Silencage genique de l'arnm codant pour l'apolipoproteine b apres administration par voie intraveineuse de nanoparticules chitosane/siarn-apob dans un modele murin d'atherosclerose

Atherosclerosis is a self-sufficient inflammatory disease whose asymptomatic progressive development leads to the majority of cardiovascular diseases causing approximately 75 000 death per year in Canada only. Apolipoprotein B (ApoB) is an essential structural component of low density lipoproteins (LDL), which are responsible for the initiation and the development of atherosclerosis. Actual medication can only slow the atherosclerotic development instead of targeting the source of the problem which is the ApoB. Hence the importance of finding a new biomedical technique such as the ApoB gene silencing using a system designed to release small interfering RNAs (siRNA) in targeted cells. We therefore hypothesized that intravenous administration of chitosan/siRNA-ApoB nanoparticles would be able to induce safe therapeutic effects in a murine model of atherosclerosis.;Analysis using Environmental Scanning Electron Microscopy and Dynamic Light Scattering (DLS) showed that spherical and homogenous chitosan/double stranded oligonucleotides (dsODN)-ApoB nanoparticles had sizes and zeta (zeta)-potentials ranging respectively from 41 to 108 nm and 15 to 23 mV. Polyacrylamide gel electrophoresis (PAGE) demonstrated the high stability of chitosan/dsODN-ApoB nanoparticles formed with chitosan 92-10 at various N:P ratios when incubated in solution of various pHs for various amounts of time. We also demonstrated using agarose gel electrophoresis that chitosan efficiently protected dsODN-ApoB from digestion by supraphysiological concentrations of desoxyribonuclease I (DNAse I) (2 units of DNAse I/mug of dsODN).;We determined using flow cytometry that chitosan (92-10-5)/dsODN-ApoB nanoparticles attained transfection levels of 55, 86 and 66 % in HepG2, HEK293 and RAW264.7 cells respectively. In the same cell lines, confocal microscopy imaging showed internalized nanoparticles, as well as released dsODN-ApoB indicative of endolysosomal release.;The relative quantification of the expression level of mRNAs coding for ApoB using qRT-PCR showed that transfecting HepG2 cells with chitosan (92-10-5)/siRNA-ApoB nanoparticles resulted in an ApoB gene silencing level of about 50 %.;Chitosan is a cationic, natural and biodegradable polymer whose intrinsic characteristics vary according to its degree of deacetylation (DDA), molecular weight (MW) and the chitosan's amine : siRNA's phosphate ratio (N:P) used to form nanoparticles. Chitosan formulations (DDA-MW-N:P) used in this research project, either 92-10-5, 80-80-5 and 80-10-10, were previously studied in gene therapy.;We evaluated the in vivo therapeutic effects of siRNA-ApoB administered in a murine model of atherosclerosis (C57BL/6 mice). Chitosan (92-10-5)/siRNA-ApoB nanoparticles were administrated intravenously (1 mg/kg) at various moments of atherosclerotic development. Using a sandwich ELISA, we determined that administration of chitosan (92-10-5)/siRNA-ApoB nanoparticles caused a reduction of circulating ApoB level of about 30 % in atherosclerotic mice over a period of six weeks, despite the continuation of a high-fat diet. Treated atherosclerotic mice reached a level of circulating ApoB comparable to that of non-atherosclerotic mice. Using a quantitative colorimetric detection test, we measured a reduction of circulating LDL/VLDL cholesterol level of about 20 % in treated atherosclerotic mice over a period of six weeks, reflecting the reduction of circulating ApoB. Our hepatotoxicity analysis suggested that chitosan (92-10-5)/siRNA-ApoB nanoparticles did not affect the hepatic functions related to atherosclerotic mice. Our histological analysis of liver sections stained with hematoxylin-eosin suggested that administration of chitosan (92-10-5)/siRNA-ApoB nanoparticles healed the steatosis of atherosclerotic mice four weeks after the end of treatment. Moreover, our histological analysis of liver sections stained with safranin-O/Fast-green/iron hematoxylin implied that hepatic lymphocytic infiltrations resulting mainly from steatosis were reduced three weeks after the end of treatment.;In conclusion, we were able to induce, for the first time, safe therapeutic effects in an animal model of atherosclerosis following intravenous administration of chitosan (92-10-5)/siRNA-ApoB nanoparticles. The physicochemical characterization of the nanoparticles, the in vitro study as well as the in vivo study showed the therapeutic potential of chitosan (92-10-5)/siRNA-ApoB nanoparticles for the treatment of atherosclerosis. Our project is a successful proof of concept that will soon be developed into a large scale in vivo study designed to evaluate the therapeutic effects of chitosan/siRNA-ApoB nanoparticles on an atherosclerotic animal model.