Investigation Of Nanometric Carriers' Gene Protection From Cleavage And Preparation And Application Of Novel Gene Carriers

Gene carriers are of great importance not only in basic research on the expression and regulation of genes and the function of genes but also in gene therapy in which genes are used as drugs for the therapy of clinical diseases. Inorganic nanoparticles as non-viral gene carriers have caught much attention because of their good stability, biocompatibility, safety, efficiency and non-immunogenicity. However, some key factors in gene transfection of these carriers are still unknown and their transfection efficiencies are not desirable. To address these problems, we have carried out some related research and achieved some progress as follows: (1) The mechanism of DNA protection of amino-modified silica nanoparticles has been deeply investigated. Protection of exogenous genes against intracellular conditions is one of key factors in gene transfection. Amino-modified silica nanoparticles as novel non-viral gene carriers have shown good DNA protection ability in many reports, but the mechanism of DNA protection has not been studied yet. Using amino-modified silica micro-particles as control, the mechanism of genes' protection from enzymatic cleavage of amino-modified silica nanoparticles has been deeply investigated from the aspects of enzymatic reaction environment and the length of DNA sequence. Results show that amino-modified nanoparticles present better genes' protection efficiency from all enzymatic cleavages than that of amino-modified micro-particles, which will provide helpful theoretic support for the development of nanometric gene carriers. (2) Two new gene transfection methods have been developed based on silica micro-particles. Based on the silica micro-particles with the size about several hundred nanometers, two new gene transfection methods have been developed. One is using the poly-L-lysine-silica micro-particles complexes which are formed through electrostatic interaction between them as gene carriers, and the other is using silica micro-particles as helpmates in the poly-L-lysine-mediated transfection. Both methods are rapid, simple and of high transfection efficiency. The latter exhibits higher efficiency than the former. This study provides great potential to apply these new gene transfection methods in the research of gene functions and gene engineering. (3) A new method has been developed to prepare needle-like hydroxyapatite particles with low-agglomeration and these hydroxyapatite particles have been successfully applied in gene transfection. Extensive attention has been focused on the preparation of hydroxyapatite particles now since this kind of material shows great application potential. Traditional liquid precipitation method is widely used, but hydroxyapatite particles prepared by this method are easily agglomerated. By changing the reaction system of traditional liquid precipitation, a new method using chemical precipitation in microemulsion has been developed to prepare needle-like hydroxyapatite particles with low-agglomeration. The new method not only retains the merits of traditional method, but also solves the problem of agglomeration. Compared with the particles prepared by traditional method, the hydroxyapatite particles prepared by the new method are of good dispersibility and superior properties. They have higher DNA binding efficiency which implies better loading efficiency and application in antimicrobial and drug carriers, and better cell biocompatibility which will widen their application in biomedicine. Moreover, the hydroxyapatite particles prepared through the new method demonstrates higher transfection efficiency when they are used as gene carriers.