes were promising antitumor drug
vehicles.
The in vitro drug release was examined using two pH values (1.4 and 7.4) at 37 C for 2
days. Fig. 4 displays the cumulative 5Fu release profiles from the CS/Cc PECs. It was found
that the 5Fu was released from the CS/Cc PECs in a sustained pH-dependent manner. The
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controlled 5Fu release from the PECs was related to the interaction of 5Fu and the –COOH
functionalities existing on the PECs. Also, the burst 5Fu release was happened in the first 12 h
which was followed by a steadily and slightly controlled release. The cumulative 5Fu release
after 48 h was nearly 87% for pH 7.4 and 70% for pH 1.4. The cumulative 5Fu release was
greater in neutral pH medium than in acidic pH. The zeta potential values of both the blank
and 5Fu-loaded CS/Cc PECs were positive confirming CS dominated the PECs and placed on
the PECs surface. In acidic condition, the CS chains at the interface are completely expanded
that create an interface to decrease the drug diffusion out of the nanocarrier. In neutral pH

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medium, the deprotonated CS chains at the interface are collapsed to the colloids cores and
could not affect the drug diffusion. Furthermore, the greater interactions of 5Fu and the CS/Cc

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PECs functional groups (particularly –COOH) are probably responsible for the enhanced 5Fu

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release from the PECs in neutral pH [30].
Nowadays, nanoparticle-based vaginal drug delivery carriers have received great
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attraction for the administration of peptide based-vaccines or microbicides in order to inhibit
and/or treat sexually transmitted diseases [31]. In this context, a direct and effective approach
was established for the vaginal application and delivery of peptide-loaded mucoadhesive
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nanoparticles [31]. This formulation was principally consisted of CS NPs encapsulated in

appropriate hydrophilic freeze-dried cylinders. The CS nanoparticles were used to carry the
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peptide drug and allow adhesion to the vaginal mucosal epithelium. Hydrophilic freeze-dried
cylinders assisted the fats release of the nanoparticles to the vaginal zone. After contacting
with the aqueous vaginal medium, the excipients of these sponge-like materials were rapidly
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dissolved and facilitated the release of their contents. The in vitro release test displayed the
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capability of the sponge-like systems and CS NPs to deliver the mucoadhesive nanoparticles
and peptide, respectively. The confocal laser scanning micrographs verified that the
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nanoparticles were able to promote the peptide penetration into the vaginal mucosa.
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Thermoresponsive nano-sized hydrogel particles were prepared as smart platforms

using CS natural polymer for curcumin delivery [32]. Chitosan backbone was grafted to poly–
(N-isopropylacrylamide) (pNIPAM) through the well-known N-ethyl-N′-(3-
(dimethylamino)propyl)carbodiimide/N-hydroxysuccinimide coupling reaction. The CS-
grafted pNIPAM (CS-g-pN) nanogels were obtained by a sonication process. Addition of
curcumin to the CS-g-pN nanogels was done by incubation route. Size, morphology of
nanogels, curcumin amounts in the nanogels and cellular uptake were examined by DLS,
TEM, fluorescent spectroscopy and confocal microscopy techniques, respectively. The
CellTiter-Blue® cell viability assay was carried out using HeLa and NIH-3T3 cells to
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evaluate the safety and the MTT assay was performed using HepG2, Caco-2, HT-29 and
MDA-231 cells to determine cytotoxic properties. It was shown that CS-g-pN with 3–60%
modification degree was easily assembled as curcumin encapsulated spherical nanogel
particles with submicron sizes. The thermoresponsive performances of CSg-pN nanogel
formulations were different because grafted pNIPAM had various length and density. The
CS-g-pN nanogels were non-toxic to HeLa and NIH-3T3 cells. All of curcumin-loaded CS-g-
pN nanogel were uptaken to NIH-3T3 cells and exhibited dose-dependent cytotoxicity against
cell lines tested. Therefore, development of such curcumin-loaded nanogels produced

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materials that could be functionalized and applied for targeted therapy and controlled delivery
of small drug molecules and biomolecules for biomedical applications.

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Folic acid-CS conjugates were used as drug delivery tools by loading of folic acid (FA)

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by chitosan-15 and chitosan-100 kDa in aqueous solution at physiological pH [33].
Thermodynamic parameters ΔH =−18 to −12 (kJ·mol−1 ), ΔS = −22 to −8 J.mol−1 .K−1 and ΔG
= −11 to −9 kJ.mol−1 displayed that FA-CS bindings occurred through van der Waals and H-
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bonding contacts. The FA-CS conjugate stability was enhanced by increasing the polymer
size. The FA loading efficiency was 35 to 55%. The TEM images demonstrated great polymer
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morphological variations upon acid interaction. The CS nanoparticles were able to in vitro
deliver folic acid.
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It is well-known that in cancer theranostics, the key strategy in nanoparticle-based

targeted delivery system is enhanced permeability and retention (EPR) influence of
macromolecules [34]. Application of varied nanoparticles offers a deeper understanding of
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diverse EPR effects which depends on their structures, chemical modifications and
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physicochemical features. Currently, the tumor microenvironment is considered as another

significant factor in tumor-targeted delivery by nanoparticles but the relationship between
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EPR effects and tumor microenvironment has not yet been completely clarified. Thus, ectopic
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subcutaneous tumor models representing dissimilar tumor microenvironments were prepared

by inoculation of U87, SCC7, HT29, A549 and PC3 cancer cell lines to athymic nude mice
[34]. The tumor-targeted delivery of self-assembled glycol CS nanoparticles was tested using
the five diverse types of tumor-bearing mice in order to recognize the correlation of the tumor
microenvironments with targeted delivery of CS NPs. The neovascularization and degree of
intratumoral extracellular matrix (ECM) were both vital in defining the tumor targeted
delivery of CS NPs. The EPR influence was increased in the tumors including great amount
of angiogenic blood vessels and little intratumoral ECM content. Thus, it was found that
different EPR impact was achieved for the tumor-targeted delivery of nanoparticles and it
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depended on the tumor microenvironment in distinct tumors. In order to overcome existing

limits in clinical nanomedicine, the tumor microenvironment of the patients and EPR effects
in clinical tumors must also be examined [34].
The in vitro CNPs cellular uptake was comparatively assessed in several cancer cells in
three pH environments (pH 6.0–7.4). In pH 6.0, 6.5 and 7.4, the CNPs were observed at the
cancer cells membranes after 30 min of incubation (Fig. 5). Besides, the in vitro CNPs
cellular uptake was time dependently tested using five different cancer cell lines. After 6 and
24 h post-incubation, the near-infrared fluorescence (NIRF) signal representing the CNPs

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distribution were detected in the cytoplasms of the cancer cells. Thus, the CNPs were
effectively internalized to the cancer cells confirming the CNPs were delivered to these five

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diverse cancer cells in vitro [34].

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The targeting efficiency of CNPs in dissimilar tumor tissues was assessed. The Cy5.5-
labeled CNPs (1 mg/mL, 200 μL/head) were injected intravenously to the tumor-bearing mice
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with the mean volume of each tumor was ~300–350 mm3 . The time-dependent whole body
NIRF images indicated the in vivo CNPs distribution in the mice that were transplanted with
five different cancer cells, Fig. 6. The NIRF intensities at tumor sites were steadily enhanced
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and the maximum intensities from tumors were achieved between 24 and 72 h. also, the
whole body NIRF images established that the CNPs were circulated in the blood during a
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long time and be successfully delivered to tumors in a targeted way. Even though the CNPs
were accumulated in the tumor cells of all tumor-grafted mice, the targeting effectiveness of
CNPs was significantly dissimilar which was depended on the tumor type. The CNPs
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provided high contrast images for SCC7, U87 and HT29 tumors confirming they were
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efficiently delivered to the tumors. However, PC3 and A549 tumors provided rather low
NIRF signals demonstrating few CNPs were delivered to these tumors [34].
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The tumors encapsulated with fibrous connective tissue was totally stained blue using
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trichrome stain and the peritumoral area was not used for the imaging in order to eliminate
such area of excess collagen in dermis. The ECM contents in the tumor tissues were observed
and each tumor type displayed diverse amounts of intratumoral ECM, Fig. 7A. The A549 and
PC3 tumors exhibited particularly high collagen amounts in the tumor nests (>2.0%) but the
SCC7 and HT29 tumors contained rather smaller ECM amounts (<0.51%). The SCC7, U87
and HT29 tumors revealed considerably smaller ECM amounts compared to the PC3 and
A549 tumors (see Fig. 7B). As the ECM amount in tumor tissues was increased, the tumor-
targeted delivery efficacy of CNPs was decreased. It was suggested that the intratumoral
ECM might be a physical barrier to inhibit the tumor-targeted CNPs delivery [34].
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A mucoadhesive polymer was synthesized by conjugation of CS to poly(ethylene

glycol) diacrylate (PEGDA) through the Michael type reaction [35]. A higher PEGDA
grafting efficacy was observed using low molecular weight PEGDA (0.7 kDa) compared to
long 10 kDa PEGDA. The acrylation percentage was determined based on the ninhydrin
reaction with CS which approved the data measured from the NMR spectra. The adhesive
properties were assessed by tensile analysis and rotating system which involved detachment
of polymer tablets from a fresh intestine sample. The CS modified with high molecular
weight PEGDA revealed improved mucoadhesive characteristics in comparison to both non-

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modified and thiolated CS. The rheology measurements of polymer/mucin mixtures
confirmed that strong interactions occurred between modified CS and mucin glycoproteins.

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Hence, this formulation could be used as a beneficial polymeric delivery system to afford

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prolonged residence time for the vehicle on the mucosa surface.
The cysteine conjugated CS/PMLA multifunctional nanoparticles were synthesized to
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be used as targeted drug delivery systems in order to eliminate Helicobacter pylori [36].
Helicobacter pylori specifically expresses urea transport protein on its membrane in order to
transfer urea to the cytoplasm urease for supplying ammonia that protects bacteria against the
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acidic environment of the stomach. Appropriate clinical topical antimicrobial agents are
necessary to discard Helicobacter pylori from the inflamed basal area. For this purpose,
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cysteine conjugated CS (Cys-CS) derivatives were obtained for their mucoadhesive and
anticoagulant features to prepare multifunctional nanoparticles. The technique was optimized
to acquire Cys-CS/PMLA nanoparticles for amoxicillin encapsulation. Results displayed that
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amoxicillin-Cys-CS/PMLA nanoparticles were satisfactorily pH-sensitive that could delay the

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amoxicillin release at gastric acid and deliver the drug in order to efficiently target the
Helicobacter pylori at its survival region. Compared to unmodified amoxicillin-CS/PMLA
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nanoparticles, efficient inhibition of Helicobacter pylori growth was witnessed for

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amoxicillin-Cys-CS/PMLA nanoparticles. Thus, the multifunctional amoxicillin-loaded

nanoparticles had shown high potency to effectively treat the Helicobacter pylori infection.
Also, these pharmacologically powerful nanocarriers could be appropriate candidates for oral
targeted delivery of diverse therapeutics/drugs for the treatment of Helicobacter pylori.
The surface charge necessitating for nanoparticles to have extended circulation and
good cell affinity have led to develop various polymeric nanoparticles for controlled drug
delivery [37]. Recently, vancomycin loaded composite nanoparticles with a CS core and
poly(lactide-co-glycolide) (PLGA) shell structure were fabricated that were pH-responsive
and had surface charge switchable properties [37]. The spherical nanocomposites were
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obtained by a modified emulsion-gelation technique with a controlled surface charge (-27.6-

31.75 mV), size (316-573 nm) and encapsulation efficiency (up to 70.8%). The composite
nanoparticles had particularly designed core-shell structures that were negatively charged in
the beginning and switched to positively charge afterward. The negative charge of particles
was slowly switched to positive charge due to the erosion of biodegradable polymer shells
and contact of the positively charged CS core. The CS hydrogels displayed multi-layer
structures which were mostly affected by CS concentration. Effects of the CS gelation
behavior on the characteristics of the nanocomposites in reaction to various CS and NH3

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concentrations were examined. Release rate was significantly declined by increasing CS
concentration. After the CS incorporation, the enhanced drug release rate was seen by orders

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of magnitude for the samples immersed in the phosphate buffer saline solution with lower pH

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value which proved a pH responsive release feature. The drug release plots of the
nanocomposites were composed of fast and slow release stages. The fast release was fitted
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with a modified first-order kinetic model but the slow release stage was described by the
classical first-order release kinetic model. Such results justified that the composite
nanoparticles were promising systems for application in controlled drug delivery.
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The key issues in drug delivery to the oromucosa include boosting the mucoadhesion of
drug formulations in order to elongate their residence time on the target site and the sustained
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drug release from the formulations [38]. In a recent investigation, the mucoadhesive CS
biopolymer and its derivatives, including high and low molecular weights as well as a
carboxylic derivative, were used as the excipients to obtain spray-dried microspheres for the
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oromucosal benzydamine hydrochloride drug delivery to enhance the delivery effectiveness

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[38]. All CS modified spray-dried powders displayed greater surface roughness, sizes below
10 μm, moisture content below 10% w/w and better flowability. Addition of CS to the
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formulations extremely extended the drug release duration. Besides, the mucoadhesive
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interaction of CS-modified microparticles was highly increased and it was further improved
by enhancement of CS the content in the formulations. Thus, it was demonstrated that adding
CS to the spray-dried microspheres elongated the drug release duration and enhanced the
mucoadhesive interaction force of microspheres to the mucosal membrane, which could be
advantageous in the treatment of patients.
In another research, the probability of preparing multifunctional oral insulin delivery
systems was examined through conjugation of different dipeptides on CS and trimethyl CS to
be employed as drug carriers [39]. The conjugates of glycyl-glycine and alanyl-alanine of CS
and trimethyl CS (on primary alcohol group of polymer located on carbon 6) were
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synthesized and nanoparticles including insulin were achieved for oral delivery. The
nanoparticles preparation conditions were optimized and their performances were assessed to
improve the insulin permeability and cytotoxicity of nanoparticles against Caco-2 cell line. In
order to estimate the effectiveness of orally administered nanoparticles, the nanoparticles with
the most permeability were investigated in male Wistar rats as animal model and their insulin
and glucose serum levels were measured. For all the conjugates, the IR and NMR spectra
established their successful formation with the favorite substitution degree. Under optimizing
preparation conditions, nanoparticles with anticipated size (157.3–197.7 nm), polydispersity

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index (0.365–0.512), zeta potential (24.35–34.37 mV), loading capacity (30.92–56.81%),
entrapment efficiency (70.60–86.52%), suitable morphology and appropriate release pattern

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were attained. Glycyl-glycine and alanylalanine conjugate nanoparticles of trimethyl CS

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presented 2.5–3.3 folds more efficient insulin permeability in Caco-2 cells than their CS
analogues. In animal model, oral administration of glycyl-glycine and alanyl-alanine
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conjugate nanoparticles of trimethyl CS established rational increase in serum insulin level
with relative bioavailability of 17.19 and 15.46% for glycyl-glycine and alanyl-alanine
conjugate nanoparticles, respectively. Also, serum glucose level was decreased compared to
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trimethyl CS nanoparticles. The glycyl-glycine and alanyl-alanine conjugate nanoparticles of

trimethyl CS could orally deliver insulin by more than one mechanism in the animal model.
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Therefore, they would be identified as auspicious candidates for biomedical applications.

In order to increase the bioavailability, aqueous stability, developing suitable delivery
systems, and enhancing the chemotherapeutic influence of curcumin, it was encapsulated in
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CS-based polyelectrolyte complexes and their antidiabetic activities were evaluated through
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in vitro α-amylase inhibition test and in vivo antihyperglycaemic potency in alloxan-induced

rats [40]. The microscopic analysis revealed curcumin nanoformulations (<50 nm) that were
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loaded in CS-alginate complex. Higher encapsulation efficiency (64–76%), loading capacity

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(20–26%) and yield (50–72%) were attained. Complexation of CS with alginate decreased the
curcumin loss (by 20%) and prolonged mean release time (by 40 min) in simulated gastric
fluid. The time dependent experiment exhibited that the α-amylase inhibition property of
curcumin was improved by the nanoencapsulation. It was indicated that oral administration of
sub-therapeutic dosage of curcumin (50 mg/kg b.wt.) nanoencapsulated in CS-based
complexes considerably decreased hyperglycaemia after 7 days of treatment. Finally, the
chemotherapeutic efficacy of curcumin was improved by its nanoencapsulation in CS-based
polyelectrolytes.
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It is known that most currently existing chemotherapy methods for cancer treatment
have limited to clinical cancer therapies which is primarily because of low drug encapsulating
efficacy and little pharmacologically effective concentrations at the tumor sites [41].
Therefore, in a recent work, a simple approach was developed to increase drug encapsulating
efficiency and local drug concentration by an injectable hydrogel prepared using thiolated
chitosan (TCS) and poly(ethylene glycol) diacrylate (PEGDA) [41]. It was shown that nearly
100% encapsulating capacity was attained for the anti-cancer drug curcumin into this system.
The curcumin interaction with PEGDA was recognized by fluorescence spectroscopy and the

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binding constant was calculated; then, this interaction was simulated with a docking
computation. In order to enhance the antitumor activity and reach favored local concentration,

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lysozyme was added to the system. A sustained curcumin release was perceived with a

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controlled lysozyme-responsive performance that quickly led to the drug concentration
reaching the therapeutic threshold. Such system exhibited proficient intracellular curcumin
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release in order to stimulate in vitro apoptosis of cancer cells. As well, the system
successfully postponed the tumor growth and decreased adverse effects in tumor-bearing nude
mice. Consequently, injectable hydrogel would be a beneficial system to be employed for
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encapsulation of various insoluble anticancer drugs.

The intracellular curcumin release was evaluated using Hoechst 33,258 stain which is a
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nuclear counter stain emitting blue fluorescence once bound to DNA. It is employed for
counterstaining, apoptosis and cell cycle tests. The living cells nuclei merely stain a
homogeneous and weak blue color whereas the apoptotic cells nuclei stain bright blue so that
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the chromatin is condensed. It is observed in Fig. 8 that the nuclei of untreated cells were
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stained elliptical blue with the staining was diffused into the nuclei that displayed nearly no
apoptotic nuclei existed in the untreated HepG2 cells (Fig. 8a). Treating with free curcumin
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(Fig. 8b) as well as the curcumin containing injectable hydrogels (Figs. 8c and 8d) lead to the
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apoptotic morphological variations including condensed chromatin, shrunken and condensed

bright blue nuclei which suggested HepG2 cells treatment with curcumin induced apoptosis.
In comparison with the curcumin incorporated injectable hydrogels without lysozyme (Fig.
8c), additional apoptotic cells were created after 24 h incubation by curcumin containing
injectable hydrogels with lysozyme (Fig. 8d) [41].
The in vivo antitumor activity of curcumin loaded TP3 was assessed in male nude mice
through their comparison with saline control, free curcumin as well as curcumin incorporated
TP0. For this purpose, the tumor volumes and body weights of tumor-bearing mice were
measured every three days during 21 days (Fig. 9). In Fig. 9a, serious side effects were
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witnessed in saline treated mice which was observed by the substantial body-weight loss (Fig.
9b). A noticeable body-weight loss was measured for the free curcumin treated tumor-bearing
nude mice (Fig. 9b). Nevertheless, a small decline in body weight was observed in mice
treated with curcumin containing TCS/PEGDA hydrogels which indicated they led to less
side effects for the tumor treatment compared to the free curcumin. It is seen in Fig. 9c that
the curcumin incorporated TP3 treated group demonstrated comparable tumor growth
inhibition in vivo relative to the free curcumin treated group so that the tumor volumes of
curcumin containing TP3 treated mice were very smaller than those of mice treated curcumin

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loaded TP0 or with saline. Moreover, the tumor volumes of the mice treated by curcumin
loaded TP0 were smaller than those treated by saline. Thus, curcumin was efficiently released

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from hydrogels to inhabit tumor growth. As the tumor volumes of the curcumin incorporated

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TP0 treated mice were not as small as those treated with curcumin containing TP3, the
curcumin loaded hydrogel with lysozyme showed rather greater tumor inhibition in vivo
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compared to the curcumin loaded hydrogel without lysozyme and this was in agreement with
the in vitro tests. The tumor inhibition rates using the free curcumin, curcumin loaded TP0
and curcumin loaded TP3 were measured to be 48.4%, 34.0% and 57.5%, respectively.
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Additionally, the tumor inhibition values of curcumin loaded TCS/PEGDA hydrogels (TP3)
were greater than 40% which was considered as an effective treatment [41].
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Histological analysis by hematoxylin and eosin (H&E) staining was performed in order
to further assess the antitumor effectiveness and the possible toxicity of curcumin containing
TP3 (Fig. 10). Numerous tumor cells were seen in the saline treated tumor tissues
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demonstrating fast tumor growth. Nonetheless, pyknotic cells with highly condensed nuclei
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existing in the curcumin incorporated TP3 treated tumor tissues showed much more dead or
apoptotic cells. Large necrotic areas found in the curcumin treated tumor tissues exhibited the
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tumor growth inhibition by the curcumin containing TP3. As well, tissue sections from
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curcumin loaded TP3 treated mice organs were evaluated for their probable toxicity. It is seen
in Fig. 10 that there were no apparent pathological variations in the main organs for the
curcumin containing TP3 treated group which confirmed there was no substantial in vivo
toxicity for the curcumin loaded TP3 [41].
The CS NPs coated with zein was developed as an auspicious encapsulation system to
be used for delivery of epigallocatechin gallate (EGCG) [42]. Factors affecting nanoparticle
preparation were studied including zein/CS weight ratio, zein concentration and EGCG
encapsulation percentage. The structural and physicochemical analyses results indicated that
the hydrogen bonds and electrostatic interactions were the main forces occurring in
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nanoparticles creation. The transmission electron microscopy (TEM) displayed a spherical

morphology along with smooth surface of the nanoparticles. An initial burst EGCG release
was observed from the nanoparticles followed by a slow release. The EGCG release from
zein/CS nanoparticles (zein/CS NPs) having higher 2,2-diphenyl-1-picrylhydrazyl (DPPH)
scavenging ability was relatively greater compared with that of nanoparticles without zein
coating in 95% ethanol fatty simulant. Therefore, it was pointed out that the EGCG
controlled-release from zein/CS and its antioxidant activity in 95% ethanol fatty simulant
could long-term protect fatty foods against oxidation.

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An electrostatic deposition technique was used for the encapsulation of quercetin into
CS/lecithin polymeric nanocapsules in order to preserve quercetin degradation and to increase

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its biocompatibility [43]. The morphology, size, storage stability, encapsulation efficiency,

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anti-proliferative and anti-oxidant activities of quercetin containing nanocapsules (Q-NCs)
were studied. The spherical homogeneous Q-NPs had small particle size and great
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encapsulation efficiency (71.14%). The antioxidant activity and storage stability was
enhanced relative to those of native quercetin. The MTT assay and trypan blue exclusion
assay displayed the inhibitory influence of Q-CPs on HepG2 cells that were similar to that of
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free quercetin. Hence, these nanocapsules provided systems for both protection and carriage
of numerous hydrophobic materials in vivo and food products.
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HepG2 cells were treated with different concentrations (2-10 mg/mL) of NPs, native
quercetin and Q-NPs for 24 h and the MTT assay was carried out to estimate their anti-
proliferative activities. Noticeable anti-proliferative influence was not perceived in Fig. 11 for
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the NPs even using 10 mg/mL concentration. Both of the native quercetin and Q-NPs induced
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dose-dependent cytotoxicity to the HepG2 cells. When using 10 mg/mL concentration, the
cell viability percentages for the Q-NPs and native quercetin were 40.92% and 46.67%,
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respectively. The cytotoxicity to HepG2 cells was further examined using trypan blue
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staining. As trypan blue cannot pass the cytoplasm of living cells, the blue color can be an
indicator of membrane damage and cellular destruction. In consistent with the MTT assay,
blue spots were observed for the quercetin and Q-NPs treated cells (Figs. 11d and 11e)
confirming their strong cancer cell killing capability. Furthermore, noticeable morphological
differences were not witnessed between the NPs treated cells (Fig. 11c) and the untreated cells
(Fig. 11b).
It is known that the molecular oxygen (O 2 ) is essential for human life and it is not very
reactive but under certain condition, it can transform to the superoxide anion radical (O 2 )
which damages the human body. Thus, removal of superoxide anion radical is one of the most
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efficient defenses in a living body against different diseases. Fig. 11c reveals a concentration-
dependent scavenging ability of quercetin and Q-NPs. Using 10 mg/mL, the scavenging
capacities of native quercetin and Q-NPs were 21.39 and 31.39%, respectively [43].

2.2. Application of chitosan in gene delivery

Gene therapy has shown great potential for the treatment or prevention of various
diseases that cannot be treated with conventional methods [44]. In fact, gene therapy is of
remarkable research attention that is known as an auspicious approach to treat diseases. This

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method aims to repair or replace the direct origin of genetic diseases through insertion of
nucleic acid polymers to patient cells and it is anticipated that this will be a proficient strategy

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for the cancer treatment, viral infections, cardiovascular and genetic diseases. The

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recombinant DNA technology, discovered in the 1970s, was employed as an impressive
process for gene regulation. It was first attempted in 1990 to incorporate foreign genes to
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human cells through retroviral mediated gene transduction [45]. This was a successful effort
which was then used for nuclear gene transfer in human as a new strategy in biomedicine. So
far, numerous efforts have been performed to recognize mutations involved in human diseases
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using therapeutic genes in order to treat diseases. The low stability, toxicity of nucleic acid
drugs and low-effective transfection to cells led to gene therapy to be only an experimental
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technique that is presently in preclinical phase. Also, translation of gene therapy was not
considerably efficacious due to several problems including unsuccessful targeted gene
delivery to diseased sites and cells, gene degradation during delivery and quick clearance in
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circulation [46]. In order to solve these problems, one fruitful solution is to deliver genes
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using delivery systems such as CS. Efficient gene therapy causes proficient transfection and
stable expression of foreign genes into the target cells/tissues, which is highly associated to
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the delivery systems. Consequently, developing effectual gene delivery systems is an

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important subject in biomedical application of gene therapy.

Nowadays, various techniques are applied to introduce nucleic acids including viral and
non-viral vectors but viral vectors can cause diverse human infections. Hence, a natural
cationic polymer like CS is extensively used as a non-viral vector system in safe and
promising nucleic acid delivery. It was found that CS nanofibers could be utilized in
controlled drug delivery application. Moreover, graft copolymerized CS revealed valuable
properties for the intracellular delivery of genetic agents [47].
Chitosan nanoparticles have appeared as favorable candidates for gene delivery but
slow dissociation of the nanoparticles in cytoplasm is a shortcoming in CS usage. The CS-
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mediated gene delivery was done using polyelectrolyte complexes (PECs) of CS and
carboxymethyl dextran (CMD) in order to transfer the micro RNA-145 (miR-145) [48]. The
optimized nano PEC preparation process and influences of CS molecular weight and CMD to
CS molar ratio (CMD:CS) on the physical properties and in vitro efficiency of the nano PECs
were investigated. The size of the nano PECs was dependent to the preparation route,
CMD:CS ratio and CS molecular weight. As well, the CS molecular weight and CMD:CS
ratio influenced the miR-145 PECs stability in presence of heparin. In vitro experiments
specified diverse gene transfection efficacies of the nano PECs having different compositions.

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Thus, these nano PECs vectors have outstanding capacity for gene delivery with a satisfactory
balance between the dissociation rate and complex stability.

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Fig. 12 exhibits the gel retardation assay results. The plasmid concentration of 10 µg/ml

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was completely preserved by all of the nano PECs whereas for the CMD:CS ratio of 5 and the
plasmid concentration of 15 µg/ml, they did not entirely retain the gene. Also, the nano PECs
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kept the gene (10 µg/ml) in heparin presence which established the stability of the miR-145
PECs against biological polyanions. However, increasing the DNA concentration led to the
release of the DNA smear from miR-145 PECs of CS9 (CMD:CS ratio=1) on the gel after
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incubation with heparin [48].

As the plasmid expresses both GFP and miR-145, the GFP positive cells were assessed
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by flowcytometery analysis and fluorescent microscopy in order to determine the gene

delivery efficiency using the PECs. Fig. 13A exhibits that the GFP was expressed in MCF-7
cells after transfection by the miR-145 PECs. The most GFP amounts was expressed in the
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cells incubated with the miR-145 PECs of CS45 having the CMD:CS ratio of 5. Also, the
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GFP expression was noticeable in the cells transfected with the miR-145 PECs of CS18
(CMD:CS ratio=1). Fig. 13B displays the GFP quantity in positive cells after transfection
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using the diverse PEC formulations. It seemed that GFP was expressed in all samples
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however the most amount of GFP positive cells was measured following transfection by the
nano PECs of CS45 and CS18 having the CMD:CS ratios of 1 and 5. Substantial differences
were not detected between the samples transfected with the nano PECs of CS9 with the
different CMD:CS ratios but an obvious increase in GFP expression was occurred by
increasing the CMD:CS ratio of CS45 PECs. Although it was not a great difference among
the nano PECs of CS18 with CMD:CS ratios of 1 and 5, they caused higher GFP expression
in the cells compared to that of the nano PECs having a CMD:CS ratio of 0.2. Fig. 14 reveals
that the Cy5 PECs uptake was suitable demonstrating the appropriateness of the system for
the drug/gene delivery [48].
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Prostate cancer is the most common malignancy in men. Also, CS is of great attention
as a suitable biopolymer to encapsulate small interfering RNA (siRNA) due to its cationic
nature which can proficiently form nanoparticles containing encapsulated siRNA molecules.
Besides, the biocompatibility and biodegradability of CS have led to its application in the in
vivo delivery of siRNAs therapeutics. It was reported that CS-carboxymethyl dextran (CMD)
nanoparticles efficiently encapsulated the anticancer drugs SN38 and Snail-specific siRNA
[49]. Physicochemical, growth inhibition and anti-migration characteristics of the co-delivery
SN38-Snail siRNA CMD-CS nanoparticles were examined in prostate cancer cells. It was

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found that in the CSNP-CMD-SN38-siRNA treated cells, the mRNA level of snail was
reduced from 1.00 to 0.30 (±0.14) and 0.09 (±0.04) after 24h and 48h, respectively. The

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folding induction of E-cadherin and Claudin-1 was increased from 1.00 to 3.12 (±0.62), 3.02

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(±0.28) after 24h and 5.6 (±0.91), 4.42 (±0.51) after 48h, respectively. As well, dual delivery
of SN38 and Snail-specific siRNA by an appropriate nanocerrier (CS NPs) declined the
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viability, proliferation and migration of PC-3 cells. Thus, the encapsulated SN38 and Snail-
specific siRNA in CSNPs can be used as a promising anticancer drug delivery system to treat
the prostate cancer.
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The modified CS was applied as a potential vector in gene delivery to gonadotropin-

releasing hormone receptor (GnRHR)-expressing cells [50]. This gene carrier is especially
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valuable for gene therapy to cancers associated to the reproductive system, gene disorders of
sexual development and fertility and contraception control. For this purpose, a decapeptide
GnRH was effectively conjugated to CS and characterized by proton nuclear magnetic
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resonance spectroscopy (1 H NMR) and attenuated total reflectance Fourier transform infrared
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spectroscopy (ATR-FTIR). The synthesized compounds, GnRH-conjugated chitosan (GnRH-

CS), was capable of condensing DNA to produce positively charged nanoparticles and
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specially to deliver the plasmid DNA into targeted cells in both three-dimensional (3D) and
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two-dimensional (2D) cell culture systems. Notably, GnRH-CS displayed higher transfection
efficiency relative to unmodified CS. Hence, GnRH-conjugated CS was considered as a
favorable carrier for application in targeted DNA carriage to GnRHR-expressing cells.
The cell viability of PEI treated cells was considerably declined by increasing the
concentration (Fig. 15a) which was related to its high cytotoxicity. However, cytotoxicity was
not seen using the concentration ranges of unmodified CS or GnRH-conjugated CS. For both
of the GnRH-CS1 and GnRH-CS2, similar results were obtained. Fig. 15b displays numerous
dead cells (red fluorescence) in cells treated by 200 µg/ml of PEI polymer relative to the
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normal viable cells (green fluorescence) treated by the same concentration of unmodified CS
or GnRH-CS [50].
The targeting ability of the GnRH-CS/pDNA complex was examined in order to
approve that transfection was specific and facilitated by interactions between the GnRH
ligand and GnRH receptor. Targeted gene delivery to transiently transfected HEK293T which
expressed GnRHR (targeted cells) and non-transfected HEK293T (non-targeted cells lacking
GnRHR) using the GnRH-CS was compared to unmodified CS obtained by increasing weight
ratios of polymers and pDNA transporting a GFP reporter gene. Fig. 16a exhibits that

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substantial differences were not seen in GFP expression by the targeted and non-targeted cells
treated with unmodified CS/pDNA complex at any weight ratios signifying incapability of

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unmodified CS to distinguish between targeted and non-targeted cells. Nevertheles, GnRH-

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CS could specifically deliver a GFP gene to the targeted cells. Using GnRH-CS/pDNA
complexes for the cell transfection, GFP expression was completely occurred in targeted cells.
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GFP expression was not happened in non-targeted cells at any weight ratios, see Fig. 16a.
The specificity of GnRH-CS/pDNA complexes was assessed in a 3D multicellular
spheroid along with 2D monolayer cultures. Comparable results were attained in both 2D and
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3D cell cultures (see Fig. 16b). These results prove that gene delivery using the GnRH-CS is
specific, targeted and dependent on the GnRH receptor [50].
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Chitosan microparticles (CSM) can be achieved to be used in delivery of several

intracellular payloads. Recently, the effect of cell culture conditions on CSM size and the
influence of CS on CD59 expression in primary human smooth muscle cells were examined
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[15]. It was revealed that particle concentration and incubation time in biological buffers
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increased particle size. The CSM size was suddenly augmented when pH was adjusted
between 7.0 and 7.5. A CSM loaded by a plasmid with a gene for CD59 (pCSM) was used to
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transfect cells. The CD59 mRNA and the number of CD59-positive cells were both enhanced
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subsequent to the pCSM treatment. Surprisingly, CSM enlarged the number of CD59-positive
cells. The CS alone increased CD59 expression more effectively than both CSM and pCSM.
Therefore, it was confirmed that CS was really bioactive and using the CS alone as control
proved that the CS as the delivery systems had activity which was different to that of the
payload.
As there are still two drawbacks in human gene therapy including lack of biosafety and
unsatisfactory delivery efficacy of gene-carriers, very biocompatible CS functionalized
Prussian blue (PB) nanoparticles (designated as CS-PB NPs) was produced for photo-
controllable gene delivery [51]. The positive charge, ultra-small size (∼3 nm) and great
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physiological stability of CS-PB nanoparticles made it appropriate as a nonviral vector.

Furthermore, the CS-PB nanoparticles efficiently converted the near infrared (NIR) light to
heat as a result of their strong absorption in the NIR area, which aided the nanoparticles
uptake by cells. Under the NIR light radiation, the CS-PB NPs exhibited higher gene
transfection efficacy that was much greater than that of free polyethylenimine. All of in vitro
and in vivo tests established that the CS-PB NPs had outstanding biocompatibility.
Consequently, the CS-PB could be used as a photo-controllable nano-vector for joint gene and
photo-thermal therapy.

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Chitosan nanoparticles modified with 10 and 30% urocanic acid (CUA) through
carbodiimide crosslinking were produced as effective gene delivery carriers [52]. The in vitro

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transfection efficacy CUA polyplex was evaluated using 3T3 and HeLa cells. The DNA

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loading efficiency was measured for the CUA complexes using diverse N:P ratios (1, 2, 4, 6,
8, and 10). The DNA loading efficiency was calculated equal to >85% for CS, CUA10 and
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CUA30% and the DNA protection capability of CUA10 and CUA30 nanoparticles was
verified by incubation with HindIII and NheI. The cell viability and cell toxicity data
confirmed the non-toxic nature of CUA10 and CUA30 nanoparticles. The in vitro transfection
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potency of CUA10 and CUA30 polyplexes was examined for EGFP expression in HeLa and
3T3 cells and a relative maximum transfection (approximately 10%) was acquired using
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CUA10 and CUA30 after 96 h transfection. Also, the biocompatibility and viability of CUA
gene carrier in transgenic chickens was validated. The in vitro transfection and in vivo
embryonic viability tests additionally established that the CUA was a promising gene carrier
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due to its enhanced biocompatibility and DNA protection capacity.

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With the aim of increasing gene transfer effectiveness and expression stability that are
significant factors in a fruitful gene therapy, a combined system have was developed for gene
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transfer which combined the well-recognized non-viral CS polymeric vector with the
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characteristics of phiC31-integrase that promoted a moderately non-immunogenic, site-

specific integration, with constant gene expression [53]. Also, to solve one of the main
challenges in adeno-associated virus mediated gene transfer (the delivery of large genes), the
capability of the prepared non-viral vectors were examined by incorporation of a large (8 Kb)
transgene. Polyplexes were completely characterized for their surface charge, size,
morphology, pDNA complexation, transgene expression and transfection effectiveness in
vitro by means of HEK293 cells. Co-transfection with integrase was accomplished through
complexation in a single polyplex or using two distinct polyplex compounds. Transgene
expression of CEP290 and GFP that were 8 and 1Kb, respectively, was assessed by flow
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cytometry, fluorescence microscopy and Western blot analysis. The DNA complexation
proficiency, particle size and morphology were in agreement with gene delivery in all
formulations. Conversely, transfection effectiveness and transgene expression were changed
with polyplex and polymer size. Subsequent to delivery by means of CS polyplexes, great
levels of GFP expression were still observed after 16 weeks post-transfection. The over-
expression of the large transgene was distinguished at least 6 weeks post-transfection.
Polyplexes containing phiC1 integrase established sustained gene expression for both of large
(CEP290, 8 Kb) and small (GFP, 1 Kb) genes. Thus, such a combined approach using

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integrase and polymer could overcome the size drawback existing in commonly applied
adeno-associated virus mediated gene transfer methods, in addition to preserving high safety

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with sustained and prolonged gene expression which led to obtain a suitable candidate for

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gene delivery [53].
In another research, a nanocarrier system was developed for non-invasive delivery to
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brain using molecules that were beneficial for gene therapy [54]. Manganese-containing
nanoparticles (mNPs) transporting anti-eGFP siRNA were examined in culture of eGFP-
expressing NIH3T3 cells of mouse fibroblast. After that, the optimum mNPs were tested in
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vivo in mice. Subsequent to intranasal instillation, mNPs were observed by 7T MRI all over
brain at 24 and 48 h. the mNPs were active in considerably reducing GFP mRNA expression
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in Tg GFP+ mice in olfactory bulb, hippocampus, striatum and cortex. Intranasal instillation
of mNPS incorporated with dsDNA encoding RFP caused the RFP expression in multiple
brain sections. The mNPs transporting siRNA or dsDNA were able to deliver the payload
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from nose to brain. Consequently, this methodology for delivery of gene therapeutics to
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human would have a significant influence on decreasing of neurodegenerative maladies.

In vivo assays confirmed the mNPs accumulation in olfactory bulb along with other
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brain regions upon intranasal installation. The magnetic resonance imaging of anesthetized
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mice 24 and 48 h following intranasal injection of mNPs that carried anti-GFP siRNA
displayed NPs accumulation in several brain regions which was specified by enhanced
manganese (Mn) signal intensity in T1 -weighted images that was quantified in four brain
regions using parcellation software (Fig. 17). All of the analyzed regions including
hippocampus, olfactory bulb, corpus striatum and cerebral cortex demonstrated substantial
increase in the Mn signal with peaking at 24 h, Figs. 17E–17F. It was likely that the peak Mn
signal was sharper in olfactory bulb if scanning was done before 24 h. The highest variation
in the Mn signal was observed in the cerebral cortex, where the signal was enhanced to a
mean of 97±17% of baseline at 24 h and decreased to 48.6±17.4% after 48 h. In other regions,
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the peak variations in Mn signals were rather lower however they were similar to the cerebral
cortex (hippocampus=89.1±19.7%, olfactory bulb=68.6±19.4% and striatum=77.3±14.4%).
All of four brain regions revealed peak Mn signal at 24 h which was followed by a decrease
after 48 h [54].
In order to find the extent of gene expression in several brain regions after the intranasal
administration, dsDNA encoding RFP was packaged to mNPs. After 48 h intranasal
instillation, the RFP was observed in olfactory bulb, cortex, striatum and hippocampus (see
Fig. 18). The fluorescence signal related to the RFP expressed gene was co-expressed in

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tyrosine hydroxylase (TH+) neurons in the zona glomerulosa of the olfactory bulb, Fig. 18A.
The RFP was also expressed in other cell types which are not recognized. In the ventral

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striatum, the RFP co-expression of in neurons (NeuN+cells) indicated the perinuclear pattern

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of its expression (Fig. 18C). Striatal RFP DNA levels were greater than those of other brain
regions after 48 h of the nanoparticles intranasal administration (Fig. 18D) [54].
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2.3. Application of chitosan in cell/virus/phage/bacteria encapsulation
Encapsulation is enveloping active agents or core materials in a coating using the
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embedding ability of a polymeric matrix and recently it has attracted extensive attraction [55].
The advantageous biodegradable, nontoxic and non-immunogenic polymers can be used to
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encapsulate, protect and improve the biocompatibility of bioactive components. Numerous

efficient encapsulation methods have so far been reported including chelation, microemulsion,
liposomes and hydrogels. Chitosan is a polycationic biopolymer that can form polyelectrolyte
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complexes with oppositely charged macromolecules through intermolecular electrostatic

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deposition. It has been employed as a coating material to encapsulate various bioactive

agents. For instance, the encapsulated CS materials are used in food industry. In fact, by
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increasing the healthcare cost, there is a growing consumer demand for functional foods that
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are food products fortified with some ingredients possessing positive influences on human
body such as improving the overall body conditions (e.g., probiotics and vitamins) and
modifying risk factors for cancer, coronary heart disease, obesity, osteoporosis, type 2
diabetes and periodontal disease [56].
It is known that stiffness or elasticity of a substrate can affect the phenotypic and
functional properties of chondrocytes [57]. Recently, the influence of changing stiffness
characteristic of a two-component injectable hydrogel fabricated using CS and oxidized
hyaluronic acid (OHA) was examined on the functionality and growth of encapsulated
chondrocytes [57]. For this purpose, three diverse gel ratios were obtained including 10:1,
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10:3 and 10:5 CS-OHA. The stiffness values of the gels were estimated from the force
displacement curves by means of atomic force microscopy (AFM). Rabbit articular
chondrocytes were harvested and the cells gathered from Passage 2 to 4 were utilized for the
encapsulation. The viability and ECM creation of encapsulated chondrocytes were measured
at days 7, 14 and 28 post cultures. It was found that when the ratio of hyaluronic acid
dialdehyde component was enhanced, the gel stiffness was improved from 130.78±19.83 to
181.47±19.77 kPa which was further verified by the decline in gelling time. Moreover, an
increase was seen in the number of viable encapsulated cells that preserved their spherical

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phenotype in less stiff gels but ECM markers (collagen type II and glycosaminoglycans)
expression was decreased relative to that of stiffer gels. Therefore, it was concluded that the

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gel stiffness considerably affected the chondrocyte microenvironment to preserve their

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phenotypic integrity and ECM fabrication.
The effect of hydrogels with different stiffness values on chondrocyte encapsulation
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was examined using rabbit articular chondrocytes encapsulated in the gels and cultured for 7
days. Live-dead imaging was accomplished to establish the viability and adhesion of cells in
the gels (Fig. 19). It was observed in the live-dead images that the chondrocytes cultured in
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the less stiff 10:1 CS-HDA gel exhibited a spherical morphology but the two other
compositions displayed more cell spreading and a flat morphology at day 7. Also, more dense
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and uniform cell distribution was seen in the less stiff gel having more viable cells (live cells
have green color) compared to the stiffer gels at day 7. Nevertheless, the chondrocytes were
aggregated having a spherical morphology as the culture time was enhanced to 14 and 28
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days. Comparable spherical cell aggregated were found for the 10:1 and 10:3 CS-HDA
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scaffolds at day 14 that was increased at day 28. The stiffer gel 10:5 CS-HDA revealed
spherical cellular aggregates which were more spread at day 28 with suitable cell viability.
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This was attributed to the increased hyaluronic acid amount in the stiffer gels which is a main
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factor to promote cartilage homeostasis and cell proliferation [57].

The immunostaining of Collagen type II, the characteristic ECM marker of functional
chondrocytes and the negative ECM marker collagen type I on the chondrocyte encapsulated
hydrogels were accomplished and it was shown that at 7 day culture, the hydrogels 10:3 CS-
HDA and 10:5 CS-HDA illustrated a greater formation of Collagen type II compared to the
less stiff gel (10:1 CS-HDA) (Fig. 20a). This was related to the the stiffness or Young’s
modulus of the stiffer gels that was close to those of the native cartilage of rabbits (0.2–0.9
MPa). Such results were supported by the histological collagen staining on the sections of the
gels having various stiffness values, see Fig. 21a. In all the three gels, Collagen type I was not
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expressed upon immunostaining which specified formation of more hyaline like cartilage
(Fig. 20b). the glycosaminoglycans were stained by Alcian blue on cryo sections of the three
gels (Fig. 21b) [57].
In another work, emulsion crosslinking process was employed to achieve CS-genipin
microgels which were used as injectable microporous scaffolds [58]. The microgels were
characterized by swelling test, light scattering analysis and rheometry of densely-packed
microgel solutions. It was indicated that once CS became highly deprotonated above the pK a,
repulsive forces were decreased and intermolecular attractions led to aggregation of pH-

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responsive chains which caused microgel–microgel aggregation. The microgel made using the
most CS and least cross-linker contents revealed maximum yield stress and a storage modulus

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of 16 kPa when it was condensed at pH=7.4. Two oppositely-charged growth factors were

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encapsulated in the microgels and endothelial cells in order to proliferate as three-dimensional
microgel scaffolds. Thus, such CS microgels could be used as injectable microporous
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scaffolds in regenerative medicine.
Several carboxymethyl cellulose-CS (CMC-CS hybrid micro- and macroparticles were
produced in aqueous medium either through dropwise addition or by nozzle-spray method
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[59]. These systems were either chemically or physically crosslinked by means of genipin as
the reticulation material. The macroparticles (~2 mm) principally showed core-shell structures
but the microparticles (~5 μm) were actually homogeneous. The crosslinked particles were
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thermally resistant and robust with low sensitivity to pH variations. Conversely, the physical
systems were pH-sensitive and presented a notable swelling at pH=7.4 whereas low swelling
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was witnessed at pH=2.4. Besides, model probiotic bacterium (Lactobacillus rhamnosus GG)
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was encapsulated in the CMC-CS based particles having suitable viability count. Thus, such
systems were promising candidates for probiotic encapsulation and effective delivery in the
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intestinal tract to modulate gut microbiota and improve human health.

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Currently, researches on phages employed to decrease pathogenic bacteria have raised

great attraction but they likely lose their bioactivity in food by the existence of acidic
materials, evaporate compounds and enzymes [60]. In order to increase the stability of
phages, a CS edible film incorporated with liposome-encapsulated phage was produced [60].
The properties of liposome-encapsulated phage and the CS film loaded by liposome-
encapsulated phage were examined. The encapsulation efficiency of phages in liposome was
57.66±0.12%. As well, appropriate physical characteristics were achieved for the CS film.
The CS film containing liposome-encapsulated phage demonstrated great antibacterial
potency against Escherichia coli O157:H7 without affecting the physical properties of beef.
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Therefore, CS film embedded with liposome-encapsulated phage could be used as a favorable

antibacterial packaging material for beef preservation.
In another work, maximizing efficiency of inactivated avian influenza vaccine was
performed by means of safe adjuvants [61]. Chitosan nanoparticles, having an average size of
150 nm and zeta potential of 11.5 mV, were obtained using ionic gelation process. After
encapsulation of avian influenza vaccine, their average size was increased to 397 nm and zeta
potential was decreased to 4.29 mV. The utmost hemagglutination inhibition (HI) antibody
titers were revealed in chicken group vaccinated by inactivated avian influenza virus (AIV)-

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CS and then the group vaccinated by inactivated AIV-CS nanoparticles followed by the group
vaccinated using oil inactivated AIV vaccine, using chicken antigen at two weeks after second

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vaccination. When using duck antigen, the maximum HI antibody titers were observed for the

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chicken group vaccinated with inactivated AIV oil emulsion vaccine, then chicken group
vaccinated with AIV-CS nanoparticles, followed by the group vaccinated with AIV–CS.
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Chicken in the group vaccinated with AIV-CS nanoparticles illustrated the most appropriate
data for the lymphocyte proliferation. The phagocytic activity percentage and phagocytic
index of AIV-CS nanoparticles and AIV–CS groups after three days post first vaccination
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were considerably enhanced compared to other groups but at 14 days post first vaccination,
group vaccinated with AIV-CS nanoparticles presented substantial proliferation in phagocytic
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index and phagocytic activity [61].

Adipose-derived stem cells (ASCs) can potentially treat ischemic diseases but poor
delivery methods bring about low cellular survival or cells dispersal from target sites. The
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ASCs contain some angiogenic growth factors that can be used to treat ischemic tissue [62].
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Nonetheless, transplantation of dissociated ASCs normally results in quick cell death.

Consequently, it was intended to prepare a thermosensitive CS/gelatin hydrogel that was able
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to ASC therapeutic angiogenesis release in a sustained manner [62]. The viability of the
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encapsulated ASCs was greatly improved after blending gelatin in the CS thermosensitive
hydrogel. The steady gelatin degradation during in vitro culturing caused sustained ASCs
release from the CS-gelatin hydrogel. The in vitro wound healing assay demonstrated
noticeably faster cell migration through co-culturing fibroblasts with ASCs encapsulated in
CS-gelatin hydrogel relative to pure CS hydrogel. As well, very greater concentrations of
vascular endothelial growth factor were measured in the supernatant of ASC-encapsulated
CS-gelatin hydrogels. Co-culturing SVEC4-10 endothelial cells with ASC-encapsulated CS-
gelatin hydrogels led to substantially greater tube-like structures which designated the
hydrogel capability for the angiogenesis promotion. Chick embryo chorioallantoic membrane
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assay and mice wound healing model proved much higher capillary density subsequent to
application of ASC-encapsulated CS-gelatin hydrogel. Relative to ASC alone or ASC-
encapsulated CS hydrogel, extra ASCs were measured in the wound tissue after five days
post-wounding using ASC-encapsulated CS-gelatin hydrogel. Hence, CS-gelatin
thermosensitive hydrogels not only preserved ASC survival, but they also supported sustained
ASCs release for therapeutic angiogenesis application, thus revealed high clinical promise for
the treatment of ischemic diseases.
ASCs labeled by green fluorescent dye were encapsulated in hydrogels and located at

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the periphery layer of Hs68 fibroblasts labeled with a red fluorescent dye. Then, the
fibroblasts were examined by the in vitro wound healing test through scratching the confluent

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cells using a pipette tip (Fig. 22A). The cells images at the hydrogel border and the scratched

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wound were analyzed after 24 and 48 h. Significantly greater ASCs was released from the
CS/gelatin hydrogel compared to the CS hydrogel (53.0±5.3 ASCs per power field versus
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13.3±2.9 at 24 h with p<0.001 and 61.3±7.4 ASCs per power field versus 19.3±6.1 at 48 h
with p=0.002, Fig. 22B). Similar to the in vitro scratched wound model, the migrated
fibroblasts covered a considerably bigger wound area (17.5±1.3%) in the ASC-encapsulated
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CS/gelatin hydrogel compared to other groups at 24 h. The artificially generated in vitro

wound was entirely healed after 48 h using the ASC-encapsulated CS/gelatin hydrogel but
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cell-free zones were still seen in other groups (Fig. 22C).

Enclosed by ASC-encapsulated hydrogels, the endothelial cells started to create a
vascular network in 4 h (Fig. 23A). The in vitro tube generation by the endothelial cells was
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quantified through counting the number of branch points per power field. Much more branch
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points were counted when ASCs were encapsulated to the hydrogels (p<0.005) compared to
hydrogels alone. The difference between ASC-encapsulated CS/gelatin hydrogel and ASC-
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encapsulated CS hydrogel tended to be significant (19.0±2.0 versus 14.8±1.5 branch points

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per power field, p=0.082, Fig. 23B) [62].

The capillary formation on chorioallantoic membrane (CAM) was studied to examine
the angiogenesis ability of ASC-encapsulated hydrogels (Fig. 24A). Following incubation by
CS or CS/gelatin hydrogels alone, the measured capillary areas on CAMs were 6.6±1.0% and
6.9±1.6% respectively. Addition of the ASC-encapsulated CS hydrogel did not significantly
affect the increased capillary area on CAM (9.1±1.2%) but the ASC-encapsulated CS/gelatin
hydrogel exhibited a considerably higher capillary area on CAM (14.0±2.0%, p<0.01 relative
to other groups, Fig. 24B). Also, adding ASC-encapsulated CS/gelatin hydrogel on CAM
provided significantly more blood vessel branch points that were 152.5±23.3 branch points
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per power field, p<0.05 compared to 64.7±17.6 for CS hydrogel, 51.7±21.1 for CS/gelatin
hydrogel and 99.8±18.7 for ASC-encapsulated CS hydrogel, Fig. 24C.
A murine model of cutaneous wound healing was used in order to assess the angiogenic
ability of ASC-encapsulated hydrogels in vivo. Immunofluorescent staining of HNA and
endothelial lineage-specific marker CD31 was accomplished in wound sections that were
harvested at day 5 after wound formation. Co-localization of HNA and CD31
immunofluorescence revealed the differentiation of transplanted ASCs to the endothelial
lineage. The HNA immunofluorescence was not seen in wounds treated using the hydrogel

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alone (Fig. 25A). Treatment by the ASC-encapsulated CS/gelatin hydrogel led to generation
of considerably more HNA+cells in the wound area on post-wounding day 5 relative to those

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treated with ASC-encapsulated chitosan hydrogel (131±12 vs. 81±16 cells per power field,

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p=0.009) or those only treated by ASCs (131±12 versus 49±10 cells per power field, p=0.001;
Fig. 25B). The wounds contained ASC-encapsulated CS/gelatin hydrogel displayed a
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significantly greater area of CD31+cells compared to the hydrogel alone (11.0±3.2% versus
2.5±0.9% of power field, p=0.003) or the ASC alone (11.0±3.2% versus 2.7±1.2% of power
field, p = 0.003). The ASC encapsulated CS hydrogel showed a significant difference with the
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hydrogel only (7.3±0.6% vs. 2.5±0.9% of power field, p=0.06) or the ASC only groups
(7.3±0.6% versus 2.7±1.2% of power field with p=0.07, Fig. 25C) [62].
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Xanthan-CS hydrogels were prepared and rheologically characterized in the simulated

gastrointestinal conditions and then they were employed for encapsulation of anaerobic
bacterial Bifidobacterium BB01 existing in yogurt [63]. The ability such system (xanthan-CS-
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xanthan) was examined on the viability of B. bifidum BB01 in yogurt during 21 days storage
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at 4 and 25 ºC. In this system, CS was used as the inner layer in order to coat the
xanthanprobiotic and xanthan was applied as the outer layer to coat CS-xanthan-probiotic
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particles. Also, the probiotic survival was explored in bile salt solution and the simulated
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gastrointestinal conditions. It was pointed out that xanthan-CS-xanthan microcapsules (XCX)

and xanthan-CS microcapsules (XC) highly enhanced the cell survival of B. BB01 in yogurt
during 21 days storage (at 4 and 25 ºC) compared to free cells. All of the microcapsules
displayed greater probiotic cell survival in bile salt solution and simulated gastric fluid
relative to that of free cells. The XC microcapsules demonstrated improved release profiles
than XCX microcapsules in the simulated intestinal fluid. Thence, it was recommended that
the XCX and XC encapsulation systems could effectively be used to enhance bacterial
survival both in the gastrointestinal condition and during the yogurt storage.
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2.4. Application of chitosan in binding to protein drugs

Proteins and peptides having health improving features and therapeutic activities have
attracted considerable attention. Various health-inducing functions of such bioactive agents
include anticancer, anti-lipidaemic, antidiabetic, antibacterial and mineral binding
characteristics. Protein drugs with biomedical anticancer and antibiotic activities are known as
promising therapeutics [64]. The protein drugs are low bioavailable, colloidal instable due to
aggregation and greatly susceptible to cleavage by proteases. Besides, because of instability
and short half-life of protein drugs in plasma, patients need numerous injections in order to

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maintain effective concentration in the therapeutic window. Hence, sustained release of
protein and peptide drugs is a solution to decrease painful and frequent injections. Up to now,

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several controlled-release delivery systems have been examined like microspheres, liposomes,

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nanoparticles and cross-linked hydrogels as systems to enhance the protein and peptide
delivery [65]. Chitosan encapsulation systems are known as one of the most favorable
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matrices for protein/peptide delivery that is related to its permeation-enhancing influence of
CS.
The CS-bioactive glass (BG) composites have been prepared as bioactive coatings for
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orthopedic applications [66]. It was shown that the bioactivity was increased as a result of the
induced calcium-phosphate/hydroxyapatite creation on the surface when the coating was
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degraded. In a recent research, protein adsorption and its effect was examined on calcium-
phosphate precipitation over such composite coatings. For this purpose, 316L stainless steel
substrates were coated with CS and CS-BG and the coated samples were immersed in two
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diverse bovine serum albumin (BSA) containing solutions, i.e. H2 O (pH~7.2) and simulated
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body fluid (SBF). Also, samples were immersed in H2 O and in SBF without BSA to explore
the impact of protein adsorption on calcium-phosphate precipitation. The surface analysis
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verified that the BSA adsorption took place on all samples and the protein adsorption was
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affected by the existence of Ca2+ and PO 4 3− ions. Moreover, the bioactivity as the
hydroxyapatite pre-stage formation was considerably enhanced on CS-BG composite coating
compared to the bare stainless steel surface. Nonetheless, calcium-phosphate precipitation in
SBF was diminished due to the BSA presence [66].
Chitosan/tripolyphosphate (TPP) micro- and nanogels are extensively used as vehicles
to deliver protein drugs and vaccines [67]. It is known that the protein uptake by such
particles is improved through stronger protein/particle binding but factors controlling their
uptake amount (including CS, TPP and protein concentrations) have not been fully
investigated. Hence, it was indicated that some differences in the association efficacies (AE-
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values) for protein uptake probably revealed the mainly ignored variations in the particle yield
(XAgg), which was defined as the added CS fraction that was self-assembled as particles and
(similar to the AE) changed with the CS, TPP and protein concentrations. At first, factors
affected XAgg were systematically discovered. Then, it was pointed out that the AE was
scaled nearly linearly with the XAgg (which was increased by the TPP and protein-to-CS
ratios) until all CS was aggregated as particles. The data collected for different TPP and
protein concentrations were presented as a single AE  XAgg curve for individual protein

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type. Additional analysis of protein/particle binding illustrated the increase in AE with XAgg
which indicated an enhancement in binding sites within the particles and a decline in soluble
(non-particulate) CS molecules which produced soluble protein/CS complexes and competed

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with the CS/TPP particles for the unassociated protein. Therefore, it was found that analyzing

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the various parameters affecting the CS/TPP particle yields can be used to optimize the
protein uptake [67].
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An interferometric fiber sensor was experimentally fabricated for detection of hexa-
histidine tagged microcin (His-MccS) [68]. Such intermodal fiber sensor was prepared using a
no-core fiber functionalized by a CS-nickel (Ni) film in order to directly detect microcin small
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peptide. The fiber intermodal sensor worked based on the refractive index variations because
selective adsorption was happened at the CS-Ni film. Due to the strong affinity existing
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between Ni2+ ions and histidine, immobilized Ni2+ ions in the CS film acted as binding sites
for the straight detection of hexa-histidine tagged microcin. A comparative assay was
performed for sensor evaluation using diverse target sizes including full proteins trypsin, BSA
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and human serum albumin (HSA) having high histidine amount on their surfaces and His-
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MccS (peptide, 11.6 kDa). It was found that selectivity was achieved for the His-MccS
compared to trypsin, HSA and BSA. The most important result was fast detection of small
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His-MccS biomolecule relative to other standard detection techniques. This sensor exhibited
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His-MccS detection sensitivity of 0.0308 nm/(ng/ml) in the range of 0–78 ng/ml with a
concentration detection limit of 0.8368 ng/mL.
Amyloid precursor protein (APP) proteolysis is necessary to produce β-amyloid
peptides (Aβ) which form senile plaques in Alzheimer’s disease (AD) brains [69]. The β-site
amyloid protein precursor cleaves enzyme 1 (BACE1) which is the rate limiting enzyme in
creating Aβ from APP; thus BACE1 inhibition is an interesting approach to discover anti-AD
drugs. Also, chitosan oligosaccharides (COS) have exhibited numerous biological activities.
Hence, the possible inhibitory influence of COS was examined on both BACE1 expression in
HEK293 APPswe cells and BACE1 enzymatic activity in vitro. It was found that the cell
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apoptosis was decreased depending on COS dose (100–500 μg/ml) and effectively suppressed
the secretion of both Aβ40 and Aβ42. Additionally, treatment with COS caused a remarkable
decline in BACE1 mRNA and protein expression level, eIF2α phosphorylation and BACE1
enzymatic effect. Consequently, it was pointed out that COS improved Aβ-associated
neurotoxicity that could be attributed to decreased BACE1 enzymatic expression/activity.
As CS-protein conjugates are commonly applied in therapeutic drug delivery, the
bindings of CS nanoparticles with trypsin and trypsin inhibitor was studied by thermodynamic
analysis and spectroscopic methods [70]. The thermodynamic parameters exhibited that CS-

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protein binding was occurred mainly through van der Waals and hydrogen bonding contacts
with trypsin inhibitor which formed a more stable conjugate than trypsin. When the CS size

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was increased, a more stable polymer-protein conjugate was obtained. The CS complexation

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led to more perturbations in trypsin inhibitor structure than trypsin along with decrease in
protein α-helix and main increase in random structure. The negative value of Gibbs free
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energy (G) specified that protein-CS complexation was spontaneous at room temperature.
As a result, the CS nanoparticles could be employed to transport trypsin inhibitor and trypsin.
Exploiting the valuable properties of both polysaccharides and proteins is of great
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importance due to a combination of these two types of biopolymers yield small emulsion
droplets with favorite physical stability [71]. The effect of CS addition to a BSA solution at
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diverse pH values was evaluated and the resulting conformational variations were examined
by UV–Vis absorption spectra as well as fluorescence spectra. Results displayed that the
BSA/CS ratio and pH significantly influenced the interaction between CS and BSA.
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Furthermore, the secondary structure and the microenvironment of BSA were altered by the
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CS addition. Also, CS quenched the intrinsic BSA fluorescence. Hence, these experimental
data provided a theoretical guidance to design other helpful ingredients required in the food
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industry.
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The CS stabilized-albumin nanoparticles study was designed and evaluated as NELL-1

protein carriers (CS-NNPs) [72]. The CS-NNPs were achieved through desolvation process
and stabilized using CS by electrostatic interactions. The CS-NNPs were characterized for
particle size, surface morphology, surface charge and drug loading efficiency. Fluorescein
isothiocyanate-labeled CS was applied to approve the homogeneity of CS coating on the BSA
nanoparticles. The NELL-1 bioactivity in CS-NNPs and the release kinetics were examined in
vitro. The mean particle size and the surface charge using 0.075, 0.15 and 0.3 wt% of CS,
respectively, were equal to 368.663±15.470, 382.881±18.767, 390.480±11.465 nm and
+25.03±1.42, +30.27±1.80, +31.03±2.05 mV, respectively. The drug entrapment efficiency
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changed from 87.83 to 89.30%. The CS-NNPs obtained using 0.15 wt% of CS could fruitfully
control the NELL-1 release and preserve a sustained release up to eight days. Besides, more
than 82.67±8.74% bioactivity of the loaded protein was well-maintained in CS-NNPs.
Therefore, it was recommended that CS-NNPs could be used as auspicious protein delivery
nanocarriers to preserve both the sustained release kinetics and the bioactivity of released
NELL-1.
A comprehensive research was done on the interactions of CS nanoparticles (15, 100
and 200 kDa with identical 90% deacetylation degree) and two model proteins (BSA and

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HAS) in order to correlate the CS molecular weight with its binding affinity to protein [73].
The influence of CS on the protein secondary structure and the effect of protein complexation

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on the morphology of CS nanoparticles were conferred. It was revealed that the three CS

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nanoparticles interacted with BSA to produce CS-BSA complexes, mostly by hydrophobic
contacts and the affinity was changed as 200>100>15 kDa. Nevertheless, HSA-CS
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complexation was primarily happened through electrostatic interactions and the stability order
was 100>200>15 kDa. Besides, the contacts occurred between protein and polymer caused a
partial protein conformational variation via a great decrease in α-helix from 63% (free BSA)
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to 57% (CS-BSA) and from 57% (free HSA) to 51% (CS-HSA). In conclusion, the TEM
micrographs clearly exhibited that the binding of serum albumins to the CS nanoparticles
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induced a substantial modification in protein conformation and the polymer shape.

The tryptophan fluorescence quenching in protein is can be used to explain the
parameters of drug–protein binding. BSA contains two tryptophan residues including Trp-212
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located in a hydrophobic binding pocket and Trp-134 situated on the protein surface, Fig. 26.
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HSA includes only one tryptophan residue (Trp-214) buried within the protein molecule (see
Fig. 26). When the binding happens near such Trp, inherent fluorescence quenching by
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tryptophan is occurred. Fig. 27 displays the influence of the binding of CS-15, CS-100 and
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CS-200 kDa on the BSA and HSA fluorescence intensity. Apparently, the HSA and BSA
fluorescence intensity is slowly decreased by increasing the CS concentration which can be
related to the formation of complexes between CS-15, CS-100 and CS-200 and HAS/BSA
[73].

2.5. Application of chitosan in tissue engineering

Tissue engineering is related to repairing damaged or diseased tissues and organs
through controlling the biological microenvironment. In fact, tissue engineering includes
several steps that are cell proliferation, differentiation and synthesis of extracellular matrix
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(ECM) [74]. An ideal tissue engineering scaffold is a template used for three-dimensional
(3D) tissue growth in order to mimic the natural tissue microenvironment by offering a porous
structure for the tissue growth, oxygen diffusion and nutrients delivery. Also, it can interact
with the neighbouring cells and preserve the phenotype of the renewed tissue. A perfect
scaffold must be biodegradable, biocompatible and promotes cell adhesion, proliferation as
well as retains the metabolic action of cells. Besides, the scaffolds with appropriate
pluripotent stem cells, angiogenic effect and prolonged nutrient resource will accomplish the
regeneration of numerous tissues [75]. An implantable scaffold must exhibit high

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compatibility with body, suitable mechanical properties, morphology, porosity, healing and
tissue replacement ability [75].

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As a result of its outstanding characteristics, CS has the capability to create scaffolds

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with excellent interconnected porosity and favorite shapes like sponges, hydrogels, two-
dimensional sheets/fibers and 3D porous structures to be employed for the enhanced cell
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viability through supplying sufficient oxygen and nutrients [76]. The CS-based scaffold
materials display controlled delivery of loaded therapeutics and growth factors indicating they
are pertinent candidates for regenerative applications and tissue engineering [76]. Thus, CS
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scaffolds could substitute for damaged or missing tissues to stimulate cell attachment and
proliferation. As well, the existence of primary amino and hydroxyl groups on the CS chains
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leads to its chemical modification to achieve different derivatives with preferred

functionalities and features.
Fibrous scaffolds with different ratios of CS to poly (lactic acid) (PLA) were obtained
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by electrospinning method [77]. Subsequent to crosslinking using the glutaraldehyde vapor,

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the mechanical properties, structures, hydrophilicity and in-fiber chemical interactions of the
scaffolds were evaluated. It was exhibited that the fiber diameter was diminished with the CS
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concentration whereas hydrophilicity and mechanical properties were enhanced. Furthermore,

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scaffolds with aligned fibers had greater mechanical strengths and biocompatibility compared
to scaffolds containing random fibers. Particularly, scaffolds having aligned fibers produced
with PLA:CS ratio of 7:1 supported cardiomyocyte viability, prompted cell elongation, and
enhanced creation of sarcomeric α-actinin and troponin I. Hence, it was found that composite
scaffolds made using PLA-CS fibers had high potential to engineer cardiac tissue and to
accelerate the myocardia regeneration.
Fig. 28a exhibits that cardiomyocytes grown on random fibers and tissue culture plates
have lost structural polarity, round morphology and express α-actinin diffusely. On the other
hand, cells that were seeded on aligned PLA/CS nanofibers maintained very polar
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morphology and more plentifully accumulated α-actinin almost parallel to the nanofibers.
Particularly, cardiomyocytes grown on aligned fibers with 7:1 PLA:CS more amply express
α-actin than other fibers. The elongated morphology and alignment of the cells revealed that
an anisotropic tissue having great ability to contract has been formed. Furthermore, Tn-I
caused a similar effect, especially the highly parallel Tn-I arrangement is aligned on the A7
fiber having a shuttle-like shape (see Fig. 28b). The PLA/CS fibers afford suitable biological
and chemical properties to support cardiomyocytes to deliver more intercellular guidance
[77].

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It is well known that CS-based porous structures have widely been investigated around
the world as promising tissue engineering scaffolds [78]. Although there are differences in CS

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polymers obtained using squid pens or crustacean shells, with the former is more reactive and

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simply accessible with a higher degree of deacetylation (DD), in most efforts the crab or
shrimp CS are used due to they are easily obtainable in commercial sources. Hence, in a
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recent study, high potential of CS materials achieved from squid pens was examined for their
biomedical applications [78]. Using freeze-dried scaffolds produced for soft tissue
engineering, the effect of the DD of CS polymer and the freezing temperature during
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processing were explored on their performances. In this procedure, CS was attained by

deacetylation of β-chitin earlier isolated from endoskeleton of giant squid Dosidicus gigas
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(DD=91.2%) and it was compared to a commercially existing batch acquired from crab shells
(DD=76.6%). The CS solutions were frozen at −80 or −196 °C and additionally freeze-dried
to get 3D porous structures. The scaffolds prepared at −196 °C had a compact structure with
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smaller pores, but those obtained at −80 °C exhibited a lamellar structure with larger pores.
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The compressive modulus was changed from 0.7 to 8.8 MPa. All scaffolds were stable up to
four weeks in phosphate buffer saline (PBS) and in the presence of lysozyme. In addition, the
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squid CS scaffolds processed at −80 °C supported ATDC5 chondrocyte-like cells adhesion

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and proliferation. Consequently, it was proposed that these squid CS scaffolds could be
exploited for application in cartilage tissue engineering.
In order to enhance the hydrophilicity of CS fiber, N-carboxyethyl CS fiber was
developed by Michael addition between CS fiber and acrylic acid and the structure was
characterized by 1 H NMR spectrum [79]. The N-substitution degree, measured from the 1 H
NMR, ranged from 0.10 to 0.51 by changing the molar ratio of CS to acrylic acid. Some
characteristics of N-carboxyethyl CS fiber such as crystallinity, mechanical and thermal
properties and in vitro degradation were explored. It was found that creation the carboxyethyl
group onto the CS chain ruined the intra/intermolecular hydrogen bonds which led to
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hydrophilicity enhancement. Indirect cytotoxicity evaluation of carboxyethyl CS fibers was

accomplished using L929 cell line indicating the N-carboxyethyl CS fiber was nontoxic to
these cells. Hence, the N-carboxyethyl CS fibers could be employed as potential scaffold
materials in tissue engineering.
Some gelatin-carboxymethyl CS scaffolds were fabricated, characterized and applied in
dermal tissue engineering [80]. The influence of carboxymethyl CS and gelatin ratio was
assessed on their physical, chemical and biological properties and drug release kinetics. The
scaffolds were produced using freeze drying process and characterized through FTIR and

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SEM analysis techniques. It was found that the scaffolds were greatly porous with pore size
changing from 90 to 170 μm, highly adsorbed (400–1100%) and retained (>300%) water. The

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scaffolds degradation mediated by collagenase depended on the gelatin content in the

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formulation. Also, a minor but significant alteration was perceived in their biological features.
All of scaffolds stimulated adhesion, spreading, growth and proliferation of 3T3 mouse
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fibroblasts. As well, the cells seeded onto the scaffolds revealed expression of collagen type I,
HIF1α and VEGF, reflecting a sign for their growth and proliferation accompanied by activity
to promote angiogenesis throughout wound healing. Besides, the scaffolds exhibited sustained
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release of both bovine serum albumin and ampicillin which established their appropriateness
as therapeutic delivery vehicles. Overall, it was recommended that gelatin-carboxymethyl CS
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scaffolds could be employed as suitable materials in dermal tissue engineering.

It has been recognized that electroactive scaffolds fabricated using conductive polymers
are able to support tissue regeneration and repair; nevertheless, such polymers are non-
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degradable which cannot be eliminated from body [81]. In order to tackle this drawback of
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conductive polymers, an injectable electroactive hydrogel including pyrrole oligomers was

developed which showed both exceptional properties of electrical conductivity and
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biodegradability. The pyrrole oligomers were first synthesized by chemical polymerization,

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which were amorphous with a non-globular morphology. Next, three diverse compositions of
injectable CS/beta glycerophosphate hydrogels were synthesized comprising various
concentrations of pyrrole oligomers and their morphology, chemical structure, swelling ratio,
conductivity, in vitro biodegradation and gelation time were investigated. It was revealed that
an increase in oligopyrrole amount diminished the pore size and improved the swelling ratio,
gelation time, degradation time and conductivity. Among all of the hydrogels, the sample
having a pyrrole oligomer:CS ratio of 0.1 (w/w) displayed the most noticeable
biocompatibility, biodegradability, swelling ratio, electro-activity and pore size. Thus, it was
selected as an ideal electroactive hydrogel [81].
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In another research, CS-co-hyaluronic acid cryogels were prepared using glutaraldehyde

crosslinker at subzero temperature and used as tissue-engineering scaffolds [82]. The cryogels
contained different ratios of CS and hyaluronic acid (0, 10, 20, 30 and 50 wt% hyaluronic
acid). The morphological investigation displayed that the macroporous cryogels had 90–95%
porosity and 150–200 μm pore size. The biomaterial and mechanical properties of pure CS
were particularly enhanced through fabrication of copolymer with hyaluronic acid in diverse
concentrations. These cryogels demonstrated biodegradable nature and fast swelling behavior.
It was proved that the swelling ratio, flexibility and durability were increased after

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copolymerization of hyaluronic acid. The MTT cell viability test established that the cryogel
scaffolds presented the highest ability in the proliferation of both 3T3 and SaOS-2 cells by

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increasing the hyaluronic acid content and they had no significant cytotoxicity influences on

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3T3 SAOS-2 and fibroblast cells. The SEM micrographs of the cryogels displayed that their
very porous networks were contributed to adhesion and spreading of SaOs-2 and 3T3 cells.
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Consequently, the CS-hyaluronic acid cryogel scaffolds could be used in both in vitro and in
vivo tissue engineering applications [82].
The CS and hyaluronic acid cryogel tissue scaffolds were applied for cell proliferation
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and growth. The SEM images in Fig. 29 display the seeded 3T3 mouse fibroblast cell and the
SAOS-2 bone cancer (osteosarcoma) cells after 24 h. the attachment efficacies of seeded 3T3
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and SAOS-2 cells on scaffolds were observed by SEM micrographs (Fig. 30) indicating
growth of cells in the pores and their adherence to scaffolds surfaces. The CS scaffolds limitat
the survival of cells and cells are migrated to the pores because of its more rigid structure and
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narrower copolymer pores when comparing CS cryogel scaffolds with CS copolymer

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scaffolds. For the CS copolymers with hyaluronic acid, the cells simply migrate to the pores
owing to their large pores and specific flexible structure. Besides, the seeded cells on the
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scaffold diagnose the hydrophilic nature of CS and hyaluronic acid thus they can well be
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adhered. The pores permit cells to migrate toward the cryogel matrix. Also, pore size affects
the metabolic activity of cells including waste removal and nutrient supply. Nevertheless, the
cell viability may be increased by increasing the hyaluronic acid ratio [82].
Several photocrosslinkable water-soluble maleilated CS and methacrylated poly (vinyl
alcohol) were synthesized and then maleilated CS-methacrylated poly (vinyl alcohol) (MCS-
MPVA) hydrogels were achieved by UV irradiation [83]. Some hydrogels properties were
evaluated including morphology, rheology, swelling and mechanical characteristics. It was
found that the MCS-MPVA hydrogels indicated rapid gel formation rate (whole
transformation to gel in 150 s), enhanced compressive strength at 0.169±0.011 MPa and fast
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absorbent capability. These features could be adjusted by controlling the weight ratio of MCS
to MPVA. Also, the indirect cytotoxicity test established that the photocrosslinked hydrogels
were compatible to L929 cells of mouse fibroblasts signifying their ability as tissue
engineering scaffolds.
Recently, chitosan-hydroxyapatite (CS-HA) nanocomposites exhibiting intercalated
structures were fabricated [84]. For this purpose, hydroxyapatite was synthesized by the sol-
gel method and formic acid was used as solvent to get stable dispersions of nano-sized HA
particles in the polymeric solution. The CS-HA dispersions were prepared using of 5, 10 and

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20 wt% of HA. Self-assembly of HA nanoparticles throughout drying the films formed
homogeneous CS-HA nanocomposites. The AFM and SEM images verified the existence of

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evenly distributed HA nanoparticles in the CS matrix. The XRD patterns and cross-sectional

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SEM micrographs displayed that layered nanocomposites were achieved. Complete CS
degradation in the thermogravimetric analysis (TGA) resulted in the formation of nanoporous
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3D scaffolds comprising hydroxyapatite, calcium pyrophosphate and β-tricalcium phosphate.
The CS-HA composites could be known as auspicious materials for application in bone tissue
engineering.
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Diverse quantities (0.5, 1 and 2) of resol resin (RS) were added to the CS-
hydroxyapatite (CS-HA) in order to develop tri-constituent CS-HA-RS nanoensembles by a
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facile co-precipitation process [85]. The TEM, SEM images indicated irregular interconnected
rough morphologies with homogenous distribution of needle like particles with average sizes
changing from 12 to 19 nm. The TGA and mechanical analysis prove that the CS-HA-RS
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nanocomposites had higher thermal stability and mechanical strength compared to the CS-HA
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(binary) composite. The CS-HA-1RS nanocomposite exhibited improved protein adsorption

and alkaline phosphate activity with exceptional apatite formation capability than CS-HA-RS
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(0.5, 2) and CS-HA nanocomposites. Accordingly, the CS-HA-1RS nanocomposite was

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chosen to test as a bare implant to repair critical-size calvarium defect (8 mm) in albino rat.
The radiological and histopathological tests pointed out that CS-HA-1RS stimulated the bone
regeneration as soon as two weeks post-implantation signifying extraordinarily quicker
healing of calvarial defect compared to Cerabone. Hence, the CS-HA-1RS could be used as a
potential biomaterial in bone tissue engineering.
Different steps occurred in surgeries are presented in Fig. 31(i). All animals displayed
uneventful wound healing in one week at selected times post-implantation. Post-surgical
edema was happened at surgical site of each rat that was vanished during 2–4 days after the
procedure. All of twenty-four rats did not reveal any signs of infection. The general health,
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activity, weight and exertion were normal in the entire follow up phases. Bone formation was
not observed in time period of 4 weeks and 8 weeks. The only difference noticed between
temporal groups was increased collagen fiber (obviously seen in Sirius red [SR] stain) and
vascularity at the defect site, Figs. 31 (ii)a, 32a.
The biopsy results for the tissues are in well agreement with the X-RAY & RVG data
obtained prior to the histopathological processings, Fig. 33(i)a,d–g,j. The radiographic
analysis illustrated that the bone defect of the sham control group was 18.20±0.18% and
31.60±1.54% (gain in bone density=GBD) that was repaired at 4 weeks and 8 weeks,

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respectively, Fig. 33(ii). For the positive control group for which the defect was filled by the
commercial formulation Cerabone (CB group), the complete absence of bone formation was

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seen at the end of 4 weeks, Fig. 31(ii)b. The defect area was filled only by irregularly

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arranged collagen fibers resulting in a loculated shape containing clumps of CB particles
enclosed by a delicate fibrous tissue signifying CB only experiences partial integration, Fig.
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33(i)b,h. Nevertheless, the GBD was only 40.81±1.12% [Fig. 33(ii)] that may only be related
to the existence of non-dispersed CB at the bone defect site but it was not corresponded to
actual bone creation verified by the histopathological data. However, promising results were
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achieved using CS-HA-1RS nanocomposite scaffold so that superior bone healing was
noticed in comparison to both the sham control and CB groups in four weeks. Such results
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were in consistent with the X-ray and RVG analysis data, Fig. 33(i)c,i. Also, the immature
lamellar bone was found in the site filled with CS-HA-1RS suggestng its highly strong
osteoregenerative ability which supported new bone formation as well as rapidly remodeled it
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into lamellar bone during a very short time of four weeks, Fig. 31(ii)c. The bone formed was
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very look like to normal mature host bone (this was occurred without previous cell seeding or
using any growth factors). The whole amount of formed bone in the CS-HA-1RS group was
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greatly enhanced with GBD (76.04±1.13%) at four weeks, Fig. 33(ii).

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After 8 weeks and in contrary to the CB group, the CS-HA-1RS group presented more
progressive calcification and the defect was almost filled by freshly created mature bone
analogous to the host bone, Fig. 32(b,c). The growing free border situated near the defect
center was enclosed by well-ordered osteoblasts and random osteoclasts compared to those in
the CB group that did not display full sealing of the bone defects confirmed by the H & E as
well as the SR stained sections. An extraordinary new bone generation and remodeling were
seen about CS-HA-1RS implant along with the ingrowth of recently formed mature lamellar
bone without encapsulation of fibrous tissue signifying a higher bone regeneration relative to
CB implants. Remarkably, the CB was not totally degraded even after 8 weeks demonstrating
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its very slow dispersion rate (see Fig. 32b). Additionally, the new bone formation zone during
8 weeks in the CS-HA-1RS was considerably bigger than that in CB group which was
observed in its radiographs in Fig. 33(i)e,k,f,l giving the GBD values of 92.69±0.66% and
77.69±0.93%, respectively, see Fig. 33(ii). Consequently, in vivo tests obviously point out
that the CS-HA-1RS nanocomposite scaffold has improved osteointegration and
osteoinduction.
The harvested calvaria of three groups including control, CB and CS-HA-1RS after 8
weeks post implantation are observed in Fig. 33i.(m–o). In addition, inflammation or necrosis

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was not noticed on both CS-HA-1RS and CB biopsy samples signifying the implants were
well tolerated without any evident toxic influence in the neighboring tissues. To exclude any

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probability of harmful effects on the vital tissues (kidney and liver) by the systemic

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absorption of the new scaffold, such tissues were examined by histopathological test and
compared to sham control group, Fig. 32(d–g). The kidneys and livers were harvested after 8
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weeks and fixed in Karnovsky fixative for two weeks and finally processed by paraffin
embedding. The renal corpuscles and renal tubules in the CS-HA-1RS group were greatly
similar to control group. As well, the hepatocytes and sinusoids in both CS-HA-1RS and
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control group were very comparable. The kidney parenchyma seemed normal having intact
renal corpuscles without congestion. Renal tubules possessed intact lining and no renal casts.
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Liver exhibited intact histoarchitecture and normal hepatocytes and sinusoids. Apparent
necrosis or congestion was not noticed which verified the nanocomposite scaffold was not
hepatotoxic or nephrotoxic, Fig. 32(e,g).
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Because the CS-HA-1RS nanocomposite scaffold led to lamellar bone formation in four
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weeks and whole mature bone was created after 8 weeks, it was mandatory to perfoem an
extra short-term test to display the lowest initial time (latent period) necessary for
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osteogenesis to start and detect the transition phase of bone formation to further recognize its
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mechanistic action. Hence, the experiments were done for two weeks using the CS-HA-1RS,
Fig. 34(a–e). This test was not replicated for control and CB groups as there was no bone
formation even after four weeks. After two weeks post-implantation, initial phase of new
bone generation was witnessed at the margin of the calvarial defect evidently indicating the
fibrous tissue, woven bone and lamellar bone (new mature bone), Fig. 34a. The in situ bone
defect site (top view) and the bone defect site inside (bottom view) are revealed in Fig.
34(b,c). The freshly created lamellar bone had a comparable density to the normal bone which
was obvious from its radiographs approving the new bone was formed originally from the
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host bone, grew and extended to the defect center, Fig. 34(d,e). The bone density degree was
61.14±4.15% [85].
Photocrosslinkable hydrogels obtained using natural materials are promising for
application as scaffolds in tissue engineering but their drawbacks weak are formation and
poor mechanical characteristics [86]. Recently, photo-clickable thiol-ene hydrogels based on
CS were synthesized by photopolymerization of maleic chitosan (MCS) and thiol-terminated
poly (vinyl alcohol) (TPVA) by means of a biocompatible photoinitiator [260]. It was shown
that the rheological and absorbing properties of the MCS-TPVA hydrogels could be tuned by

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changing the TPVA amount in the feed. Strong intermolecular hydrogen bonds were formed
between TPVA and MCS. The MCS-TPVA hydrogel (MT-3) displayed a fast gelation (<120

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s), enhanced stiffness (G'=∼5500 Pa) and compressive strength (0.285±0.014 MPa) that were

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vital for the hydrogel scaffolds, mainly for the injectable hydrogel scaffolds. Also, the
photocrosslinked MCS-TPVA hydrogels was cytocompatible which promoted the L929 cells
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attachment and proliferation confirming they were promising tissue engineering scaffolds.
In another work, composite nanofibers were produced by electrospinning method using
magnesium oxide (MgO), poly(ε-caprolactone) (PCL) and CS with diameters ranged as 0.7–
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1.3 μm [87]. The physicochemical properties including morphology, mechanical strength and
integrity in aqueous environment were evaluated. The cellular compatibility was examined by
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cell viability tests and microscopy imaging and it was revealed that the nanofibrous
membranes supported the viability and attachment of 3T3 cells. Accordingly, such
nanofibrous composites could mimic the physical structure and function of tissue extracellular
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matrix indicating they could be employed in tissue engineering purposes.

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Fabrication and characterization of bioactive scaffolds prepared using CS,

carboxymethyl chitosan (CMC) and magnesium gluconate (MgG) were accomplished [88].
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The scaffolds were prepared by subsequent freezing promoted phase separation and
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lyophilizing polyelectrolyte of CS, CMC and MgG complexes. The scaffolds revealed
uniform porosity with greatly interconnected pores having sizes in the range of 50-250 mm.
The elastic moduli up to 5 MPa and compressive strengths up to 400 kPa were measured.
Also, the scaffolds were remained intact, retained their original 3D frameworks under in vitro
testing conditions. Such scaffolds did not display cytotoxicity to 3T3 fibroblast and osteoblast
cells. Therefore, these scaffolds were efficient and appropriate for tissue engineering.
In another work, CS composite scaffolds were developed with controllable internal
architectures for bone tissue engineering [89]. The CS based composites were produced
through changing montmorillonite (MMT) and HA contents in order to obtain macro-
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spherical 3D scaffolds upon direct agglomeration of the sintered macrospheres. The

physicochemical, biological and mechanical properties of the CS, CS-MMT, CS-HA and CS-
MMT-HA 3D scaffolds were characterized. The reinforcement using HA and MMT caused
decreased degradation rate and swelling. Also, compared to pure CS, the CS-HA-MMT
composites demonstrated enhanced protein adsorption and hemocompatibility. Sintering of
the macrospheres adjusted the swelling capacities of the 3D scaffolds which were much
significant to maintain their mechanical strengths. The CS/HA/MMT composite scaffold
exhibited 14 folds enhancement in the compressive strength in comparison to the pure CS

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scaffold. As well, the scaffolds supported the MG 63 cell proliferation. As a result, it was
concluded that the CS-HA-MMT composite macrospheric 3D scaffolds could find practical

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applications in bone tissue regeneration.

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2.6. Application of chitosan in preparation of implants
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Short-term contact of implant with blood can lead to adsorption of plasma proteins,
calcium, platelet and bacterial adhesions on the implant surface. The adhered platelets could
be then activated resulting in the coagulation cascade and after that thrombosis. At long-term,
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calcification of implants (heart valves, breast implants and stents) may be happened by
growing calcium phosphates or other calcium salts deposits. Moreover, implants suffer from
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high plaque accumulation which is commonly related to the two most important pathogens,
i.e. Porphyromonas gingivalis and Streptococcus mutans [90]. For instance, colonization
occurs on the fixed appliances in prosthodontic therapy due to biofilm forming bacteria which
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could destroy the periodontal tissue [90]. Problems including thrombosis, calcification and
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bacterial growth can interfere with the implant function, decrease its lifetime and bring about
implant explanation. Therefore, researches on interaction of blood with biomaterials were
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focused on surface treatment to inhibit calcification and thrombosis [91]. In order to enhance
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the lifetime (short and long terms) of clinical implants, they should be coated with a
biocompatible material as a bio-interface that is able to overcome the above-mentioned
challenges. Among various polymers, CS appears as an extremely promising substance due to
its versatility and potential to solve these complications [92].
Numerous metal coated implants have up to now been examined against dental
pathogens leading to biofilm formation and failure of dental implants [90]. Nanoparticles can
be used accompanied by native biomolecules in order to improve the activity of such
bioactive compounds. In an effort, the efficacy of Ag conjugated CS nanoparticles was
estimated as a prospective coating material for titanium dental implants [93]. The bioactive
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CS was obtained from A. flavus Af09 and conjugated with Ag nanoparticles. The Ag-CS
nanoparticle revealed good growth inhibition influence against two key dental pathogens
including P. gingivalis and S. mutans. The Ag-CS inhibited the adhesion of these two bacteria
and decreased the biofilm formation. Also, the nanoparticle inhibited the quorum sensing
production in these bacteria. The naturally extracted CS did not exhibit cell cytotoxicity
confirming its biocompatibility. Hence, coating the titanium dental implants by the Ag-CS
could be an advantage as the corrosion resistance of dental implants to increase the
passivating property of the implants [93].

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Lower biofilm formations were observed upon treatment by gentamicin and CS
compared to those conjugated with Ag which were significantly lower than the positive

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control. Images obtained by congored staining evidently exhibited a decrease in biofilm

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thickness so that a noteworthy reduction in biofilm formation was realized as S mutans was
grown in the existence of CS conjugated with Ag nanoparticle (Fig. 35a). The image analysis
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using the ImageJ software displayed 60% biomass decrease upon treatment with antibiotic
and nanoparticles, Fig. 35b. The treatment of HGF cells did not demonstrate any cytotoxic
effects for the Ag-CS. Also, substantial differences were not observed in the proportion of
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live cells in AOEB staining (see Fig. 36) [93].

It was attempted to fabricate CS-based hydrogel implants to be used in peripheral
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nervous tissue regeneration [13]. The fabrication method was based on electrodeposition
using a solution of CS and organic acid. The solution was supplemented with hydroxyapatite
to enhance the mechanical strength of the implant. Also, the hydroxyapatite was acted as a
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source of calcium ions. The effect of the polymer and the additive concentrations were
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assessed on mechanical, chemical and biological characteristics of the implant. The

physicochemical properties of the prepared structure were highly dependent to the initial
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solution composition. The in vitro cytotoxic and pro-inflammatory assays displayed the
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biocompatibility of implants.
The directly compressed tablets were developed for implantable delivery of risedronate
sodium to treat osteoporosis and compare the mechanism and kinetics of drug release from
biodegradable CS and non-degradable polyvinylchloride (PVC) polymer matrices [94]. The
compositions and procedure parameters were optimized by a mixed 2 and 3 level full factorial
design. Critical Quality Attributes (CQA) were studied including diametral breaking hardness,
porosity and drug dissolution speed. It was revealed that there were substantial differences
between the behaviors of the two polymers. The CS displayed poor compressibility which
caused weak mechanical properties and rapid disintegration of the CS-based tablets.
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However, despite its quick disintegration, the CS-based matrices demonstrated one-week-long
continuous drug release, which could be related to strong drug-carrier interactions. The
existence of intermolecular hydrogen bonds was established by FT-IR and near infrared (NIR)
spectra. Conversely, the PVC-based composites presented outstanding compressibility,
suitable tablet hardness and little porosity. The tablets stayed intact throughout the dissolution
and showed a slower release rate than those of CS-based matrices. The NIR spectra did not
illustrate intermolecular interactions which suggested the dissolution rate was happened due
to the porosity of tablets whereas the FT-IR spectra provided some details about the molecular

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interactions occurred during the drug release mechanism.
Several CS–carbon nanotube implants were developed as tubular hydrogels that were

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enriched by calcium ions [95]. The hydrogels were intended to be used in tissue engineering

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particularly peripheral nervous tissue regeneration. The fabrication method was based on the
electrodeposition which showed important benefits over current approaches so that the
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implants could quickly be acquired at any requisite dimensions. Therefore, this could be
considered as an effective method to treat patients having peripheral nerve injuries. Both
single walled and multiwalled carbon nanotubes improved the mechanical features of the
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tubular hydrogels. Also, controlled existence of calcium ions added using hydroxyapatite
enhanced the regenerative response. The in vitro cytotoxic tests on mouse cell lines
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(mHippoE-18 hippocampal and L929 fibroblasts cells) and pro-inflammatory assays on THP-
1XBlue™ cells exhibited that the implants were biocompatible. Consequently, the immune-
and nervous-safety of implants could be evaluated on animal models.
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2.7. Application of chitosan in preparation of contact lenses

Drug delivery using ocular therapeutics is a challenge which is due to the anatomical
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and physiological restrictions of the eye which does not allow achieving correct therapeutic
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concentration at the desired site of action. Hence, clinicians recommend using numerous
dosages, which causes nonconformity by patients and less economical. To tackle these
problems, other ocular delivery systems have been explored including ocuserts, in situ gels,
liposomes and nanoparticles. A principally attractive form of these delivery systems is contact
lenses which are thin and curved plastic disks designed to protect the cornea by clinging to the
eye surface through surface tension [96].
Currently, therapeutic ophthalmic lenses have been developed that are able to release
suitable drugs in a long time in order to circumvent the ineffective and tedious eye drop
administration. In this context, numerous efforts have been done by researchers to improve
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drug delivery using soft contact lenses (SCLs) [97]. Also, in case of cataracts which are one
of the most common eye diseases, therapeutic intraocular lenses (IOLs) are fabricated to
avoid postoperative infectious problems after surgery [98]. Indeed, one of the foremost
complications in the application of drug-loaded ophthalmic lenses to replace the topical usage
of eye drops is controlling the drug release. Usually, the drug release from such devices
occurs as an early burst release followed by decline in drug release to levels under therapeutic
dose. To solve this challenge, some approaches are proposed to ensure a sustained medication
delivery to the eye throughout the requisite time period at a controlled rate. These approaches

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include addition of chemicals to reversibly interact with the drug, using nanocarriers such as
liposomes, micelles and nanoparticles, incorporation of diffusion barriers to the drugs like

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vitamin E aggregates [99]. Another method is the implementation of coatings in commercial

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lenses whose production procedures and properties have been optimized. Presently, coatings
are employed in commercial SCLs to enhance the surface wettability and lubricity which have
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led to higher comfort to the users. One of objectives in such coatings is to prevent the
adsorption of proteins and microorganisms from the ocular tear fluid in order to evade eye
infections by the users of contact lenses. Various coatings are provided to SCLs that are
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fundamentally based on polyelectrolyte multilayers achieved by the grafting/adsorption of

particular molecules and immobilization of liposomes at the lens surface [100]. Moreover,
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natural polymers like CS and alginate can be used for application as coatings in contact
lenses.
In another attempt, an extended wear therapeutic contact lens (TCL) was fabricated by
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molecular imprinting method for the sustained delivery of timolol maleate (TML) [14]. The
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designed TCL was composed of a TML imprinted copolymer of carboxymethyl CS-g-

hydroxyethyl methacrylate-g-polyacrylamide (CmCS-g-HEMA-g-pAAm) which was
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embedded to a poly HEMA matrix (pHEMA). The TML reloading to the lens was proved by
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UV–visible spectra which exhibited the outstanding reloading capacity was 6.53 μg
TML/TCL. The in vitro drug release after each cycle in lacrimal fluid was fitted to Higuchi
model which proposed the diffusion release mechanism was occurred with no polymer
degradation. As well, the TML release kinetics pointed out a sustained drug delivery which
could efficiently attain the TML therapeutic index and provided a onetime glaucoma
treatment. The biological activity of eluted drug subsequent to each cycle and cell viability of
the TCL were substantiated by means of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,3-
bis(2-methoxynitro-5-sulfophenyl)-5-(phenylaminocarbonyl)-2H-tetrazolium hydroxide
(XTT) assay, respectively.
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Balb/3T3 Clone A31 cell lines are frequently applied to assess cell viability in
ophthalmic preparations because they look like the retinal receptors. Cell viability was
examined using XTT assay which measured mitochondrial activity. The cell viabilities of the
disks were calculated to be 77.5, 74.1, 71.8, 67.2 and 61.0%, respectively for 1.50, 3.00, 6.25,
12.50 and 25.00 µg/mL of TCL. Such values exhibited outstanding cell viability and notably
the control and sample displayed nearly the same values (Fig. 37). Thus, results proved the
biological approval of the prepared TCL and its practical usefulness [14].
Despite the fact that hydrogel contact lens is of great attention as delivery carrier in the

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arena of oculopathy therapy, traditional hydrogel does not illustrate desired drug
encapsulation and controlled release properties because hydrophilic polymer chains do not

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effectively interact with drug molecules [101]. Hence, functional hydrogels were synthesized

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to deliver ophthalmic drug for oculopathy therapy. For this purpose, functional monomer of
mono-GMA-β-CD and functional crosslinker of MA-β-CD were introduced to hydrogel
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through copolymerization reaction. The contact angle and equilibrium swelling ratio of
hydrogels were affected by MA-β-CD ratio and mono-GMA-β-CD ratio, respectively. All of
hydrogels displayed comparable water loss, appropriate transparency and rheological property
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of a typical elastomer. Also, the viscoelasticity and surface morphology of hydrogels were
influenced by mono-GMA-β-CD and MA-β-CD ratios. Functional hydrogel comprising β-CD
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domain revealed superior protein resistance capability and considerably greater equilibrium
encapsulated drug quantity compared with traditional hydrogel. In addition to the
performance, the drug release from hydrogel was controlled by means of both mono-GMA-β-
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CD and MA-β-CD ratios. Preliminary in vivo assessment indicated that functional hydrogel
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contact lens had enhanced influence and effectiveness on dropping intraocular tension relative
to commercial eye drop. Consequently, it was inferred that functional contact lens was
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promising for application in oculopathy therapy [101].

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The cornea was investigated by slit lamp after it was cultured for 35 days (Fig. 38).
Slight edema and some new vessels about marginal corneal were noticed for the cornea
treated for 35 days using the drug encapsulated hydrogel contact lens (see Fig. 38a). In
untreated cornea, numerous new vessels were homogeneously scattered on the cornea surface,
Fig. 8b. The results were also established by HE stain images (Figs. 38c and 38d). The cornea
treated using the drug encapsulated hydrogel contact lens for 35 days revealed that evident
hyperplasia was mostly observed on marginal corneal as it is shown in Fig. 38c. The untreated
cornea exhibited that the hyperplasia was homogeneously scattered over the cornea surface
(Fig. 38d). New vessels on the surface of untreated cornea were attributed to intraocular
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hypertension. The slight edema of cornea treated with contact lenses was a problem that was
induced through protein deposition and cornea anoxia which might be overcome by
improving the usage method and addition of care solution. Because the hydrogel contact lens
was fabricated without postprocessing of margin, the irregular margin might have stimulated
the tissue which triggered hyperplasia. Thus, the symptom might be relieved by improving the
postprocessing method. Consequently, it was concluded that the contact lens had a bright
outlook for application in oculopathy therapy [101].
Glaucoma is generally treated by eye drops but this method is not efficient due to fast

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drug clearance (low residence time) from the ocular surface. On the other hand, contact lenses
are perfectly suitable for controlled drug delivery to cornea; however, it should be taken into

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account that introduction of any drug loaded particulate formulation could influence the

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physical and optical properties of contact lenses [102]. Thus, timolol maleate (TM) loaded
ethyl cellulose nanoparticle-laden ring was implanted in hydrogel contact lenses to allow a
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controlled drug delivery at therapeutic rates without affecting critical lens characteristics
[102]. The TM-implant lenses were achieved through dispersion of TM encapsulated ethyl
cellulose nanoparticles into acrylate hydrogel created as ring or sandwich systems. The TM-
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ethyl cellulose nanoparticles were obtained by double emulsion process using diverse ratios of
ethyl cellulose to TM. The XRD patterns showed the TM transformation to the amorphous
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phase. The in vitro release kinetic data presented a sustained drug release happened within the
therapeutic window for 168 h using 150 μg loading. The cytotoxicity and ocular irritation
assays established the safety of TM-implant contact lenses. The in vivo pharmacokinetic tests
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in rabbit tear fluid exhibited substantial rise in mean residence time and area under curve
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using the TM-implant contact lenses compared to eye drop therapy. The in vivo
pharmacodynamic results in rabbit model proved sustained decrease in intra ocular pressure
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for 192 h. Accordingly, this investigation validated the auspicious potential of implantation
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technique to treat glaucoma by means of contact lenses which could be used as a platform for
treatment of other ocular diseases.
Since the corneal tissue is the most frequently transplanted tissue around the world,
recently it was intended to produce a drug-eluting contact lens to be employed as a bandage
after keratoprosthesis [103]. Films were prepared by means of poly(vinyl alcohol) (PVA) and
CS that were crosslinked by glyoxal (GL) to yield the PVA and PVA-CS films. Also,
vancomycin chlorhydrate (VA) drug was incorporated in such systems through soaking. The
thermal behavior, drug release profile, cytotoxicity, biodegradation, hydrophilicity and
swelling capability of the samples were evaluated. The PVA and PVA-CS films were
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transparent, flexible having smooth surfaces, hydrophilic and could load and release
vancomycin for more than 8 h. The biodegradation test in artificial lachrymal fluid using
lysozyme at 37 ºC revealed that mass loss was greater for the samples including CS.
Furthermore, the samples achieved using CS presented the pore formation which were
confirmed by the SEM micrographs. All samples illustrated biocompatible characters when
they were in contact with cornea endothelial cells for 24 h. In conclusion, the 70PVA-30CS
film could combine the required properties to achieve vancomycin eluting contact lenses in
order to avoid inflammation upon corneal substitution.

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2.8. Application of chitosan in wound healing

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Wound healing is a complex physiological reaction in a living organism to chemical,

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physical, mechanical and/or thermal injuries [104]. It is a dynamic procedure where cells and
matrix components act together to facilitate wound regeneration and restore tissue integrity.
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Wound healing occurs through dynamic and overlapping phases including inflammatory
(homeostasis and inflammation), proliferative (granulation, contraction and epithelialization)
and remodeling (maturation) [105]. Nevertheless, when the process does not proceed
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normally, the healing would not continue afar the inflammatory phase and this is recognized
by the accumulation of great quantities of macrophages and neutrophils along with the
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exudation of inflammation mediators such as reactive nitrogen species, reactive oxygen

species, cytokines and their analogues. Deficiency in wound healing cascade can result from
critical size skin injury, burn, chemical damage, secondary microbial infections and problems
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happening due to pathological states such as diabetes [106]. In order to stimulate wound
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healing, the wound have to be covered using a suitable non-toxic and semi-permeable
dressing to protect it against external microbial and mechanical stresses. As well, if the wound
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area maintains a moist environment, the healing process will be initiated [107].
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Biopolymers are biocompatible and have the characteristic to swell through liquid
uptake by their polymeric networks. Hence, they offer apposite matrixes to stimulate healing
cascade with mimicking in milieu moist medium. Among diverse biopolymers, CS is widely
used in wound healing as a result of its structural similarity to glycosaminoglycans which is a
vital wound repairing macromolecule existing as a component in extracellular matrix (ECM)
[108]. Since CS is a biodegradable polymer, its degradation products can initiate synthesis of
ECM components signifying its non-toxic nature for in vivo application. Furthermore, it is
one of the few polymers showing outstanding antibacterial feature [109]. In addition to a
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simple dressing and film forming ability, it is also employed as complex artificial matrices in
tissue engineering.
Recently, a water-soluble CS derivative, N-succinyl-chitosan (NSC) was synthesized
using succinic anhydride, hydrochloric acid and alkaline CS; then, its capacity to accelerate
the wound healing progression was evaluated [110]. The NSC cytotoxicity was examined on
L929 cells and its antibacterial activity was assessed through measurement of the inhibition
zone. It was found that the NSC solubility was noticeably enhanced relative to CS and NSC
was non-toxic having suitable antibacterial potency. The animal wound healing experiment

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specified that NSC significantly decreased the healing time in comparison to CS.
Histopathological investigation proposed that the principal mechanisms of these phenomena

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were associated to NSC capability to stimulate the development of granulation tissue and

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increasing epithelialization. Overall, it was concluded that the NSC had the potential to be
used in wound healing.
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Healing of full-thickness (FT) wounds necessitates further support from native or
synthetic matrices in order to assist tissue regeneration [111]. Particularly, a matrix with
optimum hydrophilic-hydrophobic balance is required to experience satisfactory swelling and
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to diminish bacterial adhesion. In this context, polyurethane diol dispersion (PUD) and the
antibacterial CS were blended in diverse ratios which were self-organized to form
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macroporous hydrogel scaffolds (MHS) at room temperature upon drying [111]. The AFM
and SEM images of MHS indicated the macroporosity on top and cracked surfaces. The FTIR
spectra displayed that the inter/intra-molecular hydrogen bond interactions formed between
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the two polymers were responsible for phase separation which was detected in micrographs of
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blend solutions obtained in the course of the drying process. Also, the influence of phase
separation on in vitro degradation (hydrolytic, enzymatic and pH dependent) and mechanical
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characteristics of MHS were examined which proved it would be an appropriate material for
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wound healing. The in vitro cytocompatibility was established via the proliferation of primary
rat fibroblast cells on MHS. Moreover, the MHS was used in in vivo full-thickness wound
healing on Wistar rats and the results were compared to the similar Tegaderm™ polyurethane
comprising commercial dressing. The MHS-treated wounds revealed faster healing with
improved wound contraction, greater collagen synthesis and vascularization in wound area
than Tegaderm™. Consequently, it was verified that the MHS was an auspicious sample for
application in full-thickness wound healing processes [111].
After 18 days, the subcutaneously implanted C7P3 was retrieved for histological tests
(Fig. 39a–c). The H&E staining in Fig. 39d illustrated that the C7P3 was capable to integrate
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with the neighboring tissues by cell infiltration and exhibited the occurrence of matured blood
vessels (indicated with an arrow) after 18 days. Any signs of acute inflammations were not
seen. Also, toluidine blue stained sections (Fig. 39f) demonstrated small number of mast cells
infiltration in C7P3. Such slight inflammatory response was related to the normal wound
healing course. The MT staining (Fig. 39e) proved collagen deposition and capsular
layer/fibrosis was not observed around the C7P3tissue.
C7P3 was also evaluated for FT in vivo wound healing test in a rat model. The C7P3
was adhered to the wound bed because of its inherent great protein adsorption capacity which

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resulted in biological dressing fixation and avoiding wound exposure to the external medium
(Fig. 39a). Fig. 40a reveals wound healing kinetics and the of wound closure area is observed

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in Fig. 40b. The control group displayed 40±1.92%, 65±3.12% and 82±3.91% wound closure

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on days 7, 14 and 21 but the C7P3 treated groups exhibited improved wound closure values
equal to 55±2.54%, 88±3.41%, and 100±4.12%, respectively. The accelerated wound healing
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properties of the C7P3 scaffold was corresponded to the exceptional combinatorial
characteristics like interconnected porous structure, great protein/water adsorption and pH
sensitive degradation [111].
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The influence of policaju (POLI)-CS hydrogel prepared using POLI from cashew tree
(Anacardium occidentale L.) gum and CS was estimated accompanied by low level laser
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therapy (LLLT) in wound healing [112]. For this purpose, sixty male Wistar rats were
allocated to four groups including POLI-CS hydrogel (H), LLLT (L), POLI-CS with LLLT
(HL) and saline control. The macroscopic assessments were performed by clinical tests and
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area measurements along with microscopic analysis through histological principles. The H
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and HL revealed additional esthetical scar tissue and greater wound contraction than control.
The histopathological analysis illustrated higher existence of fibrin-leukocyte crust in HL and
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L at day 3, more collagen formation in H, L and HL, low focal necrosis at 7 and 14 days in H,
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poor neutrophilic exudate in H, L and HL, regression of the vascular neoformation at day 7 in
H and identical variation in HL and L. Hence, it was established that POLI-CS caused highly
effective healing process and modulated the inflammation and its application along with
LLLT potentiated the process.
Some chitosan-bentonite nanocomposite (CBN) films were developed using solvent
casting process to be employed in wound healing [113]. The physicochemical properties such
as folding endurance, thickness, water absorption ability and water vapour transmission rate
(WVTR) of the films were examined. The FTIR spectra established the interactions occurred
between positively charged CS and negatively charged bentonite. The surface morphology of
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the composite films was observed by SEM images. The WVTR, water absorption ratio,
thickness and folding durability of the CBN films were measured equal to 1093±20.5–
1954±51 gm−2 day−1 , 1232±14.58–1688±18.52, 17.50±5–42.50±9.75 μm, and 145.25±2.21–
289.50±0.57 respectively. Because bentonite was very hydrophilic, it highly enhanced the
water absorption capabilities of the nanocomposite films. Furthermore, the bentonite
existence in the films improved the mechanical strength. Besides, the antibacterial potency of
the films was assessed against Gram-positive and Gram-negative microorganisms. All CBN
films exhibited suitable inhibition activity against all the bacteria compared to control. Hence,

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it was recommended that the CBN films were promising samples as wound dressing
materials.

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It was aimed to achieve a composite dressing using collagen, CS and alginate to

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promote wound healing and avoid seawater immersion [114]. The chitosan-collagen-alginate
(CCA) cushion was obtained by paint coating and freeze-drying which was then attached to
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polyurethane in order to create CCA composite dressing. Also, the porosity, swelling,
degradation and mechanical characteristics of CCA cushion were examined. The effect CCA
composite dressing on wound healing and seawater prevention was tested using rat wound
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model. The preliminary biosecurity was estimated through hemocompatibility and

cytotoxicity experiments. Results proved that CCA cushion had appropriate mechanical and
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water absorption features. The wound healing ratio was higher in rats treated by the CCA
composite dressing than in rats treated using gauze and CS. After five days, the healing rates
using the CCA composite dressing, gauze and CS were measured to be 48.49±1.07%,
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28.02±6.4% and 38.97±8.53%, respectively. The histological images of rats treated by the
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CCA composite dressing more fibroblast and intact re-epithelialization were perceived and
the expressions of bFGF, EGF, CD31 and TGF-β were significantly augmented. As well, the
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CCA composite dressing did not have substantial cytotoxicity but it showed suitable
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hemocompatibility. Thus, it was found that CCA composite dressing prevented seawater
immersion, supported wound healing and it exhibited an appropriate biosecurity.
Fig. 41 indicates the wound healing of CCA composite dressing in three treated groups.
It was found that after soaking in seawater for 4 h, the dressing covered the wound was yet
dry. Thus, the dressing could be utilized for a wounded person working on sea in order to
limit the risk of wound seawater immersion syndrome. Also, the wound sites in CCA
composite dressing group were healed quicker than both of the gauze and the CS groups (see
Fig. 41A). The wounds used the CCA dressing group showed slight inflammation or infection
and they appeared as moist and neat. On day 3 after surgery, noticeable differences were not
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observed among three groups because the wounds were moist without any infections. On days
5 and 8, the CCA dressing treated group exhibited more effective wound healing compared to
the gauze-treated and the CS-treated groups. From the 11th day, all of the wounds began to
contract from the edges and the wound covered using the CCA dressing was contracted more
rapidly.
Fig. 41B illustrates the wound healing rates indicating on the third day after surgery, the
healing rate in CCA treated group was 27.89±6.04% that was greater compared to those of the
gauze negative control (12.46±2.7%) and the CS positive control (23.52±6.13%). On the fifth

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day, the measured healing rates in CCA treated, CS and gauze groups were 48.49±1.07%,
38.97±8.53%, 28.02±6.4%, respectively confirming the rate in CCA group was very greater

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than that of the gauze group. On days 8 and 11, the healing rates in CCA group were

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56.69±7.41% and 82.85±7.23%, respectively but those in the CS group were 60.41±3.65%
and 84.11±4.24%, and in gauze group were 43.67±6.05% and 70.78±6.06%. These results
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exhibit that the healing rates in CCA and CS dressing group were greater than that of gauze
group. The healing rates on the 13th day for all three groups were around 90% and there were
not any differences among the three groups. Hence, the CCA composite dressing have
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improved wound healing efficiency in the early stages of wound healing than CS dressing and
gauze.
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The histological data are provided in Fig. 42 showing in day 5 after surgery, the wound
areas in CCA treated group contained much more akaryocytes than CS and gauze groups. On
the eighth day, noticeable granulation tissue growth and partial fibroblasts were seen in the
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CCA and CS groups. The CCA and CS groups illustrated granulation tissue and the
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neogenetic epidermis formation in the 11th day. The epidermis and dermis were repaired in
the all groups in the 13th day and it was looked like normal skin in the CCA treated group.
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Consequently, the CCA composite dressing revealed high capability for re-epithelialization,
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well-organized granulation tissue and epidermis/dermis repair [114].

Lupeol entrapped chitosan-gelatin hydrogel (LCGH) films were achieved by means of
solution casting technique through blending CS and gelatin using glycerol plasticizer and
glutaraldehyde crosslinker [115]. The LCGH films were characterized by SEM, FTIR,
differential scanning calorimetry (DSC), equilibrium water content (EWC), WVTR and in
vitro release analyses. The SEM images proved the existence of the homogeneous porous
structure in both blank and LCGH films. The FTIR and DSC confirmed the presence of lupeol
in hydrogels. The LCGH film was smooth, non-brittle and flexible which displayed
outstanding swelling capacity. The EWC (85.40%) and WVTR (2228±31.8) were suitable for
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a perfect wound dressing. Also, the biological properties of lupeol were evaluated using
antibacterial and antioxidant assays. The antioxidant assay established that lupeol and LCGH
film had exceptional antioxidant activities due to scavenging radicals with an enhanced
constant rate which was increased with time upon the continuous lupeol release. The disc
diffusion method exhibited that the antibacterial activity of lupeol in LCGH film was
preserved. The cell viability was estimated by the MTT assay using NIH/3T3 fibroblast cells
and it was found that the CGH film obviously had satisfactory non-toxicity and cell viability.
Hence, it was illustrated that CS/gelatin hydrogel film was a perfect delivery system for

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sustained lupeol release and the LCGH film was an auspicious wound healing material [115].
The RGD peptide sequences can regulate cellular actions through interaction with α5 β1 ,

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αv β5 and αv β3 integrin, which influences the wound healing [108]. In a recent work, the

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RGDC peptide was immobilized onto CS derivative 1,6-diaminohexane-O-carboxymethyl-
N,N,N-trimethyl chitosan (DAH-CMTMC) in order to demonstrate the RGDC-supporting
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adhesion for improved wound healing [116]. The effectiveness of N-methylation, O-
carboxymethylation and spacer grafting was both qualitatively and quantitatively studied
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using the FTIR and H NMR spectra which showed >0.85 substitution degree for O-
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carboxymethylation and 0.38 for N-methylation. Also, the glass transition temperatures were
measured for the CS derivatives. The peptide immobilization was done by the sulfhydryl
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groups by means of sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method).

The RGDC immobilized peptide onto the DAH-CMTMC was ~15.3 μg/mg for the CS
derivative that was proved by the amino acid analysis. A substantial increase in human dermal
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fibroblast viability in vitro during 7 days (viability >140%) proposed that the RGDC-
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functionalized CS led to greater wound healing. Besides, proliferation and bio-adhesion

assays justified that coating of the RGDC-functionalized CS derivatives exhibited in vitro
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wound healing through increasing fibroblast adhesion and proliferation. The results revealed
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that the RGDC peptide-functionalized CS offered an ideal substrate for both fibroblast
proliferation and adhesion.
It was aimed to prepare CS hydrogels containing nerolidol to optimize their
antimicrobial and wound healing properties [117]. The hydrogels were achieved by addition
of 2 or 4% of the nerolidol to the CS solution. The TGA, DSC and FTIR analysis confirmed
the nerolidol incorporation in the hydrogels. Also, direct contact of hydrogels and
Staphylococcus aureus bacterium exhibited a synergistic influence in the materials which led
to complete bacterial growth inhibition. The hydrogel comprising 2% nerolidol displayed
exceptional healing activity. The emergence of re-epithelialization and collagen
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reorganization was witnessed on the day 7 of treatment. Hence, the hydrogels ascertained to
be favorable as healing and antibacterial substances.
In another study, a promising wound dressing was developed using CS cross-linked
with genipin by introducing partially oxidized Bletilla striata polysaccharide [118]. The
prepared material was coded with CSGB which presented lower gelling time, more
homogeneous aperture distribution, greater water retention, required mechanical strength and
higher L929 cell proliferation relative to the CS only cross-linked by genipin. Because free
amino groups of CS were partially blocked, the CSGB practically did not indicate

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antibacterial potency, consequently a bilayer composite of CS-silver nanoparticles (CS-AgG)
was produced on CSGB in order to inhibit microbial growth. The in vivo tests specified that

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both CSGB and bilayer wound dressing considerably enhanced the healing rate of cutaneous

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wounds in mice. The bilayer demonstrated superior mature epidermization with fewer
inflammatory cells on day 7. Accordingly, the bilayer composite was highly promising for
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wound dressing application.
Recently, CS–hyaluronic acid composite sponge scaffold was developed that was
supplemented with Andrographolide (AND) lipid nanocarriers [119]. One of nanocarriers
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named NLC4 showed the highest desirability value; hence it was selected as the best
nanocarrier. It had a spherical shape that was 253 nm in diameter, 83.04% entrapment
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efficacy and a prolonged AND release up to 48h. The NLC4 was incorporated into a CS–
hyaluronic acid gel and lyophilized for 24 h to acquire CS–hyaluronic acid/NLC4
nanocomposite sponge in order to increase AND carriage to wound sites. The sponge
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morphology was characterized by the SEM micrograph. The nanocomposites exhibited

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56.22% porosity and improved swelling character. The in vivo evaluation in rats established
that the CS–hyaluronic acid/NLC4 sponge boosted the wound healing without scar and
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amended tissue quality.

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It is known that severe oxidative stress in chronic wounds hinders the wound healing.
Thus, an antioxidant-loaded hydrogel was fabricated to heal diabetic wounds [17]. For this
purpose, composite hydrogels composed of CS, heparin and poly (γ-glutamic acid) in diverse
ratios were produced by electrostatic interactions. The hydrogels displayed good 3D network
structures and their porosities were diminished as the crosslinking density was improved. The
hydrogels indicated suitable swelling capability, usual viscoelastic character and satisfactory
mechanical feature in rheological test. The fibroblast proliferation test established that the
hydrogels were cytocompatible. Superoxide dismutase was loaded to the hydrogel to achieve
a wound dressing with antioxidant activity. It was shown that the dressing applied on diabetic
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rat models accelerated wound healing through stimulating wound closure and collagen
deposition. Overall, the hydrogel demonstrated appropriate physical properties and could
effectively support repairing chronic trauma in diabetic rats confirming it would be a
promising wound dressing.
A composite sponge was prepared through physical mixing of hydroxybutyl CS and CS
to get a porous spongy material by vacuum freeze-drying method [120]. The macroporous and
hydrophilic hydroxybutyl CS composite sponge was produced by the introduction of CS to
hydroxybutyl CS. The composite sponge exhibited greater porosity of ~85%, superior water

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absorption of near 25 times, improved softness and less blood-clotting index compared to
those of CS and hydroxybutyl CS sponges. The composite sponge with satisfactory

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hydrophilic nature absorbed moisture from blood to raise the blood concentration and

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viscosity, and became a semi-swelling viscous colloid which could block the capillaries. The
cytocompatibility experiments using HUVEC and L929 cells established that composite
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sponge was not cytotoxic and could stimulate the fibroblasts growth. The composite sponge
was made up in order to attain higher antibacterial potency (>99.99% reduction) due to the
hydroxybutyl CS had drawbacks and poor antibacterial activity. Ultimately, the in vivo tests
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performed on Sprague–Dawley rats illustrated that epithelial cells were attached to the
composite sponge and penetrated to the interior. Furthermore, it was evidenced that the
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composite sponge had a superior capacity to stimulate wound healing and faster skin glands
creation and re-epithelialization.
Repairing dermal wounds mainly in the diabetic population is an important healthcare
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problem [121]. The poor wound healing in diabetic wounds can be ascribed to low levels of
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endogenous growth factors such as vascular endothelial growth factor (VEGF) that generally
stimulate multiple phases of wound healing. Recently, CS scaffolds were produced by freeze
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drying and incorporated with plasmid DNA encoding perlecan domain I and VEGF189 and
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their capacity to support dermal wound healing was investigated in vivo. The plasmid DNA
encoding perlecan domain I and VEGF189 loaded scaffolds stimulated dermal wound healing
in both normal and diabetic rats. Such treatment enhanced number of blood vessels and sub-
epithelial connective tissue matrix components in the wound beds relative to wounds treated
with CS scaffolds including control DNA or wounded controls. Therefore, it was suggested
that the CS scaffolds embedded with plasmid DNA encoding VEGF189 and perlecan domain
I had the potential to promote angiogenesis and wound healing.
It has been known that wound dressings should preserve a moist and alkaline
environment to create a protecting barrier against secondary infections and mechanical stress
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and to promote granulation [122]. In an effort, a polymer-based sponge composed of CS-

sodium hyaluronate-resveratrol (CHR) was synthesized and its regenerative ability was
evaluated [122]. The porosity, density and cytotoxicity of the sponges were studied. The in
vivo test was carried out on the CHR polymer to decide its potential to stimulate tissue
regeneration on a reproducible and measurable skin wound created in an animal model. The
skin punch biopsies were harvested from the healed area and were evaluated by the
histopathological experiments. Results confirmed that the CHR polymer enhanced the
granulation formation and assisted wound healing along with a bacteriostatic effect.

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Several flexible and transparent CS-based membranes incorporated with antibacterial
drugs were achieved by a solvent evaporation casting method using a CS floccule suspension

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[123]. Glycerin was added as a plasticizer to the CS floccule suspension in order to increase

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the mechanical properties. The mechanism of membrane creation was attributed to the inter-
and intra-hydrogen bonds formed between CS and glycerol species. Results displayed that
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glycerol incorporation had an important effect on the characteristics of the CS membranes.
Increasing the glycerol amount significantly improved the swelling rate, tensile strength,
wettability and water vapor permeability of membranes. The in vitro enzymatic degradation
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test exhibited that the CS membrane provided long-term stability irrespective of the glycerol
content. Tetracycline hydrochloride and silver sulfadiazine (AgSD) as the water-soluble and
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water-insoluble drugs, respectively, were incorporated to the membranes in order to increase

antibacterial features. The controlled-release and inhibition zone data pointed out that the
glycerol reinforced CS membranes loaded by drugs would be promising materials to treat
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bacterial infections as wound dressings.

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A wound healing material was produced by means of two marine biomaterials including
CS and squid ink polysaccharide as carriers and calcium chloride as initiator for coagulation
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[124]. According to the central composite design using the response surface methodology, the
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appearance quality and water adsorption ability of composite sponges were used as evaluation
indices in order to achieve the optimized preparation conditions and also to estimate the
properties of the squid ink polysaccharide-CS (SIP-CS) sponge. The optimized SIP-CS
formulation was obtained as CS concentration=2.29%, squid ink polysaccharide
concentration=0.55% and calcium chloride concentration=2.82% in a volume ratio of 15:5:2.
The SIP-CS was favorable and stuck on the wound, had spongy nature, great tackiness and
absorptivity. Rabbit ear arterial, hepatic and femoral artery hemorrhage tests pointed out that
compared to CS dressing and absorbable gelatin, shorter hemostatic times and smaller
bleeding volume was measured. Besides, the SIP-CS absorbed high quantity of hemocytes
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which led to fast hemostasis. Healing zones and wound pathological pieces in scalded New
Zealand rabbits illustrated that the SIP-CS supported wound healing more quickly than CS
and superior than commercially accessible burn cream.
Amphiphilic CS salt, i.e. CS oleate (CS-OA), was used to physically stabilize
lemongrass antimicrobial nanoemulsions (NE) by a mild spontaneous emulsification method
[125]. Both oleic acid and CS are known as effective materials in wound healing, thus CS-OA
was applied to encapsulate alpha tocopherol (αTph) in NEs that will be used to heal skin
wounds. One developed NE formulation displayed ~220 nm dimensions, αTph concentration

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of up to 1 mg/ml and 36% drug loading. The two CS-OA and αTph NE supported
proliferation of keratinocytes and fibroblast cells, and the ex vivo skin biopsies proved that the

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CS-OA was suitable having antioxidant activity for topical use in wound healing. The αTph

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stability was more enhanced through encapsulation by NE spray drying as a powder (up to
~90% αTph residual after three months). The spray drying procedure was optimized to
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increase αTph recovery and powder yield. The powder was simply re-suspended to deliver the
NE that could entirely release αTph.
Moist wounds can heal more quickly than dry wounds and hydrogel wound dressings
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are appropriate materials to heal the moist wounds because they have hyperhydrous structures
[126]. Chitosan is a favored candidate to prepare hydrogel wound dressings due to its
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exceptional biological characteristics that can promote wound healing. Some physically-
crosslinked CS cryogels were developed only by freeze-thawing a CS-gluconic acid conjugate
(CG) aqueous solution for application in wound treatment [126]. The CG cryogels were
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disinfected by immersion in 70% ethanol before their employment onto wounds. The effect of
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autoclave sterilization (121 ºC, 20 min) on the features of CG cryogel was studied because
whole sterilization is one of the important necessities for medical devices. It was established
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that the optimum gluconic acid content in CG (the number of the loaded gluconic acid units
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per 100 glucosamine units of CS) was 11 for autoclaving. Increasing the CG cryogel
crosslinking level upon autoclaving improved the resistance of the gels against the enzymatic
degradation. Additionally, the autoclaved CG cryogels preserved promising biological
characteristics of the pre-autoclaved CG cryogels so that they revealed identical hemostatic
efficiency in repairing full-thickness skin wounds. Hence, the autoclavable CG cryogels were
highly promising as practical wound dressings.
Halloysite is a natural nanotubular clay mineral known as halloysite nanotubes, HNTs,
which is chemically similar to kaolinite and as a result of its appropriate biocompatibility, it is
an interesting nanomaterial for numerous biological applications [136]. It was aimed to
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develop a nanocomposite based on CS oligosaccharides and HNTs to increase healing in the

chronic wounds treatment [127]. A 1:0.05 %wt ratio HTNs/CS oligosaccharide
nanocomposite was prepared by mixing the HTNs powder in 1% w/w aqueous CS
oligosaccharide solution and spontaneous ionic interactions producing a composition
containing 98.6 %w/w HTNs and 1.4 %w/w CS oligosaccharide. Both HTNs and HTNs/CS
oligosaccharide nanocomposite displayed satisfactory in vitro biocompatibility to normal
human dermal fibroblasts up to 300 μg/ml concentration and improved in vitro fibroblast
motility by stimulating both proliferation and migration. The HTNs/CS oligosaccharide

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nanocomposite and its two individual components were examined for their healing capability
in a murine (rat) model. The HTNs/CS oligosaccharide led to superior skin reepithelization

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and reorganization compared to separate HNTs or CS oligosaccharide. Consequently, the

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nanocomposite could be used as a medical agent for wound healing application.
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2.9. Application of chitosan in bioimaging
Molecular imaging is a promising non-invasive method to evaluate biochemical and
biological processes occurred in living organisms. Using the X-ray technique in medical
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imaging, several noninvasive methods have been developed and effectively employed in
various applications such as clinical diagnosis, drug discovery and cellular biology [12].
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Hence, these technologies can increase understanding the drug activity and disease in the
course of preclinical and clinical drug design/development. Molecular imaging procedures
have advantage over more conventional readouts like immunohistochemistry which could be
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described as they can be carried out in the intact organism with enough temporal and spatial
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resolution to study biological processes in vivo. Besides, molecular imaging leads to a non-
invasive and repetitive assay of identical living matter by means of the same or different
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biological imaging tests at diverse time points. Consequently, it exploits the statistical
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influence of longitudinal assays and decreases the cost and number of necessary animals
[128].
Typically, molecular imaging uses particular molecular probes and inherent tissue
properties as the basis of image contrast. It has the capability to recognize a combination of
data on cell biology, initial detection and characterization of diseases and treatment
evaluation. Also, it can help to choose effective drugs that probably appear to be fruitful but
to stop using drugs that seem to be failed. Chitosan is a non-antigenic and hydrophilic
biopolymer which is non-toxic to mammalian cells; besides, CS composites are biomaterials
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possessing appropriate physicochemical, mechanical and functional properties that can be

used in biomedical imaging applications [129].
Recently, a CS-based biosensing material modified with carbon dots (CDs) was
prepared for vitamin D2 detection [130]. The CDs were synthesized by microwave pyrolysis
method and characterized by TEM images, Raman, FTIR and UV-VIS spectra. Then, the CDs
were added to the 1% acetic acid CS solution in order to afford the carbon dots-CS (CD-CS)
composite and a thin film of the CD-CS composite was deposited on indium-doped tin oxide
(ITO) glass substrate (CD-CS/ITO) through drop casting process. The composite film surface

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was studied by AFM, static contact angle and cyclic voltammetry measurements. The CD-
CS/ITO surface was additionally modified by immobilizing vitamin D2 antibody (Ab-VD2 )

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and bovine serum albumin to achieve BSA/Ab-VD2 /CD-CS/ITO bioelectrode. The

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electrochemical response of the bioelectrode to vitamin D2 antigen (Ag-VD2 ) was performed
via differential pulse voltammetry. The biosensing electrode displayed linearity using 10–50
ng.mL−1 of Ag-VD2 . The sensitivity was 0.2 μA.ng−1 mL cm−2 , detection limit was 1.35 ng
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mL−1 and the shelf-life of biosensor was ~25 days.
It is known that the naturally plentiful CS biopolymer demonstrates pH-sensitive feature
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within a narrow pH range. Binding hydrophobic moieties to CS chains produces modified CS

polymer with further adjustable pH sensitivity [131]. In this context, near-infrared (NIR)
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photoluminescent Ag2 S QDs capped by long-chain carboxylic acid were synthesized and
conjugated with CS through esterification reaction [131]. The doxorubicin (DOX) anticancer
drug having affinity for the hydrophobic oleoyl groups was encapsulated into the QDs to
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achieve Ag2 S(DOX)@CS nanospheres. The in vitro and in vivo tests exhibited that the
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nanospheres had great antitumor efficiency and released DOX at lower pH in tumor cells. As
well, the strong NIR signal resulting from the entrapped Ag2 S QDs led to real-time
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monitoring the distribution of nanospheres in body.

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To examine the antitumor activity of Ag2 S(DOX)@CS nanospheres, free DOX,

Ag2 S@CS nanospheres, Ag2 S(DOX)@CS nanospheres and PBS control were intravenously
injected every other day to BALB/c mice having HeLa tumor via tail vein. The body weight
and tumor volume of the treated mice were measured in 12 days. Fig. 43a exhibits that the
average tumor volume of the Ag2 S@CS treated mice was similar that of the mice treated by
PBS at day 12 post-injection. In mice treated using the Ag2 S(DOX)@CS, the tumor growth
was highly inhibited during the same period reflected by 48.5% decrease in tumor volume
relative to the control. The Ag2 S(DOX)@CS nanospheres were delivered to the tumor place
by the influence of enhanced permeability and retention (EPR) and then they were
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internalized the tumor cells. The DOX release was triggered as the nanospheres were entered
the cellular compartments including early/late endosomes and lysosomes. The nanospheres
sensitivity to the pH variations expedited fast leakage of the nanospheres from the
endolysosomal system and decreased the multiple drug resistance (MDR) effect. Fig. 43b
indicates that the mice treated differently exhibited only minor body weight changes after 12
days which proved the Ag2 S(DOX)@CS nanospheres could be well-tolerated without having
severe side effects.
Distribution of the Ag element in the tumor bearing mice was studied at diverse time

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intervals after injection of Ag2 S(DOX)@CS nanospheres, Fig. 43c. The Ag amount in the
tumor was very greater than those in five major organs signifying the nanospheres intended to

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be accumulated in the tumor and this was related to the EPR influence. The Ag amounts in

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liver and spleen were greater compared to those of stomach, heart and kidney reflecting rather
higher affinity of the nanospheres for spleen and liver. Furthermore, the Ag amounts in the
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tumor and the organs started to drop after 6 h upon metabolism. Compared to those in the
organs, decreasing the Ag level in the tumor was less noticeable demonstrating extended
retention of the nanospheres in the tumor.
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The non-invasive in vivo NIR imaging of nude mice was performed in order to examine
the distribution of Ag2 S(DOX)@CS nanospheres employed for therapeutic purposes in a
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living body. The nanospheres were dispersed in PBS and administered to anesthetized nude
mice through the tail vein injection. At various time intervals post-injection, the mice were
imaged upon 808 nm excitation. The NIR fluorescence related to the nanospheres in the mice
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was simply monitored, Fig. 43c. The nanospheres were distributed in the mice bodies by
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blood circulation after 6 h post-injection, Fig. 43c (i). The fluorescence from the tumor was
stronger compaed to those of other areas in the mice bodies including spleen, liver and heart.
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After 12 h post-injection, the fluorescence intensity was yet higher signifying the nanospheres
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were accumulated in the tumor through the EPR effect (see Fig. 43c(ii)). The fluorescence
signals of the mice bodies were yet observed after 24 h along with decreased intensities (Fig.
43c (iii)) which were correlated to the nanospheres clearance through metabolism.
Nevertheless, the fluorescence intensity was still high at the tumor site illuminating the
nanospheres could remain in the tumor site for an extended time. Additionally, a tumor was
harvested from a mouse at 24 h post-injection and imaged, Fig. 43c (iv). Five key organs of
this mouse were harvested (see Fig. 43c (v)). The in vivo and ex vivo images established that
the Ag2 S(DOX)@CS nanospheres were accumulated in the tumor after injection to a mouse
body and released the anticancer drug in intracellular environment to inhibit the tumor [131].
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Highly fluorescent graphene quantum dots (GQDs)-CS (GQDs-CS) hybrid xerogels

were simply synthesized and their morphology was controllable through changing the GQDs
contents within the xerogels [132]. The GQDs-CS demonstrated a three-dimensional porous
network as the GQDs content was 43wt% in the xerogel that was valuable for drug loading
and sustained release. It was found that the GQDs-CS could be used for in vivo imaging due
to it exhibited strong blue, green and red luminescence by excitation of different wavelengths.
Besides, the pH-induced protonation and deprotonation of the –NH2 groups on CS chains
could lead to a pH-dependent drug delivery by the GQDs-CS hybrid xerogel.

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The imaging flow cytometry as an effective statistical method was used to study the
degradation influence on the biological properties of trimethyl CS (TMC)-based nanoparticles

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(NPs) [133]. High transfection efficiency requires an exact balance between NPs stability and

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degradation. The biodegradation rate of the TMC NPs was changed through variation of the
degree of acetylation (from 4 to 21%) of the polymer producing NPs with diverse enzymatic
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degradation rates. Although degree of acetylation did not influence the NP size, charge and its
capability to protect plasmid DNA, TMC NPs with an intermediate acetylation degree of 16%
presented the highest transfection efficacy. For all of formulations, key steps of the NP-
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mediated gene delivery process were monitored including NP-cell membrane association,
internalization and intracellular transferring such as plasmid DNA transport to the nucleus.
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The NP cytotoxicity was determined via quantification of cell apoptosis. Hence, it was proved
that the biodegradation rate of the NPs affected their intracellular trafficking and accordingly
their efficacy to transfect cells confirming changing the polymer acetylation degree can
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modulate the NPs to achieve diverse degradation rates and bioactivities. In addition, this
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approach ascertained to be an appreciated tool in the early phases of nucleic acid vector
design.
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3. Pharmaceutical application of chitosan in food industry

3.1. Application of chitosan in food protein binding
Food enrichment with proteins and peptides as bioactive compounds has gained great
attention but there are some problems in introducing such functions into food, one of which is
their instability in the human gastrointestinal tract (GI). Encapsulation technology can be used
a valuable method to overcome this challenge [134]. It was recommended that, for systemic
absorption into the human GI tract, proteins/peptides must be carried to a lower part of the
intestine (ideally the colon) where lower protease activity would expedite preservation of the
proteins/peptides and intact bioactivity prior to the systemic absorption. Nevertheless, a large
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peptide difficulty moves through the mucosal barrier to be absorbed intact by the colon.
Consequently, it is needed to use membrane disruptors and/or absorption enhancers. In a
recent study, the capabilities of encapsulation systems were examined to recognize gastric
protection and sustained release in the intestine [134]. For this purpose, bovine serum albumin
(BSA), whey protein isolate (WPI), insulin and a casein hydrolysate were encapsulated in CS-
polyphosphoric acid (PPA) beads. Then, the in vitro protein release from the beads was
assessed in simulated intestinal fluid (SIF, pH=7) and simulated gastric fluid (SGF, pH=3).
Great entrapment efficiencies were attained for intact proteins (>95% in all cases) whereas

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lower entrapment was measured for the casein hydrolysate (~50%) which were perhaps due to
physical/steric entrapment of the proteins in such CS-PPA beads. Inhibited BSA release was

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obtained with low PPA concentration in both SIF and SGF. The WPI and insulin were slowly

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released in SIF and efficiently preserved in SGF. Peptides from casein hydrolysate were
incompletely (~35%) while rapidly released in SGF with no additional release in SIF. Thus, it
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was indicated that CS-PPA beads were promising systems for lower gastrointestinal delivery
of bioactive proteins.
Ferritins are proteins which can store thousands of iron ions in their internal cavities
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[135]. Every ferritin contains same or dissimilar twenty-four subunits that are assembled as a
shell-like structure having an inner size of 8 nm and an external diameter of 12 nm. Due to the
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nano-sized inner cavity as well as the reversible assembly property of the ferritin, small
bioactive molecules can be encapsulated in the ferritin cage. Subsequent to the encapsulation,
food nutrients can be functionalized through the ferritin shell in order to investigate their
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solubilization, stabilization and targeted delivery characteristics. The inner and outer surfaces
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of ferritin cage offer twenty interfaces for the encapsulation and transport of food nutrients.
However, traditional methods for the production of ferritin-nutrients shell-core nanoparticles
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commonly use acid/alkaline pH transition which possibly leads to the loss of activity for the
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food nutrients or the creation of insoluble aggregates. Thus, in order to overcome these
drawbacks, a facile one-step process was applied to achieve red bean seed ferritin (RBF)-
EGC-CS (REC) nanoparticle by means of thermal treatment at 55 °C [135]. It was pointed out
that the apoRBF was relatively uncoiled with 5.3% decrease in α-helix content which was due
to 55 °C treatment. The EGC molecules spontaneously permeated to the ferritin inner cavity
with 11.8% (w/w) encapsulation ratio. Also, the thermal treatment assisted the attachment of
CS to the outer surface of the ferritin via electrostatic interactions (binding constant=4.7×10 5
M-1 ). The transmission electron microscope and dynamic light scattering data proved that the
REC was 12 nm in diameter and 7.3 nm in hydrodynamic radius that was mono-dispersedly
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distributed. As well, the CS decorated onto the apoRBF enhanced the EGC stability through
weakening the apoRBF degradation by digestive enzymes in simulated gastrointestinal tract.
Hence, the REC shell-core nanoparticle can encapsulate and deliver functional molecules
under benign conditions without great pH variations.
The complex coacervate formation was examined between CS and canola protein
isolate (CPI) extract achieved from canola meal [16]. The yield of CPI-CS complex
coacervates were affected by various factors including pH, CPI-to-CS ratio and strength of the
electrostatic interaction. Also, the thermal characteristics of the transglutaminase cross-linked

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and un-cross-linked complex coacervates were investigated. The optimum complex
coacervation between CS and CPI was happened in the pH range of 5.8-6.2 using the CPI-to-

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CS mass ratio of 16. The highest denaturation temperature and the denaturation enthalpy of

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CPI in CPI-CS complex were greater than those of the free CPI demonstrating the CPI
thermally stability was increased upon complexation. The thermal stability of the coacervates
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was more improved once they were cross-linked using the transglutaminase. Higher CPI
thermal stability in CPI-CS coacervates specified that CPI-CS coacervates would be
appropriate candidates to encapsulate thermally sensitive pharmaceutics and food ingredients.
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Thymol nanoemulsions were achieved by spontaneous emulsification, ultrasound and a

combination of both procedures [136]. The most appropriate nanoemulsion in terms of
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polydispersion and size was spontaneous emulsification where thymol was effectively
encapsulated that could inhibit Botrytis cinerea using 110 ppm of thymol. Also, 10% dilution
of this nanoemulsion in water was employed to obtain quinoa-CS film having a
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heterogeneous and porous microstructure. The tensile strength of the film was considerably
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lower whereas its mean elongation at break was comparable to that of the control film. As
well, the water vapor permeability of the film was analogous to that of the control film. The
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influence of nanoemulsion-thymol-quinoa protein/CS coating was assessed on mould growth

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in inoculated cherry tomatoes. The tomatoes having this coating and inoculated with Botrytis
cinerea displayed a great decrease in fungal growth after 7 days at 5 °C compared to the
control samples (tomatoes without coating and those coated with quinoa protein/CS).

3.2. Application of chitosan in preparation of antimicrobial food additives

Food borne diseases are one of important reasons for illness and death worldwide
although great progress has been achieved in understanding the infections caused by
pathogenic microorganisms, performing more exact control measures and strict controls to
produce commercial foods [137]. Moreover, food decomposition as a result of microbial
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action will lead to economic losses. Thus, food preservation is a challenge in food industry
which results in using usual food preservatives like benzoates, nitrites and sodium
metabisulfite having an extensive history of safe application [137]. Nevertheless, occasional
allergic reactions observed in sensitive persons and the possible creation of toxic by-products
using several preservatives that may be carcinogenic (such as nitrosamines from nitrites)
result in worries among users due to their probable negative health effects. Therefore, there is
a growing consumer demand to use foods having no or lower concentration chemical
preservatives or consuming preservatives with restricted risks.

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Antimicrobial films and coatings are promising materials to control foodborne
pathogens infecting food products [138]. Natural antimicrobial agents are commonly used as

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food preservatives in order to increase microbiological quality and safety and to prolong the

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food shelf life. Numerous natural compounds are applied the food industry as antimicrobials
against pathogenic microorganisms and spoilage [138]. The antimicrobial compounds can be
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categorized to six groups considering the succeeding criteria including (1) biosynthesis; such
compounds are synthesized by ribosomes or they are primary/secondary metabolites; (2)
biological source; created by bacteria, animals (vertebrates and invertebrates) and plants; (3)
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biological functions like antifungal, antibacterial, antiparasitic and insecticide compounds; (4)
molecular features; based on size, charge and hydrophobicity; (5) structure and composition;
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the chemical materials and biomolecules with diverse topologies and (6) molecular
objectives; either intracellular or extracellular.
Diverse antimicrobial compounds formed by plants and bacteria are employed in food
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products as biopreservatives since they can prolong the food shelf life. For instance, bacteria
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can produce antimicrobial materials to inhibit other bacteria, thus they are beneficial to
control bacterial death and against pathogenic microorganisms in food. Such antimicrobial
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agents are primarily formed through Gram-positive bacteria such as lactic acid bacteria [139].
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Furthermore, natural antimicrobials are of great attention due to they are known to be healthy
and safe by customers. Some natural antimicrobials showing suitable antimicrobial activities
are essential oils including cinnamon oil, eugenol and thyme oil [140]. Antimicrobial
films/coatings loaded by essential oils have frequently been prepared. For instance, coatings
fabricated using with 1 g/100 g CS and 3 g/100 g lemon oil considerably decreased the fungal
decay of strawberries stored at 5 ºC for 3 days, compared to that of uncoated strawberries
[141].
Nisin is the most generally employed bacteriocin in commercial processes. As well,
pediocin has been applied in food products. However, several factors unfortunately decrease
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its antimicrobial efficacy in foods [142]. Different factors affect the antimicrobial activity of
bacteriocins including the appearance of bacteriocin-resistant bacteria, conditions
destabilizing the biological effect of proteins like the existence of proteases and oxidation
routes, inactivation through other additives, binding to food constituents (for example, protein
surfaces and fat particles), low solubility, non-homogenous distribution in the food (if the
antimicrobial compound dispersed uneven in the food matrix, it will leave different bare areas
that are prone to the microorganism growth) and pH effect on bacteriocin efficacy and
stability.

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It is known that combination of lauric arginate (LAE) and cinnamon oil (CO) leads to
synergistic antimicrobial influence on Gram-positive bacteria whereas antagonistic impact on

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Gram-negative bacteria [143]. Also, it was found that ethylenediaminetetraacetate (EDTA)

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could increase the LAE activity and overcome the antagonistic influence of combined LAE-
CO. hence, recently, physical and antimicrobial characteristics of CS films containing LAE,
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CO and EDTA were investigated [143]. The thickness of CS films was significantly increased
by incorporation of antimicrobial materials. The yellow color of films was enhanced but their
water solubility was decreased when the CO concentration was increased. The water vapor
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permeability of films was greater after incorporating the antimicrobials. Further, the presence
of antimicrobials in CS films dropped the tensile strength while did not affected the
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elongation amount. The inhibition zones of films loaded by antimicrobials against foodborne
pathogens were much greater relative to those of films only containing CS. the EDTA
addition improved the antimicrobial activities of films including LAE but binding to CO
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decreased the LAE diffusion from films to inhibit the bacterial growth. Thus, such
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antimicrobial films containing LAE, CO and EDTA displayed capability to enhance the food
safety.
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In the recent years, using films and coatings to preserve foods is of great interest due to
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the development of biopolymeric materials including essential oils as antimicrobial agents in

foods in order to substitute synthetic additives [144]. In this context, the influences of
composition parameters on antimicrobial potency and characteristics of films fabricated using
modified CS comprising diverse types of carvacrol nanoemulsions were investigated [144].
Also, the concentrations of biopolymer and carvacrol nanoemulsion in the film forming
dispersions were changed to increase the antimicrobial effect against two model
microorganisms (Listeria innocua and Escherichia coli). The surface hydrophobicity was
varied to obtain the optimum conditions for the prepared systems. It was indicated that
emulsion formulations had a substantial influence on the inherent antimicrobial activity and
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their interactions with the modified CS matrix could affect properties of films and their
bactericidal activities. The two most active emulsions achieved by a combination of glycerol
monooleate and polysorbate 20, and whey protein isolates, respectively, added to the
modified CS films noticeably enlarged the inhibition zones against L. innocua and E. coli
from 7.2-7.4 mm (modified CS alone) to 13.4-16.1 mm, along with preserving the surface
hydrophobicity of the films. Compared to the film containing pure carvacrol, the
incorporation of essential oil nanoemulsions into the films created more homogeneous films
having better appearance.

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In another work, the CS based films were developed by incorporation of diverse
concentrations of flour plus microparticles of olive pomace flour [10]. The mechanical,

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barrier, antioxidant and optical properties of the films were evaluated. Also, the protective

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influence of films was assessed against nut oxidation. Addition of the olive residue flour into
the CS matrix changed the morphology and produced more rough and heterogeneous films.
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The incorporation of 10% of olive microparticles highly increased the tensile strength
(22.40±0.22 MPa) of films without changing their original features. The flour and the olive
microparticles enhanced the antioxidant activity of the films which was related to the flour
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concentration or microparticles loaded into the films. The films containing 30% flour or
microparticles were efficient as protective packaging against the nuts oxidation during 31
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days. Therefore, the fabricated packages were sustainable considering the materials used for
their preparation, their biodegradability and the antioxidants naturally attained from waste.
Some gelatin-CS films were prepared containing nanoemulsions loaded by several
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active compounds including N1: canola oil, N2 : α-tocopherol/cinnamaldehyde, N 3 : α-

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tocopherol/garlic oil and N4 : α-tocopherol/cinnamaldehyde and garlic oil [145]. The films
were fabricated through the casting technique using 5 g N 1,2,3,4 /100 g biopolymers and
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glycerol as a plasticizer. Then, the physicochemical, antioxidant and antimicrobial properties

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of the films were assessed. The moisture content (18% w/w) and thermal characteristics of all
films were comparable. The water solubility and light transmission at 280 nm of the
nanoemulsion containing films were substantially decreased compared to those of the control.
The film incorporated with N 1 was the roughest and exhibited the greatest opacity, elongation
at break and stiffness decrease, while the films loaded with N 3 and N 4 illustrated the lowest
tensile strength and swelling capacity, respectively. Also, films containing nanoencapsulated
active agents were highly active against Pseudomonas aeruginosa and displayed great
antioxidant activity. Consequently, it was revealed that such nanoemulsion loaded films had
the ability to be used as packaging materials to enhance the food shelf life.
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Because nanoencapsulation may enhance the antimicrobial capacity of nisin when used
in foods such as lean beef, nisin-loaded nanoparticles were achieved through ionic gelation of
alginate and complexation with CS [146]. Also, response surface methodology was applied to
find optimized formulation. For the nanoparticles, the z-average was obtained equal to
66.4±8.9 nm, the encapsulation efficiency was 36.1±0.6% and the zeta potential was
measured to be -31.7±2.6 mV. The in vitro test on growth inhibition of Listeria
monocytogenes at 4 °C exhibited sustained antimicrobial ability of nisin-loaded nanoparticles
for 21 days. Besides, 400 and 800 IU/g encapsulated nisin could inhibit L. monocytogenes

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growth in refrigerated vacuum-sealed beef samples for 10 and 24 days, respectively, but free
nisin inhibited the microbial growth merely for 4 and 17 days, respectively. Overall,

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experimental design was employed to afford nisin-loaded nanoparticles that could be applied

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in food industry as an antimicrobial agent in order to prolong the shelf life of lean beef.
As edible films and coatings not only act as barriers of water vapor, gases and volatile
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compounds but also they can carry functional ingredients, composite edible films were
prepared using CS and zein as and supplemented with dicarboxylic acids (succinic acid and
adipic acid) and phenolic compounds (gallic acid and ferulic acid) [147]. The composite films
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illustrated superior mechanical and water vapor barrier characteristics. The recovery
percentage of dicarboxylic acids and phenolic compounds from composite films were 48-65%
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and 71-84%, respectively. As well, the composite films revealed antioxidant activities. The
antimicrobial potencies of composite films were proved using Escherichia coli and
Staphylococcus aureus microorganisms.
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It is known that practical application of CS-essential oil blend films has been limited
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because the extraction of essential oils from plants is not economic [148]. Thus, it was
attempted to prepare CS films blended with low cost and commercially accessible fats and
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oils that are consumed in daily human foods such as olive, sunflower and corn oils, butter and
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animal fats [148]. It was shown that the biological, physicochemical and mechanical features
of CS blend films were affected by adding the oils and fats. The CS-olive oil film exhibited
superior surface morphology and greater thermal stability compared to the films with other
unsaturated oils. The tensile strength, Young’s modulus and elongation at break were
enhanced by 57.2, 25.1 and 31.7% for CS-olive oil film, respectively. The highest
antibacterial activity was observed for the CS-olive oil blend film that was nearly equal to that
of commercial antibiotic gentamicin. Hence, the edible films prepared by incorporation of
natural fats and oils in CS could be used as low cost and environmentally friendly packaging
materials.
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Nisin-loaded CS-monomethyl fumaric acid (CM-N) nanoparticles were ahieved as a

direct food additive [149]. For this purpose, CS was modified by monomethyl fumaric acid
(MFA) using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. The CS-loaded nisin (CS-N)
and CM-N nanoparticles were formed by ionic interactions occurred between the positively
charged amino groups of CS and CS-MFA and negatively charged tripolyphosphate ions. The
CS-MFA exhibited 8.38±0.02% substitution of the amino groups. The CS-N and CM-N
nanoparticles yields were 81.64 and 76.83% and nisin encapsulation efficiencies were
71.48±0.48 and 60.32±0.63%, respectively. The average particle sizes of CS-N and CM-N

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nanoparticles were 134.3 and 207.9 nm and the zeta potential values of CS-N and CM-N
nanoparticles were +39.4 and +31.5 mV, respectively. The antibacterial activity of the CM-N

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against foodborne pathogens in orange juice after 48 h incubation was significantly greater

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than those of other samples. Accordingly, the CM-N nanoparticles were suitable materials for
application in food industry as antimicrobial agents.
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In another study, CS-based edible films were fabricated through supplementation of
Berberis crataegina DC.'s seed oil and fruit extract to the CS matrix [150]. The films were
characterized by both physiochemical (DSC, SEM, UV–vis, FT-IR, contact angle and
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mechanical test) and biological (anti-quorum sensing, antioxidant and antimicrobial) methods.
The CS-fruit extract film indicated greater thermal stability, antimicrobial, antioxidant and
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anti-quorum sensing potencies than other films. Also, incorporation of B. crataegina's seed oil
and fruit extract to the CS film outstandingly diminished the UV–vis transmittance whereas
improved the tensile strengths. The hydrophobicity of the CS-seed oil film was higher
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(92.64±4.17) than the CS control film (84.67±1.50) but CS-fruit extract film displayed
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somewhat lower hydrophobicity (73.82±7.42) compared to the CS film. The total

extraordinary thermal stability, antimicrobial and antioxidant capability of the CS-fruit extract
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film showed that B. crataegina's fruit extract could be employed as an efficient ingredient to
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produce edible films with improved physicochemical and biological features.

The antibacterial characteristics of emulsion-encapsulated and unencapsulated
isoeugenol were investigated against biofilms of S. aureus, Lis. monocytogenes, Leu.
mesenteroides and P. fluorescens in tryptic soy broth and carrot juice [151]. The emulsion
encapsulation improved the antimicrobial capacity of isoeugenol against biofilms in media
but did not act in carrot juice. Some isoeugenol emulsions coated by CS disrupted the
structures of biofilms. Moreover, addition of the surfactant Tween 80 (which is frequently
used for the dispersion of oils in foods) decreased the antibacterial activity of isoeugenol.
Thus, it is found that common food additives such as surfactants may show an opposite
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influence on the antibacterial action of preservatives. Isoeugenol proved to be a promising

agent as a food preservative due to it acted nearly well against planktonic bacteria and
biofilms.
Antimicrobial edible films and coatings were fabricated using micro-emulsions to
diminish populations of foodborne pathogens in foods [152]. Corn-bio-fiber gum (C-BFG)
was applied as an emulsifier along with CS and allyl isothiocyanate (AIT) and lauric arginate
ester (LAE) were acted as antimicrobial agents. Micro-emulsions were prepared using a
solution composed of 1% CS, 0.5% C-BFG and 1–4% AIT or LAE. The coatings and films

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created by means of the micro-emulsions exhibited micro-pore sizes ranged from 100 to 300
nm and micro-channels that efficiently trapped the antimicrobials and facilitated their release

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from the center to the surface of the films/coatings to enhance their antimicrobial

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effectiveness. The coatings and films containing 1% AIT dropped population of Listeria
innocua by more than 5, 2 and 3 log CFU in culture medium, ready-to-eat meat and
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strawberry, respectively. The coatings and films loaded by 1% LAE decreased the populations
of Escherichia coli O157:H7 and Salmonella spp. to above 5 and 2 log CFU in Tryptic soy
broth and strawberry, respectively. Hence, effective antimicrobial materials were developed to
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lessen food borne pathogens on ready-to-eat meat, strawberries or other foods.

The influences of guarana, cinnamon, rosemary and boldo-do-chile ethanolic extracts
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and diverse gelatin:CS (GEL:CS) ratios on the microstructural, optical, barrier, mechanical,
antioxidant and antimicrobial features of the films were investigated [153]. The two gelatin
and CS polymers were homogeneously blended in the film matrixes. Increasing the CS
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proportion enhanced the elasticity of the films and diminished the water vapor permeability
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which was not considerably decreased by adding the extracts. The blend films exhibited
suitable antioxidant activities and exceptional growth inhibitions both against Staphylococcus
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aureus and Escherichia coli bacteria confirming such films could be alternative active
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packaging materials in the food industry.

The antimicrobial activities of pure polymeric films (CS100 and GEL100) and
GEL50:CS50 blended films containing diverse extracts against E. coli and S. aureus are
presented in Fig. 44. It is seen that the chloramphenicol as antibiotic control has the utmost
inhibition activity among other films. The inhibition zone diameter for pure CS films (CS100)
revealed growth inhibitions against both E. coli and S. aureus bacteria but the pure GEL films
did not exhibit any antimicrobial activities [153].
Antimicrobial biodegradable films were fabricated using thermoplastic corn starch and
CS oligomers to develop a package prototype for perishable food products [154]. The
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diffusion assays established that the CS oligomers could migrate from the active film to the
aqueous simulant media. Furthermore, oligomers could diffuse from the matrix irrespective of
the aqueous medium acidity. The experimental of diffusion assay data were fitted to a
mathematical model by estimation of diffusion coefficients at three pH values equal to 3, 5,
and 7. The active film was applied to make sachets to package perishable foods like ricotta,
strawberries, and flavored breads, which were stored under controlled conditions for 7 days.
Antimicrobial capability of active sachets was verified by yeast and molds counting in the
stored foods. It was illustrated that incorporation of CS oligomers into the packaging

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materials could more effectively inhibit microbial growth compared to the spraying
procedure.

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3.3. Application of chitosan in preparation of antibacterial food packaging materials
The ever increasing population has led to growth the food demand, thus it is required to
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extend active packaging technology in order to enhance the safety, quality shelf-life and of
foods [155]. The active packaging materials not only act as inert barriers to external
conditions but also they can decrease food waste through scavenging/absorbing (moisture,
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oxygen, ethylene, carbon dioxide, odors, UV light and flavors), emitting/releasing

(antioxidants, ethanol, carbon dioxide, sulphur dioxide, preservatives, pesticides, flavors),
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removing (removal of food component such as cholesterol and lactose) and antimicrobial
characteristics as well as controlling the food quality and temperature [156] in order to
efficiently preserve food.
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Nowadays, development of active food packaging using bio-based functional packaging

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materials incorporated with natural ingredients and active compounds has received great
interest [157]. It is notable that the growing obligation for sustainability has resulted in the
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progress in preparation of biodegradable packages using biopolymers containing bioactive

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compounds acquired from waste materials to give additional value to such products. The
biopolymers have numerous benefits compared to polyethylenes such as nontoxicity, fast
biodegradability, biocompatibility with other biopolymers, simple interaction with food,
capability to serve as vehicles for antioxidants and antimicrobials. Among several natural
polymers employed as biodegradable packages, CS is a functional prominent biopolymer due
to its exceptional film forming capacity, great mechanical strength, appropriate barrier
property along with intrinsic antioxidant and antimicrobial features [158]. In fact, the bio-
based packaging materials showing antimicrobial and antioxidant features have become
common because microbial and oxidation pollutions are foremost difficulties decreasing food
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safety and quality. Moreover, the plant polyphenols can be used as substitutes to synthetic
antioxidant and antimicrobial agents.
In order to fabricate environmentally benign packaging materials, portable CS-ZnO
nanocomposite pouches were achieved by a simple process through changing the ZnO amount
[159]. The films were much superior compared to bare CS and some conventional films. Two
bacteria generally contaminated the packed meat were nominated to illuminate the
antimicrobial effects of the CS-ZnO films. It was found that the antimicrobial efficacy was
linearly associated to the quantity of ZnO nanoparticles added to the composite. The optimum

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film displayed outstanding antimicrobial potency; thus it was fabricated as a packaging pouch
for raw meat. The pouch presented substantial activity against the microbes in raw meat due

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to total microbial growth inhibition on the sixth day of storage at 4ºC. Hence, the pouch could

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be used as a top-notch material compared to polyethylene bag applied to extend the raw meat
shelf life.
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The capability of the optimized C-2 film was evaluated as a packaging material to
preserve raw meat. Before the test, the C-2 films were preserved in air tight polyethylene bags
during 6 months. The C-2 films were prepared as flexible pocket resembling bags by means
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of cotton yarn using a home-made weaving device (Fig. 45). The shelf life efficacy of the
composite bag was compared to that of synthetic plastic bag (low density polythene)
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popularly utilized to pack, store and transport meat and other foods in the marketplace. The
composite and plastic bags were sterilized through autoclaving at 121 C for 15 min. Then,
equal quantity of meat was placed into the bags so that each set of bags contained three
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duplicates of C-2 bag (i.e. A1 SET) and polythene bag (B1 SET) and incubated for 4 days at 4
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C. After four days incubation, aerobic plate count was done. This procedure was repeated for
A2 and B2 sets incubated for 5 days and followed for A3 and B3 sets incubated for 6 days
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(see Fig. 46) [159].

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Recently, CS films incorporated with 0, 2.5, 5, 10 and 20% w/w propolis extract (high
in polyphenols) were fabricated [160]. The elongation at break, tensile strength, total phenolic
content and antioxidant capacity of films were increased but their water vapor permeability
and oxygen permeability were decreased by increasing the propolis amount. Also, increasing
propolis extract concentration endowed with a deeper orange color to the films compared to
light yellow color of the control film. The capability of the films to inhibit Salmonella
Enteritidis, Staphylococcus aureus, and Pseudomonas aeruginosa Escherichia coli was
assessed using agar diffusion method. The CS films containing propolis extract inhibited all
of bacteria beneath the films. The FTIR spectra of the films were changed upon adding
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propolis extract indicating some interactions were happened between CS and propolis
polyphenols. The appropriate oxygen and moisture barrier, mechanical properties as well as
the antimicrobial and antioxidant activities of the films proved adding propolis extract to the
CS films was beneficial and the films could be served as active food packaging materials.
In another study, flexible, homogeneous and transparent films were prepared using CS
and ellagic acid [161]. For this purpose, different ratios of ellagic acid (0.5, 1.0, 2.5 and 5.0%
w/w) were used relative to CS to estimate their UV-blocking features. Additionally, the
photochemical stability of the films was investigated through artificial solar light radiation.

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These films displayed UVA- and UVB-barrier ability, high mechanical characteristics
(Young’s modulus=3.21-3.57 GPa), moderate water vapor permeability (WVP=2.82-3.70 g

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mm m-2 day-1 kPa-1 ), thermal stability up to 215-220 ºC and the highest antioxidant capacity

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of about 28% by scavenging 2,2-diphenyl-1-picrylhydrazyl hydrate (DPPH). All of CS-ellagic
acid films exhibited antimicrobial activity against food-borne pathogens including Gram-
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negative Pseudomonas aeruginosa and Gram-positive Staphylococcus aureus bacteria.
Some blended films were achieved using poly(vinyl alcohol) (PVA) containing CS by
solution casting and electrospraying techniques [162]. The influence of CS amount was
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evaluated on the mechanical, thermal, water vapor permeation, oxygen permeability and
antibacterial (against Gram-positive Staphylococcus aureus and Gram-negative Escherichia
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coli strains). Relative to the pure PVA film, the PVA-CS films displayed superior elongation
at break, less oxygen permeability, improved water barrier properties and higher antibacterial
activity, particularly when the PVA:CS weight ratio was 75:25.
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An active film was developed using CS and kombucha tea (KT) by the solvent casting
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method and the influence of KT encapsulation (1–3% w/w) was studied on the functional and
physical features of CS film [163]. The antimicrobial ability of the CS-KT film was assessed
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against Escherichia coli and Staphylococcus aureus bacteria by agar diffusion technique and
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its antioxidant activity was evaluated by the DPPH scavenging test. It was found that the KT
addition to the CS films enhanced the water vapor permeability from 256.7 to 132.1 g
cm−2 h−1 KPa−1 mm and improved the antioxidant activity up to 59%. Additionally, the KT
incorporation to the CS film augmented the protective influence of the film against ultraviolet
irradiation. The FTIR spectra exhibited that chemical interactions were occurred between CS
chains and the polyphenols of KT. The CS-KT film successfully acted as an active packaging
and prolonged the shelf life of the minced beef which was established by the inhibition of
lipid oxidation and microbial growth (from 5.36 to 2.11 log CFU.g−1 ) during four days of
storage. The CS-KT film not only preserved the quality of the minced beef but also
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considerably retarded microbial growth and extended the shelf life of the minced beef meat up
to three days.
Several CS films containing thinned young apple polyphenols (YAP) were prepared and
their mechanical, physical and bioactivity characteristics of were investigated [164]. It was
indicated that adding YAP significantly increased the density, thickness, opacity, solubility
and swelling degree of CS films but the water vapor permeability, water content and
mechanical characteristics of the film were diminished. Also, the antimicrobial and
antioxidant features of the CS films were noticeably improved through YAP addition. The

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FTIR and NMR spectra confirmed that the interactions occurred between CS chains and YAP
probably had a non-covalent nature. The thermal stability of the films was declined when

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YAP was introduced. The XRD patterns of the YAP-CS films revealed that the crystalline

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degree was changed kept pace with that of their thermal stability.
A CS-TiO2 composite film was produced by incorporating TiO2 nano-powder in CS
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which exhibited high microbicidal activity against food-borne pathogenic microbes and
anticipated to be an auspicious food packaging compound [165]. The SEM image of the
composite film displayed that the TiO 2 nano-powder was uniformly dispersed in the CS
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matrix. TiO 2 incorporation improved the hydrophilicity, mechanical properties but diminished
the visible light transmittance. The CS-TiO 2 film illustrated high antimicrobial capacity
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against four strains including Staphylococcus aureus, Escherichia coli, Aspergillus niger and
Candida albicans bacteria after incubation for 12 h. Thus, it was provoked that cellular
substances were leaked through the damaged bacterial membranes which was led to their
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death. Moreover, the CS-TiO2 film was used in packaging red grapes to avoid microbial
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infection and prolong their shelf life.

In another work, cellulose nanofiber (CNF) was modified by rosin and employed to
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reinforce the polylactic acid (PLA) matrix and the film was coated by CS to obtain a two-
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layer composite film to be used in antimicrobial food packaging [166]. The FT-IR spectra of
rosin modified CNF (R-CNF) exhibited a peak at 1730 cm−1 confirming the esterification of
CNF by rosin. The dispersion of R-CNF in the PLA matrix was more appropriate than CNF
and the R-CNF loading significantly affected the mechanical properties of the film. It was
observed that a percolation network was created using 8% R-CNF loading where the film
demonstrated the optimum mechanical features. The antimicrobial assay revealed that the R-
CNF/PLA/CS film had exceptional antimicrobial capacity against B. subtilis and E. coli
microorganisms that was recognized as the synergistic antimicrobial effects of rosin and CS.
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Antimicrobial performance of low/medium-molecular weight CS and organic acids

including sorbic acid and benzoic acid and commercially accessible nano-sized sorbic- and
benzoic- acid solubilisate equivalents was evaluated and compared with those of commercial
mixtures of organic acids applied as meat coatings (Articoat DLP-02® and Sulac-01®) [167].
The antimicrobial tests exhibited that both low and medium molecular weight CS displayed
the highest antimicrobial capacity against all tested bacteria with mean minimum inhibitory
concentration values of 0.010 and 0.015% w/v, respectively. Hence, it was found that the CS
molecular weight affected its antimicrobial activity. Moreover, the nano-sized solubilisates of

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sorbic acid and benzoic acid revealed considerably greater antimicrobial activities compared
to their non-nano equivalents.

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The influence of CS concentration on the characteristics of biocomposites incorporated

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with plasticized poly(lactic acid) (PLA) with tributyl o-acetyl citrate was investigated [168]. It
was indicated that the tributyl o-acetyl citrate decreased the PLA brittleness and the
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sheets/films acquired from plasticized PLA-CS biocomposites exhibited acceptable
mechanical, transparence and thermal features. The PLA-CS biocomposites were non-toxic
for growth of radish and cucumber seeds. The migration tests were accomplished on two food
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simulants and one method was modified to avoid using high temperatures near the glass
transition of PLA. It was found that the biocomposites were appropriate materials for the
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packaging of non-fatty foods with pH> 4.5 at refrigeration temperature. All of the PLA-based
materials presented satisfactory antifungal capacity against Penicillium corylophilum
CBMF1, Aspergillus brasiliensis ATCC 16404 and Fusarium graminearum G87 at the
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contact surface which was increased by the CS addition. It was observed that only
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biocomposites comprising CS displayed significant decrease in E. coli and S. aureus

microorganisms.
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In another work, antimicrobial characteristics of CS and CS-ZnO nanocomposite

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coatings were examined on polyethylene films [169]. Oxygen plasma pretreatment of

polyethylene films improved adhesion of 2% CS and nanocomposite coating solutions onto
the packaging films. The SEM micrographs indicated uniform coatings were formed on the
polyethylene surfaces. Addition of ZnO nanoparticles to the CS matrix caused 42% solubility
increase, the swelling was decreased by 80% but the water contact angle was enhanced from
60 to 95° relative to the CS coating. As well, the polyethylene coated by CS-ZnO
nanocomposite films totally deactivated and inhibited the growth of food pathogens whereas
the CS-coated films only exhibited 10-fold decrease in the viable cells of Escherichia coli,
Salmonella enterica and Staphylococcus aureus after 24 h incubation relative to the control.
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The propolis and CS were combined to achieve a completely bio-based active food
packaging material [170]. To do this, propolis glycolic extract was added as antioxidant and
antimicrobial agent having polyphenols. Two CS polymers with diverse molecular weights
were employed as antimicrobial, wet strength additive substitute and polyphenols carrier. The
paper was produced using carboxymethyl-/microfibrillated-cellulose at two pH values to
examine the polyphenols preservation and paper strength. It was shown that using CS instead
of the most frequently used wet strength resin (polyamine polyamide epichlorohydrin)
improved the polyphenols retention more than 10 times. The paper sheets obtained at pH=7

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using the highest molecular weight CS by microfibrillated cellulose incorporation exhibited
the highest wet resistance (13.3±1.2%) and wet strength (7.4±0.5 Nm/g). The antimicrobial

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activity of the paper was established on thinly sliced raw veal meat and a decrease was

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realized for the inoculated L. innocua after 48 h at 4 °C.
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4. Application of chitosan in textiles industry (preparation of antibacterial textiles)
Currently, there is an increasing requirement to provide healthy, safe and comfortable
living environment and protection from the infection by pathogenic microorganisms [171].
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Therefore, the demand for healthcare/medical textiles and antimicrobial products is ever-
increasing. Thus far, numerous chemicals have been used to endow with antibacterial capacity
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to textiles including organometallics, inorganic salts, iodo-phors (materials that gradually

release iodine), onium salts, phenols and thiophenols, heterocyclics with anionic groups,
antibiotics, ureas and related compounds, nitro compounds, amines and formaldehyde
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analogues [172]. Nevertheless, some of such chemicals are toxic to humans and cannot be
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simply degraded in the nature.

The textile industry seeks environmental processes to be accomplished without using
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toxic textile chemicals. Since CS is an exceptional material for an eco-friendly textile

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industry, it is employed in health-care products and in hygiene fabric engineering such as

bandages, artificial implants and medical sutures [173]. As well, CS has the capacity to be
utilized in the textile field as a dye fixing agent, thickener and binder for pigment printing of
cellulosic fabric and to improve the fastness characteristics of dyed textiles [173]. It was
reported that the properties of cotton fabric were enhanced by nanochitosan incorporation and
consuming natural bioactive compounds including CS as antimicrobial and ecological
finishing of textiles.
The CS nanoparticles were prepared by ionic gelation method through the interaction of
CS and sodium tripolyphosphate in acidic medium [18]. The CS NPs were characterized for
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their size, morphology and zeta potential and then they were applied on cotton fabric by the
pad-dry-cure procedure. The nanofinish process was optimized by Taguchi approach and the
responses were bending length, recovery angle, fabric tensile strength, absorption time and
antibacterial performance against some bacteria. The smallest average particle size was 115
nm and highest zeta potential was +31.3 mV using 0.2%w/v of CS. The SEM image equipped
with energy dispersive X-ray (EDX) established the existence of CS NPs on the treated fabric.
The treated fabrics displayed durable and satisfactory antibacterial capacities with suitable
textile features.

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A series of CS based water dispersible polyurethanes (WDPUs) were synthesized in
three steps [174]. In first step, the NCO end capped PU prepolymer was obtained from the

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reaction of dimethylolpropionic acid, polyethylene glycol and isophorone diisocyanate. In

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second step, the neutralization was performed using triethylamine to achieve neutralized NCO
terminated PU-prepolymer and in the last step, chain extension was accomplished via CS
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addition to make the dispersion formation by adding calculated quantity of water. The
structures of CS-WDPUs were established by the FTIR spectra. The antimicrobial capacities
of the bare weave poly-cotton printed and dyed textile swatches were assessed after
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CSWDPUs application. It was found that the CS incorporation into the PU backbone
evidently improved the antibacterial capability of WDPUs. Hence, such synthesized CS-
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WDPUs were produced as sustainable antimicrobial finishes using CS as a natural bioactive

agent to be used on polyester/cotton textiles.
An antibacterial coating was developed for cotton fabrics using core-shell particles
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composed of poly(n-butyl acrylate) (PBA) cores and CS shells [175]. The spherical particles
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were achieved by a surfactant-free emulsion copolymerization of n-butyl acrylate in aqueous

CS solution prompted in presence of a little quantity of tert-butyl hydroperoxide. The PBA-
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CS core-shell particles exhibited a narrow particle size distribution, an average particle

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diameter of ~300 nm and very positive surface charges. The TEM images obviously showed
the well-defined core-shell morphology for the particles so that the PBA cores were coated by
the CS shells. Each particle contained both the PBA homopolymer and the CS-g-PBA
copolymer that were characterized by the 1 H NMR and FTIR spectra. The cotton fabric was
coated by the PBA-CS particles using a common pad-dry-cure process and its antibacterial
efficacy was quantitatively assessed against Staphylococcus aureus by the shake flask
technique. The cotton treated with PBA-CS particles illustrated an outstanding antibacterial
capacity and bacterial reduction was greater than 99%.
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Chitosan can be used as a safe antibacterial material on textiles however there is a

limitation for its durability. Thus, CS was extracted from shrimp shells and employed as
antibacterial exhaust finishing agent on grafted bamboo rayon [176]. Then, the antibacterial
activity of CS bound bamboo rayon was estimated against Gram negative and Gram positive
bacteria. The product exhibited antibacterial capacity against both types of microorganisms
which was durable until 30 washes.
In another study, CS extracted from waste shrimp shells was applied in finishing
formulation for cotton fabric, accompanied by dimethylol dihydroxy ethylene urea and other

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chemicals to impart multiple properties like wrinkle free, flame retardant and antibacterial
features [177]. The finished fabrics were examined for textile properties such as bending

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length, tensile strength, yellowness index and functional characteristics including antibacterial

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capacity, crease recovery angle, flame retardancy and ecological properties like formaldehyde
release. The finished fabric exhibited exceptional crease recovery, antibacterial activity and
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flame retardancy which were preserved to an adequate level even after 20 washes. In addition
to the formaldehyde scavenging property, CS obviously endowed with multifunctional
properties to the cotton fabric.
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Some polyurethane (PU) dispersions were synthesized by a two-step polymerization

reaction [178]. To do this, a PU prepolymer with NCO termini was achieved using isophorone
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diisocyanate, poly caprolactone diols and dimthylolpropanoicacid. The PU prepolymer chain

was extended using various mole ratios of low molecular weight CS and the aqueous
emulsion was obtained through addition of appropriate water amount. The structures of CS
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based PU dispersions were established by the FTIR spectra. The aqueous CS-PU emulsions
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were applied on the diverse quality bare weave poly-cotton dyed and printed fabric pieces by
means of pad-dry-cure technique. The physical characteristics of the treated and untreated
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fabric samples including pilling resistance, stiffness, air permeability, crease recovery angle,
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tear and tensile strength were evaluated. It was found that the CS incorporation had a
prominent influence on the properties of treated fabrics.
In another research, environmental biosynthesis of chitosan–neem seed (CS-NS)
composite was accomplished through co-precipitation technique using aqueous neem seed
extract [179]. Cotton fabrics were treated using citric acid and glutaraldehyde as two diverse
crosslinking materials and the synthesized composite was then coated on cotton fabric via
chemical binding between the cellulose structure and the composite coating. The antibacterial
performance of the CS-NS composite coated cotton fabric and CS-NS composite coated
cotton fabric along with crosslinking agents were assessed against the Gram negative and
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Gram-positive microorganisms using agar well diffusion procedure. It was confirmed that CS-
NS composite with crosslinked coated cotton fabric had greater antibacterial potency than
cotton fabric without crosslinking. Thus, the CS-NS composite could be used to prepare
medical textiles.
Fig. 47(a–e) exhibits the morphologies of untreated cotton fabric, CS-NS composite,
CS-NS composite coated cotton, CS-NS composite coated cotton with glutaraldehyde and
CS-NS composite coated cotton with citric acid. The CS-NS composite in Fig. 47(a) displays
a multilayered, rough and non-even surface. The untreated cotton demonstrates that there is

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no deposition of composites, Fig. 47(b). The CS-NS composite coated cotton shows a layer
resembling structure and several places demonstrate the composite deposition in Fig. 47(c).

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The CS-NS composite coated cotton with glutaraldehye, Fig. 47(d), indicates a rough surface

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and good composite deposition over the cotton surface because of using crosslinker. Besides,
Fig. 47(d) illustrates extra roughness than that of the composite coated cotton along with
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numerous spherical particles dispersed over the fabric surface. The CS-NS composite coated
cotton with citric acid in Fig. 47(e) reveals that the composite is well bound to the cotton
fabric upon using citric acid [179].
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Durable cotton/polyester blended fabrics with multifunctional features were produced

by introducing CS and several metal oxide nanoparticles (MONPs), i.e., TiO 2 , ZnO and SiO 2
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to the fabric surface by means of citric acid/sodium hypophosphite as ester–crosslinking and

generating anchoring/binding sites (COOH groups) on the ester-crosslinked fabrics surfaces
[180]. The surface morphology and the existence of MONPs and CS active ingredients on the
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coated fabrics were confirmed by the SEM images and EDX analysis. The effect of different
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finishing formulations on functional and performance properties including antibacterial

activity, wettability, self-cleaning, UV-protection, resiliency and durability to wash were
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examined. It was found that the improvement degree of the imparted functional properties
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was controlled by the loaded composite type and changed in the order of CS-TiO 2 NPs> CS-
ZnONPs>SiO 2 NPs>CS alone, plus substrate kind as cotton/polyester
(65/35)>cotton/polyester (50/50). Furthermore, the durability of the CS-TiO 2 NPs loaded
substrates was slightly diminished after 15 washing cycles demonstrating the durable fixation
of the composite components on the ester-crosslinked substrates. Hence, such bioactive
multifunctional textiles could be employed to produce ecological protective textiles.
To prepare durable antimicrobial cotton fabric, carboxymethyl CS was covalently
bound to cotton fibers through esterification using the cellulose hydroxyl groups and the
silver nanoparticles were attached to the fiber surface via the coordination bonds with the
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amino groups of carboxymethyl CS [181]. The finished cotton fabrics revealed exceptional
laundering durability and outstanding antibacterial activities. After 50 sequential laundering,
the modified cotton fabrics exhibited acceptable bacterial decrease rates against both E. coli
and S. aureus, which were greater than 94%. Therefore, these antimicrobial cotton textiles
had lessened safety risk and decreased environmental influence aroused from the silver
nanoparticles.
The environmental synthesis of CS-based nanocomposites was carried out by
interactions of AgNPs and clay with CS to yield CS-AgNPs and CS-AgNPs-clay

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nanocomposites which were applied on cotton fabric to endow with them fabulous properties
[182]. The CS-AgNPs-clay nanocomposites explicitly proved that their usage in one–step

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treatment process on cotton fabrics caused noticeable appropriate properties such as uniform

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morphology, great strength, improved thermal stability, effective composite deposition on the
surface of cotton fabrics, high water absorption, flame retardancy, antimicrobial capacity, UV
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protection and controlled release of fragrance. The treatment of cotton fabrics using these
nanocomposites was stable against washing after 20 washing cycles. Consequently, the
environmentally benign synthesis of CS-AgNPs-clay nanocomposites was a promising
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method to achieve multifunctional finishing textiles.

Cotton fabrics treated by the CS/AgNPs and CS/AgNPs/clay nanocomposites were
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compared to the cotton fabric only treated with CS indicating the color varies from colorless
to yellow and yellow darkness, respectively. The surface morphologies of the untreated and
treated cotton fabric using CS, CS/AgNPs and CS/AgNPs/clay studied by FE-SEM images
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are exhibited in Fig. 48 confirming the untreated sample shows a rather smooth surface, Fig.
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48(A, a) indicating there are not any nanoparticles on the surface. Treatment with CS solution
causes the CS film to cover the fabric surface (alongside the formation of CS film) and the
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surface to be rough by the influence of CS polymer, see Fig. 48(B,b). The SEM micrographs
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of cotton fabrics treated by CS/AgNPs nanocomposite are provided in Fig. 48C. It is apparent
that the AgNPs have been deposited on the cotton fabrics surface. Furthermore, the SEM
image at higher magnification (8000X) points out that the AgNPs are evenly dispersed on the
fabrics. The CS/AgNPs nanocomposites are spherical having small diameters and the cotton
fabric treated by colloidal dispersion of hybrid CS/AgNPs/clay composite is covered by
aggregated nanocomposite and a rough surface is achieved. The SEM images (Figs. 48D and
48d) exhibit the creation of aggregated clay minerals over the cotton fabrics surface [182].
A facile process was established to prepare the super-hydrophobic antibacterial textile
for biomedical purposes [183]. For the coating formulation, the spraying of nanoparticles
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dispersion on the textile was done to obtain a multiscale textured layer on top of cotton fabric.
The antibacterial activity of coating was confirmed using CS-based nanoparticles. The
nanoparticles were fabricated by electrostatic interactions of amino groups of CS and
negatively charged fluoro anions. The relative number of fluoro anions per CS unit had a
crucial role on the aggregates structures in the coating, the wettability and durability of
coatings in contact with aqueous environment.
The surface of wool fabric was modified using anhydrides in order to graft the CS
polymer [184]. The weight gain, antifelting and antibacterial characteristics of the CS grafted-

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acylated wool fabric were evaluated. The wool fabrics were acylated by two anhydrides,
succinic anhydride and phthalic anhydride in two diverse solvents including N,N-dimethyl

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formamide and dimethylsulfoxide. The influences of solvents, anhydrides, anhydride

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concentration, reaction time and liquor ratio on wool acylation were explored. The CS was
grafted to the acylated wool and the influences of CS concentration, pH and reaction time on
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grafting CS to the acylated wool were assessed. The FTIR, DSC, SEM and weight gain
analyses proved that CS was grafted to the acylated wool by the covalent bonds. The grafted
samples exhibited antibacterial activities owing to the antibacterial potency of CS. Moreover,
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the CS grafted-acylated wool fabrics showed antifelting feature.

The patchouli oil loaded CS–gelatin microcapsules were developed through the
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complex coacervation technique [185]. The morphology and surface were investigated by
SEM micrographs displaying the microcapsules had regular spherical shapes in the range of
1-20 μm. The thermal stability analysis exhibited that the microcapsules were stable below
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190 ºC indicating the fabrics finish should be performed at 160 ºC. The loading capacity and
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encapsulation efficiency of the microcapsules were 30.31 and 50.69%, respectively. The
microcapsules were grafted to cotton fabrics using the 2D resin dimethylol dihydroxyethylene
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urea as the crosslinking agent. The SEM images illustrated that the microcapsules were not
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only grafted onto the fabrics surfaces but also introduced in the fibers spaces. Furthermore,
the creation of ether bonds among 2D resin and hydroxyl groups of cotton and/or hydroxyl
moieties of the microcapsules was confirmed by FTIR spectra. The antibacterial capacities of
the fabrics against Escherichia coli and Staphylococcus aureus were ~65% even after 25
times washing verifying their potential applications as antibacterial masks, bacteriostatic
sheets and health-care clothes.
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5. Molecular dynamic (MD) simulations on pharmaceutical applications of chitosan

Molecular dynamic simulation is a useful tool for researchers to investigate the
structural and dynamical changes in atomic-scale resolution which are hardly observed in
experimental efforts [186,187]. Moreover, the MD simulation is a powerful method and a
valuable complement to experiment that can allow to avoid expensive experiments. Hence,
MD simulations are commonly applied on drug delivery systems in order to examine the drug
encapsulation at the molecular level [108].
Follicle-stimulating hormone (FSH) is used in modern ovarian stimulation methods but

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it must be daily administered due to it has a short half-life. The cholesterol (ChS) modified
CS nanogels are known as favorable controlled release protein delivery systems because they

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are able to decrease irreversible denaturation and aggregation of proteins [188]. The MD

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simulations were carried out for up to 200 ns on FSH encapsulation into ChS-CS nanogels
and it was found that the main driving force for the creation of the ChS-CS nanogels was
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hydrophobic interactions occurred in water between the ChS–ChS fragments. Also, the ChS-
CS nanogel could form by the hydrogen bonds along with the hydrophobic interactions. Fig.
49 exhibits the morphology and structure of the ChS-CS nanogel. The solvent-exposed
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hydrophobic patch in FSH is indicated in Fig. 50. The FSH encapsulation in the ChS-CS
nanogels was a slow process which was motivated by the hydrophobic interactions happened
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between the FSH hydrophobic patch and the nanogel hydrophobic nanodomains. The total
binding energy was decomposed per residue basis to recognize main residues that were
contributed to the adsorption on ChS-CS. It was shown that the residues Phe17, Phe18, Val70,
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Met71, and Phe74 were the most important residues contributed to the FSH absorption to the
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ChS-CS nanogel by the non-polar solvation energy and the van der Waals interaction. All of
these amino acids were positioned within the hydrophobic patch of FSHα confirming the most
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substantial contributions to the adsorption were from the hydrophobic patch region.
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Moreover, the total binding free energy for the FSH/CS-CTS nanogel was positive (+8.86).
Considering the important role of drug delivery in the treatment of the central nervous
system diseases, selecting an appropriate carrier is very crucial to have a higher drug
efficiency. Thus, CS and poly(n-butylcyanoacrylate) (PBCA) were chosen as carriers in brain
drug delivery because they have indicated good biodegradability, biocompatibility and low
toxicity [189]. For this purpose, MD simulations were performed using these polymers with
diverse polymerization degrees and Tacrine as the most familiar drug used for the treatment
of Alzheimer's disease. Figs. 51 and 52 reveal the structures of various CS-Tacrine and
PBCA-Tacrine systems, respectively which confirm the interaction between the Tacrine
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molecule and PBCA chains becomes stronger when the polymer chain length is increased
whereas it is decreased in the CS/Tacrine.
Recently, sorption of L- and D-Tyrosine (Tyr) from aqueous solutions onto chiral CS
membranes was evaluated by docking and MD calculations and a high adsorption with a
noticeable enantioselectivity to L-Tyr, was established [190]. Furthermore, different
enantiomers affinities were found for two adsorption regions within the polymeric matrix. It
was indicated that Tyr adsorption decreased the membrane crystallinity and rearranged the
chains. As well, the hydrated to anhydrous polymorph ratio was changed upon the adsorption

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due to water bound to CS was altered. The energy balance between the hydrogen bond
formation, desolvation and the conformational changes caused an endothermic spontaneous

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process.

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The stability and solubility of the CS film were measured by evaluating the interchain
distances using a cut-off value of 0.35 nm. It was found that the sixteen CS chains remained
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close enough to each other so that they were assumed as a part of a single aggregate film
during the entire simulation time. Fig. 53 indicates the simulation of CS-NaOH film in which
three stages of the MD simulations from a crystal-like structure (0 ns) to a relax solvated film
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(50 ns) are depicted. Also, distances between the closest atoms in neighboring chains are
computed horizontally and vertically. Although there were some fluctuations particularly
between chains B and C (Fig. 3A–C), the total chain’s connectivity held the structure
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together. The distance between chains B and C showed the deviation from the average
minimum distance of 0.2 nm occurred for most of chains. The temporary separations created
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holes/microcavities that allowed tyrosine and solvent molecules to move deeper in the film
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core. Therefore, swelling occurred and it was verified by increasing the number of water
molecules near the central chains of the CS film in the first 5 ns of the simulation. Although
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the acetylation degree (20%) was low to have a soluble CS film, the modelled neutral
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environment lacked the electrostatic repulsion of protonated amino groups between CS

polysaccharide chains, thus the polymer was remained aggregated as a solid membrane.
The binding interactions of both tyrosine enantiomers (L-Tyr and D-Tyr) with CS film
molecular docking calculations were carried out. Fig. 54 reveals the best energy ranked
docking complexes for D-Tyr and L-Tyr. All tyrosine/CS-NaOH complexes exhibited the
amino acid bound in a cleft between central film chains. Two possible binding regions were
observed in this cleft including region 1 (placed deeper in the CS film core) and region 2
(located in the vicinity of the film surface) [190].
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It is known that nanotechnology-based drug delivery systems are employed to increase

the bioavailability and biological properties. Also, CS incorporated curcumin can be used as a
biocompatible alternative for metal nanoparticles to prevent biofilm formation of
Streptococcus mutans and plaque on teeth. The interactions between CS carrier and curcumin
(a natural antibacterial agent) were examined by the simulation method [191]. The root mean
square deviation (RMSD=26.81±0.1 Å) and the root mean square fluctuations
(RMSF=1.13±0.02 Å) for all of the complex atoms were relaxed after 4 ns. It was established
that during the first interval (10 ns), a stable binding was occurred between the two sections.

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All bindings were disappeared from 10 to 20 ns but the curcumin was entrapped within the
CS nanoparticles and no release was happened until 20 ns after which the curcumin started to

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release which confirmed the CS nanoparticle could carry the curcumin.

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Chitosan oligosaccharide (COS) derivatives are attractive drug delivery systems
because of their eminent low toxicity, outstanding biodegradability and biocompatibility. In
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an effort, salicylic acid-grafted chitosan oligosaccharide (COS/SA) was used to prepare
paclitaxel (PTX) loaded nanoparticles [192]. Also, to realize the mechanism of the action of
the PTX encapsulated COS/SA, MD simulations were conducted. The van der Waals and
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hydrophobic interactions were known as the major driving forces in the drug encapsulation.
The hydrogen-bonding and electrostatic interactions have useful effects in the COS/SA
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aggregation. The solvent accessible surface area (SASA) and radial distribution function data
showed that the COS/SA nanoparticles were extremely water soluble that could considerably
increase the aqueous solubility of a hydrophobic drug.
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Different drug loading systems were examined and the best theoretical drug loading was
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obtained to be 10%w/w. A 20 ns run of three systems was performed under the same
conditions to investigate drug loading in which forty COS/SA chains were located near 5, 7,
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and 9 PTX molecules that were related to drug loadings of 7.3%, 10%, 12% (w/w),
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respectively. For the 12% drug loaded system, the 20 ns MD trajectory revealed that the 40
COS/SA chains did not spontaneously form a compact aggregated structure (Fig. 55). The
average SASAs values for the nanoparticles in these three systems were 391.04, 391.59, and
432.41 nm2 , respectively. The SASAs at drug loadings of 7.3% and 10% nanoparticles were
practically comparable but the SASA at the drug loading of 12% was significantly enhanced
in comparison with the two other systems. Consequently, the best theoretical PTX drug
loading for COS/SA drug carrier was ~10%. The ratio of PTX to COS/SA was experimentally
fixed at 20% but the measured drug content was only 3.45%. The difference between the
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simulation and the observation was related to the influence of the experimental operating
conditions [192].
The influence of the CS nanoparticles was studied on the structure, dynamics and
enzyme activity of trypsin [193]. It was found that the enzyme activity in complex with the
nanoparticles was somewhat improved indicating the interactions between the enzyme and the
nanoparticles but fluorescence spectroscopy did not demonstrate significant variations in the
trypsin conformation in the presence of nanoparticles. The MD simulations were done to
investigate the mechanism of interactions occurred between the nanoparticles and trypsin. The

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binding free energy data proved that the nonpolar interactions were the main forces for the
creation of stable nanoparticle-trypsin complex.

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It was found that the total, hydrophilic and hydrophobic SASA values of the CS

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nanoparticles were rapidly decreased in the beginning the MD simulations and then reached
the average value of 113.5, 15.7 and 97.8 nm2 , respectively (Fig. 56A). The decline in SASA
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of CS nanoparticles in the presence of water confirmed induced compactness of the molecules
which caused restrictions in the interactions between the CS chains and water molecules.
Also, the total number of hydrogen bonds formed between the CS and water molecules was
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diminished with time whereas the total number of the CS-CS hydrogen bonds was enhanced
(Fig. 56B). in order to examine the influence of the CS nanoparticles on the water structure,
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the RDF between water oxygen atoms was obtained. Fig. 56C displays that the peak at 0.28
nm in the presence of the nanoparticles was sharper than that of the pure water. It was
proposed that the CS nanoparticles increased the hydrogen bonds between the water
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molecules, hence strengthened the water structure and stabilized the nanoparticles [193].
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Chitosan is a biocompatible and biodegradable carbohydrate biopolymer which is one

of the most useful polymers in the pharmaceutical science. Moreover, various protein and
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peptide drugs are applied as therapeutic agents which may be exposed to temperature stresses
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in transporting and/or storage stages. Such stresses may cause the protein structure unstable,
alter the active structure and destroy its therapeutic function thus limit their usage as fruitful
drugs. To overcome such problems related to the protein drugs, diverse materials like natural
or synthetic polymers can be utilized to prepare protein loaded biocompatible
nano/microspheres. Recently, MD simulations were performed to investigate the effect of CS
with different deacetylation degrees on the stability of Interferon αII structure at high
temperature and to compare the data with those of commonly used biocompatible synthetic
polymers including polylactic-co-glycolic acid and polyethylene glycol [194]. Final structures
of IFNαII-polymer complexes for all systems after 50 ns of simulation are displayed in Fig.
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57. The conformational variations occurred at high temperature (343 K) both in the presence
and absence of polymers were compared to results obtained for the protein at normal
temperature (300 K). It was indicated that low deacetylated CS and polylactic co-glycolic acid
were more effective in protein stability at high temperature but polyethyleneglycol was
penetrated into the protein and showed some instability of protein conformation.

6 Conclusion
Polymeric pharmaceutical carriers (such as chitosan) are of great attention due to they

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have numerous advantages mainly in enhancing the effectiveness and safety of the
pharmaceutics. Such systems can encapsulate both hydrophilic and hydrophobic active

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substances. Additionally, they can result in higher stability for the therapeutics against

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enzymatic and chemical degradation, greater drug impact in the target organ and more
bioavailability. They can hold active compounds such as drugs, vaccines, peptides, proteins,
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genes and oligonucleotides that may be adsorbed, encapsulated, covalently and/or
electrostatically bound to their surfaces or inside their matrices. Indeed, these materials
especially polysaccharides have developed medical treatments. It is noteworthy that the CS-
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based systems are very appealing vehicles that are capable of releasing their active
encapsulated compounds with the desired rate at targeted site in the body. Herein, the most
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important pharmaceutical applications of CS were discussed in numerous biomedical areas

like wound healing, tissue engineering, drug/gene delivery, protein binding, cell
encapsulation, preparation of implants and contact lenses, bioimaging, food additives, food
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packaging and antibacterial textiles. Also, recent studies about the molecular dynamics
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simulation of chitosan in the pharmaceutical applications were reviewed. As most of the CS

applications are yet at laboratory phase, additional investigations are necessary to utilize them
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as commercial pharmaceutics in clinical applications. Finally, it could be concluded that the

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CS-based materials are promising candidates for application in various pharmaceutical fields.

Acknowledgments
Authors would like to appreciatively express their thanks to the research office of
Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran for the financial
support of this work.

Conflict of interests
The authors do not have any personal or financial conflicts of interests.
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References
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and antifungal evaluation of diethoxyphosphoryl polyaminoethyl chitosan derivatives,
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Drug delivery Gene delivery Cell encapsulation Protein binding

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OH
H
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O
Wound healing H
H O Food additives
HO
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NH2
H H
Chitosan (CS) n

Contact lens Food packaging

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Implants Tissue engineering Antibacterial textiles Bioimaging

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Scheme 1. A schematic indicating pharmaceutical applications of chitosan in various areas.

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Fig. 1. Het-Cam images for ocular irritation study treated with (A) 0.1N NaOH,
(B) Normal saline (0.9% NaCl), (C) LFX-CS-NPopt in situ gel system.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 650–659).
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Fig. 2. Comparative histopathology images of treated groups (A) control,

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(B) LFX-CS-NPopt in situ gel.

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Fig. 3. Comparative confocal laser microscopy images of

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(A) LFX-CS-NPopt in situ gel, (B) LFX solution.

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(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 650–659).
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Fig. 4. In vitro release of 5Fu from CS/Cc PECs (0.5 mg/mL solutions of CS and Cc,
1:1(w/w) CS/Cc mass ratio, and 0 mM NaCl at pH 3.0) at pH 1.4 and pH 7.4. Each value is
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expressed by means ± SD (n=3).

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(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 397–405).
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Fig. 5. In vitro cellular uptake of CNPs in SCC7, U87, HT29, PC3, and A549 cells depending on
various pH conditions. Confocal fluorescence microscopic images of each type of cancer cells
after 30 min of treatment with CNPs (25 μg/mL) in the RPMI media (pH 6.0, 6.5, and 7.4).
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(Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).
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Fig. 6. Distribution of CNPs in the mice transplanted SCC7, U87, HT29, PC3, A549 tumors.
Time-dependent non-invasive in vivo NIRF imaging of the tumor-bearing mice after
the injection of NIRF dye labeled CNPs (200 μg/μL head).
(Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).
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Fig. 7. Distribution of the intratumoral extracellular matrix in various tumors.

Collagen matrix in tumor tissues stained blue with Masson's trichrome stain (bar = 150 μm).
(B) Quantitative analysis of ECM contents in SCC7, U87, HT29, PC3, and A549 tumors;
ECM contents in tumor tissue =blue stained collagen area / total area ×100 (%).
(Reprinted with permission from Elsevier, J. Control. Release 267 (2017) 223–231).
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Fig. 8. HepG2 cells determined using Hoechst 33,258 stain. (a) cells untreated; (b) cells
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treated with free curcumin; (c) cells treated with curcumin loaded TCS/PEGDA injectable
hydrogels without lysozyme (TP0); (d) cells treated with curcumin loaded TCS/PEGDA
injectable hydrogels with lysozyme (TP3).
(Reprinted with permission from Elsevier, Mater. Sci. Eng. C 83 (2018) 121–129).
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Fig. 9. In vivo antitumor effect of curcumin loaded TP3 on tumor-bearing nude mice. The
optical pictures of tumor-bearing nude mice recorded at 0 day and 21 day of treatment (a),
body weight changes (b) and tumor volume changes (c) from 0 day to 21 day.
(Reprinted with permission from Elsevier, Mater. Sci. Eng. C 83 (2018) 121–129).
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Fig. 10. H&E staining of tumor, heart, liver, lung and kidney tissue sections
from tumor-bearing nude mice (scale bar: 50 μm).
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Fig. 11. (a) Relative cell viability of HepG2 cells treated with various concentrations of NPs
(black), quercetin (red) and Q-NPs (blue). Trypan blue staining images of HepG2 cells
without treatment (b), treated with 2 mg mL_1 of NPs (c), native quercetin (d), and Q-NPs
(e). Data are shown as means ± SD of five separate experiments. Significant difference
between treated and negative control groups is indicated at P<0.05 levels.
(Reprinted with permission from Elsevier, LWT - Food Sci. Technol. 85 (2017) 37-44).
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Fig. 12. Gel retardation assays. (A) Gene retardation with the concentration of 10 µg/ml.
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(B) Gene retardation with the concentration of 15 µg/ml, and stability of them against heparin.
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miR-145 PECs of CS9 with CMD:CS ratios of 0.2 (1), 1 (2), 5 (3); CS18 with CMD:CS ratio
of 0.2 (4), 1 (5), 5 (6); CS45 with CMD:CS ratio of 0.2 (7), 1 (8), 5 (9). The numbers 0, 0.1,
0.2, and 0.4 are the heparin concentrations (µg/ml). DL: DNA ladder 1 KD. C: naked plasmid.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 159 (2017) 66–75).
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Fig. 13. Green fluorescent protein (GFP) expression after transfection by miR-145 PECs. and
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DAPI staining of nucleus. (A) Confocal microscopy of non-transfected cells (a), and
transfected cells by the nano PEC of CS45 with the CMD:CS 5 (b) and CS18 with the
CMD:CS 1 (c); (B) Flowcytometery analysis. The miR-145 plasmid expresses both miR-
145and GFP. CS9: chitosan 9 KD, CS18: chitosan 18 KD, CS45: chitosan 45 KD, CMD:CS:
the ratio of carboxymethyl dextran to chitosan. Data are presented as mean ± SD.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 159 (2017) 66–75).
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Fig. 14. Confocal microscopy of MCF7 treated with the Cy5 PECs composed of CS18
with the CMD:CS ratios of 0.2 (A) and 1 (B). The merge image of nucleolus staining by
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DAPI and Cy5 PECs in the cells.

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(Reprinted with permission from Elsevier, Carbohydr. Polym. 159 (2017) 66–75).
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Fig. 15. (a) Following 24 h incubation with polymers, cell viability was determined by the
CellTiter-Glo®cell viability assay. Mean cell viability was normalized to non-treated
controls, with the mean of n=3+SEM, from one representative experiments of three
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independent experiments. Statistical analysis was performed using two way ANOVA with
tukey’s post hoc test, n.s.-not significant, *p<0.05, **p<0.01,***p<0.001. (b) Morphological
characteristics of human fibroblast cells were visualized under the fluorescence microscope.
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Cells were also stained with the reagents in the LIVE/DEAD®Cell Viability/Cytotoxicity
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Assay Kit and visualized under the fluorescence microscope. Dead and live cells fluoresce
red-orange and green, respectively.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).
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Fig. 15. Continued.

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Fig. 16. Evaluation of the specificity of targeted cell transfection by GnRH-CS/pDNA complexes.
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(a) Assessment of targeted gene transfer by GnRH-CS/pDNA compared to the unmodified

CS/pDNA complexes carrying the GFP reporter gene using a range of polymer concentrations in
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the targeted cells expressing GnRH receptor and the non-targeted cells lacking GnRH receptor.
(b) Transfection of 3D spheroids by polyplexes. The spheroids were transfected with polyplexes
(i.e. GnRH-CS/pDNA and unmodified CS/pDNA).Representative images showing GFP expression
in the monolayer cultures and spheroids were taken at day 3 post transfection.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).
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Fig. 16. Continued.

(Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 311–320).
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Fig. 17. Mn-containing NPs were visualized and tracked by MRI. (A) Baseline T1-weighted
image of coronal section through olfactory bulb and (B) baseline horizontal section showing
the anatomical regions of interest. (C) T1-weighted MR of mouse 24 h after administration of
mNPs showing enhanced Mn signal in coronal section of olfactory bulb, and (D) T1-weighted
image signal in horizontal section including olfactory bulb, cerebral cortex, striatum and
hippocampus. (E, F) Parcellation of brain regions (to demarcate brain structures) was
performed to quantify Mn signal at 24 and 48 h. (G) Olfactory bulb; H) Cerebral Cortex; (I)
Hippocampus; (J) Corpus Striatum. Mean Mn signal (±SEM, n = 3) was increased in all brain
regions at 24 and 48 hrs compared to control mice (ie “no contrast”) after intranasal
instillation. One-way ANOVA was performed for each brain region using Matlab Statistics
Toolbox (Mathworks, Inc.).
(Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).
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Fig. 17. Continued.

(Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).
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Fig. 18. RFP expression in mouse brains following intranasal instillation of mNPs containing dsDNA
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encoding RFP. Mice were euthanized at 24 h for fluorescence histology and at 48 h for measurement
of RFP DNA regional brain concentration. (A) Olfactory bulb. Left panel: Structure of the olfactory
bulb is visualized with DAPI staining, which stains all cell nuclei. Middle panel: Tyrosine hydroxylase
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(TH) immunostaining in green identifies dopaminergic neurons located in the glomerulosa layer. Red
identifies cells that express red fluorescent protein (RFP), indicating that the plasmid DNA
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encapsulated in the manganese-containing nanoparticles is expressed. Right panel: Merged image of

DAPI, TH and RFP. onl=olfactory nerve layer; gl=glomerular layer; opl=outer plexiform layer;
ml=mitral cell layer; gr=granular cell layer. (B) Corpus striatum. Left panel: neurons immunostained
with antibodies against neuron specific nuclear protein (NeuN); Middle panel: RFP expression; right
panel: merged image of NeuN and RFP. (C) Ventral striatum. Left panel: DAPI-stained nuclei and
RFP expression; Right panel: magnified yellow box from panel on left shows RFP expression in
cytoplasm distributed in a perinuclear pattern in many, but not all cells. (D) Regional brain
concentrations of RFP DNA was determined at 48 hrs after nasal instillation Mn-nanoparticles
containing DNA encoding RFP as described in methods section. Data are expressed as mean and SEM
for each brain region (n = 4 mice). Y-axis indicates concentration of DNA (pg/mg tissue). Highest
mean concentration of RFP DNA was observed in striatum with the lowest concentration found in
olfactory bulb at this time point.
(Reprinted with permission from Elsevier, J. Drug Deliv. Sci. Technol. 43 (2018) 453–460).
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Fig. 19. Confocal laser microscopy images of chondrocytes that were stained in the three gels
(a)10:1 CS-HDA (b) 10:3 CS-HDA and (c) 10:5 CS-HDA with Calcein AM/ethidiumbromide
(LIVE/DEAD assay) for ascertaining the viability of the cells that were encapsulated. Live
cells fluoresce green and red cells fluoresce red. Scale bar = 10 µm.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).
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Fig. 20. (a) Fluorescence images showing Type II Collagen staining of chondrocytes
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encapsulated in 10:1 CS-HDA, 10:3 CS-HDA and 10:5 CS-HDA gels at 7 day, 14 day and 28
day. Cells were stained for type II collagen green and nuclei (blue). Scale bar for 7 day = 10
µm, 14 day = 5 µm and 28 day = 2 µm. (b) Fluorescence images showing Type I Collagen
staining of chondrocytes encapsulated in 10:1 CS-HDA, 10:3 CS-HDA and 10:5 CS-HDA
gels at 7 day (a, b and c),14 day (d–f) and 28 day (g–i). Cells were stained for Type I collagen
(red) and nuclei (blue). Scale bar for (a–c) = 10 µm and for (d–i) = 20 µm. (Reprinted with
permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1925–1935).
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Fig. 20. Continued.

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Fig. 21. (a) Sirius red staining for collagen performed on the chondrocyte encapsulated gels at
7 day (a, b and c),14 day (d–f) and 28 day (g–i). Scale bar = 50 µm. (b) Alcian blue staining
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for collagen performed on the chondrocyte encapsulated gels at 7 day (a, b and c),14 day (d, e,
f) and 28 day (g, h, i). Scale bar = 50 µm.
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Fig. 21. Continued.

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Fig. 22. Release of ASCs from the border of the hydrogels placed on a layer of Hs68 fibroblast
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and in vitro wound healing assay. (A) Scheme of the experimental design. (B) Double
immunofluorescence of ASCs (green) and fibroblasts (red), the dash line represents the border of
chitosan or chitosan/gelatin hydrogels. The number of ASC migrated beyond the dash line was
estimated at 24 and 48 h after applying the hydrogel (image analysis performed on 4 random
fields per sample; n=3 samples/condition). (C) After scratching confluent fibroblasts with a
pipette tip, the cell-free zone was photographed at 24 and 48 h. Area in the cell-free zone
covered by the migrated cells was measured (scale bars: 100 µm; image analysis performed on 4
random fields per sample; n=3 samples/condition; *p<0.05, compared to the group treated with
chitosan hydrogel; # p<0.05 between the indicated two groups).
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Fig. 22. Continued.

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Fig. 23. In vitro tube formation assay. (A) Images of endothelial cells after 4 h incubation
with hydrogels or ASC-encapsulated hydrogels at the periphery of each well (scale bars: 100
µm). (B) Branching points per power field were compared among different groups (image
analysis performed on 4 random fields per sample; n=3 samples/condition; *p<0.05,
compared to the group treated with chitosan or chitosan/gelatin hydrogel).
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Fig. 24. Chick embryo chorioallantoic membrane (CAM) assay. (A) Photographs of CAMs
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after treatment with hydrogels or ASC-encapsulated hydrogels, which were loaded on the
CAMs of day 9 chick embryos. After 72 h of incubation, CAMs were excised and
photographed (scale bars: 100 lm). (B) Blood vessel formation on CAM was quantified by
measuring the area covered by capillaries and counting the number of blood vessel branch
points (image analysis performed on 10 random fields per sample; n=3 samples/condition;
*p<0.05, compared to any of the other groups).
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Fig. 24. Continued.

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Fig. 25. Delivery of ASC-encapsulated chitosan/gelatin hydrogel enhanced angiogenesis in a

mice cutaneous wound model. (A) Double immunofluorescent staining of HNA and an
endothelial marker CD31 in the day 5 wound sections. White arrows indicate co-localization
of the HNA and CD31 immunofluorescence, indicating human ASCs incorporated into
vasculature structure (scale bar: 100 µm). (B) The number of HNA + cells per power field
was significantly higher in wound sections that received ASC encapsulated chitosan/gelati n
hydrogel. (C) The ratio of CD31 + area was also significantly larger in the group of ASC-
encapsulated chitosan/gelatin hydrogel (image analysis performed on 10 random fields per
sample; n=4 samples/condition; #p<0.05, between the indicated two groups).
(Reprinted with permission from Elsevier, Acta Biomater. 51 (2017) 258–267).
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Fig. 26. Chemical structures of chitosan and BSA and HSA with tryptophan residues in green color.
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(Reprinted with permission from Elsevier, Colloid. Surf. B Biointerfaces 125 (2015) 309–317).
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Fig. 27. Fluorescence emission spectra of BSA or HSA (10 µM) in acetate buffer pH 5–6 at
298.15 K, in the presence of chitosan nanoparticles with the following concentration:0 (a), 25
(b), 50 (c), 80 (d) and 120 µM (e) for chitosan-15; 0 (a), 5 (b), 10 (c), 15 (d), 20 (e) and 30
µM (f) for chitosan-100 and chitosan-200. (Reprinted with permission from Elsevier, Colloid.
Surf. B Biointerfaces 125 (2015) 309–317).
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Fig. 28. (a) Typical immunofluorescent staining images of α-actinin, and (b) Tn-I synthesized by
cardiomyocytes after incubation for 15 days on composite PLA/chitosan electrospun nanofibers.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 1130–1137).
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Fig. 29. Scanning electron micrographs of 3T3 on (A) 4G2C, (B) 4G2C10HY, (C) 4G2C20HY, (D)
4G2C30HY and (E) 4G2C50HY cryogels matrix after 10 days of culture at 5000x magnification.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 366–378).
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Fig. 30. Scanning electron micrographs of SaOS-2 on (A) 4G2C, (B) 4G2C10HY,
(C) 4G2C20HY, (D) 4G2C30HY and (E) 4G2C50HY cryogels matrix
after 10 days of culture at 5000x magnification.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 366–378).
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Fig. 31. (i) Steps of surgical procedures: (a) Exposed rat calvarium (b) Selected area on
parietal bone (c) Round critical size defect (8 mm). Defect filled with (d) CHA-1RS
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nanocomposite scaffold (e) Cerabone (f) sham control. (g) Periostium sutured with vicryl (h)
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Skin stitched with silk. (ii) Histopathology of bone: Overviews and magnified images of the
black-lined squares of the sections at 4 weeks post-implantation (a-c): (a) Sham control group
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(b) Cerabone Group, (c) CHA-1RS group. Stains H & E and SR; initial magnification x200.
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[OB: Osteoblasts, CF: Collagen Fibre, CB: Cerabone, IB: Immature bone, MB: Mature Bone,
NC: Nanocomposite scaffold, FT: Fibrous tissue]. (Reprinted with permission from Elsevier,
Carbohydr. Polym. 179 (2018) 317–327).
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Fig. 31. Continued.

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Fig. 32. Histopathology of bone: Overviews and magnified images of the black-lined squares of the sections at 8
weeks post-implantation (a-c): (a) Sham control group (b) Cerabone group (c) CHA-1RS group. Stain H & E
and SR. Initial magnification x200. Histopathological results of Kidney (d,e) and liver (f,g). Left panel (d & f)
are from control group and right panel (e & g) from CHA-1RS group. H & E stain, initial magnification x200.
[OC: Osteoclasts, OB: Osteoblasts, CF: Collagen Fibre, CB: Cerabone, MB: Mature Bone, FT: Fibrous tissue].
(Reprinted with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).
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Fig. 33. (i) Radiographs: X ray and RVG 4 weeks: (a,g) sham control, (b,h) cerabone, (c,i) CHA-1RS
group, and 8 weeks post-implantation: (d,j) sham control, (e,k) cerabone, (f,l) CHA-1RS respectively.
Harvested calvaria viewed from inside: (m) Sham control, (n) Cerabone, (o) CHA-1RS after 8 weeks
post-implantation with arrows indicating defect site. (ii) Quantitative analysis of the Gain in Bone density
(GBD)% of CHA-1RS, CB and Sham control groups after the time period of 4 and 8 weeks. (Reprinted
with permission from Elsevier, Carbohydr. Polym. 179 (2018) 317–327).
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Fig. 33. Continued.

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Fig. 34. Histopathology of bone from CHA-1RS group after 2 weeks post implantation:
Overview and magnified images of the black-lined squares (a) Stain H & E and SR, initial
magnification x200. (b) Bone defect site in situ (Top view) and (c) the bone defect site as seen
from inside (Bottom view) and (d) its X-Ray and (e) RVG radiographs with arrows indicating
defect site. [OB: Osteoblasts, NC: Nanocomposite scaffold, HB: Host Bone, WB: Woven
Bone, CT: Connective Tissue, DM: Defect Margin]. (Reprinted with permission from
Elsevier, Carbohydr. Polym. 179 (2018) 317–327).
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Fig. 35. (a) Biofilm formation by S. mutans in the presence of chitosan and conjugate Ag
nanoparticle. (b) ImageJ analysis of biofilm biomass.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 790-797).
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Fig. 36. Phase contrast Image of HGF before and after treatment with nanoparticle materials.
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(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 108 (2018) 790-797).
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Fig. 37. Cell viability studies using XTT assay.

(Reprinted with permission from Elsevier, Eur. J. Pharm. Biopharm. 109 (2016) 61–71).
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Fig. 38. Slit-lamp photograph of (a) eye after wearing functional contact lens for 35 days;
(b) untreated eye after 35 days. Light microscope observation (HE stain) of (c) eye after wearing
functional contact lens for 35 days; and (d) untreated eye after 35 days eye at day 35 after surgery.
(Reprinted with permission from Elsevier, J. Mech. Behav. Biomed. Mater. 64 (2016) 43–52).
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Fig. 39. In vivo biocompatibility studies of C7P3 by subcutaneous implantation in rat model
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(a) subcutaneous pouch (b) implantation of C7P3 scaffold (c) retrieval of tissue integrated
with C7P3 in back of albino Wister rat; (d) H&E (Hematoxylin & Eosin); (e)MT (Masson
Trichrome) and (f) TB (Toluidine Blue) staining of subcutaneous tissue harvested from the
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implanted site; Black line demarcates between host and scaffold, black arrows represent blood
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vessel and star represents mast cells.

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Fig. 40. Photographs (a) and closure rate (b) of wounds treated with Tegaderm™ (control)
and C7P3 during the wound healing process for 21 days. Arrow represents implanted
scaffold, Y-error bars represent standard deviation.
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Fig. 41. The healing promoting effect of CCA composite dressing on seawater immersion
wound of rat. A: The wound healing image of the dressings. B: The wound healingratio
statistics of the dressings. compared to gauze group *P<0.05), ** P<0.01.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 93–104).
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Fig. 42. The effects of different dressings on the healing process of rat dorsalis skin wounds
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on the fifth, eighth, 11th , and 13th days post-surgery examined by H&E staining (×200).
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 107 (2018) 93–104).
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Fig. 43. (a) Average volume of mice-bearing HeLa tumors at different time points after
intravenous injection of PBS, free DOX, Ag2 S@CS and Ag2 S(DOX)@CS nanospheres via
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tail vein. Statistical significance: *P<0.05. (b) Body weight changes of the tumor-born mice
after treatment with PBS, free DOX, Ag2 S@CS and Ag2 S(DOX)@CS nanospheres. The data
are presented as average±standard deviation (n=3). (c) ICP-MS analysis of tumor and five
major organs of the mice sacrificed at different time points after injection of
Ag2 S(DOX)@CS. The data are presented as average±standard deviation (n=5). Statistical
significance: *P<0.05. (d) In vivo NIR images of a nude mouse at 6 h (i), 12 h (ii)and 24 h
(iii) after injection of the Ag2 S(DOX)@CS nanospheres; ex vivo NIR image of the tumor (iv)
and the organs (v) harvested from the sacrificed nude mouse.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 157 (2017) 325–334).
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Fig. 43. Continued.

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Fig. 44. Inhibition of Staphylococcus aureus and Escherichia coli following antibiotic disk
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diffusion assay. GEL:gelatin, CS:chitosan, R:Rosemary,C:cinnamon, B:boldo and G:guaraná.

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A:GEL50:CS50 films with extracts added against S. aureus B: GEL50:CS50 films with
extracts added against E. coli.
(Reprinted with permission from Elsevier, Food Biosci. 16 (2016) 17–25).
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Fig. 45. Image of flexible antimicrobial C-2 pouch.

(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 97 (2017) 382–391).
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Fig. 46. Image of packed meat samples in C-2 pouches (A1, A2 & A3) and plastic bags (B1, B2 & B3).
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 97 (2017) 382–391).
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Fig. 47. HR-SEM images of (a) CS-NS composite (b) Untreated cotton fabric (c) CS-NS
composite coated cotton (d) CS-NS composite coated cotton fabric with glutaraldehyde
and (e) CS-NS composite coated cotton fabric with citric acid.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 104 (2017) 1890–1896).
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Fig. 48. SEM at low and high magnification of (A) untreated cotton fabrics, cotton fabric
treated with (B) CS solution, (C) CS/AgNPs nanocomposite
and (D) CS/AgNPs/clay nanocomposite.
(Reprinted with permission from Elsevier, Carbohydr. Polym. 182 (2018) 29–41).
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Fig. 49. The morphology and structure of the ChS-CS nanogel. It is observed that there is
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more than one hydrophobic nanodomain in the nanogel structure. Additionally, the
complexation process was mainly occurred by the hydrophobic interactions between the FSH
hydrophobic patch and the cholesterol groups in the hydrophobic nanodomains existing in the
nanogel. (Reprinted with permission from Elsevier, Eur. J. Pharm. Sci. 107 (2017) 126-137).
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Fig. 50. The solvent-exposed hydrophobic patch in FSH. (Reprinted with permission from
Elsevier, Eur. J. Pharm. Sci. 107 (2017) 126-137).
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Fig. 51. The structures of various CS-Tacrine systems in which the polymer chains and
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Tacrine molecules are indicated in green and pink colors, respectively.

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Fig. 52. The structures of different PBCA-Tacrine systems in which the polymer chains and
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Tacrine molecules are indicated in blue and pink colors, respectively. (Reprinted with
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permission from Elsevier, Eur. J. Pharm. Sci. 82 (2016) 79–85).

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Fig. 53. Simulation of CS-NaOH film. [A] Three stages of the MD simulation from a crystal-
like structure (0 ns) to a relax solvated film (50 ns). Polysaccharide chains are labelled with
letters from A to P. Distances between the closest atoms of neighbor chains computed
horizontally [B] or vertically [C]. (Reprinted with permission from Elsevier, Carbohydr.
Polym. 206 (2019) 57–64).
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Fig. 53. Continued.

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Fig. 54. Best energy ranked docking complexes for D-Tyr (purple sticks) [A] and L-Tyr
(turquoise sticks) [B]. Dashed yellow lines represent hydrogen bonds.
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Fig. 55. The last snapshot of the system after 20 ns indicating

drug loading values of (a) 7.3%, (b) 10% and (c) 12%.
(Reprinted with permission from Elsevier, Biomaterials 34 (2013) 1843-1851).
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Figure 56. (A) The total, hydrophobic and hydrophilic SASA values of the chitosan
nanoparticles during the MD simulation. (B) The total number of hydrogen bonds between the
chitosan molecules and the water molecules to compare with the total number of the chitosan-
chitosan hydrogen bonds. (C) Radial distribution function (RDF) between water oxygen
atoms to study the effect of the chitosan nanoparticles on the water structure.
(Reprinted with permission from Elsevier, Int. J. Biol. Macromol. 103 (2017) 902-909).
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Fig. 57. Final 3D structure of protein-polymer complexes; (A) before simulation and (B) after
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simulation. (Reprinted with permission from Elsevier, Carbohydr. Polym. 203 (2019) 52-59).
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Highlights

 Numerous pharmaceutical applications of chitosan-based compounds were reviewed.

 Chitosan was applied in pharmaceutics/drug/gene delivery and cell encapsulation.

 Chitosan was used in wound healing, tissue engineering, bioimaging and food

industry.

 Chitosan was applied in binding to protein drugs, contact lenses and implants.

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 Molecular dynamics simulations were done on pharmaceutical applications of

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chitosan.

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