In the United States, 20.8 million people (7% of the population) suffer from diabetes. About 75% of all newly diagnosed cases of type I diabetes occurs in individuals younger than 18 years of age. Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells. The major goal of the proposed research is to develop a nanoparticulate gene delivery vector for highly efficient nuclear delivery of plasmid encoding interleukin-10 (IL-10) for prevention of type 1 diabetes. We hypothesize that the combination of cationic polymer and poly (lactide-co-glycolide) (PLGA) in the presence of a cationic surfactant will produce nanoparticles with high positive zeta potential that will facilitate efficient loading of negatively charged plasmid DNA encoding IL-10 gene on the surface;and the positively charged nanoparticles loaded with plasmid DNA are biocompatible and can efficiently transfect the cells and express the protein both in vitro and in vivo. To test our hypotheses, we plan to study the following specific aims: (1). To synthesize methacrylate copolymers using monomers, 2- dimethyl amino ethyl methacrylate (DMAEMA) and methylmethacrylate (MMA) with increasing molar ratio of DMAEMA. The copolymers will be characterized for weight average molecular weight by gel permeation 1 chromatography and number average molecular weight by H NMR. (2). To prepare cationic nanoparticles using a blend of cationic polymer and PLGA by double emulsion solvent evaporation technique, using cetyl trimethyl ammonium bromide as a cationic surfactant. The nanoparticles will be characterized for size, shape, charge density, plasmid loading efficiency, buffering ability, and structural integrity of plasmid DNA by dynamic light scattering, electron microscopy, zeta potential measurement, UV spectrophotometer, titrimetric, and gel electrophoresis, respectively. (3). To study the cellular internalization in Human Embryonic Kidney (HEK 293) cells by confocal microscopy, using cationic nanoparticles loaded with coumarin 6. In vitro transfection efficiency of cationic nanoparticles in HEK 293 cells will be studied, using a therapeutic plasmid encoding IL-10. The expression of and IL-10 will be quantified by enzyme-linked immunosorbent assay. (4). To evaluate in vitro and in vivo in mice biocompatibility of cationic nanoparticles, using an MTT assay and light microscopy, respectively. (5). To study the efficiency of cationic nanoparticles to deliver plasmid encoding Interleukin-10 in vivo in mice and its ability to prevent the onset of type 1diabetes. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes.
Type 1 diabetes is a chronic autoimmune disease affecting 0.3% of world's population. It results from selective destruction of pancreatic 2-cells mediated by T lymphocytes which leads to gradual reduction in body's ability to produce insulin. The gene delivery vectors need special features to overcome extracellular and intracellular barriers, and ensure efficient DNA delivery to the nucleus. The use of cationic polymer and cationic surfactant will synergistically enhance the positive zeta potential of nanoparticles and their transfection efficiency. The study would be conducted in vitro and in vivo in animal model. The proposed study will contribute towards the development of a high efficiency and low toxicity non-viral gene delivery vehicle in order to deliver plasmid encoding IL-10 gene for prevention of type 1 diabetes.
