摘要
AIM:To prepare thymidine kinase gene (TK gene) nanopartides and to investigate the expression of TK gene.METHODS: Poly(D,L-lactic-co-glycolic acid) (PLGA), a biodegradable and biocompatible polymer, was used to prepare recombinant plasmid p^EGFP-AFP nanoparticles by a double-emulsion evaporation technique. Characteristics of the nanoparticles were investigated in this study, including morphology, entrapment efficiency, and tissue distribution.The expression of TK gene was also investigated by MTT assay, by which the viable cells were determined alter the addition of ganciclovir (GCV).The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cellswere assessed by flow cytometry.RESULTS: The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72±12nm.The mean entrapment efficiency was 91.25%. A total of 80.14% DNA was found to be localized in the livers after 1-h injection with ^32P-DNA-PLGA nanoparticles in mouse caudal vein. The expression of DNA encapsulated in nanopartides was much higher than that in naked DNA, and human hepatocellular carcinoma SMMC-7721 cells were more sensitive to GCV than human normal parenchymal Chang liver cells.CONCLUSION:The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.
AIM:To prepare thymidine kinase gene (TK gene) nanoparticles and to investigate the expression of TK gene. METHODS:Poly(D,L-lactic-co-glycolic acid) (PLGA),a biodegradable and biocompatible polymer,was used to prepare recombinant plasmid p^(EGFP-AFP) nanoparticles by a double-emulsion evaporation technique.Characteristics of the nanoparticles were investigated in this study,including morphology,entrapment efficiency,and tissue distribution. The expression of TK gene was also investigated by MTT assay,by which the viable cells were determined after the addition of ganciclovir (GCV).The enhanced green fluorescent protein (EGFP) expression in human hepatocellular carcinoma SMMC-7721 cells and normal parenchymal Chang liver cells were assessed by flow cytometry. RESULTS:The prepared plasmid-nanoparticles had regular spherical surface and narrow particle size span with a mean diameter of 72±12 nm.The mean entrapment efficiency was 91.25%.A total of 80.14% DNA was found to be localized in the livers after 1-h injection with ^(32)P-DNA-PLGA nanoparticles in mouse caudal vein.The expression of DNA encapsulated in nanoparticles was much higher than that in naked DNA,and human hepatocellular carcinoma SMMC- 7721 cells were more sensitive to GCV than human normal parenchymal Chang liver ceils. CONCLUSION:The enhanced transfection efficiency and stronger ability to protect plasmid DNA from being degraded by nucleases are due to nanoparticles encapsulation.
