Chitosan And Its Derivatives As A Gene Vector Biosafety Evaluation

Since the beginning of 1990's, nano-science and technology gained rapiddevelopment, and has been widely applied in the areas of electronics, information andcommunication, semiconductor, biology and medicine. More and more countries havebeen putting huge investment in the research and development of nanotechnology forthe purpose of taking advantage of the opportunities which could bring hugecommercial benefits.However, the bio-safety of nanomaterials has been drawing increasing attention ofscientists and environmental protection organization. Preliminary studies showed thatsome kinds of nano-scale materials might have negative impact on human beings.Greater effort is urgently needed on the evaluation of the bio-safety of nano-scalematerials for the healthy development of nanotechnology.Our research group has been conducting research on the chitosan and itsderivatives as gene carder for several years. Based on our previous study,chitosan-DNA nanoparticles, arginine-modified chitosan-DNA nanoparticles, andN-Alkylated chitosan-DNA nanoparticles were selected for the evaluation of theirimpact on the function of macrophages. The research includes the following threeparts.1. Endotoxin-free plasmid DNA was prepared and used to form nanoparticles withchitosan, arginine-mdified chitosan, and N-Alkylated chitosan, respectively. Thenanoparticles were prepared by coacervation process and N/P ratio was 4:1. Thenanoparticles were characterized with transmission electron microscope (TEM), atomic forcemicroscope (AFM), and dynamic light scattering (DLS).2. The murine monocyte/macrophage cell line RAW264.7 cells were incubatedwith three kinds of nanoparticles at the concentration of 0.1, 1, 10 and 20μg/ml(based on the content of DNA). The cytokines IL-1β, IL-6, IL-10, IL-12, and TNF-αin the cell culture media at lh, 6h, 24h after co-incubation were assessed byenzyme-linked immunosorbent assay (ELISA). The results indicated exposure ofRAW264.7 macrophage to CS-DNA nanoparticles and CS-Arg-DNA nanoparticlesdidn't enhance the secretion of cytokines IL- 1β, IL-6, IL-10, IL- 12, TNF-α, even withthe highest concentrations of nanoparticles (20μg/ml in term of DNA content) up to24 hours.3.1 ml nanoparticles (50μg/ml) were intraperitoneally injected into six groups ofmice, The peritoneal macrophages were harvested after 24h and subsequentlyco-incubated with freshly prepared chicken red blood cells (CRBC) for 30min in vitro.The phagocytic rate (PR) and phagocytic index (PI) were calculated by direct visualenumeration using a light microscope. The data revealed that phagocytosis ofperitoneal macrophages were not significantly affected after exposure to DNA nanoparticles.