Évaluation de l'efficacité des nanoparticules chitosane-siARN à inhiber, in vitro, l'expression du gène MDR1 responsable de la résistance à la chimiothérapie
Year: 2014
Type: dissertation
Abstract: RESUME
Depuis la decouverte de la Moutarde azotee, premier agent anticancereux, la chimiotherapie occupe une place importante dans le traitement de plusieurs types de cancer. Malgre le progres et l’avenement des medicaments anticancereux, la chimiotherapie est souvent limitee par des mecanismes de resistance developpes par les cellules cancereuses. Parmi les mecanismes les plus repandus est l’expulsion des medicaments anticancereux a l’exterieur des cellules. Ceci aboutit a une diminution de la concentration intracellulaire du medicament affectant ainsi son activite anti tumoral. L’expulsion des medicaments anticancereux est causee par la surexpression des proteines membranaires dont la plus connue est la glycoproteine P (Pgp). L’hydrolyse de l’ATP par cette proteine fournit de l’energie necessaire pour assurer un transport actif a travers la membrane plasmique de plusieurs types de molecules anticancereuses independamment de leur structure. Codee par le gene MDR1, la surexpression de la Pgp est detectee chez plus que 50% des patientes atteintes de cancer du sein. Contrairement aux inhibiteurs chimiques, les petits ARN interferents (MDR1-siRNA) sont capables d’inhiber plus specifiquement la Pgp et contourner la chimioresistance. Ces petits ARN doubles brins de 21 nucleotides peuvent lier specifiquement la sequence d’ARN messager du gene MDR1 et engendrer sa degradation inhibant ainsi la traduction de la Pgp. Cependant, l’efficacite des MDR1-siRNA est limitee par leur vulnerabilite dans le milieu physiologique et leur incapacite a franchir les barrieres cellulaires. D’ou la necessite d’un systeme de livraison pour assurer le potentiel therapeutique des MDR1-siRNA. Dans ce present travail, le systeme de livraison utilise est le chitosane, un polymere naturel, biodegradable et non toxique.
Le chitosane est caracterise par son degre de desacetylation (DDA), sa masse moleculaire (MM) et son ratio amine phosphate (N : P). Pour des fins de simplicites, ces parametres sont decrits de la facon suivante [DDA-MM-N:P]. Etant donne sa charge cationique, le chitosane est capable d’interagir electrostatiquement avec les MDR1-siRNA et former des nanoparticules chitosane/MDR1-siRNA. La taille et la forme de nanoparticules a ete evaluees par diffusion dynamique de la lumiere et aussi par microscopie electronique a balayage (ESEM). La mesure du potentiel zeta caracterise la charge des nanoparticules chitosane/MDR1-siRNA. La formation des nanoparticules et leur stabilite en fonction du pH a ete evaluees avec le recours au reactif RiboGreen et suite a une migration sur gel de polyacrylamide. La capacite des nanoparticules a----------ABSTRACT
Since the discovery of nitrogen mustard, the first anticancer agent, chemotherapy has palyed an important role in the treatment of several types of cancer. Despite the progress and the advencement of anti-cancer drugs, chemotherapy is often limited by resistance mechanisms developed by cancer cells. Among the most common mechanisms is the expulsion of anticancer drugs out of the cells, resulting in a decreased intracellular concentration and lowered antitumor activity. The expulsion of anticancer drugs is mediated by the over-expression of the transmembrane transport protein where the best characterized is P glycoprotein (Pgp). This protein utilize the energy of ATP to actively carry out structurally and functionally unrelated anticancer agents across the plasma membrane. Encoded by the MDR1 gene, the predominant role of Pgp is detected in more than 50 % of breast cancer patient. Unlike chemical inhibitors, small interfering RNAs (MDR1-siRNA) provide a more specific downregulation of Pgp and reversion of the drug resistance. These small double-stranded RNA (21 nucleotides) are able to bind to a specific mRNA sequence of the MDR1 gene, causing its degradation and thereby disabling translation of Pgp. However, the efficiency of MDR1-siRNA is limited by their vulnerability in the physiological medium and their inability to cross cell barriers. For this purpose, a delivery system is needed to potentiate the effect of siRNA. In this study, the delivery system used is chitosan, a natural, biodegradable and non-toxic polymer.
Chitosan is characterized by its degree of deacetylation (DDA), molecular weight (MM) and amine to phosphate (N: P) ratio. For simplicity, these parameters are described as follows [DDA- MM- N:P]. Chitosan is able to interact electrostatically with the MDR1-siRNA due to its cationic charge, leading to chitosan/MDR1-siRNA nanoparticles. The nanoparticles size and morphology was evaluated by dynamic light diffusions (DLS) and scanning electron microscopy (ESEM). Nanoparticles surface charge was characterized by measuring the zeta potential. Furthermore, the formation of chitosan nanoparticles and their stability under variable pH was evaluated by RiboGreen and polyacrylamide gel electrophoresis. The nanoparticles ability to bypass cellular membrane was analyzed by confocal microscopy and flow cytometry (FACS). The efficacy of nanoparticles in gene silencing was assessed by quantitative RT-PCR, and confirmed at the phenotypic level by western blot analysis. Whereas, the toxicity profile of chitosan nanoparticles was evaluated using Alamar Blue proliferation assay.
Author: Mohamed Benhammadi
Language: French
Subfield: Oncology
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W1193075568