Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 116

complexes. Mannose receptor-mediated gene transfer thus enhances the transfection
efficiency of pDNA-chitosan complexes.
Chitosan was modified with mannose to target primary APCs such as dendritic
cells (DCs) owing to the high density of mannose receptors expressed on the surface of
immature DCs. After (i.m.) immunization, the microspheres induced significantly
enhanced serum antibody and cytotoxic T lymphocyte responses in comparison with
naked DNA [64].
The mean particle diameter and average zeta potential of the galactosylated chitosan
(GC)/DNA complex were 350 nm and +22.1 mV, respectively. The GC-DNA nanoparticle
was tested to transfect HEK293 cells, and the viability of HEK293 cells was not affected by
the GC-DNA nanoparticle compared to that of the control [65].
Recently, some peptide sequences known as protein transduction domains or membrane
translocalization signals were identified and introduced for the delivery of plasmid DNA
[66,67]. Interestingly, it is known that these sequences usually contain positively charged
amino acid residues such as arginine and lysine, which have been reported to be able to
enhance transportation into cells by several groups [68,69]. Oligo-arginine conjugates dem-
onstrated characteristics similar to cell-penetrating peptides in cell translocation, and the
transfection efficiency in HeLa cells could be highly improved by conjugating oligo-arginine
to PEGylated lipids [70]. The polyamidoamine dendrimer conjugated with l-arginine was
also found to enhance gene delivery potency compared with native dendrimer [71].
Arginine-rich peptides have attracted considerable attention due to their distinct inter-
nalization mechanism. It was reported that arginine and guanidino moieties were able to
translocate through cell membranes and played a critical role in the process of membrane
permeation. Arginine was conjugated to the backbone of chitosan to form a novel chitosan
derivative, arginine-modified chitosan (Arg-CS). Arg-CS-DNA complexes were prepared
according to the method of the coacervation process. Arg-CS was characterized by FTIR
and 13 C NMR. Arg-CS-DNA polyelectrolyte complexes were investigated by agarose gel
retardation, DLS, and atomic force microscopy (AFM). Arg-CS-DNA complexes started to
form at an N:P ratio of 2:1, and the size of particles varied from 100 to 180 nm. The cytotox-
icities of Arg-CS and their complexes with plasmid DNA were determined by the MTT
assay for HeLa cells, and the results suggested that Arg-CS-DNA complexes were slightly
less toxic than Arg-CS. Moreover, the derivative alone and their complexes showed signifi-
cantly lower toxicity than PEI and PEI-DNA complexes, respectively. Taking HeLa cells as
target cells and using pGL3-control as reporter gene, the luciferase expression mediated
by Arg-CS was greatly enhanced by about 100-fold compared with the luciferase expres-
sion mediated by chitosan in different pH media. These results suggest that Arg-CS is a
promising candidate as a safe and efficient vector for gene delivery and transfection [72].
Arg-CS/DNA self-assembled nanoparticles (ACSNs) showed that particle size and zeta
potential were 200-400 nm and 0.23-12.25 mV, respectively. The transfection efficiency of
ACSNs was much higher than that of CS/DNA self-assembled nanoparticles. The average
cell viability of ACSNs was over 90% [73].
Folic acid (FA) is appealing as a ligand for targeting the cell membrane and allowing
nanoparticle endocytosis via the folate receptor (FR) for higher transfection yields. The
high affinity of folate to bind its receptor (1 nM) [72] and folate's small size allow its use in
specific cell targeting. Moreover, the ability of FA to bind its receptor to allow endocytosis
is not altered by covalent conjugation of small molecules [74]. FR is overexpressed on many
human cancer cell surfaces [75], and the nonepithelial isoform of FR (FRX) is expressed on
activated synovial macrophages present in large numbers in arthritic joints [76]. Many
researchers have used FA as a ligand with cationic liposome and other polymers to target