Preparation and evaluation of injectable Chitosan-based implant for intratumoral gene delivery

Abstract

In this study Diethylmethyl chitosan (DEMC) and Triethyl chitosan (TEC), quaternized derivatives of chitosan, were synthesized. For in vitro and in vivo gene delivery we used Aspc-1 cell line (pancreatic cancer cell line). In order to evaluate polymer’s efficiency on AsPC-1 cells, enhanced green fluorescent protein (EGFP) has been used as a reporter molecule for gene expression, because it emits green fluorescence after blue-light excitation.The aim of this study was to evaluate DEMC and TEC as transfecting reagents of pEGFP, as a model plasmid, for intratumoral gene delivery to human pancreatic cancer cells (AsPC-1). Also we used mathematics in in vitro studies.In order to reach our study aims briefly, at first the system was optimized using HEK 293T (human embryonic kidney) cell line. Then polyplexes were prepared at different polymer to plasmid (N/P) ratios. The polyplexes’ stability, size and zeta potential were determined. After cell transfection to AsPC-1 cell line, fluorescence microscopy was used to detect green fluorescent protein. Also flow cytometry was used to determine the percent of transfection at different N/P ratios compared to non-treated cells. Cytotoxicity of the derivatives was also determined. These polymers were compared to a polymeric commercial transfecting reagent (Arrest-In) as control. The best N/P ratio particle size and shape was evaluated via atomic force microcopy (AFM). Considering that mathematical models can be used to understand and predict consequences associated with nanomedicine, the relation between TEC/pDNA and DEMC/pDNA complexes charge ratio, cell transfection and toxicity was evaluated for the first time with Lagrange’s interpolation polynomial method.13In in vivo experiments, nude mice were subcutaneously injected with AsPC-1 cell line and the created tumors were used to determine the effect of DEMC and TEC on intratumoral gene delivery via fluorescence microscopy, flow cytometry and immunohistochemistry