Study of novel polymeric liposome as a nonviral vector for transfection of VEGF siRNA into RPE cells

Abstract

Background It is a key step to transfect the therapeutic gene into target tissue safely and effectively. Because of its potential pathogenicity, immunogenicity and mutagenicity, viral vector is limited in clinical application. Therefore, nonviral vector is becoming a study hotpot due to its security, low price and flexibility. However, the study of octadecyl-quaternized lysine modified chitosan/cholesterol (OQLCS/chol) as genetic vector is rare. Objective This study was to investigate the optimal conditions for gene transfection by new polymeric liposome-OQLCS/chol. Methods Human retinal pigment epithelial (RPE) cells were cultured in modified 1640 medium containing fetal bovine serum. Plasmid DNA was prepared and extracted based on the sequence of human vascular endothelial growth factor (VEGF) from GeneBank. The loading efficiency of OQLCS/chol lipidosome to plasmid DNA was clarified by electrophoresis to select the optimal tranfected conditions, including the mass ratio, action time as well as with or without blood serum. The dissolution curve of OQLCS/chol lipidosome was drafted by supercentrifuge process. The RPE cells were transfected by OQLCS/chol lipidosome and Lipo2000, respectively, and the transfected efficiency was detected by green fluorescence protein (GFP) and compared between the OQLCS/chol lipidosome group and Lipo2000 group. The in vitro survival rate of the cells was evaluated by cell counting kit-8(CCK-8). Results The average size of OQLCS/chol liposome nanoparticles was 134 nm, and the Zeta potential was + 39.64 mV. About 70%of total encapsulated plasmid DNA was rapidly released within initial 5 days followed by a constant and sustained release for 14 days. This new polymeric liposome was safe for biological use under the 20 μg/ml, with the optimal OQLCS/chol: plasmid DNA of ≥2∶1 and incorporation time of 30-40 minutes in the free-serum medium. The transfected cell rate was 71.05% in the OQLCS/chol liposome group, and that in the Lipo2000 group was 70.58%, showing a significant difference between the two groups (χ2=0.534, P=0.465). Conclusions OQLCS/chol loposome is synthesized successfully with uniform size, better stability, less cytotoxicity, higher Zeta potential and cellular uptake efficiency in comparison with Lipo2000. This novel polymeric liposome loaded plasmid DNA can transfect VEGF siRNA into RPE cells efficiently like a Lipo2000. Key words: Gene delivery; Nanoparticles; Non-viral vectors; Small interfering RNA; Transfection; Retinal pigment epithelial cells