MICROFLUIDIC SYSTEMS FOR INCORPORATION OF SMALL INTERFERING RNA (siRNA) IN CATIONIC LIPOSOMES AND FOR IN VITRO ANIMAL CELL TRANSFECTION TARGETING GENE THERAPY

Abstract

This research project aims the technological development of microfluidic systems to incorporate small interfering RNA (siRNA-silencer) inside of nano-scaled cationic liposomes (CLs), in order to get non-viral vectors for applications in gene therapy. Gene therapy is a promising technique that introduces a correct copy of the defective gene for the treatment of genetic diseases. The production of CLs-siRNA complexes by using microfluidics technology, which processes fluids in small amounts, enables the continuous operation as a single-phase system. The project will be divided into three stages: In the first stage, microfluidic device will be designed and the electrostatic complexation between conventional CLs and siRNA in microchannels will be studied and compared to bulk process, which is a conventional technique to produce CLs/siRNA complexes. In the second stage, the liposome composition will be modified with the inclusion of lipid derivatives that contain polyethylene glycol-distearoyl phosphatidylethanolamine with folate (DSPE-PEG-Folate) in order to generate Stealth liposomes with specific targeting to cells with folate receptors on their surface. The efficiency of the new liposome composition will be compared with the conventional ones. At the end of the first two stages, the physicochemical and biochemical properties of the complexes will be evaluated. In the final stage, the most appropriate lipid structures containing siRNA that will have been produced in previous stages will be transfected as a dynamic transfection analysis within microfluidic systems into HeLa Reporter Cells (HeLa) that stably express Green Fluorescent Protein (GFP). The expression level of GFP will be visualized and evaluated by using fluorescence microscope and afterwards, the transfection efficiency will be compared with the conventional method carried out in wells.