Plasmid DNA encapsulated PLGA nanoparticles as a model non-viral vector for gene therapy

B78

Purpose: In gene therapy, while viruses have been most commonly used as vectors to effectively deliver the genes to cells, this approach remains fraught with the potential for viral toxicity. Non-viral vectors are therefore highly sought after. The purpose of this study was to investigate one such approach by developing biocompatible and biodegradable polymeric nanoparticles of pDNA (pEGFN1-Luc) as a non-viral gene delivery system.
 Methods: PLGA 50:50, a biodegradable and biocompatible polymer, was used to encapsulate pDNA using a modified solvent evaporation technique. A fixed amount of calcium chloride was added with the pDNA or with the external aqueous phase. The nanoparticles were evaluated for surface morphology, particle size, zeta potential, efficiency of encapsulation, structural integrity of pDNA, and transfection efficiency. The efficacy of DNA delivery and gene expression after addition of nanoparticles to Huh-7 cells was examined in a kinetic study by fluorescence imaging.
 Results: SEM photographs showed that the pDNA loaded nanoparticles were smooth and spherical, and the zeta potential varied between -16 mV and +0.67 mV. An analysis of the particle size distribution revealed that 90% of the particles were less than 1 µm and the median size varied from 500 nm 1086 nm. The nanoparticles containing calcium chloride pretreated pDNA showed higher encapsulation efficiency compared with the nanoparticles prepared with naked pDNA and calcium chloride in the external aqueous phase. The agarose gel electrophoresis showed that the encapsulated pDNA maintained its structural integrity during and after the encapsulation process. The results of the in vitro transfection assay showed that the nanoparticles prepared with the calcium chloride pretreated pDNA had significantly higher GFP and luciferase expression.
 Conclusion: When using the solvent evaporation technique to prepare pDNA loaded nano-particles, pretreatment of pDNA with calcium chloride results in positively charged nanoparticles which showed higher encapsulation efficiency. These nanoparticles also showed higher transfection efficiency.
 Acknowledgement: This work was funded in part by the NIH/NIGMS grant # GM08008-32, NASA grant # NCC3-946-S4, Louisiana Board of Regents RC/EEP (2006-16), NIH grant# 5P20CA118768-02, and Louisiana Cancer Research Consortium.