Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 111

parameters for nanoparticle synthesis, including concentrations of DNA, chitosan, and
sodium sulfate, temperature of the solutions, pH value of the buffer, and MWs of chitosan
and DNA. At an amino group to phosphate group ratio (N/P ratio) between 3 and 8 and a
chitosan concentration of 100 mg/mL, the size of particles was optimized to 100-250 nm
with a narrow distribution, with a composition of 35.6% and 64.4% by weight for DNA
and chitosan, respectively. The surface charge of these particles was slightly positive
with a zeta potential of 112-118 mV at pH lower than 6.0, and became nearly neutral at
pH 7.2. In this system, the transfection efficiency of chitosan-DNA nanoparticles was
cell type dependent. They also developed three different schemes to conjugate transferrin
or knob protein to the nanoparticle surface. Transferrin conjugation only yielded a maxi-
mum 4-fold increase in 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 polyethylene glycol (PEG) on the nanoparticles allowed lyophi-
lization without aggregation and without loss of bioactivity for at least 1 month in storage.
The clearance of PEGylated nanoparticles in mice following intravenous (i.v.) administra-
tion was slower than unmodified nanoparticles at 15 min, and with higher depositions in
kidney and liver. However, no difference was observed at the 1 h time point.
A study of the condensation of depolymerized chitosans with DNA was carried out.
High-molecular-weight (HMW) chitosan was depolymerized by oxidative degradation
with NaNO 2 at room temperature to obtain 11 samples of chitosan derivatives of varying
MWs with a view to assessing their effective MW range for gene delivery applications. The
results showed that chitosans with very low MWs and high charge density exhibited a
strong binding affinity to DNA compared to HMW chitosans [35-36].
Chitosan was used to transfer luciferase plasmid into tumor cells. Chitosan largely
enhanced the transfection efficiency of luciferase plasmid (pGL3). Transfection efficiencies
of the pGL3-chitosan complexes were dependent on pH of the culture medium, stoichiom-
etry of pGL3, chitosan, serum, and molecular mass of chitosan. The transfection efficiency
at pH 6.9 was higher than that at pH 7.6. The optimum charge ratio of pGL3:chitosan was
1:5. Chitosan of 15 and 52 kDa largely promoted luciferase activities. The transfection
efficiency mediated by chitosan of >100 kDa was less than that mediated by chitosan of
15 and 52 kDa. Heptamer (1.3 kDa) did not show any gene expression. Chitosan showed
resistance to serum [37].
In an approach to study the transfection mechanism of plasmid-chitosan complexes as
well as the relationship between transfection activity and cell uptake, Ishii et al. [38] used
fluorescein isothiocyanate-labeled plasmid and Texas Red-labeled chitosan. They observed
that there are several factors that contribute to transfection activity, MW of chitosan, stoi-
chiometry of the complex, as well as serum concentration and pH of the transfection
medium. The level of transfection with plasmid-chitosan complexes was found to be high-
est when the molecular mass of chitosan was 40 or 84 kDa, the ratio of chitosan nitrogen to
DNA phosphate (N/P ratio) was 5, and the transfection medium contained 10% serum at
pH 7.0. While investigating the transfection mechanism, they found that plasmid-chitosan
complexes most likely condense to form large aggregates, which adsorb on the cell surface.
After this, plasmid-chitosan complexes are endocytosed, and possibly released from
endosomes due to the swelling of lysosomes along with the swelling of the plasmid-
chitosan complex, causing the endosome to rupture. Finally, these complexes were observed
to accumulate in the nucleus.
Probing for a solution to track the efficiency of DNA delivery, Lee et al. [39] used fluo-
rescence resonance energy transfer (FRET) to monitor the molecular dissociation of a
chitosan-DNA complex with different MWs of chitosan. Chitosan with different MWs