Gene Delivery Using Chemical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 178

presence of 10-20% serum was significantly higher than in serum-free medium [351] .
The chitosan polyplexes were less cytotoxic than Lipofectin TM , Lipofectamine TM , or
PEI [346,351,352] . When evaluated in vitro in 293 cells, the IC50 (630 mg/ml) of the
DNA-chitosan complex was found to be significantly higher than IC50 (75 mg/ml)
of the DNA-PEI. Moreover, chitosan had demonstrated variable transfection results
depending upon the type of cell line used for in vitro studies [348,353] . The DNA-
chitosan nanoparticles displayed higher transfection efficiency for HEK293 cells in
comparison to MG3 cells and mesenchymal stem cells [352] .
Several structural modifications were done to improve transfection efficiency of
chitosan polyplexes. Attempts have been made to improve the cationic property of
chitosan by N-quaternization of chitosan terminal amine groups. Quaternization leads
to higher transfection efficiency despite higher cytotoxicity [354,355] . The higher
cytotoxicity can be minimized with incorporation of PEG in trimethyl chitosan to
give PEG-grafted chitosan [356] . Another strategy explored to improve cationization
was incorporating PLL in chitosan, which exhibited superior transfection efficiency
and reduced cytotoxicity as compared to both polylysine and 25 kDa PEI [357] .
To minimize the aggregation and improve interaction with cellular interaction,
hydrophobic moieties such as stearic acid [358] or deoxycholic acid [359] have been
conjugated to chitosan. Stearic acid-chitosan complexes demonstrated about eight-
fold higher transfection efficiency as compared to underivatized chitosan polyplexes.
Deoxycholic acid derivatives displayed varied transfection efficiency depending upon
molecular weight of the chitosan. Both low- (5 kDa) and high- (200 kDa) molecular-
weight-deoxycholic acid-chitosan polyplexes exhibited lower gene transfer activity
because of complex instability and reduced DNA release, respectively. However, the
40 kDa analogue demonstrated highest transfection efficiency.
Several approaches were employed to achieve cell-specific targeting using chi-
tosan polyplexes. Conjugation of chitosan with transferrin selectively targets tumor
cells. The transferrin-conjugated chitosan nanoparticles displayed a fourfold increase
in the transfection efficiency compared to conventional DNA-chitosan nanoparticles
when evaluated against HEK293 cells [360] . In another study, chitosan was attached
with a monosaccharide or disaccharide side chain [361] . It was known that hepato-
cytes express asialoglycoprotein receptors that have affinity for glycoprotein having
a terminal galactose moiety. Galactosylated or lactosylated chitosan was prepared to
target hepatocyte cells. Galactosylated chitosan- g -dextran, synthesized by coupling
lactobionic acid bearing a galactose group with chitosan for liver targeting and by
attaching dextran to galactosylated chitosan for stability in water, displayed effi-
cient transfection efficiency in HepG2 cells. However, lactosylated chitosans did not
improve transfection efficiency in HepG2 [353] . It was discussed that the result was
probably caused by reduction in surface charge of the complexes after lactosylation
leads to interparticular aggregation.
Galactosylated chitosan with polyethylene-glycol and poly(vinylpyarolidone) graft-
ing gives galactosylated chitosan-graft-polyethylene-glycol (GC-PEG) and galactosyl-
ated chitosan-graft-poly(vinylpyarolidone) (GC-PVP), respectively, which displayed
efficient DNA delivery to HepG2 cells [362] . The hydrophilic nature of PVP and PEG
prevent enzymatic degradation of the DNA complexes by plasma protein [363] .