Abstract
The aim of this study was to optimize transfection efficiency (TE) of the depolymerized low molecular weight (LW) chitosan with molecular weight (Mw) at 16 kDa and 54% degree of deacetylation (DDA) on three primary cells of fibroblast (F), dental pulp (P), and periodontal ligament (PDL). The effect of low frequency ultrasound treatment on the chitosan-DNA complexes prior transfection on TE was also evaluated. This LW chitosan required high N/P ratio (>34) to bind DNA completely. An N/P ratio above 56 tended to improve TE in most primary cells nearly at the level of Lipofectamine. Ultrasonication can reduce the aggregation and sizes of the chitosan-DNA microparticles. It increased TE of F cells at an N/P ratio above 34, which was higher than Lipofectamine. However, this ultrasound treatment caused loss of TE in KB cells. MTT assay of these chitosan-DNA complexes revealed no significant cytotoxicity to both KB and F cells. This LW chitosan has potential for further development into a safer alternative to gene delivery systems in various cells of interest; however the optimal conditions have to be adjusted, depending on each cell source.
Highlights
An efficient gene delivery system for a wide range of cell types is central to the development of regenerative biomedicine and tissue engineering therapy
There are many studies concerning factors related to enhancing transfection efficiency (TE) of this non-viral vehicle, including varying its molecular weight, degree of deacetylation (DDA), molar ratio between the amino positive charge of chitosan/ negative charge of phosphate group from DNA (N/P), and the pH
Most of the information has been gained from cancer or immortalized cell lines and limited studies have been performed on primary cell lines
Summary
An efficient gene delivery system for a wide range of cell types is central to the development of regenerative biomedicine and tissue engineering therapy. Non-viral vectors offer high safe and versatile alternative to viral counters have gain increasing interest. They have limited application due to the lack of transfection efficiency (TE) [1], especially with non-cancer or somatic cells. Chitosan (poly[(-(1-4)-2-amino-2-deoxy-D-glucopyranose]), a nontoxic biodegradable biopolymer, has been widely used as a pharmaceutical excipient due to its biocompatibility, good bioadhesiveness and general availability [2] This biopolymer has been broadly studied for drug delivery [3] and as a scaffold for tissue engineering, as well as potential use as a non-viral vehicle for gene delivery [4,5]. Most of the information has been gained from cancer or immortalized cell lines and limited studies have been performed on primary cell lines
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