Review

Received: 13 November 2010

Revised: 16 December 2010

Accepted: 17 December 2010

Published online in Wiley Online Library: 16 March 2011

(wileyonlinelibrary.com) DOI 10.1002/pi.3056

Chitosan nanoparticles: a contribution

to nanomedicine
Hazel Peniche and Carlos Peniche
Abstract
Chitosan is a cationic polysaccharide that finds diverse applications in medicine and pharmacy because of its excellent
biological qualities: it is biocompatible, biodegradable, mucoadhesive and non-toxic, and exhibits antimicrobial, antiviral
and immunoadjuvant properties. It can be easily processed in diverse forms, such as films, threads, tablets, membranes
and microparticles/nanoparticles, allowing the design of a variety of medical and pharmacological devices adaptable to end
purposes. In particular, chitosan nanoparticles have become of great interest as polymeric platforms for the development of new
pharmacological and therapeutic drug release systems with improved biodistribution and increased specificity and sensitivity,
and reduced pharmacological toxicity. Chitosan nanoparticles have been found appropriate for non-invasive routes of drug
administration: oral, nasal, pulmonary and ocular routes. These applications are facilitated by the absorption-enhancing effect
of chitosan. Additionally, chitosan nanoparticles have been proposed as non-viral vectors in gene therapy and have shown
adjuvant effect in vaccines. This paper reviews the main procedures developed for preparing chitosan nanoparticles. Moreover,
it illustrates the state of the art of chitosan nanoparticle applications in drug delivery.
c 2011 Society of Chemical Industry


Keywords: chitosan; nanoparticles; drug delivery; coacervation; vaccines; gene therapy

INTRODUCTION

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nanocapsules.4 Nanospheres have a monolithic-type structure in

which the drug is dispersed inside the polymer matrix or adsorbed
onto its surface. Nanocapsules exhibit a membrane-wall structure
with the drug entrapped in their core or adsorbed onto their
external surface. The term nanoparticle has a wider connotation
and is often used to preserve generality. The polymeric matrix
for nanoparticles can be of natural or synthetic origin. Natural
polymers are usually preferred because of their biocompatibility,
biodegradability and non-toxicity.
Chitosan is a natural polysaccharide composed essentially of
(1 4)-linked glucosamine units together with some proportion
of N-acetylglucosamine units (Fig. 1). Chitosan occurs rarely in
nature, but it is generally obtained by extensive deacetylation
of chitin, a homopolymer of (1 4)-linked N-acetyl-Dglucosamine, present in the shells of crustaceans and molluscs, the
cell walls of fungi and the cuticle of insects.5 The microstructure
of chitosan depends on whether the deacetylation reaction is
carried out under homogeneous or heterogeneous conditions.6
This structural difference is very important in determining many
properties of chitosan, such as solubility. Chitosan obtained by
a heterogeneous procedure is not soluble in water, although it
can become soluble in acidic conditions, whereas water-soluble
chitosan can be prepared by homogeneous deacetylation of
chitin.7 The acetylation of highly deacetylated chitin can also
produce soluble chitosan.8,9 As a result, chitosan is available in a

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Correspondence to: Carlos Peniche, Centro de Biomateriales, Universidad de

La Habana, Avenida Universidad, s/n, e/G y Ronda, 10600 La Habana, Cuba.
E-mail: peniche@reduniv.edu.cu
Centro de Biomateriales, Universidad de La Habana, Avenida Universidad, s/n,
e/G y Ronda, 10600 La Habana, Cuba

c 2011 Society of Chemical Industry

883

The remarkable achievements attained in the fields of nanoscience

and nanotechnology have led to applications in the most diverse
fields of human activity. The miniaturization of devices to nanometric size dramatically increases their potentialities. Nanostructured
materials have applications in semiconductors, optoelectronics,
semiconductor lasers, bioprocessing, image colouring, atomic
force microscopy, ceramics and catalysis.1 The keystone of the
unique properties of nanomaterials resides in their size, surface
structures and interparticle interactions.2
The application of nanotechnology to medicine is referred to
as nanomedicine. Nanomedicine has been broadly defined as the
process of diagnosing, treating and preventing disease and traumatic injury, relieving pain, and preserving and improving human
health, using molecular tools and molecular knowledge of the human body.3 In principle, nanomedicine focuses on two main fields:
(a) development of new diagnostic systems (implantable medical
devices, nanobiosensors, lab-on-a-chip platforms for diagnostic
applications and development of nanoparticles and nanoconjugates for imaging diagnosis); and (b) new pharmacological and
therapeutic release systems (development, biofunctionalization
and validation of nanoconjugates with therapeutic capacity,
improvement of the biodistribution and controlled release of
drugs, increase of their specificity and sensitivity and reduction of
their pharmacological toxicity).2 The materials usually employed
for preparing nanostructured systems for pharmacological and
therapeutic release include: organic nanoparticles (dendrimers,
polymers and functionalized fullerenes), organic/inorganic ensembles (gold, silver and ferric oxide nanoparticles), liposomes,
proteins and peptide-based nanoparticles.
Nanoparticles are solid colloidal particles with diameters
ranging from 1 to 1000 nm. They are classified as nanospheres and

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not all the techniques developed for obtaining microparticles can

be used to yield particles of nanometric size. The aim of this work
is to review the procedures developed for preparing chitosan
nanoparticles and to illustrate their application in drug delivery
systems.

Dr. Carlos Peniche is a Professor at

the University of Havana (Cuba). He
received his Ph.D. degree (1973) from
the University of Essex (UK) and his D.Sc.
degree (2006) from the University of
Havana. He leads a research group on
polymeric biomaterials at the Centre
of Biomaterials of the University of
Havana. He is Academic of the Cuban
Academy of Sciences.

METHODS OF PREPARATION OF CHITOSAN

NANOPARTICLES
The procedure selected to prepare chitosan nanoparticles must
take into account the natural tendency of nanoparticles to
agglomerate due to their high surface free energy. Various
methods have been reported for the preparation of chitosan
nanoparticles, but the selection of a particular one must consider,
among others, the nature of the drug to be loaded into the
particles, the type of delivery device, the route of administration
and the target site. The methods employed to generate chitosan
nanoparticles include ionotropic gelation, complex coacervation,
emulsion and microemulsion techniques, and self-assembling of
hydrophobically modified chitosan.

M.Sc. Hazel Peniche received her M.Sc.

from the University of Havana (Cuba)
in 2002. During her master study she
prepared superparamagnetic chitosan
microparticles for enzyme immobilization. Currently she is engaged in the
preparation of chitosan based nanostructured systems as part of her doctoral study. She is a junior researcher at
the Biomaterials Centre of the University of Havana.
wide range of molecular weights and degrees of acetylation. Moreover, chitosan can be chemically modified through its structural
NH2 and OH groups generating derivatives with improved
properties providing flexibility in formulation development.
Chitosan is biocompatible, biodegradable, non-toxic and
mucoadhesive, which make it attractive for applications in
medicine and pharmacy (Table 1). It is degraded by lysozyme in
serum10 and lipase, an enzyme present in the saliva and in human
gastric and pancreatic fluids.11 The products of the enzymatic
degradation of chitosan are non-toxic. Chitosan increases cell
membrane permeability, both in vitro12,13 and in vivo.14 From
a biopharmaceutical viewpoint, chitosan has the potential of
serving as an absorption enhancer across intestinal epithelia
prolonging the residence time of delivery systems at absorption
sites,15 and has the ability to open the tight junctions of
cell membranes. Chitosan has antimicrobial,16 antiviral17 and
antitumoral activity.18 The immunoadjuvant activity of chitosan
has also been recognized.19 21
All these interesting characteristics have led to the development
of numerous applications of chitosan and its derivatives in
pharmacy, as matrices in compressed tablets, membranes and
microparticles for drug delivery.22 24 The techniques employed
to prepare chitosan microparticles for drug encapsulation include
ionotropic gelation, spray drying, emulsion phase separation,
simple and complex coacervation, chitosan coatings, amongst
others, and there are various reviews on the subject.25,26 However,

H OH

Ionotropic gelation
Ionotropic gelation consists of the ionic crosslinking of chitosan
with multivalent counter-ions such as Fe(CN)6 4 , Fe(CN)6 3 , citrate
and sodium tripolyphosphate (TPP). The procedure is very simple;
it is carried out in aqueous medium, and does not involve the
use of organic solvents. The nanoparticles can be obtained by the
addition of a dilute chitosan acid solution to a solution of TPP
or vice versa, with stirring. In any case, the size of the particles
is strongly dependent on the concentration of both chitosan
and TPP.
The technique was first reported in 1997 by Calvo et al.27 for
the preparation of chitosan, chitosanpoly(ethylene oxide) and
chitosanpoly(ethylene oxide)poly(propylene oxide) nanoparticles. Chitosan particles of nanometric size were obtained for
chitosan concentrations up to 2.8 g L1 and TPP concentrations
from 0.21 to 0.43 g L1 . The size and surface charge of particles
can be modified by varying the ratio of chitosan and stabilizer.
Janes et al.28 prepared doxorubicin-loaded chitosan nanoparticles
in the following way. Chitosan or chitosandoxorubicin complex
was dissolved at 0.175% (w/v) with 1% (v/v) acetic acid and then
adjusted to pH = 4.74.8 with 10 N NaOH. To 500 mL of this
solution, 100 mL of 0.291% (w/v) TPP was added under magnetic
stirring, leading to the immediate formation of nanoparticles. This
procedure has been frequently reported in the literature for the
preparation of drug-loaded chitosan nanoparticles.29 33 The main
problematic aspects of the technique are time-stability of the colloidal dispersion which may require the addition of stabilizers, and
the need of using very dilute solutions which may be inconvenient
when large amounts of nanoparticles are required.

H OH
H O

HO

H Peniche, C Peniche

H O
O

H
H
O

NH

HO

H
H

NH2

C
CH3
(a)

(b)

884

Figure 1. Structural units of chitosan: (a) N-acetylglucosamine unit; (b)

glucosamine unit.

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Complex coacervation
Complex coacervation is achieved by mixing two oppositely
charged polyelectrolytes. The polyelectrolyte complex (sometimes
called coacervate complex) separates into a polymer-rich phase
that coexists with a very dilute phase. The polyelectrolyte complex
produced forms an insoluble film or barrier that covers the
particles.
Hu et al.34 prepared chitosanpoly(acrylic acid) (PAA) nanoparticles by the dropwise addition of dilute chitosan solutions (0.02

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wt%) into 0.002 wt% PAA aqueous solutions under magnetic stirring, and vice versa. In both cases, particles with a coreshell
structure were formed, but transmission electron micrographs of
dried particles revealed a solid structure for the particles obtained
by adding chitosan into PAA solutions whereas there were cavities
in the cores of the particles obtained by adding PAA into chitosan
solutions. The zeta potential of the nanoparticles was also dependent on the order of addition. Nanoparticles with a positive charge
were produced by adding PAA into chitosan solutions, whereas
negatively charged nanoparticles were generated by adding chitosan into PAA solutions. Recently Davidenko et al.35 studied the
influence of the pH of the chitosan and PAA solutions, their concentrations and the purification procedure on the particle dimensions
and their size distribution. They obtained nanoparticles for higher
polyelectrolyte concentrations (up to 0.1% (w/w).
Hu et al. also prepared chitosanPAA nanoparticles by template
polymerization of PAA in a chitosan solution at 70 C using K2 S2 O8
as initiator.34,36 After polymerization, aggregates were separated
by filtration and the nanoparticles in the supernatant solution were
characterized. In a later work the hollow nanospheres obtained by
this procedure were crosslinked with glutaraldehyde and loaded
with doxorubicin.36
Chitosan nanoparticles encapsulating siRNA were prepared by
complex coacervation of chitosan and polyguluronate (PG), which
is isolated from alginate and is strongly related to ionic interactions
of negatively charged alginate. Various physicochemical properties of chitosanPG nanoparticles including size, surface charge,
morphology and interaction with siRNA were characterized. The
mean diameter of siRNA-loaded chitosan-based nanoparticles
ranged from 110 to 430 nm, depending on the weight ratio
between chitosan and siRNA.37
Carboxymethylcellulose and alginate have also been
complexed with chitosan to prepare nanoparticles. Chitosancarboxymethylcellulose nanoparticles were subsequently
coated with plasmid DNA (pDNA),38 whereas chitosanalginate
nanoparticles were loaded with insulin.39 Nanoparticles have also
been obtained in which the polyion is the active principle itself (heparin, DNA). Alonso and co-workers patented a procedure
for obtaining chitosanheparin nanoparticles crosslinked with
TPP.40 Chitosanheparin particles have also been prepared by
others.41,42 Fig. 2 shows a scanning electron micrograph of chitosanheparin nanoparticles prepared by complex coacervation
(Peniche C, unpublished). The particle size of these nanoparticles
was 14633 nm. There are numerous reports on the preparation of

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Chitosan-based system
Nanoparticles
Liposomes
Superparamagnetic particles

Chitosannanoapatite
composites

Applications
Drug delivery; gene therapy;
vaccines
Drug delivery; vaccines
(immunoadjuvant)
Magnetic hyperthermia (therapy);
magnetic cell labelling and
separation (therapy); targeted
drug delivery (nanomedicine);
contrast agent in magnetic
resonance imaging (diagnosis)
Bone restoration (orthopaedics,
maxillar surgery)

chitosanDNA nanoparticles43 47 and quaternized chitosanDNA

nanoparticles48 using complex coacervation. Nanoparticles are
spontaneously formed after addition of DNA solution into chitosan dissolved in acetic acid solution, under mechanical stirring
at room temperature. The size of the complexes can be varied from
50 to 700 nm. The particles have a negative charge for chitosan
nitrogen to DNA phosphate ratio (N/P ratio) below 2. At N/P = 2,
the particles are neutral and become positively charged for higher
N/P values, reaching +35 mV at N/P = 5.49
It can be concluded that, similarly to ionotropic gelation,
complex coacervation is a very mild and useful procedure for
obtaining nanoparticles, but it is necessary to start with very dilute
solutions and to control the pH carefully during preparation,
purification and storage to avoid aggregation. Chitosan molecular
weight (MW) and degree of acetylation (DA), the stoichiometry of
the reaction mixture (in terms of ionic groups of the polycation to
the ionic groups of the polyanion) and the order of addition also
have an influence on nanoparticle composition, particle charge
and stability.
Microemulsion
The principle underlying this technique is to prepare the
nanoparticles in the aqueous core or the micellar droplets formed
in a water-in-oil reverse microemulsion.50 The microemulsion is
formed by dispersing an aqueous chitosan solution containing
the drug in the continuous oil phase, composed of an organic
solvent (n-hexane, isooctane) and surfactant, usually sodium bis(2ethylhexyl)sulfosuccinate (AOT), with continuous stirring on a
vortex mixer to avoid any turbidity. Then, a crosslinking agent is
added with stirring and the organic solvent is evaporated. The
dry mass obtained has to be freed from the surfactant, either
by washing with 20% (v/v) acetonewater solution first and
distilled water afterwards,51 or by dissolving the pellet in water
and adding a salt for precipitating AOT. In this latter case the
solution is dialysed and lyophilized to a dry powder.52 The method
produces nanoparticles of less than 200 nm and very narrow size
distributions. The major drawbacks of this technique are the use
of organic solvents, the involved preparation procedure and the
complexity of the cleansing step.
Emulsion-droplet coalescence
This method was developed by Tokumitsu et al.53 and consists of
preparing a stable water-in-oil emulsion of a chitosan solution in
liquid paraffin oil, and a similar one but with NaOH solution as the

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885

Figure 2. Scanning electron micrograph of chitosanheparin nanoparticles obtained by complex coacervation.

Table 1. Nanostructured chitosan systems for pharmacological and

therapeutic applications

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aqueous phase. By mixing both emulsions with vigorous stirring,
the droplets coalesce and small chitosan particles precipitate.
Using this procedure those authors prepared gadopentetic acidloaded chitosan nanoparticles. Particle size and drug loading are
influenced by the same variables affecting these parameters in
simple coacervation: degree of acetylation and molecular weight
of chitosan and type of drug encapsulated.
Emulsion-solvent extraction
In this method an oil-in-water emulsion is obtained by adding an
organic solvent partially miscible in water into a chitosan solution
containing a stabilizing agent (i.e. poloxamer) under mechanical
stirring, followed by high-pressure homogenization. The emulsion
is then diluted with a large amount of water to extract the organic
solvent. Nanoparticles are formed as a result of the diffusion of the
organic solvent into water. The method is suitable for hydrophobic
drugs and allows a high percentage of drug loading. The most
important shortcomings of this method include the use of organic
solvents and the high shear forces used during nanoparticle
preparation.54
Self-assembling of hydrophobically modified chitosan
Chitosan-based nanoparticles have also been obtained by hydrophobic modification of a soluble derivative of chitosan.
Self-assembled nanoparticles based on hydrophobically modified glycol chitosan (HGC) were prepared as carriers for
paclitaxel.55 HGC conjugates were prepared by chemically linking 5-cholanic acid to glycol chitosan chains using 1-ethyl-3(3-dimethylaminopropyl)-carbodiimide chemistry. In phosphatebuffered saline (pH = 7.4), the synthesized HGC conjugates formed
nanoparticles with a diameter of 200 nm and exhibited high thermodynamic stability as reflected by their low critical aggregation
concentration (0.03 mg mL1 ). The same principle for preparing
chitosan-based nanoparticles was used by Chiu et al.56 using a hydrophobically modified chitosan (N-palmitoylchitosan (NPCHI)).
By conducting molecular dynamic simulations, it was found that a
degree of substitution of 5% of palmitoyl groups on the chitosan
backbone was sufficient to allow NPCHI to form nanoparticles,
due to a significant increase in the intra- and intermolecular hydrophobic interactions. It was suggested that the prepared NPCHI
nanoparticles may serve as a carrier for intracellular delivery of
therapeutic agents.

PROTEIN AND DRUG DELIVERY APPLICATIONS

OF CHITOSAN NANOPARTICLES

886

Oral administration
The oral route of administration is the most frequently used way for
administering drugs, although it may have serious inconveniences
for peptides and protein drugs.57 The primary reasons for this are
that during the transit through the gastrointestinal tract they
are exposed to enzymatic degradation, and they also have to
overcome the mucus layer and epithelial absorption barriers.
This results in their low bioavailability by the oral route.58,59 The
use of polymeric nanoparticles for encapsulating peptides and
protein drugs is a promising approach for protecting the biological
molecules from enzymatic degradation. In addition, it has been
stated that particles smaller than 500 nm can pass across the
intestinal mucus layer by endocytocis.60 Chitosan nanoparticles
have the advantage of being mucoadhesive, which increases the
residence time in the intestine improving drug bioavailability.61

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H Peniche, C Peniche
Furthermore, chitosan is an adsorption promoter: it opens the
tight junctions of cell membranes increasing the transport of a
drug across the mucosal epithelial membrane.62

Andersson and Lofroth

obtained a heparinchitosan complex
using the microemulsion method and concluded that it could
be an interesting system for the oral administration of drugs.41
Alginatechitosan nanoparticles administered orally to diabetic
rats were found effective for oral insulin delivery.63 Zang et al.64
reported that water-soluble chitosan nanoparticles enhance and
prolong the intestinal absorption of bovine serum albumin, which
makes them a potential protein delivery system.64
Nasal and pulmonary administration
Pulmonary and nasal routes are the other mucosal pathways
that are attracting considerable attention as alternative routes
for peptide and protein delivery since they involve very large
surface areas and less intracellular and extracellular enzymatic
degradation. Chitosan was demonstrated to promote the nasal
absorption of insulin in rats and sheep. Fernandez-Urrusuno
et al.65 found that chitosan nanoparticles were efficient vehicles
for the transport of insulin through the nasal mucosa. It should be
noted, however, that insulinchitosan powder (chitosan blended
with insulin using a pestle and mortar), was shown to have
greater bioavailability than chitosan nanoparticles containing
insulin.66 Poly(ethylene glycol)-grafted chitosan (PEG-g-chitosan)
nanoparticles obtained by ionotropic gelation administered
intranasally to rabbits enhanced the absorption of insulin by the
nasal mucosa to a greater extent than a suspension of insulinPEGg-chitosan and control insulin solution.67
Amidi et al.68 showed the potential of N-trimethylchitosan
nanoparticles as a new delivery system for the transport of proteins through the nasal mucosa. Hybrid chitosancyclodextrin
nanoparticles have also demonstrated their potential for enhancing the transport of complex molecules across the nasal barrier.69
Enrquez de Salamanca et al.70 reported chitosan-based nanoparticles that are able to provide a controlled release of encapsulated
insulin, evidencing release profiles that are dependent on their
lipid composition. Moreover, the satisfactory in vivo results obtained by those authors confirmed the potential of these newly
developed drug delivery systems as drug carriers through distinct
mucosal routes.
Ocular administration
The main problems of conventional ocular therapy are short residence time, drug drainage and frequent instillation. Nanoparticles
seem to be promising vehicles for designing new controlled delivery systems to improve the ocular bioavailability of drugs for
ophthalmic diseases.71,72
Enrquez de Salamanca et al.73 reported chitosan nanoparticles
that readily penetrate conjunctival epithelial cells and are well
tolerated at the ocular surface of rabbits. These carriers hold
promise as a drug delivery system for the ocular mucosa. More
recently nanoparticles consisting of hyaluronic acid and chitosan,
which exhibit very low cytotoxicity, and have the ability to enter
the corneal epithelial cells by CD44 receptor-mediated endocytic
uptake, have been described.74 De la Fuente et al.75 reported
that their group has designed and developed a delivery platform
based on chitosan nanoparticles, which suits the requirements of
the topical ocular route. These nanosystems have been specifically
adapted for the delivery of hydrophilic and lipophilic drugs and
also polynucleotides onto the eye surface.

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A recent review by Paolicelly et al.76 provides an updated

overview of the advances made in ocular delivery of bioactive
molecules by means of chitosan-based nanosystems, and their
potential relevance in clinical use.
Delivery of vaccines
The immunoadjuvant effect of chitosan has been used profitably in the development of various systems for vaccine
administration.25,77 81 In fact, it has been shown that chitosan
offers a potentially safer alternative to Freund immunoadjuvant
and Al(OH)3 in liposome-based vaccine delivery systems.21 Cui
and Mumper45 reported that several different chitosan-based
nanoparticles containing pDNA resulted in both detectable and
quantifiable levels of luciferase expression in mouse skin 24 h
after topical application and significant antigen-specific IgG titre
to expressed -galactosidase at 28 days. Madrigal-Carballo et al.82
studied the effect of chitosan nanoparticles on the uptake and antigen presentation of the model protein hen-egg white lysozyme
to peritoneal macrophages isolated from mice. Their results suggest that a chitosan nanoparticle system is a potential candidate
for an oral vaccine delivery system. The immunological effects
and membrane interactions of chitosan nanoparticles were also
studied by Pattani et al.83 using various molecular markers such
as nitric oxide production, IL-6 gene expression and lymphocyte
proliferation involved in the wound-healing process. The results
indicate the immunoactivating nature of chitosan nanoparticles
and their strong membrane interactive potential.
Bivas-Benita et al.84 indicated that pulmonary delivery of DNA
vaccines against tuberculosis may provide an advantageous
delivery route compared to intramuscular immunization, and that
increased immunogenicity can be achieved by delivery of this type
of DNA encapsulated in chitosan nanoparticles.

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CONCLUSIONS
It has been shown that chitosan nanoparticles exhibit excellent
qualities for drug delivery applications. This is due to the
outstanding biological properties of chitosan. It is a biocompatible,
biodegradable, non-toxic, cationic polymer that possesses good
mucoadhesivity and the important capacity for increasing the
penetration of drugs across mucosal barriers. Therefore, it has
been shown to be a suitable polymer matrix for designing noninvasive routes of drug administration, such as oral, mucosal
(nasal, pulmonary) and ocular routes. In all cases the use
of chitosan facilitates or improves drug bioavailability. The
benefits of using chitosan nanoparticles as non-viral vectors in
gene therapy and their adjuvant effect in vaccine formulations
have also been documented. Several methods for obtaining
chitosan nanoparticles have been developed, and some of them,
such as ionotropic gelation and complex coacervation, involve
very mild preparation conditions. Consequently, an increasing
number of scientific reports on new applications of chitosanbased nanoparticles for drug delivery, gene therapy and vaccine
administration at the nanoscale is foreseen in the near future.

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887

Gene therapy
Increasingly, nucleic acids are being applied for both vaccination and therapeutic gene expression, and chitosan nanoparticles
have been proposed as promising non-viral gene carriers. Chitosanalginate coreshell nanoparticles obtained using a waterin-oil reverse microemulsion template were used to encapsulate
pDNA for gene delivery via the cell endocytosis pathway.51 However, as has been stated above, chitosanDNA nanoparticles may
be readily formed by complex coacervation between the positively charged amine groups on chitosan and negatively charged
phosphate groups on DNA.46,85 The effect of chitosan molecular
weight and charge ratio on particle formation was tested by depolymerizing 102 103 mol g1 , 89.4% deacetylated chitin into
smaller oligomers.86 The influence of the degree of acetylation of
chitosan on nanoparticle formation has also been investigated. For
chitosans of the same molecular weight (390 103 mol g1 ), increasing the degree of polymer acetylation required increasing the
amount of chitosan in order to achieve complete DNA complexation. For chitosan with a molecular weight of 390 103 mol g1 ,
the N/P ratio to achieve complete DNA complexation for degrees
of acetylation of 10, 30 and 28% was 3.3 : 1, 5.0 : 1 and 9.0 : 1,
respectively. The size and morphology of these nanoparticle formulations were not significantly different. The increased degree of
acetylation results in a decrease in overall luciferase expression levels in HEK293, HeLa and SW756 cells due to particle destabilization
in the presence of serum proteins.87,88
Protection of encapsulated pDNA from nuclease attack offered
by chitosan nanoparticles was confirmed by Bozkir and Saka89

by assessing degradation in the presence of DNase I, and the

transformation of the plasmids with incubated nanoparticles was
examined by -galactosidase assay. Model pDNA existed as a
mixture of both supercoiled (84.2%) and open circular (15.8%)
forms.
The use of siRNA as a potential therapeutic for treatment of
various diseases is limited due to its rapid degradation and low
intracellular association in vitro and in vivo. Lee et al.37 reported
that chitosanPG nanoparticles showed low cytotoxicity and
were useful in delivering siRNA to HEK 293 FT and HeLa cells.
Those authors considered these nanoparticles promising for siRNA
delivery due to their low cytotoxicity and ability to transport siRNA
into cells.
The in vitro and in vivo transfection efficiency of chitosan
and its derivatives as vectors for gene therapy has also been
investigated.90 Zheng et al.91 prepared three types of chitosan
nanoparticles. These were a 60% trimethylated chitosan oligomer
(TMCO-60%, with a degree of substitution of 60% and MW =
221103 mol g1 ) and two chitosan samples differing in molecular
weight and DA (chitosan-43: MW = 43 103 45 103 mol g1 ,
DA = 13%; and chitosan-230: MW = 230 103 mol g1 , DA =
90%). Those authors used them to encapsulate pDNA encoding
green fluorescent protein (GFP) using the complex coacervation
technique. An in vivo study, carried out by oral administration
of pDNAchitosan nanoparticles to mice, showed outstanding
GFP expression in the gastric and upper intestinal mucosa.
GFP expression in the mucosa of the stomach and duodenum,
and jejunum, ileum and large intestine was found, respectively,
in 100, 88.9, 77.8 and 66.7% of the nude mice examined.
TMCO-60%pDNA nanoparticles had a higher in vitro and in vivo
transfection activity with minimal toxicity, which made them an
attractive non-viral vector for gene therapy via oral administration.
More recently PEG-g-chitosan nanoparticles prepared by
ionotropic gelation with TPP were investigated as potential gene
carriers.92 High and long-lasting gene expression levels for these
nanoparticles were reported. Moreover, PEG-g-chitosanTPP
nanoparticles also mediated high gene expression levels in vivo,
following nasal administration. These results indicate the potential of PEG-g-chitosanTPP nanoparticles as transmucosal gene
delivery systems.

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