Bioactivities of Chitosan and Its Derivatives - Chitosan Based Hydrogels: Functions and Applications - page 112

was complexed with plasmid DNA and complex formation was monitored using dynamic
light scattering (DLS) and a gel retardation assay. Plasmid DNA and chitosan were sepa-
rately labeled with quantum dots and Texas Red, respectively, and the dissociation of the
complex was subsequently monitored using confocal microscopy and fluorescence spec-
troscopy. As the chitosan MW in the chitosan-DNA complex increased, the Texas Red-
labeled chitosan gradually lost FRET-induced fluorescence light. This observation was
noted when HEK293 cells incubated with the chitosan-DNA complex were examined with
confocal microscopy. This suggested that the dissociation of the chitosan-DNA complex
was more significant in the HMW chitosan-DNA complex. Fluorescence spectroscopy also
determined the molecular dissociation of the chitosan-DNA complex at pH 7.4 and 5.0 and
confirmed that the dissociation occurred in acidic environments. This finding suggested
that the HMW chitosan-DNA complex can more easily be dissociated in lysosomes com-
pared to a low-molecular-weight (LMW) complex. Furthermore, the HMW chitosan-DNA
complex showed superior transfection efficiency in relation to the LMW complex. Therefore,
it could be concluded that the dissociation of the chitosan-DNA complex is a critical event
in obtaining the high transfection efficiency of the gene carrier-DNA complex [39].
3.1.1.3 Chitosan Derivatives as a Gene Delivery Matrix
Although chitosan has been widely used in gene delivery, further developed applications
of chitosan for gene delivery are limited because of its poor water solubility and low trans-
fection efficiency. Its low transfection efficiency problem remains to be solved. For this
target, various modifications of the side chains of chitosan and optimizations of chitosan
formulation have been performed.
Chitosan modified with betaine could increase its ability to facilitate DNA uptake and
its cytotoxicity, both of which showed an influence on transfection efficiency. It was able to
increase the cellular uptake and transfection efficiency of complex nanoparticles in COS-7
cells to increase betaine substitution of CsB; however, the higher sensitivity of MDA-MB-468
cells to CsBs led to decreased transfection efficiency because of the increased cytotoxicity
with increasing betaine substitution [40].
Quaternized modifications of chitosan are another technique with characteristics that
might be useful in DNA condensing and efficient gene delivery [41]. The transfection
efficiency was compared with DOTAP ( N -[1-(2,3-dioleoyloxy)propyl]- N , N , N -trimethylam-
monium sulfate) lipoplexes. Additionally, their effect on the viability of respective cell
cultures was investigated using the 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyl tetrazo-
lium bromide (MTT) assay. Their observations suggested that quaternized chitosan oli-
gomers were able to condense DNA and form complexes with a size ranging from 200 to
500 nm. Chitoplexes proved to transfect COS-1 cells, but to a lesser extent than DOTAP-
DNA lipoplexes. The quaternized oligomer derivatives appeared to be superior to oligo-
meric chitosan.
Chitosan, trimethyl chitosan, or polyethyleneglycol-graft-trimethyl chitosan-DNA
complexes were characterized with respect to physicochemical properties such as hydro-
dynamic diameter, condensation efficiency, and DNA release [42-44]. Further, the cyto-
toxicity of these polymers and the uptake and transfection efficiency of polyplexes in vitro
were evaluated. Under conditions found in cell culture, the formation of aggregates and
strongly decreased DNA condensation efficiency were observed in the case of chitosan
polyplexes. These characteristics resulted in only 7% cellular uptake in NIH/3T3 cells
and low transfection efficiencies in four different cell lines. By contrast, quaternization
of chitosan strongly reduced aggregation tendency and pH dependency of DNA