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Chitosan-based nanoparticles as drug delivery systems: A review on two

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DOI: 10.1080/1061186X.2018.1512112

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JOURNAL OF DRUG TARGETING
2019, VOL. 27, NO. 4, 379–393
https://doi.org/10.1080/1061186X.2018.1512112

REVIEW ARTICLE

Chitosan-based nanoparticles as drug delivery systems: a review on two decades

of research
Sweet Naskar, Ketousetuo Kuotsu and Suraj Sharma
Department of Pharmaceutical Technology, Jadavpur University, Kolkata, India

ABSTRACT ARTICLE HISTORY

Chitosan (CS) is one of the most functional natural biopolymer widely used in the pharmaceutical field due Received 8 May 2018
to its biocompatibility and biodegradability. These privileges lead to its application in the synthesis of nano- Revised 7 August 2018
particles for the drug during the last two decades. This article gives rise to a general review of the different Accepted 8 August 2018
chitosan nanoparticles (CSNPs) preparation techniques: Ionic gelation, emulsion cross-linking, spray-drying,
KEYWORDS
emulsion-droplet coalescence method, nanoprecipitation, reverse micellar method, desolvation method, Chitosan nanoparticles;
modified ionic gelation with radial polymerisation and emulsion solvent diffusion, from the point of view of drug delivery;
the methodological and mechanistic aspects involved. The physicochemical behaviour of CSNPs including Pharmaceutical applications;
drug loading, drug release, particles size, zeta potential and stability are briefly discussed. This review also Derivatives of Chitosan
directs to bring an outline of the major applications of CSNPs in drug delivery according to drug and route nanoparticles; Chitosan
of administration. Finally, derivatives of CSNPs and CS nano-complexes are also discussed. nanocomplexes

Abbreviations: CSF: Cerebrospinal fluid; 5-FU: 5- Fluorouracil; CyA: Cyclosporin A; FITC-BSA: Fluorescein
isothiocyanate labeled bovine serum albumin; pDNA: Plasmid deoxyribonucleic acid; CS- NAC: Chitosan N-
acetyl-L-cysteine; Glycol CS-TGA: Glycol chitosan thioglycolic acid; CS-TGA: Chitosan thioglycolic acid; CS-
TBA: Chitosan $- thiobutylamidine; pSEAP: Recombinant secreted alkaline phosphate; OVA: Ovalbumin;
Thiolated HA: Thiolated hyaluronic acid; SVN: Survivin; SiRNA: Small interfering ribonucleic acid.

Introduction crystalline structure due to the inter and intra-molecular hydrogen

Biodegradable polymeric nanoparticles (NPs) can act as efficient
drug delivery vehicles for controlled and targeted drug [1].
Chitosan (CS) is a polysaccharide, used as NPs in the pharmaceut-
ical field since two decades ago [2]. CS is a biodegradable,
biocompatible and non-toxic biopolymer, made up of b-1 ! 4
linked 2-amino-2-deoxy-glucopyranose and 2-acetamido-2-deoxy-
b-D-glucopyranose residues. Chitin, a biopolymer extracted from
the exoskeleton of crustaceans, such as crabs and shrimp is used
to prepare CS, is produced by alkaline N-deacetylation [3–5]. CS
shows its antibacterial activity only in an acidic medium above pH
6.5 [6].
Source and physicochemical properties of CS
CS is a polysaccharide composed of b-1, 4-linked D-glucosamine
residue. It is derived from the deacetylation of chitin, a naturally
occurring polymer found in the exoskeletons of crustaceans and
insects [6]. CS has been officially agreed by FDA for the use of
wound dressings as well as in dietary applications in Japan, Italy
and Finland [7]. It is produced on a commercial scale by exhaust-
ive deacetylation of chitin with the concentrated solution of
sodium hydroxide [8,9]. CS, a non-antigenic hydrophilic polymer,
containing one amino group and two hydroxyl groups in the
restating hexosaminic residue [10,11]. The amino groups make CS
a natural polyelectrolyte that readily dissolves in an acidic solu-
tion. The insolubility of CS in the solution and the degree of
acetylation (DA) effect on the density of it. CS possesses a
bonds between the hydroxyl and amino groups. Due to the pres-
ence of N-acetyl groups, CS shows a slight degree of hydrophobic
behaviour [12–18].
Preparation of chitosan nanoparticles (CSNPs)
CSNPs have been fully recorded in the literature as a carrier sys-
tem for various drug delivery systems.Since first reported in 1994
by Ohya et al. [19], various techniques have been used to
develop CSNPs.
Ionic gelation
In this method, firstly, CS is dissolved in an aqueous acidic solu-
tion to obtain the cation of CS. After that, this solution is added
dropwise to anionic tripolyphosphate (TPP) solution under con-
stant stirring. CS undergoes ionic gelation due to the complex-
ation between polyanion TPP and cationic CS by electrostatic
forces and precipitates to form spherical particles [20–36]. The
method is schematically represented in Figure 1.
Emulsion cross-linking
In this method, firstly, an aqueous CS solution is emulsified in the
oil phase to prepare water-in-oil (W/O) emulsion and after that
aqueous droplets are stabilised using a suitable surfactant. Then,
the stable emulsion is cross-linked with the most versatile cross-

CONTACT Ketousetuo Kuotsu ketousetuoju@yahoo.in Department of Pharmaceutical Technology, Jadavpur University, Kolkata 700032, West Bengal, India
This article has been republished with minor changes. These changes do not impact the academic content of the article.
ß 2018 Informa UK Limited, trading as Taylor & Francis Group
380 S. NASKAR ET AL.
Figure 1. Representation of preparation of CSNPs by ionic gelation method.
Figure 2. Representation of preparation of CSNPs by emulsion crosslink-
ing method.
Figure 3. Representation of preparation of CSNPs by spray drying method.
linking agent, i.e. glutaraldehyde. Here, cross-linking reaction took
place between the amino groups of CS with the aldehyde groups
of glutaraldehyde and precipitates to form particles [19,37,38].
This method is schematically shown in Figure 2.
Figure 4. Representation of preparation of CSNPs by emulsion-droplet coales-
cence method.
Spray-drying
In this method, CSNPs are prepared by use of a nanospray dryer.
The method depends on the drying of atomised droplets in a hot
air stream. Firstly, a CS solution is prepared by dissolving purified
CS powder in an acetic acid solution and stores it until overnight.
Small droplets are formed due to atomisation. The solvent is
evaporated from small droplets instantaneously leading to the
formation of NPs. This method is schematically shown in
Figure 3 [39–42].
Emulsion droplet coalescence method
In this method, at first, two stable emulsions are formed. At firstly,
a stable emulsion containing an aqueous solution of CS along
with drug is brought about in liquid paraffin oil. After that,
another stable emulsion containing CS aqueous solution of NaOH
is carried out in liquid paraffin oil. Then two emulsions are mixed
under high-speed stirring. When these two emulsions are mixed,
then each emulsion droplets would come into conflict at random
and coalesce, as a result of that precipitating CS droplet to form
small size particles [43,44]. The method is schematically shown in
Figure 4.
Nanoprecipitation
In this method, firstly, CS is dissolved in a suitable solvent to form
the diffusing phase. This phase is then added to the dispersing
phase i.e. methanol under magnetic stirring condition. The diffus-
ing phase is added to the dispersing phase by means of a needle
positioned 2 cm above the surface at 0.86 ml min1 using a peri-
staltic pump. Then the addition of a very small amount of

Figure 5. Representation of preparation of CSNPs by nanoprecipitation method.
Figure 6. Representation of preparation of CSNPs by reverse micellar method.
polysorbate-80 to the non-solvent phase provides smaller NPs
regardless of the non-solvent to solvent volume ratio [45]. The
method is schematically represented in Figure 5.
Reverse micellisation method
In reverse micellisation method, W/O droplets are known as
reverse micelles [46,47]. Firstly, a W/O emulsion is prepared using
a lipophilic surfactant [sodium bis(ethyl hexyl) sulfosuccinate or
cetyl trimethyl ammonium bromide] that is dissolved in an appro-
priate organic solvent, such as n-hexane. After that, an aqueous
CS solution, drug and glutaraldehyde are added to the organic
phase under continuous stirring to avoid turbidity. Finally, extrac-
tion of NPs is done [48–50]. This method is schematically shown
in Figure 6.
Desolvation/simple coacervation/phase separation
In this method, at first, a CS solution is prepared using a suitable
solvent. After that, the solvent competing agent of greater hydro-
philicity (e.g. sodium sulphate) is added to this CS solution. When
saltly enters in contact with the aqueous environment of CS solu-
tion then a progressive elimination of salvation water surrounding
CS occurs as a result of the higher affinity of water for the salt.
This step leads to the polymer insolubilisation and as a result, pre-
cipitation occurs [51–53]. Sodium sulphate [19,54–56] and acetone
[57] are used as precipitating agents in this process. Polysorbate-
80 is used as a stabilising agent in the preparation medium, to
stabilise the nanoparticle suspension. After that, glutaraldehyde is
JOURNAL OF DRUG TARGETING 381
Figure 7. Representation of preparation of CSNPs by desolvation method.
Figure 8. Representation of preparation of CSNPs by modified ionic gelation with
radical polymerisation method.
added to the formulation, in order to harden the NPs [51]. This
method is schematically shown in Figure 7.
Modified ionic gelation with radical polymerisation
In this method, under stirring condition, an aqueous acrylic acid
monomer solution is added to an aqueous CS solution at room
temperature. Sometimes, polyethylene glycol (PEG) or polyether
(PEG–polypropylene glycol–PEG) is also added to the reaction
medium, either separately into the monomer solution or the fol-
lowing mixing with CS. Ionic gelation occurs due to ionic inter-
action between the cation of CS and anion of acrylic acid
monomer and radical polymerisation of the latter is started with
potassium persulfate under stream nitrogen at 60–70  C. After, the
polymerisation reaction the formed NPs suspension is allowed to
settle overnight. Finally, the dialysis process is used to remove
unreacted monomers [58–60]. This method is schematically shown
in Figure 8.
Emulsion solvent diffusion
In this method, firstly, the organic phase (e.g. methylene chloride
and acetone) containing the hydrophobic drug is added to an
aqueous solution containing CS and a stabiliser (e.g. poloxamer
and lecithin) to prepare an oil-in-water (O/W) emulsion under stir-
ring condition. Then the formed O/W emulsion is subjected to
high-pressure homogenisation and methylene chloride is removed
under reduced pressure at room temperature. At this stage,
382 S. NASKAR ET AL.
Figure 9. Representation of preparation of CSNPs by emulsion solvent diffu-
sion method.
acetone is diffused into the aqueous phase, decreasing CS solubil-
ity and thus, NPs are formed by polymer precipitation. An extra
amount of water is usually added in order to permit the complete
diffusion of acetone. Ultimately, centrifugation is used to separate
NPs [61]. This method is schematically shown in Figure 9.
Cross linker of CSNPs
Aldehyde
Due to low-cost production, great fixation and the formation of a
thick cationic emulsion, glutaraldehyde is used as a versatile cross-
linker [62,63]. From the mechanistic point, a covalent crosslinking
takes place between the aldehyde groups of glutaraldehyde and
the amino groups of CS, as a result, Nps formation takes place
[48]. Overt toxicity and balanced drug integrity are the drawbacks
of glutaraldehyde [64]. As per the report, the particle size has also
been found to vary with the amount of glutaraldehyde [52].
Natural occurring a,b-unsaturated aldehyde, cinnamaldehyde is
another cross-linker used to prepared CSNPs [65]. Baicalein-loaded
CSNPs were successfully prepared by chemical cross-linking with
cinnamaldehyde as a cross-linking agent, by cross-linking method
in a W/O emulsion system [66]. Vanillin (3-methoxy-4-hydroxyben-
zaldehyde) is another important phenolic aldehyde is used to pre-
pare CSNPs [67]. From the mechanistic point, chemical
crosslinking takes place between the aldehyde groups of vanillin
and the amino groups of CS, as a result, NPs formation take
place [68].
Tripolyphosphate
TPP is a non-toxic, polyanion crosslinker. It has multivalent anions
in its structure [69]. The amino groups of CS undergo reversible
physical cross-linking through electrostatic interaction with the
negatively charged TPP molecules, as a result, NPs formation takes
place [70,71]. Several advantages have been progressively indi-
cated for TPP, including the reversible physical cross-linking by
electrostatic interactionmakes it possible to prevent the toxicity of
reagents [72].
Genipin
Natural crosslinker genipin is obtained from gardenia fruit that
has been utilised in herbal medicine and fabrication of food dyes.
The cytotoxicity of genipin is approximately 10,000 times less than
glutaraldehyde [73,74]. Firstly, Song et al. [75] proposed the mech-
anism of proteins crosslinking reaction by genipin. Kumar et al.
[76] synthesised ciprofloxacin-loaded genipin cross-linked CS/hep-
arin nanoparticles (CIPRO-GP-CS/Hep NPs). They prepared these
NPs by ionic gelation method. The antibacterial activity of the pre-
pared NPs was evaluated against Escherichia coli MTCC 443
by them.
Citric acid
Thermal cross-linking is another aspect of physical cross-linking in
which citric acid (CA), a commonly used cross-linking agent is
added to an aqueous CS acidic solution in a constant molar ratio
between CA and CS [77]. Bagheri et al. [78] modified CS with carb-
oxyl functional groups using CA as a cross-linking agent at the dif-
ferent molar ratio of CS to CA.
Other crosslinkers
Sometimes, malic acid, tartaric acid, CA and succinic acid were
used as a cross-linking agent for the preparation of CSNPs. The
carboxylic groups of di and tricarboxylic acids undergo chemical
cross-linking with the amino groups of CS, as a result, NPs forma-
tion take place [79]. Sodium sulphate is another important cross-
linking agent used for the preparation of CSNPs.The sulphate
anion of sodium sulphate undergo crosslinking with the positively
charged functional groups of CS [80].
Physicochemical behaviour of CSNPs
Drug loading & drug release
Drugs were loaded into CSNPs may be either incorporated the
drug at the time of NPs production or absorbed the drug after
the formation of NPs by incubating them in the drug solution.
Diffusion and desorption are involved in the drug release from
CSNPs [81]. The protein release from CSNPs may be due to three
predominant mechanisms including desorption, diffusion and
release due to polymer erosion of CSNPs [82]. Many factors were
found to influence the drug release rate from CSNPs including
solubility, diffusion and biodegradation of the matrix materials,
the choice of polymer matrices, the drug loading capacity and
size of NPs [1]. The equation which is used to describe drug
release mechanism
Mt
¼ ktn ;
M1
Mt
where M1 is equal to the drug release in the fraction at time t, k
is equal to the constant which signify the macromolecular poly-
mer system properties.The n value is equal to the analysis of drug
release mechanism from the drug loaded NPs [83,84].
Particle size &surface charge
Most CSNPs prepared by the above methods have recorded mean
sizes ranging from 100 to 400 nm. Particle size is one of the most
important features related to obtaining optimal in vitro efficacy of
CSNPs [85,86]. Particle size and its distribution were affected by
encapsulation efficiency and zeta potential [87]. Decrease the par-
ticles size could increase the specific surface area and the ratio of
surface to volume, which could increase the dissolution and thus
increase the bioavailability of poorly water-soluble drugs [88].
Molecular weight and degree of deacetylation of CS are factors
found to affect particle size and surface charge [89]. Particle size
and zeta potential are important factors which may influence the
antifungal activity of NPs [90].

Zeta potential/surface charge is an important parameter of NPs
for the determination of their interaction in vivo with the nega-
tively charged tumour cell membrane, stability, mucoadhesives
and permeation enhancing effect [91–93]. Zeta potential has the
greater influence on the particle stability in suspension. The less
zeta potential, the less likely the suspension to be stable [94].
Stability
The stability of NPs is an important factor in the pharmaceutical
field until the loss of therapeutic efficacy [95]. Agglomerations of
particles, bridging flocculation, coagulation are the predominant
factors for the physical instability of NPs [96]. Chemical stability of
NPs depends on the following conditions like temperature, pH of
the medium, composition of the formulation, type of polymer
used and the molecular weight of the polymer used in the formu-
lation. Most of the drugs sensitive to pH, hence pH should be
optimised to control the leakage of the drug [51]. CS chains pos-
sess glucosamine groups that may be deprotonated if the pH
increases [97]. It was recorded that CSNPs prepared using sodium
TPP and glycidoxy propyl trimethoxysilane cross-linking were pH
sensitive [98]. Other research demonstrated that camptothecin-
loaded N-isopropyl acrylamide-CSNPs were sensitive to tumour pH
when the charge ratio of N-isopropylacrylamide: CS was 4:1 [99].
Cytotoxicity study& cellular uptake
Cytotoxicity of CSNPs has been carried out to determine cell via-
bility. Cell viability and cell cytotoxicity have been carried out by a
simple non-radioactive colorimetric MTT (3–(4,5-dimethylthiazol-2-
yl)-2, 5 diphenyltetrazolium bromide) assay [100]. Corsi et al. [101]
prepared CS–DNA NPs and assessed the effect of prepared NPs
cell viability on human osteosarcoma cells, MSCs and HEK293,
compared to a commercial lipid, Lipofect AMINETM2000 (LF). They
measured the cell viability by MTT assay. In another study,
Mansouri et al. [102] synthesised FA–CS–DNA NPs and evaluated
their effect on cell viability compared to LifofectAMINETM2000 (LF),
a commercially available lipid vector [102]. The cytotoxic activities
of the CSNPs and copper (II)-loaded CSNPs was investigated by Qi
et al. [103] and a relationship between physiochemical properties
and activity is suggested. They used different cell lines for this
study. That author suggested that CSNPs and copper (II)-loaded
CSNPs showed much higher cytotoxicity toward BEL7402, BGC823
and Colo320 tumour cells while showing a slight effect on L-02
human normal liver cells.
Efficient cellular uptake is an important parameter for NPs.
Confocal microscopic analysis is used to check the uptake of
CSNPs into the cell nucleus [100] after 4 h exposure with 1% w/v
of CSNPs. Necrotic or autophagic cell death, possibly caused by
cell membrane damage is evoked by electron micrographs [104].
Pharmaceutical applications of CSNPs
A specific administration route is highly essential for delivery of
drugs to the site of its action [105]. A detail study of such admin-
istration to drug delivery is outlined below.
Mucosal delivery
Nanocarriers have appeared as an effective strategy for mucosal
drug delivery due to small steric obstruction and protection of
freight therapeutics at both extracellular and intracellular range
JOURNAL OF DRUG TARGETING 383
[105]. Due to the mucoadhesive property, CS has been used
extensively as a carrier for mucosal drug delivery [106–108].
Baltzley et al. [109] studied the probable use of CSNPs as an intra-
nasal delivery system to amplify olanzapine systemic bioavailabil-
ity. They prepared these NPs through ionotropic gelation method.
They studied particle size, drug loading and in vitro release. Al-
Ghananeem et al. [110] studied the possible use of CSNPs as an
intranasal delivery system to amplify didanosine systemic and
brain targeting efficiency. They prepared these NPs through iono-
tropic gelation method and they studied prepared NPs size, drug
loading and in vitro release. Thus, both the intranasal route of
administration and formulation of didanosine in CSNPs aug-
mented the delivery of didanosine to CSF and brain.
Cancer drug delivery
Due to compatibility and biodegradability of CS, CSNPs are an
emerging field in the drug delivery of cancer [111]. CSNPs have
been largely used for the delivery of anti-cancer drugs including
methotrexate [112], 5-flourouracil [113,114], cysplatin [115], epiru-
bicin [116], curcumin [117,118], mitomycin C [119], docetaxel
[120], doxorubicin (DOX) [49,121], paclitaxel [122] and tamoxifen
[123], focussing to augment anti-tumour efficacy, control release
and direct the drugs to the tumour thus reducing the toxicity.
In those studies, CSNPs were developed for: (i) to release 50%
of the methotrexate loaded in 48 h [112], (ii) to sustain release of
5FU compared with the 5FU solution [113], (iii) to investigate NPs
were able to release the drug cysplatin in a sustained manner for
a prolonged period of time [115], (iv) entrapment of epirubicin
into cholesterol-modified CS and show pH-dependent release of
drug in vitro [116], (v) encapsulation of doxorubicin in CS-based
NPs could be released into the cells in its active form [121], (vi)
encapsulation of mitomycin C as intravesical chemotherapeutic
agent and selectively incorporate NPs containing molecules in the
bladder cancer cell line, but not for normal bladder epithelial cells
[119], (vii) the reduction of drug toxicity compared with free drug
and reduce tumour volume in mice [120], (viii) carrier/vehicle/pro-
drug of cancer therapy [117,122,124–126], (ix) encapsulation of
dextran–doxorubicin conjugate in CSNPs [49], (x) to control the
release of drug and to increase tamoxifen chemotherapeutic effi-
ciency [123], (xi) chemotherapy of breast cancer in which the side
effects of traditional chemo treatment could be reduced [114].
Anti-microbial drug delivery
Nowadays, CSNPs have been extensively utilised for drug delivery
applications in various infectious diseases. The functional groups
help to aim loaded drugs to the site of infection. Slow biodegrad-
ation of CS permits controlled and sustained release of loaded
moieties, reduces the dosing frequency and suitable for raising
patient compliance in infectious diseases [127]. CSNPs have been
extensively used for the delivery of anti-microbial drugs including
cefazolin [128], rifampicin [129–131], daptomycin [132], ofloxacin
[133], amphoterecin B [134–136], vancomycin [137,138], ciprofloxa-
cin [139], amoxicillin [140], isoniazid [130,139] and tetracyc-
line [141].
In those studies, studies, CSNPs were developed for: (i) better
encapsulation efficiency (EE), good stability, excellent activity
against Klebseliapnominia, Paeroginosa and lactamase positive E.
coli [128], (ii) better entrapment efficiency and good corneal per-
meability [132], (iii) rapid bactericidal activity and reduced dose
frequency [129], (iv) better corneal permeation [133], (v) greater
efficacy and better safety than commercial products [134], (vi)
384 S. NASKAR ET AL.
high entrapment efficiency, sustained release, prolong residence
time [135], (vii) to sustain release, low toxicity, preferential alveolar
uptake and greater effectiveness [130], (viii) to sustain drug
release [137], (ix) better internalisation, low dose and reducing
dose frequency [142], (x) greater efficacy and complete removal of
H. pylori [140], (xi) selective macrophage uptake and greater effi-
cacy [131], (xii) prolonged residence time and better corneal per-
meability [136], (xiii) to increase stability, targeted drug delivery
and control the release of drug [138], (xiv) higher mucoadhesion
higher swelling [139], (xv) productive intracellular delivery and
dose reduction [141].
Colon-targeted drug delivery
Nowadays, CS is recognised as the polymer for oral site-specific
drug delivery [143]. Ulcerative colitis, crohn’s disease, pseudomem-
branous colitis and irritable bowel syndrome are the colonic dis-
eases where CSNPs has been used in recent year [144,145]. CS is
recognised as the polymer for colon-targeted delivery because it
has a tendency to dissolve in acidic pH of the stomach and get
swollen in the intestinal pH [146].
As colon-targeted drug delivery, CSNPs were developed for: (i)
improving uptake in HT-29 cell and colorectal cancer [147], (ii)
releasing of 5-aminosalicylic acid from NPs was based on the ion-
exchange mechanism [148], (iii) sustaining in vitro drug release
[149], (iv) CS–DNA NPs were more stable in the upper regions of
the small intestine suggested that higher uptake rates may occur
in the duodenum compared with the ileum and the colon [150],
(v) targeting drug delivery to colon tumour [151], (vi) in vivo fluor-
escent endoscopic detection of colorectal cancer cells [152], (vii)
the apoptosis was progressed on treatment with all-trans retinoic
acid bearing methoxy poly(ethylene glycol)-grafted CSNPs [153],
(viii) 5-FU in hyaluronic acid-coupled CSNPs was about to 60-fold
more effective than free 5-FU on HT-29 cells [154].
Ocular delivery
CS, hydrophilic natural biodegradable polymer improves stability,
precorneal retention and enhances interaction with eye mucosa.
Moreover, CS is recognised as the polymer suitable for ocular
drug delivery for the following reasons: non-toxic, low eye irrita-
tion, the sustained release, mucoadhesive, in situ gelling, transfec-
tion and permeation enhancing properties [155]. The
mucoadhesive polymer such as CS is able to form disulphide
bonds with mucus glycoproteins when found with the mucus gel
layer [156]. CSNPs have been extensively used for the delivery of
ocular drugs including CyA [34,157], FITC–BSA [158,159], indo-
methacin [160,161], pilocarpine [162,163], pDNA [164] and pred-
nisolone [165].
In those studies, CSNPs were developed for: (i) increasing the
therapeutic index of clinically challenging drugs [34], (ii) demon-
strating biocompatibility of the CSNPs [158], (iii) increasing drug
levels in the anterior segment of the eye in connection with the
uncoated systems [160], (iv) increasing the concentration of drug
detected in the anterior segment and the vitreous versus indo-
methacin eye drops [161], (v) sustaining drug release and prolong-
ing therapeutic effect of drug-loaded NPs [162,163], (vi) increasing
the drug levels detected in the aqueous humour versus those
observed with the commercial suspension [165].
Vaccine delivery
CSNPs for parenteral and mucosal delivery of antigen vaccines
have been developed [166–178]. CSNPs were widely apt for the
modern vaccinology facilitated by the oral and nasal delivery of
NPs with mucosal protective immune responses. The submicron
size of NPs promotes their uptake up by Mcells, in mucosa-
associated lymphoid tissue, i.e. gut-associated, nasal-associated
and bronchus-associated lymphoid tissue and other targeting sites
of vigorous immunological responses [167]. CS-based DNA flu vac-
cine for intranasal administration was formulated [179]. Smitha
et al. [180] developed O-carboxymethyl CSNPs encapsulated with
amidase. From this study, they concluded that the prepared NPs
can be used against S. aureus infections and CSNPs is the most
suitable candidate for the oral vaccine delivery system [180].
Pattani et al. [181] studied the immunological effects and mem-
brane interactions of CSNPs by using nitric oxide production, IL-6
gene expression and lymphocyte proliferation involved in the
wound-healing process. Bivas-Benita et al. [182] studied the pul-
monary delivery of DNA vaccines against tuberculosis. From the
results, they concluded that pulmonary delivery of DNA vaccines
may be a preferable delivery route compared to intramuscular
immunisation.
Transdermal drug delivery
Transdermal route is generally considered as ‘patient-friendly’ due
to the avoidance of gastrointestinal side effects which usually
require many oral preparations [183]. In the recent study, Al-
Kassas et al. [184] prepared CSNPs dispersed in the mucoadhesive
gel for the transdermal delivery of propranolol hydrochloride. Zeta
potential was found to increase with increasing the concentration
of CS. All prepared NPs showed 70% entrapment efficiency and
drug loading. From this study, they suggested that CSNPs dis-
persed into gel enhanced the propranolol–HCl systemic bioavail-
ability. In another study, Hafner et al. [185] investigated the
potential CSNPs as colloidal nanosystem for transder-
mal melatonin.
Anti-inflammatory drug delivery
CSNPs have been greatly used for the delivery of anti-inflamma-
tory drugs including zaltoprofen [186], hydrocortisone [97], ketoro-
lac tromethamine [188], tretinoin [189] aiming to increase anti-
inflammatory activity, improved efficacy and high phys-
ical stability.
In those studies, CSNPs were used for: (i) prolonging anti-
inflammatory activity [186], (ii) increasing anti-inflammatory activ-
ity and improving efficacy [187], (iii) showing anti-inflammatory
activity in ocular diseases [188], (iv) greater physical stability, bet-
ter potential for topical delivery and augmenting therapeutic effi-
cacy [189].
Miscellaneous drug delivery
CSNPs have also been used as delivery systems for miscellaneous
classes of drug including anti-HIV (lamivudine [190], zidovudine
[191]), anti-malarial (chloroquine [192], anti-tubercular (rifampicin
and isoniazid [130]), skeletal muscle relaxant (thiocolchicoside
[193]) and oral hypoglycaemic (insulin [194]).
In those studies, CSNPs were used for: (i) controlling drug
delivery applications [190], (ii) acting as drug delivery vehicle
against rodent parasite [192], (iii) sustaining the release of anti-
 JOURNAL OF DRUG TARGETING 385

Table 1. Main characteristics of NPs based on TCS derivatives.

Drug Polymer Gelling agent Particle size (nm) Zeta potential (mV) References
Insulin CS–NAC TPP 140–210 19–31 [199]
Calcitonin Glycol CS–TGA TPP 230–330 21–27 [200]
OVA Thiolated TMC Thiolated HA 250–350 10–20 [201]
pSEAP CS–TGA None 212–113 4–8 [202]
None CS–TBA TPP 268 4–19 [203]

Table 2. Main characteristics of NPs based on amphiphilic CS.

Drug Polymer Particle size (nm) Zeta potential (mV) References
Camptothecin Glycol CS–5b–cholanic acid 250–350 Not reported [208]
None Glycol CS–5b–cholanic acid 230–310 10–11 [206]
Doxorubicin O-carboxymethyl CS-2,2I,(ethylene dioxy)-bis-(ethylamine)-folic acid 150–200 10–20 [215]
Griseofulvin/Cyclosporin A/Ranitidine Quaternary ammonium palmitoyl glycol CS 100–500 Not reported [212]
None Glycol CS–5b–cholanic acid 300–400 10 [209]
Doxorubicin Oleoyl–CS 250–350 Not reported [211]

Table 3. Main characteristics of NPs based on quaternised CS.

Drug Polymer Gelling agent Particle size (nm) Zeta potential (mV) References
Anti-neuroexcitation peptide TMC TPP 255 32 [224]
OVA TMC TPP 300 20 [223]
Insulin TMC Poly(c-glutamic acid) 100 30 [222]
Insulin TMC TPP 250 25 [225]
OVA TMC TPP 254–300 20–61 [226]
FITC–BSA TMC TPP Not reported Not reported [227]

tubercular drugs such as isoniazid and rifampicin to the Quaternised derivatives

lungs [130].
Derivatives of CSNPs
Thiolated chitosan
Thiolated chitosan (TCS) is prepared by immobilisation of thiol
groups on the primary amino groups of CS backbone.TCS is a
new generation thiolated or thiolmer mucoadhesive polymer, pro-
vides much better mucoadhesive properties than CS [195]. TCS
NPs were expected to be themselves mucoadhesive, and hence,
fit to prepare nanocarriers such as NPs for oral drug delivery
[196,197]. Examples of the above thiolated derivatives are the fol-
lowing: CS–cysteine, CS–thiobutylamidine, CS–thioglycolic acid,
CS–polymethyl methacrylate [198], CS–sodium alginate [195] and
CS–glutathione conjugates. Main characteristics of NPs based on
TCS derivatives shown in Table 1.
Amphiphilic derivatives
Some amphiphilic derivatives such as glycol CS-5b-cholanic acid
conjugate [204–209], palmitoyl CS [210], oleoyl CS [211] were
developed by connecting hydrophobic structures to the CS back-
bone. Other amphiphilic derivatives such as quaternary ammo-
nium palmitoyl glycol CS [212] was developed from CS bearing
fixed positive charges or linoleic acid-modified O-carboxymethyl
CS [213]/deoxycholic acid-modified N,O-carboxymethyl CS [214]
were prepared from CS bearing fixed negative charges. Oleoyl CS,
hydrophobically modified CS derivative from which self-assembled
NPs was obtained [211]. O-carboxymethyl CS, an amphiphilic CS
derivative that would selfassemble into NPs when folic acid
was conjugated with O-carboxymethyl CS via the bifunctional
2,20 (ethylenedioxy)-bis-(ethylamine) [181]. Quaternary ammonium
palmitoyl glycol CS is used to enhance absorption of hydrophobic
and hydrophilic drugs in rat orally [212]. Main characteristics of
NPs based on amphiphilic CS shown in Table 2.
Trimethyl chitosan (TMC) being a quaternary derivative of CS has
shown better mucoadhesive, permeation enhancement, drug
delivery and DNA delivery properties [216]. Examples of the above
quaternised CS derivative are the followings: N-TEC [183,217],
N-diethyl methyl CS [218], N,N-dimethyl N-ethyl chitosan (DMEC)
[219] and N,N-diethyl N-methyl CS [220]. N-trimethylene chloride,
a quaternary derivative of CS has the better aqueous solubility,
intestinal permeability and greater absorption over a wide pH
range [221]. NPs loaded with insulin were developed from TMC by
PEC method using poly(c-glutamic acid) as the polyelectrolyte
complexing agent [222]. NPs loaded with OVA were developed
from TMC using unmethylated CpG DNA as an adjuvant and a
crosslinker for nasal vaccination in mice [223]. Main characteristics
of NPs based on quaternised CS shown in Table 3.
CS nanocomplexes
CS formed nano-complex with an europium containing polyanion
[CsEu6As6W63O218 (H2O)14(OH)4]25-(EuWAs) without the help of
any crosslinker through ionotropic gelation method [228]. A few
negatively charged active drugs, such as insulin or gene drugs,
when mixed with cationic CS derivatives in adequate proportions
spontaneously formed nanoparticulate dispersions of insoluble
complexes [202,225,229–232]. For the systemic delivery of SVN
siRNA, a hybrid CS nano-complex made of CS, protamine, lecithin
and thiamine pyrophosphate was successfully developed. The tar-
get gene silencing and cellular uptake efficiencies of the siRNA
nanocomplexes in prostate cancer cells (PC-3 cells) were meas-
ured. PC-3 tumour xenograft mouse model by near-infrared fluor-
escence (NIRF) imaging and tumour growth monitoring was used
to assess in vivo tumour targetability and anti-tumour efficacy by
systemic administration [233]. Park et al. [234] prepared ionically
crosslinked Ad/CS nanocomplexes for targeted cancer gene ther-
apy. They managed it by the process of electrospinning. Cheng
et al. [235] have investigated the potential of DNA/CS
386 S. NASKAR ET AL.
nanocomplexes as a carrier for DOX. The in vivo biodistribution of
FITC–CS and DNA/FITC–CS nanocomplexes after intravenous injec-
tion to the mice was evaluated by them. After 24 h post-injection,
the DNA/FITC–CS nanocomplexes were accumulated into the liver
and kidney and remained at a relatively high stable level in blood,
while the fluorescence intensity of free FITC–CS decreased rapidly
within 4 h post-injection. In vitro cytotoxicity test confirmed that
DNA/CS–DOX conjugate exhibited cytotoxic effects on HeLa,
HepG2, QGY-7703 and L02 cells. In the study, Shah et al. [236]
three different nanoassemblies were made with CS. The
three nanoassemblies are CS–[NaP5W30O110]14 (CTS–P5W30),
Cs–[TeW6O24]6 (CTS–TeW6) and CS–[V10O28]6 (CTS–V10).
Zhao et al. [237] prepared TMC–cysteine conjugate (TMC–Cys)
nanocomplexes as a nonviral gene carrier.
Limitations
Due to CS unique and versatile biological properties, it has been
regarded as a useful polymer in the pharmaceutical area.
Considerable research attempt has been done in order to develop
safe and efficient CSNPs since 1994. However, poor stability of
CSNPs regulates its practical applicability; thus, it has become a
significant challenge to form adequate shelf-life for CSNPs formu-
lations. Stability can be improved by controlling the environmen-
tal factors, temperature, launching a proper stabilising compound,
flourishing CS blends with another polymer and altering the CS
structure using chemical/ionic agents. The problem like, the solu-
bility of CS has to be resolved urgently. Only some hydrophilic
drug can be encapsulated in unmodified CSNPs. Although modi-
fied CSNPs can encapsulate the hydrophobic drug.
Drawbacks and challenges
A nasal formulation of morphine (RylomineTM) based on CS is cur-
rently in Phase 2 clinical trials (UK and EU) and Phase 3 clinical tri-
als in the USA. We predict that when it comes to the market it
will approach the subject of similar products in the near future as
well as help in discerning any unforeseen outcome in humans
[238]. Simple and mild preparation methods can be used to pre-
pare CSNPs due to water-soluble property of CS which as an ideal
property for drug delivery carriers. In addition, CSNPs can be
administered through various route, especially non-invasive routes,
i.e. peroral, nasal and ocular mucosa, which are preferable routes
of administration and it acts as a good adjuvant in vaccine
delivery.Considerable attention has been directed to the applica-
tion of CSNPs.Bioavailability and absorption of drug enclosed into
CSNPs can be improved, so they can be used to deliver protein
drugs, gene drugs and can protect itself effectively from enzyme
degradation in vivo.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The authors wish to thank the University Grants Commission,
Bahadur Shah Zafar Marg, New Delhi, India, for financial support.
References
[1] Kumari A, Yadav SK, Yadav SC. Biodegradable polymeric
nanoparticles based drug delivery systems. Colloids Surf B:
Biointerfaces. 2010;75:1–18.
[2] Rampino A, Borgogna M, Blasi P, et al. Chitosan nanopar-
ticles: preparation, size evolution and stability. Int J
Pharm. 2013;455:219–228.
[3] Diop M, Auberval N, Viciglio A, et al. Design, characterisa-
tion, and bioefficiency of insulin-chitosan nanoparticles
after stabilisation by freeze-drying or cross-linking. Int J
Pharm. 2015;491:402–408.
[4] Vimal S, Taju G, Nambi KSN, et al. Synthesis and character-
ization of CS/TPP nanoparticles for oral delivery of gene in
fish. Aquaculture 2012;358–359:14–22.
[5] Tømmeraas K, Strand SP, Christensen BE, et al. Preparation
and characterization of branched chitosans. Carbohydr
Res. 2011;83:1558–1564.
[6] Chua BY, Kobaisi MAl, Zeng W, et al. Chitosan micropar-
ticles and nanoparticles as biocompatible delivery vehicles
for peptide and protein-based immunocontraceptive
vaccines. Mol Pharmaceutics. 2012;9:81–90.
[7] Kean T, Thanou M. Biodegradation, biodistribution and
toxicity of chitosan. Adv Drug Deliv Rev. 2010;62:3–11.
[8] Kafetzopoulos D, Martinou A, Bouriotis V. Bioconversion of
chitin to chitosan: purification and characterization of chi-
tin deacetylase from Mucor rouxii. Proc Natl Acad Sci USA.
1993;90:2564–2568.
[9] Eijsink V, Hoell I, Vaaje-Kolstada G. Structure and function
of enzymes acting on chitin and chitosan. Biotechnol
Genet Eng Rev. 2010;27:331–366.
[10] Agrawal P, Strijkers GJ, Nicolay K. Chitosan-based systems
for molecular imaging. Adv Drug Deliv Rev. 2010;62:42–58.
[11] Nishimura K, Nishimura S, Seo H, et al. Macrophage activa-
tion with multiparous beads prepared from partially
deacetylated chitin. J Biomed Mater Res. 1986;20:
1359–1372.
[12] Kwon S, Park JH, Chung H, et al. Physicochemical charac-
teristics of self-assembled nanoparticles based on glycol
chitosan bearing 5^a-cholanic acid. Langmuir 2003;19:
10188–10193.
[13] Yang Y, Wang S, Wang Y, et al. Advances in self-
assembled chitosan nanomaterials for drug delivery.
Biotechnol Adv. 2014;32:1301–1316.
[14] Ding F, Deng H, Du Y, et al. Emerging chitin and chitosan
nanofibrous materials for biomedical applications.
Nanoscale 2014;6:9477–9493.
[15] Hu X, Tang Y, Wang Q, et al. Rheological behaviour of chi-
tin in NaOH/urea aqueous solution. Carbohydr Polym.
2011;83:1128–1133.
[16] Wang Q, Zhang N, Hu X, et al. Chitosan/starch fibers and
their properties for drug controlled release. Eur J Pharm
Biopharm. 2007;66:398–404.
[17] Wang Q, Jamal S, Detamore MS, et al. PLGA-chitosan/
PLGA-alginate nanoparticle blends as biodegradable col-
loidal gels for seeding human umbilical cord mesenchy-
mal stem cells. J Biomed Mater Res A 2011;96A:520–527.
[18] Qun W, Yu-min D, Li h. F, et al. Structures and properties
of chitosan-starch-sodium benzoate blend films. J Wuhan
Univ (Nat Sci Ed) 2003;49:725–730.
[19] Ohya Y, Shiratani M, Kobayashi H, et al. Release behaviour
of 5-fluorouracil from chitosan-gel nanospheres immobiliz-
ing 5-fluorouracil coated with polysaccharides and their

cell specific cytotoxicity. J Macromol Sci A. 1994;31:
629–642.
[20] Kawashima Y, Handa T, Takenaka H, et al. Novel method
for the preparation of controlled-release theophylline
granules coated with a polyelectrolyte complex of sodium
polyphosphate-chitosan. J Pharm Sci. 1985;74:264–268.
[21] Kawashima Y, Handa T, Kasai A, et al. The effects of thick-
ness and hardness of the coating film on the drug release
rate of theophylline granules coated with chitosan-sodium
tripolyphosphate complex. Chem Pharm Bull. 1985;33:
2469–2474.
[22] Fernandez-Urrusuno R, Cavlo P, Remunan LC, et al.
Enhancement of nasal absorption of insulin using chitosan
nanoparticles. Pharm Res 1999;16:1576–1581.
[23] Pan Y, Li Y, Zhao H, et al. Bioadhesive polysaccharide in
protein delivery system: chitosan nanoparticles improve
the intestinal absorption of insulin in vivo. Int J Pharm.
2002;249:139–147.
[24] Aydin RST, Pulat M. 5-Fluorouracil encapsulated chitosan
nanoparticles for pH-stimulated drug delivery: evaluation
of controlled release kinetics. J Nanomater. 2012;2012:1.
[25] Doustgani A, Faraahani EV, Imani M. Preparation of chito-
san nanoparticles loaded bydexamethasone sodium phos-
phate. Iran J Pharm Res 2008;4:111–114.
[26] Leelapornpisid P, Leesawat P, Natakarnkitkul S, et al.
Application of chitosan for preparation of arbutin nano-
particles as skin whitening. JMMM 2010;20:101–105.
[27] Saha P, Goyal AK, Rath G. Formulation and evaluation of
chitosan-based ampicillin trihydrate nanoparticles. Trop J
Pharm Res. 2010;9:483–488.
[28] Rajendran NN, Natrajan R, Kumar RS, et al. Acyclovir-
loaded chitosan nanoparticles for ocular delivery. Asian J
Pharm. 2010;4:220–226.
[29] Motwani SK, Chopra S, Talegaonkar S, et al. Chitosan-
Sodium alginate nanoparticles as submicroscopic reser-
voirs for ocular delivery: formulation, optimization and
in vitro characterization. Eur J Pharm Biopharm 2008;68:
513–525.
[30] Senthil V, Kumar RS, Nagaraju CVV, et al. Design and
development of hydrogel nanoparticles for mercaptopur-
ine. J Adv Pharm Tech Res. 2010;1:334–337.
[31] Nanjwade BK, Singh J, Parikh KA, et al. Preparation and
evaluation of carboplatin biodegradable polymeric nano-
particles. Int J Pharm. 2010;385:176–180.
[32] Meng J, Sturgis TF, Youan BC. Engineering tenofovir
loaded chitosan nanoparticles tomaximize microbicide
mucoadhesion. Eur J Pharm Sci. 2011;44:57–67.
[33] Alam S, Khan ZI, Mustafa G, et al. Development and evalu-
ation of thymoquinone encapsulated chitosan nanopar-
ticles for nose-to-brain targeting: a pharmacoscintigraphic
study. Int J Nanomed. 2012;7:5705–5718.
[34] De Campos AM, Sanchez A, Alonso MJ. Chitosan nanopar-
ticles: a new vehicle for the improvement of the delivery
of drugs to the ocular surface. Application to cyclosporin
A. Int J Pharm. 2001;224:159–168.
[35] Wu Y, Yang W, Wang C, et al. Chitosan nanoparticles as a
novel delivery system for ammonium glycyrrhizinate. Int J
Pharm. 2005;295:235–245.
[36] Bhattarai N, Ramay HR, Chou SH, et al. Chitosan and lactic
acid-grafted chitosan nanoparticles as carriers for pro-
longed drug delivery. Int J Nanomed. 2006;1:181–187.
[37] Grenha A. Chitosan nanoparticles: a survey of preparation
methods. J Drug Target. 2012;20:291–300.
JOURNAL OF DRUG TARGETING 387
[38] Songjiang Z, Lixiang W. Amyloid-beta associated with chi-
tosan nano-carrier has favorable immunogenicity and per-
meates the BBB. AAPS PharmSciTech. 2009;10:900–905.
[39] Ngan LTK, Wang SL, Hiep M, et al. Preparation of chitosan
nanoparticles by spray drying, and their antibacterial activ-
ity. Res Chem Intermed. 2014;40:2165–2175.
[40] Sinsuebpol C, Chatchawalsaisin J, Kulvanich P. Preparation
and in vivo absorption evaluation of spray dried powders
containing salmon calcitonin loaded chitosan nanopar-
ticles for pulmonary delivery. Drug Des Devel Ther.
2013;7:861–873.
[41] Li FQ, Ji RR, Chen X, et al. Cetirizine dihydrochloride
loaded microparticles design using ionotropic cross-linked
chitosan nanoparticles by spray drying method. Arch
Pharm Res. 2010;33:1967–1973.
[42] Mehrotra A, Nagarwal RC, Pandit JK. Fabrication of lomus-
tine loaded chitosan nanoparticles by spray drying and
in vitro cytostatic activity on human lungcancer cell line
L132. J Nanomed Nanotechnol. 2010;1:1–7.
[43] Tokumitsu H, Ichikawa H, Fukumori Y. Chitosan-
gadopentetic acid complex nanoparticles for gadolinium
neutron-capture therapy of cancer: preparation by novel
emulsion-droplet coalescence technique and characteriza-
tion. Pharm Res. 1999;16:1830–1835.
[44] Reddy YD, Dhachinamoorthi D, Chandra SKB. Formulation
and in vitro evaluation of antineoplastic drug loaded
nanoparticles as drug delivery system. Afr J Pharm
Pharmacol. 2013;7:1592–1604.
[45] Luque-Alcaraz AG, Lizardi-Mendoza J, Goycoolea FM, et al.
Preparation of chitosan nanoparticles by nanoprecipitation
and their ability as a drug nanocarrier. RSC Adv.
2016;6:59250–59256.
[46] Melo EP, Aires-Barros MR, Cabra JMS. Reverse micelles and
protein biotechnology. Biotechnol Annu Rev. 2001;7:
87–129.
[47] Pileni MP. Reverse micelles used as templates: a new
understanding in nanocrystal growth. J Exp Nanosci.
2006;1:13–27.
[48] Banerjee T, Mitra S, Singh AK, et al. Preparation, character-
ization and biodistribution of ultrafine chitosan
nanoparticles. Int J Pharm. 2002;243:93–105.
[49] Mitra S, Gaur U, Ghosh PC, et al. Tumour targeted delivery
of encapsulated dextran-doxorubicin conjugate using chi-
tosan nanoparticles as carrier. J Control Release. 2001;74:
317–323.
[50] Kafshgari MH, Khorram M, Mansouri M, et al. Preparation
of alginate and chitosan nanoparticles using a new
reverse micellar system. Iran Polym J. 2012;21:99–107.
[51] Alonso MJ. Nanoparticulate drug carrier technology. In:
Cohen S, Bernstein H, editors. Microparticulate systems for
the delivery of proteins and vaccines. New York: Marcel
Dekker; 1996. p. 203–242.
[52] Janes KA, Calvo P, Alonso MJ. Polysaccharide colloidal par-
ticles as delivery systems for macromolecules. Adv Drug
Deliv Rev. 2001;47:83–97.
[53] Poncelet D. Microencapsulation: fundamentals, methods
and applications. In: Blitz JP, Gun’ko VM, editors. Surface
chemistry in biomedical and environmental science.
Dordrecht: Springer; 2006. p. 23–34.
[54] Mao HQ, Roy K, Troung-Le VL, et al. Chitosan-DNA nano-
particles as gene carriers: synthesis, characterization and
transfection efficiency. J Control Release. 2001;70:399–421.
388 S. NASKAR ET AL.
[55] Borges O, Borchard G, Verhoef JC, et al. Preparation of
coated nanoparticles for a new mucosal vaccine delivery
system. Int J Pharm. 2005;299:155–166.
[56] Atyabi F, Talaie F, Dinarvand R. Thiolated chitosan nano-
particles as an oral delivery system for amikacin: in vitro
and ex vivo evaluations. J Nanosci Nanotechnol. 2009;9:
4593–4603.
[57] Agnihotri SA, Aminabhavi TM. Chitosan nanoparticles for
prolonged delivery of timolol maleate. Drug Dev Ind
Pharm. 2007;33:1254–1262.
[58] Hu Y, Jiang X, Ding Y, et al. Synthesis and characterization
of chitosan-poly(acrylic acid) nanoparticles. Biomaterials
2002;23:3193–3201.
[59] Sajeesh S, Sharma CP. Cyclodextrin-insulin complex encap-
sulated polymethacrylic acid based nanoparticles for oral
insulin delivery. Int J Pharm. 2006;325:147–154.
[60] Sajeesh S, Sharma CP. Novel pH responsive polymetha-
crylic acid-chitosan-polyethylene glycol nanoparticles for
oral peptide delivery. J Biomed Mater Res. 2006;76B:
298–305.
[61] El-Shabouri MH. Positively charged nanoparticles for
improving the oral bioavailability of cyclosporin-A. Int J
Pharm. 2002;249:101–108.
[62] Grasianto S, Siswanta, Andriani Y, et al. Retracted-glutaral-
dehyde-crosslinked chitosan-pectin nanoparticles as a
potential carrier for curcumin delivery and its in vitro
release study. Int J Drug Deliver. 2015;7:167–173.
[63] Zhao LM, Shi LE, Zhang ZL, et al. Preparation and applica-
tion of chitosan nanoparticles.and nanofibers. Braz J Chem
Eng. 2011;28:353–362.
[64] Agnihotri SA, Mallikarjuna NN, Aminabhavi TM. Recent
advances on chitosan-based micro- and nanoparticles in
drug delivery. J Control Release. 2004;100:5–28.
[65] Balaguer MP, Gomez-Estaca J, Gavara R, et al. Functional
properties of bioplastics made from wheat gliadins modi-
fied with cinnamaldehyde. J Agric Food Chem. 2011;59:
6689–6695.
[66] Babu VN, Kannan S. Enhanced delivery of baicalein using
cinnamaldehyde cross-linked chitosan nanoparticle induc-
ing apoptosis. Int J Biol Macromol. 2012;51:1103–1108.
[67] Li PW, Wang G, Yang ZM, et al. Development of drug-
loaded chitosan-vanillin nanoparticles and its cytotoxicity
against HT-29 cells. Drug Deliv. 2016;23:30–35.
[68] Wang G, Li PW, Peng Z, et al. Formulation of vanillin
crosslinked chitosan nanoparticles and its characterization.
AMR. 2011;335-336:474–477.
[69] Liu Z, Jiao Y, Wang Y, et al. Polysaccharides-based nano-
particles as drug delivery systems. Adv Drug Deliv Rev.
2008;60:1650–1662.
[70] Konecsni K, Low NH, Nickerson MT. Chitosan-tripolyphos-
phate submicron particles as the carrier of entrapped
rutin. Food Chem. 2012;134:1775–1779.
[71] Servat-Medina L, Gonzalez-Go mez A, Reyes-Ortega F, et al.
Chitosan-tripolyphosphate nanoparticles as Arrabidaea
chica standardized extract carrier: synthesis, characteriza-
tion, biocompatibility, and anti ulcerogenic activity. Int J
Nanomed. 2015;10:3897–3909.
[72] Hu B, Pan C, Sun Y, et al. Optimization of fabrication
parameters to produce chitosan-tripolyphosphate nano-
particles for delivery of tea catechins. J Agric Food Chem.
2008;56:7451–7458.
[73] Elzoghby AO, Samy WM, Elgindy NA. Novel spray-dried
genipin-crosslinked casein nanoparticles for prolonged
release of alfuzosin hydrochloride. Pharm Res. 2013;30:
512–522.
[74] Elzoghby AO, Helmy MW, Samy WM, et al. Spray-dried
casein-based micelles as a vehicle for solubilization and
controlled delivery of flutamide: formulation, characteriza-
tion, and in vivo pharmacokinetics. Eur J Pharm Biopharm.
2013;84:487–496.
[75] Song F, Zhang LM, Yang C, et al. Genipin-crosslinked
casein hydrogels for controlled drug delivery. Int J Pharm.
2009;373:41–47.
[76] Kumar GV, Su CH, Velusamy P. Ciprofloxacin loaded geni-
pin cross-linked chitosan/heparin nanoparticles for drug
delivery application. Mater Lett. 2016;180:119–122.
[77] Ahmed TA, Aljaeid BM. Preparation, characterization, and
potential application of chitosan, chitosan derivatives,
and chitosan metal nanoparticles in pharmaceutical drug
delivery. Drug Des Dev Ther. 2016;10:483–507.
[78] Bagheri M, Younesi H, Hajati S, et al. Application of chito-
san-citric acid nanoparticles for removal of chromium (VI).
Int J Biol Macromol. 2015;80:431–444.
[79] Bodnar M, Hartmann JF, Borbely J. Preparation and
characterization of chitosan-based nanoparticles.
Biomacromolecules 2005;6:2521–2527.
[80] Mayyas MA, Remawi A. Properties of chitosan nanopar-
ticles formed using sulfate anions as crosslinking bridges.
Am J Appl Sci. 2012;9:1091–1100.
[81] Ringe K, Walz C, Sabel B. Nanoparticle drug delivery to
the brain. In: Nalwa HS, editor. Encyclopedia of nano-
science and nanotechnology, vol. 7. New York: American
Scientific Publishers; 2004.
[82] Gan Q, Wang T. Chitosan nanoparticle as protein delivery
carrier—systematic examination of fabrication conditions
for efficient loading and release. Colloids Surf B:
Biointerfaces. 2007;59:24–34.
[83] Peppas NA. Analysis of Fickian and non-Fickian drug
release from polymers. Pharm Acta Helv. 1985;60:110–111.
[84] Peppas NA, Korsmeyer RW. Dynamically swelling hydro-
gels in controlled release applications. In: Peppas NA,
editor. Hydrogels in medicine and pharmacy. Boca Raton,
FL: CRC Press; 1987. p. 103–135.
[85] Panyam J, Labhasetwar V. Biodegradable nanoparticles for
drug and gene delivery to cells and tissue. Adv Drug Deliv
Rev. 2003;55:329–347.
[86] Moghimi SM, Hunter AC, Murray JC. Long-circulating and
target-specific nanoparticles: theory to practice. Pharmacol
Rev. 2001;53:283–318.
[87] Nagarwal RC, Singh PN, Kant S, et al. Chitosan nanopar-
ticles of 5-fluorouracil for ophthalmic delivery: character-
ization, in-vitro and in-vivo study. Chem Pharm Bull.
2011;59:272–278.
[88] Kreuter J. Peroral administration of nanoparticles. Adv
Drug Deliv Rev. 1991;7:71–86.
[89] Tiyaboonchai W. Chitosan nanoparticles. A promising sys-
tem for drug delivery. Naresuan Univ J. 2003;11:51–66.
[90] Ing LY, Zin NM, Sarwar A, et al. Antifungal activity of chi-
tosan nanoparticles and correlation with their physical
properties. Int J Biomater. 2012;2012:1–9.
[91] Gaserod O, Jolliffe IG, Hampson FC, et al. The enhance-
ment of the bioadhesive properties of calcium alginate
gel beads bycoating with chitosan. Int J Pharm.
1998;175:237–246.
[92] Smith J, Wood E, Dornish M. Effect of chitosan on epithe-
lial cell tight junctions. Pharm Res. 2004;21:43–49.

[93] Dong Y, Feng SS. Methoxy poly(ethylene glycol)-poly(lac-
tide) (MPEG-PLA) nanoparticles for controlled delivery of
anticancer drugs. Biomaterials 2004;25:2843–2849.
[94] Mu L, Feng SS. Fabrication, characterization and in vitro
release of paclitaxel (TaxolV) loaded poly (lactic-co-glycolic
R
acid) microspheres prepared by spray drying technique
with lipid/cholesterol emulsifiers. J Control Release.
2001;76:239–254.
[95] Abdelwahed W, Degobert G, Stainmesse S, et al. Freeze-
drying of nanoparticles: formulation, process and storage
considerations. Adv Drug Deliv Rev. 2006;58:1688–1713.
[96] Hunter RJ. Foundations of colloid science, vol. I. New York:
Oxford Science Publications; 1993. p. 489–491.
[97] Lopez-Leon T, Carvalho ELS, Seijo B, et al. Physicochemical
characterization of chitosan nanoparticles: electrokinetic
and stability behavior. J Colloid Interface Sci. 2005;283:
344–351.
[98] Pan AW, Wu BB, Wu JM. Chitosan nanoparticles cross
linked by glycidoxy propyl trimethoxy silane for pH trig-
gered release of protein. Chin Chem Lett. 2009;20:79–83.
[99] Fan L, Wu H, Zhang H, et al. Novel super pH-sensitive
nanoparticles responsive totumor extracellular pH.
Carbohydr Polym. 2008;73:390–400.
[100] Hu B, Ting Y, Zen X, et al. Cellular uptake and cytotoxicity
of chitosan-caseinophos phopeptides nanocomplexes
loaded with epigallo catechin gallate. Carbohydr Polym.
2012;89:362–370.
[101] Corsi K, Chellat F, Yahia LHocine, et al. Mesenchymal stem
cells, MG63 and HEK293 transfection using chitosan-DNA
nanoparticles. Biomaterials 2003;24:1255–1264.
[102] Mansouri S, Cuie Y, Winnik F, et al. Characterization of
folate-chitosan-DNA nanoparticles for gene therapy.
Biomaterials 2006;27:2060–2065.
[103] Qi L, Xu Z, Jiang X, et al. Cytotoxic activities of chitosan
nanoparticles and copper-loaded nanoparticles. Bioorg
Med Chem Lett. 2005;15:1397–1399.
[104] Loh JW, Yeoh G, Saunders M, et al. Uptake and cytotox-
icity of chitosan nanoparticles in human liver cells. Toxicol
Appl Pharmacol. 2010;249:148–157.
[105] Liu M, Zhang J, Shan W, et al. Developments of mucus
penetrating nanoparticles. Asian J Pharmacol. 2015;10:
275–282.
[106] Lavelle E. Targeted mucosal delivery of drugs and vac-
cines. Expert Opin Ther Pat. 2000;10:179–190.
[107] Lubben IMV, Verhoef JC, Borchard G, et al. Chitosan for
mucosal vaccination. Adv Drug Deliv Rev. 200;52:139–144.
[108] Vila A, Sanchez A, Tobıo M, et al. Design of biodegradable
particles for protein delivery. J Control Release.
2002;78:15–24.
[109] Baltzley S, Mohammad A, Malkawi AH, et al. Intranasal
drug delivery of olanzapine-loaded chitosan nanoparticles.
AAPS Pharm Sci Tech. 2014;15:1598–1602.
[110] Al-Ghananeem AM, Saeed H, Florence R, et al. Intranasal
drug delivery of didanosine-loaded chitosan nanoparticles
for brain targeting; an attractive route against infections
caused by aids viruses. J Drug Target. 2010;18:381–388.
[111] Prabaharan M. Chitosan-based nanoparticles for tumor-
targeted drug delivery. Int J Biol Macromol. 2015;72:
1313–1322.
[112] Yang X, Zhang Q, Wang Y, et al. Self-aggregated nanopar-
ticles from methoxy poly(ethylene glycol)-modified
chitosan: synthesis, characterization; aggregation and
JOURNAL OF DRUG TARGETING 389
methotrexate release in vitro. Colloids Surf B: Biointerfaces.
2008;61:125–131.
[113] Zheng Y, Yang W, Wang C, et al. Nanoparticles based on
the complex of chitosan and polyaspartic acid sodium salt:
preparation, characterization and the use for 5-fluorouracil
delivery. Eur J Pharm Biopharm. 2007;67:621–631.
[114] Anitha A, Chennazhi KP, Nair SV, et al. 5-Flourouracil
loaded N,O-carboxymethyl chitosan nanoparticles as an
anticancer nanomedicine for breast cancer. J Biomed
Nanotechnol. 2012;8:29–42.
[115] Cha J, Lee WB, Park CR, et al. Preparation and character-
ization of cisplatin-incorporated chitosan hydrogels, micro-
particles, and nanoparticles. Macromol Res. 2006;14:
573–578.
[116] Wang YS, Liu LR, Jiang Q, et al. Self-aggregated nanopar-
ticles of cholesterol-modified chitosan conjugate as a
novel carrier of epirubicin. Eur Polym J. 2007;43:43–51.
[117] Anitha A, Maya S, Deepa N, et al. Curcumin-loaded N,O-
Carboxymethyl chitosan nanoparticles for cancer drug
delivery. J Biomater Sci Polym Ed. 2012;23:1381–1400.
[118] Anitha A, Deepagan VG, Divya Rani VV, et al. Preparation,
characterization, in vitro drug release and biological stud-
ies of curcumin loaded dextran sulphate-chitosan nano-
particles. Carbohydr Polym. 2011;84:1158–1164.
[119] Bilensoy E, Sarisozen C, Esendag  li G, et al. Intravesical cat-
ionic nanoparticles of chitosan and polycaprolactone for
the delivery of mitomycin C to bladder tumors. Int J
Pharm. 2009;371:170–176.
[120] Hwang IS, Kim HY, Kwon IC, et al. Tumor targetability and
antitumor effect of docetaxel-loaded hydrophobically
modified glycol chitosan nanoparticles. J Control Release.
2008;128:23–31.
[121] Janes KA, Fresneau MP, Marazuela A, et al. Chitosan nano-
particles as delivery systems for doxorubicin. J Control
Release. 2001;73:255–267.
[122] Li F, Li J, Wen X, et al. Anti-tumor activity of paclitaxel-
loaded chitosan nanoparticles: an in vitro study. Mater Sci
Eng C. 2009;29:2392–2397.
[123] Vivek R, Nipun Babu V, Thangam R, et al. pH-responsive
drug delivery of chitosan nanoparticles as tamoxifen car-
riers for effective anti-tumor activity in breast cancer cells.
Colloids Surf B: Biointerfaces. 2013;111:117–123.
[124] Zhang X, Zhang H, Yin L, et al. A pH-sensitive nanosystem
based on carboxymethyl chitosan for tumor-targeted
delivery of daunorubicin. J Biomed Nanotechnol. 2016;12:
1688–1698.
[125] Zheng H, Yin L, Zhang X, et al. Redox sensitive shell and
core crosslinked hyaluronic acid nanocarriers for tumor-
targeted drug delivery. J Biomed Nanotechnol. 2016;12:
1641–1653.
[126] Hu R, Zheng H, Cao J, et al. Synthesis and in vitro charac-
terization of carboxymethyl chitosan-cba-doxorubicin con-
jugate nanoparticles as pH-sensitive drug delivery
systems. J Biomed Nanotechnol. 2017;13:1097–1105.
[127] Rajitha P, Gopinath D, Biswas R, et al. Chitosan nanopar-
ticles in drug therapy of infectious and inflammatory dis-
eases. Expert Opin Drug Deliv. 2016;13:1177–1194.
[128] Jamil B, Habib H, Abbasi S, et al. Cefazolin loaded chitosan
nanoparticles to cure multidrug resistant gram-negative
pathogens. Carbohydr Polym. 2015;78:1–33.
[129] Dube A, Reynolds JL, Law WC, et al. Multimodal nanopar-
ticles that provide immune modulation and intracellular
390 S. NASKAR ET AL.
drug delivery for infectious diseases. Nanomed
Nanotechnol. 2014;10:831–838.
[130] Garg T, Rath G, Goyal AK. Inhalable chitosan nanoparticles
as antitubercular drug carriers for an effective treatment
of tuberculosis. Artif Cells Nanomed Biotechnol.
2016;44:997–1001.
[131] Moretton MA, Chiappetta DA, Andrade F, et al. Hydrolyzed
galactomannan modified nanoparticles and flower-like
polymeric micelles for the active targeting of rifampicin to
macrophages. J Biomed Nanotechnol. 2013;9:1076–1087.
[132] Costa JR, Silva NC, Sarmento B, et al. Potential chitosan-
coated alginate nanoparticles for ocular delivery of dapto-
mycin. Eur J Clin Microbiol Infect Dis. 2015;34:1255–1262.
[133] € undag
Ust€ -Okur N, Go€kçe EH, Bozbıyık D_I, et al. Preparation
and in vitro-in vivo evaluation of ofloxacin loaded ophthal-
mic nano structured lipid carriers modified withchitosan
oligosaccharide lactate for the treatment of bacterial kera-
titis. Eur J Pharm Sci. 2014;63:204–215.
[134] Jain V, Gupta A, Pawar VK, et al. Chitosan-assisted
immunotherapy for intervention of experimental leish-
maniasis via amphotericin B-loaded solid lipid nanopar-
ticles. Appl Biochem Biotechnol. 2014;174:1309–1330.
[135] Zhou W, Wang Y, Jian J, et al. Self-aggregated nanoparticles
based on amphiphilic poly(lactic acid)-grafted-chitosan
copolymer for ocular delivery of amphotericin B. Int J
Nanomed. 2013;8:3715–3728.
[136] Chhonker YS, Prasad YD, Chandasana H, et al.
Amphotericin-B entrapped lecithin/chitosan nanoparticles
for prolonged ocular application. Int J Biol Macromol.
2015;72:1451–1458.
[137] Kong Z, Yu M, Cheng K, et al. Incorporation of chitosan
nanospheres into thin mineralized collagen coatings for
improving the antibacterial effect. Colloids Surf B.
Biointerfaces. 2013;111:536–541.
[138] Pornpattananangkul D, Zhang L, Olson S, et al. Bacterial
toxin-triggered drug release from gold nanoparticle-stabilized
liposomes for the treatment of bacterial infection. J Am
Chem Soc. 2011;133:4132–4139.
[139] Junise V, Saraswathi R. Development and characterization
of inhaled chitosan nanoparticles loaded with isoniazid.
JPTRM 2014;2:159–170.
[140] Lin YH, Tsai SC, Lai CH, et al. Genipin-cross-linked fucose-
chitosan/heparin nanoparticles for the eradication of
Helicobacter pylori. Biomaterials 2013;34:4466–4479.
[141] Maya S, Indulekha S, Sukhithasri V, et al. Efficacy of tetra-
cycline encapsulated O-carboxymethyl chitosan nanopar-
ticles against intracellular infections of Staphylococcus
aureus. Int J Biol Macromol. 2012;51:392–399.
[142] Gnanadhas DP, Thomas MB, Elango M, et al. Chitosan-dex-
tran sulphate nanocapsule drug delivery system as an
effective therapeutic against intra phagosomal pathogen
Salmonella. J Antimicrob Chemother. 2013;68:2576–2586.
[143] Cota-Arriola O, Cortez-Rocha MO, Burgos HA, et al.
Controlled release matrices and micro/nanoparticles of
chitosan with antimicrobial potential: development of new
strategies for microbial control in agriculture. J Sci Food
Agric 2013;93:1525–1536.
[144] Hua S, Marks E, Schneider JJ, et al. Advances in oral nano-
delivery systems for colon targeted drug delivery in
inflammatory bowel disease: selective targeting to dis-
eased versus healthy tissue. Nanomed Nanotechnol.
2015;11:1117–1132.
[145] Bagre AP, Jain K, Jain NK. Alginate coated chitosan core
shell nanoparticles for oral delivery of enoxaparin: in vitro
and in vivo assessment. Int J Pharm. 2013;456:31–40.
[146] Tozaki H, Odoriba T, Okada N, et al. Chitosan capsules for
colon-specific drug delivery: enhanced localization of
5-aminosalicylic acid in the large intestine accelerates
healing of TNBS-induced colitis in rats. J Control Release.
2002;82:51–61.
[147] Li P, Wang Y, Zeng F, et al. Synthesis and characterization
of folate conjugated chitosan and cellular uptake of its
nanoparticles in HT-29 cells. Carbohydr Res. 2011;346:
801–806.
[148] Saboktakin MR, Tabatabaie RM, Maharramov A, et al.
Synthesis and in vitro evaluation of carboxymethyl starch-
chitosan nanoparticles as drug delivery system to the
colon. Int J Biol Macromol. 2011;48:381–385.
[149] Akhlaghi SP, Saremi S, Ostad SN, et al. Discriminated
effects of thiolated chitosan-coated pMMA paclitaxel-
loaded nanoparticles on different normal and cancer cell
lines. Nanomed: Nanotechnol. 2010;6:689–697.
[150] Kadiyala I, Loo Y, Roy K, et al. Transport of chitosan-DNA
nanoparticles in human intestinal M-cell model versus
normal intestinal enterocytes. Eur J Pharm Sci. 2010;39:
103–109.
[151] Jain A, Jain SK, Ganesh N, et al. Design and development
of ligand-appended polysaccharidic nanoparticles for the
delivery of oxaliplatin in colorectal cancer. Nanomed
Nanotechnol. 2010;6:179–190.
[152] Yang SJ, Shieh MJ, Lin FH, et al. Colorectal cancer cell
detection by 5-aminolaevulinic acid-loaded chitosan nano-
particles. Cancer Lett. 2009;273:210–220.
[153] Park JS, Koh YS, Bang JY, et al. Antitumor effect of all-
trans retinoic acid-encapsulated nanoparticles of methoxy
poly (ethylene glycol)-conjugated chitosan against CT-26
colon carcinoma in vitro. J Pharm Sci. 2008;97:4011–4019.
[154] Jain A, Jain SK. In vitro and cell uptake studies for target-
ing of ligand anchored nanoparticles for colon tumors.
Eur J Pharm Sci. 2008;35:404–416.
[155] Kapanigowda UG, Nagaraja SH, Ramaiah B, et al. Improved
intraocular bioavailability of ganciclovir by mucoadhesive
polymer based ocular microspheres: development and
simulation process in wistar rats. DARU J Pharm Sci.
2015;23:11.
[156] Werle M, Bernkop SA. Thiolated chitosans: useful exci-
pients for oral drug delivery. J Pharm Pharmacol.
2008;60:273–281.
[157] Yuan X, Li H, Yuan Y. Preparation of cholesterol-modified
chitosan self-aggregated nanoparticles for delivery of
drugs to ocular surface. Carbohydr Polym. 2006;65:
337–345.
[158] De Salamanca AE, Diebold Y, Calonge M, et al. Chitosan
nanoparticles as a potential drug delivery system for the
ocular surface: toxicity, uptake mechanism and in vivo tol-
erance. Invest Ophthalmol Vis Sci. 2006;47:1416–1425.
[159] Diebold Y, Jarrın M, Saez V, et al. Ocular drug delivery by
liposome-chitosan nanoparticle complexes (LCS-NP).
Biomaterials 2007;28:1553–1564.
[160] Calvo P, Vila-Jato JL, Alonso MJ. Evaluation of cationic
polymer-coated nanocapsules as ocular drug carriers. Int J
Pharm. 1997;153:41–50.
[161] Calvo P, Alonso MJ, Vila-Jato JL, et al. Improved ocular
bioavailability of indomethacin by novel ocular drug car-
riers. J Pharm Pharmacol. 1996;48:1147–1152.

[162] Kao HJ, Lo YL, Lin HR, et al. Characterization of pilocar-
pine-loaded chitosan/Carbopol nanoparticles. J Pharm
Pharmacol. 2006;58:179–186.
[163] Lin HR, Yu SP, Kuo CJ, et al. Pilocarpine-loaded chitosan-
PAA nanosuspension for ophthalmic delivery. J Biomater
Sci Polym Ed. 2007;18:205–221.
[164] De la Fuente M, Seijo B, Alonso MJ. Bioadhesive hyalur-
onan/chitosan nanoparticles can transport genes across
the ocular mucosa and transfect ocular tissue. Gene Ther.
2008;15:668–676.
[165] Qu X, Khutoryanskiy VV, Stewart A, et al. Carbohydrate-
based micelle clusters which enhance hydrophobic
drug bioavailability by up to 1 order of magnitude.
Biomacromolecules 2006;7:3452–3459.
[166] Vila A, Sanchez A, Janes K, et al. Low molecular weight
chitosan nanoparticles as new carriers for nasal vaccine
delivery in mice. Eur J Pharm Biopharm. 2004;57:123–131.
[167] Illum L, Jabbal-Gill I, Hinchcliffe M, et al. Chitosan as a
novel nasal delivery system for vaccines. Adv Drug Deliv
Rev. 2001;51:81–96.
[168] Amidi M, Romeijn SG, Verhoef JC, et al. N-trimethyl chito-
san (TMC) nanoparticles loaded with influenza subunit
antigen for intranasal vaccination: biological properties
and immunogenicity in a mouse model. Vaccine
2007;25:144–153.
[169] Boonyo W, Junginger HE, Waranuch N, et al. Chitosan and
trimethyl chitosan chloride (TMC) as adjuvants for induc-
ing immune responses to ovalbumin in mice following
nasal administration. J Control Release. 2007;121:168–175.
[170] Svirshchevskaya EV, Alekseeva LG, Reshetov PD, et al.
Mucoadjuvant properties of lipo- and glycoconjugated
derivatives of oligochitosans. Eur J Med Chem.
2009;44:2030–2037.
[171] Sayin B, Somavarapu S, Li XW, et al. Mono-N-carboxy-
methyl chitosan (MCC) and N-trimethyl chitosan (TMC)
nanoparticles for non-invasive vaccine delivery. Int J
Pharm. 2008;363:139–148.
[172] Borges O, Cordeiro-da-Silva A, Tavares J, et al. Immune
response by nasal delivery of hepatitis B surface antigen
and codelivery of a CpG ODN in alginate coated chitosan
nanoparticles. Eur J Pharm Biopharm. 2008;69:405–416.
[173] Klas SD, Petrie CR, Warwood SJ, et al. A single immuniza-
tion with a dry powder anthrax vaccine protects rabbits
against lethal aerosol challenge. Vaccine 2008;26:
5494–5502.
[174] Florindo HF, Pandit S, Lacerda L, et al. The enhancement
of the immune response against S. equi antigens through
the intranasal administration of poly-epsilon-caprolactone-
based nanoparticles. Biomaterials 2009;30:879–891.
[175] Baudner BC, Giuliani MM, Verhoef JC, et al. The concomi-
tant use of the LTK63 mucosal adjuvant and of chitosan-
based delivery system enhances the immunogenicity and
efficacy of intranasally administered vaccines. Vaccine
2003;21:3837–3844.
[176] Amidi M, Mastrobattista E, Jiskoot W, et al. Chitosan-based
delivery systems for protein therapeutics and antigens.
Adv Drug Deliv Rev. 2010;62:59–82.
[177] Cui Z, Mumper RJ. Chitosan-based nanoparticles for top-
ical genetic immunization. J Control Release. 2001;75:
409–419.
[178] Madrigal-Carballo S, Esquivel M, Sibaja M, et al. Protein-
loaded chitosan nanoparticles modulate uptake and anti-
gen presentation of hen egg-white lysozyme by murine
JOURNAL OF DRUG TARGETING 391
peritoneal macrophages. Int J Nanoparticles 2010;3:
179–191.
[179] Xu W, Shen Y, Jiang Z, et al. Intranasal delivery of chito-
san-DNA vaccine generates mucosal SIgA and anti-
CVB3protection. Vaccine 2004;22:3603–3612.
[180] Smitha KT, Sreelakshmi M, Nisha N, et al. Amidase encap-
sulated O-carboxymethyl chitosan nanoparticles for vac-
cine delivery. Int J Biol Macromol. 2014;63:154–157.
[181] Pattani A, Patravale VB, Panicker L, et al. Immunological
effects and membrane interactions of chitosan nanopar-
ticles. Mol Pharm. 2009;6:345–352.
[182] Bivas-Benita M, Van Meijgaarden KE, Franke KLMC, et al.
Pulmonary delivery of chitosan-DNA nanoparticles enhan-
ces the immunogenicity of a DNA vaccine encoding
HLA-A0201-restricted T-cell epitopes of Mycobacterium
tuberculosis. Vaccine 2004;22:1609–1615.
[183] Tanner T, Marks R. Delivering drugs by the transdermal
route: review and comment. Skin Res Technol. 2008;14:
249–260.
[184] Al-Kassas R, Wen J, E-M, Cheng A, et al. Transdermal deliv-
ery of propranolol hydrochloride through chitosan nano-
particles dispersed in mucoadhesive gel. Carbohydr
Polym. 2016;153:176–186.
[185] Hafner A, Lovric J, Pepic I, et al. Lecithin/chitosan nanopar-
ticles for transdermal delivery of melatonin.
J Microencapsul. 2011;28:807–815.
[186] Shah HA, Patel RP. Statistical modeling of zaltoprofen
loaded biopolymeric nanoparticles: characterization and
anti-inflammatory activity of nanoparticles loaded gel. Int
J Pharma Investig. 2015;5:20–27.
[187] Katas H, Hussain Z, Ling TC. Chitosan Nanoparticles as a
percutaneous drug delivery system for hydrocortisone.
J Nanomater. 2012;2012:1:1–11.
[188] Fardet L, Flahault A, Kettaneh A, et al. Corticosteroid-
induced clinical adverse events: frequency, risk factors and
patient’s opinion. Br J Dermatol. 2007;157:142–148.
[189] Ridolfi DM, Marcato PD, Justo GZ, et al. Chitosan-solid
lipid nanoparticles as carriers for topical delivery of tretin-
oin. Colloids Surf B Biointerfaces. 2012;93:36–40.
[190] Dev A, Binulal NS, Anitha A, et al. Preparation of poly(lac-
tic acid)/chitosan nanoparticles for anti-HIV drug delivery
applications. Carbohydr Polym. 2010;80:833–838.
[191] Dahmane EM, Rhazi M, Taourirte M. Chitosan nanopar-
ticles as a new delivery system for the Anti-HIV drug
Zidovudine. Bull Korean Chem Soc. 2013;34:1333–1338.
[192] Tripathy S, Das S, Chakraborty SP, et al. Synthesis, charac-
terization of chitosan-tripolyphosphate conjugated chloro-
quine nanoparticle and its in vivo anti-malarial efficacy
against rodent parasite: a dose and duration dependent
approach. Int J Pharm. 2012;434:292–305.
[193] Nanda RK, Patil SS, Navathar DA. Chiotsan nanoparticles
loaded with Thiocolchicoside. Der Pharma Chemica
2012;4:1619–1625.
[194] Makhlof A, Tozuka Y, Takeuchi H. Design and evaluation
of novel pH-sensitive chitosan nanoparticles for oral insu-
lin delivery. Eur J Pharm Sci. 2011;42:445–451.
[195] Zhu X, Su M, Tang S, et al. Synthesis of thiolated chitosan
and preparation nanoparticles with sodium alginate for
ocular drug delivery. Mol Vis. 2012;18:1973–1982.
[196] Kast CE, Bernkop SA. Thiolated polymers-thiomers: devel-
opment and in vitro evaluation of chitosan-thioglycolic
acid conjugates. Biomaterials 2001;22:2345–2352.
392 S. NASKAR ET AL.
[197] Leitner VM, Walker GF, Bernkop-Schn€ urch A. Thiolated pol-
ymers: evidence for the formation of disulphide bonds
with mucus glycoproteins. Eur J Pharm Biopharm.
2003;56:207–214.
[198] Saremi S, Atyabi F, Akhlaghi SP, et al. Thiolated chitosan
nanoparticles for enhancing oral absorption of docetaxel:
preparation, in vitro and ex vivo evaluation. Int J
Nanomed. 2011;6:119–128.
[199] Wang X, Zheng C, Wu Z, et al. Chitosan-NAC nanoparticles
as a vehicle for nasal absorption enhancement of insulin. J
Biomed Mater Res Part B Appl Biomater. 2009;88:150–161.
[200] Makhlof A, Werle M, Tozuka Y, et al. Nanoparticles of gly-
col chitosan and its thiolated derivative significantly
improved the pulmonary delivery of calcitonin. Int J
Pharm. 2010;397:92–95.
[201] Verheul RJ, Sl€utter B, Bal S, et al. Covalently stabilized tri-
methyl chitosan-hyaluronic acid nanoparticles for nasal
and intradermal vaccination. J Control Release. 2011;156:
46–52.
[202] Martien R, Loretz B, Sandbichler AM, et al. Thiolated chito-
san nanoparticles: transfection study in the Caco-2 differ-
entiated cell culture. Nanotechnology 2008;19:045101.
[203] Bernkop-schn€ urch A, Weithaler A, Albrecht K, et al.
Thiomers: preparation and in vitro evaluation of a
mucoadhesive nanoparticulate drug delivery system. Int J
Pharm. 2006;317:76–81.
[204] Yoo HS, Lee JE, Chung H, et al. Self-assembled nanopar-
ticles containing hydrophobically modified glycol chitosan
for gene delivery. J Control Release. 2005;103:235–243.
[205] Kim JH, Kim Y-S, Kim S, et al. Hydrophobically modified
glycol chitosan nanoparticles as carriers for paclitaxel.
J Control Release. 2006;111:228–234.
[206] Park K, Kim JH, Nam YS, et al. Effect of polymer molecular
weight on the tumor targeting characteristics of self-
assembled glycol chitosan nanoparticles. J Control
Release. 2007;122:305–314.
[207] Kim J-H, Kim Y-S, Park K, et al. Self-assembled glycol chito-
san nanoparticles for the sustained and prolonged deliv-
ery of antiangiogenic small peptide drugs in cancer
therapy. Biomaterials 2008;29:1920–1930.
[208] Min KH, Park K, Kim YS, et al. Hydrophobically modified
glycol chitosan nanoparticles encapsulated camptothecin
enhance the drug stability and tumor targeting in cancer
therapy. J Control Release. 2008;127:208–208.
[209] Nam HY, Won SMK, Chung H, et al. Cellular uptake mech-
anism and intracellular fate of hydrophobically modified
glycol chitosan nanoparticles. J Control Release. 2009;135:
259–267.
[210] Chen KJ, Chiu YL, Chen YM, et al. Intracellularly monitor-
ing/imaging the release of doxorubicin from pH-responsive
nanoparticles using Fo € rster resonance energy transfer.
Biomaterials 2011;32:2586–2592.
[211] Zhang J, Chen XG, Li YY, et al. Self-assembled nanopar-
ticles based on hydrophobically modified chitosan as car-
riers for doxorubicin. Nanomed: Nanotechnol, Biol Med.
2007;3:258–265.
[212] Siew A, Le H, Thiovolet M, et al. Enhanced oral absorption
of hydrophobic and hydrophilic drugs using quaternary
ammonium palmitoylglycol chitosan nanoparticles. Mol
Pharmaceutics. 2012;9:14–28.
[213] Tan YL, Liu CG. Self-aggregated nanoparticles from linoleic
acid modified carboxymethyl chitosan: Synthesis,
characterization and application in vitro. Colloids Surf B:
Biointerface. 2009;69:178–182.
[214] Jin Y, Song Y, Zhu X, et al. Goblet cell-targeting nanopar-
ticles for oral insulin delivery and the influence of mucus
on insulin transport. Biomaterials 2012;33:1573–1582.
[215] Sahu S, Mallick SK, Santra S, et al. In vitro evaluation of
folic acid modified carboxymethyl chitosan nanoparticles
loaded with doxorubicin for targeted delivery. J Mater Sci:
Mater Med. 2010;21:1587–1597.
[216] Mourya VK, Inamdar NN. Trimethyl chitosan and its appli-
cations in drug delivery. J Mater Sci Mater Med.
2009;20:1057–1079.
[217] Avadi MR, Zohuriaan-mehr MJ, Younessi P, et al.
Optimized synthesis and characterization of N-triethylchi-
tosan. J Bioact Compat Polym. 2003;18:469–479.
[218] Avadi MR, Jalali A, Sadeghi AMM, et al. Diethyl methyl chi-
tosan as an intestinal paracellular enhancer: ex vivo and
in vivo studies. Int J Pharm. 2005;293:83–89.
[219] Bayat A, Sadeghi AMM, Avadi MR, et al. Synthesis of N,N-
dimethyl N-ethyl chitosan as a carrier for oral delivery of
peptide drugs. J Bioact Compat Polym. 2006;21:433–444.
[220] Avadi MR, Sadeghi AMM, Tahzibi A, et al. Diethylmethyl
chitosan as antimicrobial agent: synthesis, characterization
and antibacterial effects. Eur Polym J. 2004;40:1355–1361.
[221] Thanou M, Verhoef JC, Marbach P, et al. Intestinal absorp-
tion of octreotide: N-trimethyl chitosanchloride (TMC)
ameliorates the permeability and absorption properties of
the somatostatin analogue in vitro and in vivo.
J Pharmaceutical Sci. 2000;89:951–957.
[222] Mi FL, Wu YY, Lin YH, et al. Oral delivery of peptide drugs
using nanoparticles self-assembled by poly(c-glutamic
acid) and a chitosan derivative functionalized by trimethy-
lation. Bioconjugate Chem. 2008;19:1248–1255.
[223] €tter B, Jiskoot W. Dual role of CpG as immune modula-
Slu
tor and physical crosslinker in ovalbumin loaded
N-trimethyl chitosan (TMC) nanoparticles for nasal vaccin-
ation. J Control Release. 2010;148:117–121.
[224] Wang S, Jiang T, Ma M, et al. Preparation and evaluation
of anti-neuroexcitation peptide (ANEP) loaded N-trimethyl
chitosan chloride nanoparticles for brain-targeting. Int J
Pharm. 2010;386:249–255.
[225] Sadeghi AM, Dorkoosh FA, Avadi MR, et al. Preparation,
characterization and antibacterial activities of chitosan,
N-trimethyl chitosan(TMC) and N-diethylmethyl chitosan
(DEMC) nanoparticles loaded with insulin using both the
ionotropic gelation and polyelectrolyte complexation
methods. Int J Pharm. 2008;355:299–306.
[226] Amidi M, Romeijn SG, Borchard G, et al. Preparation and
characterization of protein-loaded N-trimethyl chitosan
nanoparticles as nasal delivery system. J Control Release.
2006;111:107–116.
[227] Chen F, Zhang ZR, Yuan F, et al. In vitro and in vivo study
of N-trimethyl chitosan nanoparticles for oral protein
delivery. Int J Pharm. 2008;349:226–233.
[228] Menon D, Thomas RT, Narayanan S, et al. A novel chito-
san/polyoxometalate nanocomplex for anti-cancer applica-
tions. Carbohydr Polym. 2011;84:887–893.
[229] Bayat A, Larijani B, Ahmadian S, et al. Preparation and
characterization of insulin nanoparticles using chitosan
and its quaternized derivatives. Nanomedicine 2008;4:
115–120.
[230] Bayat A, Dorkoosh FA, Dehpour AR, et al. Nanoparticles of
quaternized chitosan derivatives as a carrier for colon

delivery of insulin: ex vivo and in vivo studies. Int J
Pharm. 2008;356:259–266.
[231] Jintapattanakit A, Junyaprasert VB, Mao S, et al. Peroral
delivery of insulin using chitosan derivatives: a compara-
tive study of polyelectrolyte nanocomplexes and nanopar-
ticles. Int J Pharm. 2007;342:240–249.
[232] Lee D, Zhang W, Shirley SA, et al. Thiolated chitosan/DNA
nanocomplexes exhibit enhanced and sustained gene
delivery. Pharm Res. 2007;24:157–167.
[233] Ki MH, Kim JE, Lee YN, et al. Chitosan-based hybrid nano-
complex for siRNA delivery and its application for cancer
therapy . Pharm Res. 2014;31:3323–3334.
[234] Park Y, Kang E, Kwon OJ, et al. Ionically crosslinked Ad/
chitosan nanocomplexes processed by electrospinning for
View publication stats
JOURNAL OF DRUG TARGETING 393
targeted cancer gene therapy. J Control Release. 2010;148:
75–82.
[235] Cheng X, Zhang F, Zhou G, et al. DNA/chitosan nanocom-
plex as a novel drug carrier for doxorubicin. Drug Deliv.
2009;16:135–144.
[236] Shah HS, Al-Oweini R, Haider A, et al. Cytotoxicity and
enzyme inhibition studies of polyoxometalates and
their chitosan nanoassemblies. Toxicol Reports. 2014;1:
341–352.
[237] Zhao X, Yin L, Ding J, et al. Thiolated trimethyl chitosan
nanocomplexes as gene carriers with high in vitro and in vivo
transfection efficiency. J Control Release. 2010;144:46–54.
[238] Bellich B, D’Agostino I, Semeraro S, et al. “The good, the
bad and the ugly” of chitosans. Mar Drugs 2016;14:99.