Abstract
Purpose :
Although viral vectors have been widely used for gene therapy, their potential risk associated mainly with immunogenicity and mutagenesis has promoted the design of non-viral vectors. A typical non-viral vector contains a cationic compound that binds electrostatically the genetic material and forms a stable complex. The nucleic acid is protected, and the resulting cationic vector also facilitates the attachment to the cell surface and subsequent endocytosis. The purpose of this study is to develop positively charged D-α- tocopheryl glycol succinate 1000 (TPGS)-chitosan nanocapsules as a non viral vector for gene therapy.
Methods :
Cationic TPGS-chiotsan nanocapsules (TCNs) were synthesized by a two-step reaction. TPGS was first functionalized with a carboxylic acid group by esterification with succinic anhydride (SA). Then, the activated TPGS were combined with chitosan by ionic cross linking through a complex coacervation method. The physical characteristics of the TCNs and the TCNs/DNA complex were determined. The in vitro transfection efficiency of the TCNs/DNA encoding eGFP complex was determined in five different cell lines (HEK 293, NTM5, Huh7, ARPE and He La).
Results :
The average size and zeta potential of TCNs were 234 nm and 27 mV, respectively. Agarose gel electrophoresis experiments showed that DNA plasmids effectively bind with TCNs and are protected against DNAase. Transfection efficiency of TCNs in different cell lines was obtained as HEK 293 > NTM5 > Huh7 > ARPE > He La. The transfection efficiency of the TCNs was lower than Lipofectamime, even at different ratios of TCNs/DNA. However, the cytotoxicity of the TCNs was lower than that of Lipofectamine, making it a suitable vector for gene therapy. We confirm that the most optimal mass ratio of TCNs/DNA is 2/1.
Conclusions :
Our results suggest that cationic TCNs are a very promising non-viral gene delivery system capable of targeting the MYOC gene for myocilin associated glaucoma therapy.
This is a 2020 ARVO Annual Meeting abstract.