SILENÇAGE GÉNIQUE DE L'ARNM CODANT POUR L'APOLIPOPROTÉINE B APRÈS ADMINISTRATION PAR VOIE INTRAVEINEUSE DE NANOPARTICULES CHITOSANE/SIARN-APOB DANS UN MODÈLE MURIN D'ATHÉROSCLÉROSE

摘要: RESUME L’atherosclerose est une maladie inflammatoire auto-suffisante dont le developpement progressif asymptomatique mene a la majorite des maladies cardiovasculaires causant approximativement 75 000 deces par annee au Canada seulement. L’apolipoproteine B (ApoB) composante structurelle essentielle lipoproteines de faible densite (LDL), responsables l’initiation et du l’atherosclerose. Les medicaments actuels ne font que ralentir atherosclerotique lieu d’atteindre source probleme qui principalement l’ApoB. D’ou l’importance trouver nouvelle avenue biomedicale telle silencage genique l’ApoB en utilisant un systeme concu pour livrer systemiquement petits ARN interferents (siARN) les liberer dans cellules « cibles ». Nous avons donc hypothese recherche l’administration voie intraveineuse nanoparticules chitosane/siARN-ApoB mesure d’induire effets therapeutiques securitaires modele murin d’atherosclerose. Le chitosane polymere cationique, naturel biodegradable caracteristiques intrinseques varient fonction son degre desacetylation (DD) sa masse moleculaire (MM). De plus, ratio amine : phosphate siARN (N:P) utilise former nanoparticules, ainsi DD MM influencent grandement physico-chimiques nanoparticules. formulations (DD-MM-N:P) utilisees cadre ce projet, soit 92-10-5, 80-80-5 80-10-10 ont ete prealablement evaluees therapie genique. Les analyses microscopie electronique balayage environnemental (ESEM) diffusion dynamique lumiere (DLS) demontre chitosane/oligonucleotides double brin (ODNdb)-ApoB spheriques homogenes possedent tailles potentiels zeta (δ) variant entre 41 108 nm 15 23 mV, respectivement. L’electrophorese sur gel polyacrylamide stabilite chitosane/ODNdb-ApoB formees 92-10 divers ratios N:P lors d’incubation differentes periodes solutions pH differents. egalement electrophorese d’agarose proteger efficacement ODNdb-ApoB contre degradation concentrations supra-physiologiques desoxyribonucleases I (ADNase I) (2 unites d’ADNase I/μg d’ODNdb).----------ABSTRACT Atherosclerosis is self-sufficient inflammatory disease whose asymptomatic progressive development leads to the majority of cardiovascular diseases causing approximately death per year in only. Apolipoprotein an essential structural component low density lipoproteins which are responsible for initiation and atherosclerosis. Actual medication can only slow atherosclerotic instead targeting problem ApoB. Hence importance finding new biomedical technique such as ApoB gene silencing using system designed release small interfering RNAs (siRNA) targeted cells. We therefore hypothesized that intravenous administration chitosan/siRNA-ApoB nanoparticles would be able induce safe therapeutic effects murine model atherosclerosis. Chitosan cationic, natural polymer intrinsic characteristics vary according its degree deacetylation (DDA), molecular weight (MW) chitosan’s siRNA’s used form nanoparticles. Chitosan (DDA-MW-N:P) this research project, either 80-10-10, were previously studied therapy. Analysis Environmental Scanning Electron Microscopy Dynamic Light Scattering showed spherical homogenous chitosan/double stranded oligonucleotides (dsODN)-ApoB had sizes (δ)-potentials ranging respectively from mV. Polyacrylamide electrophoresis (PAGE) demonstrated high stability chitosan/dsODN-ApoB formed with chitosan at various when incubated solution pHs amounts time. also agarose efficiently protected dsODN-ApoB digestion by supraphysiological desoxyribonuclease (DNAse units DNAse dsODN). We determined flow cytometry (92-10-5)/dsODN-ApoB attained transfection levels 55, 86 66 % HepG2, HEK293 RAW264.7 cells respectively. In same cell lines, confocal microscopy imaging internalized nanoparticles, well released indicative endolysosomal release.