DNA complexes.
The presence of 10% fetal bovine serum did not
interfere with their transfection ability. The study
also developed three different schemes to conjugate
transferrin or KNOB protein to the nanoparticle
surface. The transferrin conjugation only yielded a
maximum of 4-fold increase in their transfection
efficiency in HEK293 cells and HeLa cells, whereas
KNOB conjugated nanoparticles could improve the
gene expression level in HeLa cells by 130-fold.
Conjugation of PEG on nanoparticles allowed
lyophilization without aggregation, and without loss
of bioactivity for at least 1 month in storage. The
clearance of PEGylated nanoparticles in mice follow-
ing i.v. administration was slower than the unmodi-
fied nanoparticles at 15 min, and with higher
depositions in kidney and liver. However, no differ-
ence was observed during the first hour.
Self-aggregates were prepared [104] by hydro-
phobic modification of CS with deoxycholic acid in
aqueous media. Self-aggregates have a small size
(mean diameter of 160 nm) with an unimodal size
distribution. Self-aggregates can form charge com-
plexes when mixed with plasmid DNA. The useful-
ness of self-aggregates/DNA complex for transfer of
genes into mammalian cells in vitro has been
suggested. Several transfection studies using chemi-
cally modified CS have been reported. Trimethyl CS
oligomers were examined for their potency as DNA
carriers [105]. Chitosan and lactosylated CS carriers
were investigated for their transfection efficiencies in
vitro [106]. Recently, galactosylated CS-g dextran
DNA complexes have been prepared [107]. Galactose
groups were chemically bound to CS for liver
specificity and dextran was grafted to increase the
stability of the complex in water. It was shown that this
system could efficiently transfect liver cells.
Chew et al. [108] studied the i.m. immunization
with full-length Der p 1 cDNA induced significant
humoral response to the left domain (approximately
corresponding to amino acids 1 116), but not to the
right domain (approximately corresponding to amino
acids 117 222) of Der p 1 allergen. Authors explored
the use of CS DNA nanoparticles for oral immuniza-
tion to induce the immune responses specific to both
left and right domains of Der p 1. DNA constructs
pDer p 1 (1 222) and pDer p 1 (114 222), which
were complexed with CS and delivered orally
followed by an i.m. injection of pDer p 1 (1 222)
after 13 weeks. Such an approach has successfully
primed Th1-skewed immune responses against both
domains of Der p 1. It was suggested that such a
strategy could be further optimized for more effica-
cious gene vaccination for full-length Der p 1.
Numerous studies have been reported on prophy-
lactic and therapeutic use of genetic vaccines for
combating a variety of infectious diseases in animal
models. Recent human clinical studies with the gene
gun have validated the concept of direct targeting of
dendritic cells (Langerhans cells) in the viable
epidermis of the skin. However, it is unclear whether
the gene gun technology or other needle-free devices
will become commercially viable. Cui and Mumper
[109] investigated the topical application of CS-
based nanoparticles containing plasmid DNA
(pDNA) as a potential approach to genetic immuni-
zation. Two types of nanoparticles were investigated:
(i) pDNA-condensed CS nanoparticles and (ii)
pDNA-coated on pre-formed cationic CS/carboxy-
methylcellulose (CMC) nanoparticles. These studies
have shown that both CS and a CS oligomer can
complex CMC to form stable cationic nanoparticles
for subsequent pDNA coating. Selected pDNA-
coated nanoparticles (with pDNA up to 400 mg/
mL) were stable to challenge with the serum.
Several different CS-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 titer to expressed h-galactosidase at
28 days.
S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 21
Borchard [110] has recently published a review on
the efficient non-viral gene delivery using cationic
polymers as DNA-condensing agents. The gene
delivery is dependent on several factors such as
complex size, complex stability, toxicity, immunoge-
nicity, protection against DNase degradation, intra-
cellular trafficking and processing of the DNA. The
review also examined the advances made in the
application of CS and CS derivatives to non-viral
gene delivery. It gives an overview of the transfection
studies performed by using CS as a transfection agent.
5.5. Topical delivery
Due to good bioadhesive property and ability to
sustain the release of the active constituents, CS has
been used in topical delivery systems. Bioadhesive CS
microspheres for topical sustained release of cetyl
pyridinium chloride have been evaluated [55].
Improved microbiological activity was shown by
these microparticulate systems. Conti et al. [111]
prepared microparticles composed of CS and designed
as powders for topical wound-healing properties.
Blank and ampicillin-loaded microspheres were pre-
pared by spray-drying technique. In vivo evaluation in
albino rats showed that both drug-loaded and blank
microspheres have shown good wound healing
properties.
5.6. Ocular delivery
De Campos et al. [112] investigated the potential of
CS nanoparticles as a new vehicle to improve the
delivery of drugs to ocular mucosa. Cyclosporin A
(CyA) was chosen as a model drug. A modified ionic
gelation technique was used to produce CyA-loaded
CS nanoparticles. These nanoparticles with a mean
size of 293 nm, a zeta potential of +37 mV, high CyA
association efficiency and loading of 73% and 9%,
respectively were obtained. The in vitro release
studies, performed under sink conditions, revealed
the fast release during the first hour followed by a
more gradual drug release during the 24-h period. The
in vivo experiments showed that after topical instilla-
tion of CyA-loaded CS nanoparticles to rabbits,
therapeutic concentrations were achieved in the
external ocular tissues (i.e., cornea and conjunctiva)
within 48 h while maintaining negligible or undetect-
able CyA levels in the inner ocular structures (i.e., iris/
ciliary body and aqueous humour), blood and plasma.
These levels were significantly higher than those
obtained following the instillation of CS solution
containing CyA and an aqueous CyA suspension. The
study indicated that CS nanoparticles could be used as
a vehicle to enhance the therapeutic index of the
clinically challenging drugs with potential application
at the extraocular level.
5.7. Chitosan as a coating material
Chitosan has good film forming properties and
hence, it is used as a coating material in drug delivery
applications. Chitosan-coated microparticles have
many advantages such as improvement of drug
payloads, bioadhesive property and prolonged drug
release properties over the uncoated particles. Chito-
san-coated microspheres composed of poly(lactic
acid) poly(caprolactone) blends have been prepared
[113]. These microspheres showed good potential for
the targeted delivery of antiproliferative agents to treat
restenosis. Shu and Zhu [73] have prepared the
alginate beads coated with CS by three different
methods. The release of brilliant blue was not only
affected by CS density on the particle surface, but also
on the preparation method and other factors. Chiou et
al. [114] have used different molecular weight
chitosans for coating the microspheres. The initial
burst release was observed in the first hour with 50%
release of lidocaine. But, 19.2% release occurred at
25th hour for the un-coated particles and 14.6% at the
90th hour for the CS-coated microspheres.
6. Chemically modified chitosans
Various chemical modifications of CS have been
studied to alter its properties. N-Trimethyl chitosan
chloride (TMC), a quaternized CS derivative, has
been proven to effectively increase the permeation of
hydrophilic macromolecular drugs across- the
mucosal epithelia by opening the tight junctions
[115]. The study investigated the intestinal absorption
of octreotide when it is co-administered with a
polycationic absorption enhancer, TMC. Chitosan
succinate and CS phthalate were synthesized and
assessed as potential matrices for colon-specific orally
S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 22
administered drug delivery applications. The prepared
matrices resisted the dissolution under acidic con-
ditions. On the other hand, improved drug release
profiles were observed in basic conditions. These
results suggested the suitability of the prepared
matrices in colon specific and orally administered
drug delivery applications [116]. In order to overcome
the low solubility of CS in neutral pH, which is the
major drawback to use this type of polymer as a
transfection agent, N-trimethylated and N-triethylated
oligosaccharides have been synthesized [105].
Lee et al. [104] synthesized the hydrophobically
modified CS containing 5.1 deoxycholic acid groups
per 100 anhydroglucose units by 1-ethyl-3-(3-dime-
thylaminopropyl) carbodiimide (EDC)-mediated cou-
pling reaction as shown in Fig. 11. Since deoxycholic
acid can form self-assemblies in aqueous media, it
was found that the modified CS also formed the self-
aggregates. The self-aggregates were characterized by
fluorescence spectroscopy and dynamic light scatter-
ing method. A charge complex was produced between
the cationically charged self-aggregates and the
negatively charged plasmid DNA. The feasibility of
self-aggregates as an in vitro delivery vehicle was
investigated for the transfection of genetic material in
mammalian cells.
Microcrystalline CS has been investigated as a gel
forming excipient [117]. Matrix granules of CS of
differing physicochemical properties loaded with
either ibuprofen or paracetamol as model drugs have
been prepared. Varying the amount or molecular
weight of the microcrystalline CS and to a lesser
extent by the degree of deacetylation controlled
release rate. Giunchedi et al. [59] prepared and
characterized a new derivative of CS: methyl
pyrrolidone CS. It randomly carries pyrrolidinone
groups covalently attached to the polysaccharide
backbone. This CS derivative combines the biocom-
Fig. 11. A scheme of the coupling mechanism between chitosan and deoxycholic acid using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) through amide linkage formation [taken from Ref. 104].
S.A. Agnihotri et al. / Journal of Controlled Release 100 (2004) 528 23
patibility of CS [118] and hydrophilic characteristics
of the pyrrolidinone moiety [119], being particularly
susceptible to the hydrolytic action of lysozyme
[120]. The microparticles were characterized by
S.E.M., particle size analyzer, DSC and in vitro
ampicillin release. Drug release characteristics
depend upon the nature of CS used.
Chen et al. [121] studied the modification of CS by
coupling with linoleic acid (LA) through 1-ethyl-3-(3-
dimethylaminopropyl) carbodiimide-mediated reac-
tion to increase its amphipathicity for improved
emulsification. The micelle formation of linoleic
acid-modified CS in 0.1 M acetic acid solution was
enhanced by O/W emulsification with methylene
chloride, an oil phase. Fluorescence spectra indicated
that without emulsification, the self-aggregation of
LA-CS occurred at the concentration of 1.0 g/L or
above, and with emulsification, self-aggregation was
greatly enhanced followed by a stable micelle
formation at 2.0 g/L. Addition of 1 M NaCl solution
promoted the self-aggregation of LA-CS particles
both with and without emulsification. The nanosize
micelles of LA-CS were formed ranging in size
between 200 and 600 nm. The LA-CS nanoparticles
were used to encapsulate the lipid soluble model
compound, retinal acetate, with 50% efficiency.
Chitosan was chemically modified [122] by graft
copolymerization of poly(ethylene glycol) diacrylate
macromonomer onto CS backbone. Microspheres
based on chitosan and polymer grafted chitosan were
prepared by a polymer dispersion technique. A
comparative study in relation to structural deviation
among CS and modified CS microspheres was
evaluated. These chemically modified CS micropar-
ticles were hydrophilic in nature and formed aggre-
gates. Chitosan derivative with galactose groups was
synthesized by introducing galactose group into the
amine group of CS [123]. The results indicated that
although acyl reaction on the part of amino groups of
CS took place, the degree of galactosylated substitu-
tion was 20%. Crystallinity, solubility, stability and
other physical properties were different from CS.
Microspheres of CS and galactosylated CS were
prepared by the physical precipitation and coacerva-
tion techniques, respectively. Microspheres of CS and
galactosylated CS were spherical in nature with an
average diameter of 0.54 and 1.05 Am and an average
zeta potential of +17 and +15 mV, respectively. It was
suggested that galactosylated CS microspheres could
be used for passive and active hepatic targeting.
7. Conclusions
Chitosan has the desired properties for safe use as a
pharmaceutical excipient. This has prompted accel-
erated research activities worldwide on chitosan micro
and nanoparticles as drug delivery vehicles. These
systems have great utility in controlled release and
targeting studies of almost all class of bioactive
molecules as discussed in this review. Recently,
chitosan is also extensively explored in gene delivery.
However, studies toward optimization of process
parameters and scale up from the laboratory to pilot
plant and then, to production level are yet to be
undertaken. Majority of studies carried out so far are
only in in vitro conditions. More in vivo studies need
to be carried out. Chemical modifications of chitosan
are important to get the desired physicochemical
properties such as solubility, hydrophilicity, etc. The
published literature indicates that in the near future,
chitosan-based particulate systems will have more
commercial status in the market than in the past.
Acknowledgements
Authors thank the University Grants Commission
(UGC), New Delhi, India for a major grant (F1-41/
2001/CPP-II) sanctioned to Karnatak University to
establish Center of Excellence in Polymer Science.
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