Characterization, Cytoxicity and Transfection Efficiency of Plasmid DNA Loaded Cationic Polymer Nanoparticles for Expressions of Interleukin 12

Abstract

PURPOSE. Interleukin-12 (Il-12) as a cytokine has been proved to possess antitumor effects via stimulating the immune system. Gene delivery systems based on using naked DNA is not desirable as naked DNA has extremely low in vivo stability and is rapidly degraded by hydrolytic nucleases. Also, the cellular uptake of naked DNA molecules is extremely inefficient. Non-viral gene delivery systems exhibit low toxicity and are easier to prepare compared to their viral counterparts. Chitosan as a polysaccharide and gelatin as a natural protein are good candidates for gene delivery since they are biodegradable, non-toxic, and biocompatible macromolecule. In this project, we aimed to prepare plasmid DNA loaded chitosan and gelatin nanoparticles for expression of Il-12 and to evaluate their physicochemical characteristics, cytotoxicities and transfection efficiencies in murine CT-26coacervation process at different N/P ratios. Gelatin-DNA nanoparticles were prepared by a pH and temperature controlled ethanol-water solvent displacement technique. Chitosan and gelatin nanoparticles were prepared using different concentrations of the polymers and pUMVC3-hIL12 DNA solutions at concentrations of 100 and 200 g/ml, and 50, 100, and 200 g DNA/10ml, respectively. All prepared nanoparticles were characterized in terms of size, zeta potential, polydispersity index, morphology, encapsulation efficiency and polyplex formation. The cytotoxicities and transfection efficiencies of the prepared polyplexes were evaluated by MTT assay and ELISA (for hIL-12, p70), respectively. RESULTS. Sizes and zeta potentials of plasmid DNA loaded chitosan nanoparticles varied from 156.83 to 966.03 nm and between 5.68 and 16.77 mV, respectively. Also, sizes of plasmid DNA loaded gelatin nanoparticles were found to be between 344.27 and 826.23 nm. Zeta potentials of the gelatin-DNA complexes varied from -188 to -944 mV, respectively. Both of the prepared chitosan and gelatin nanoparticles were spherical in shape. Encapsulation efficiencies forIXchitosan-DNA complexes were found to be 68.8391.53% and for gelatin-DNA complexes were approximately above 97%. Prepared chitosan and gelatin polyplexes were nontoxic to CT-26 cells. The transfection efficiencies of the prepared chitosan complexes were obviously higher than those of naked pDNA when N/P ratios were between 16 and 60. Maximum level of phIL-12 expression was obtained at N/P = 16 (C6) with particle size of 286.8628.26 nm (polydispersity index=0.440.066) indicating the improved transfection of pUMVC3-hIL12 about 3 times compared to that of the naked pUMVC3-hIL12. DNA-gelatin nanoparticles prepared using 0.5% gelatin solution (G14) with mean particle size of 816.8729.80 nm (polydispersity index=0.560.01) showed maximum transfection efficiency and could improve transfection of pUMVC3-hIL12 approximately 2.5 orders of magnitude compared to the naked plasmid DNA. CONCLUSIONS. Chitosan-DNA nanoparticles at N/P = 16 and gelatin-DNA nanoparticles using 0.5% gelatin solution with minimal cytotoxicity and acceptable transfection efficiency, can be used as suitable candidates for further gene delivery studies and applications.