FORMULATION OF LINOLIC ACID-BASED CATIONIC LIPOPEPTIDE WITH DOPE AND CHOLESTEROL TO IMPROVE IN-VITRO TRANSFECTION EFFICIENCY IN HEK-293T MAMMALIAN CELLS

Abstract

This invention concerns a cationic lipopeptide formula C18:2-CHS-PKKKRKV with cholesterol and DOPE in molar ratios of 1:1:2, 2:1:2, and 3:1:2 for the purpose of delivering genetic material to HEK-293T mammalian cells. The lipopeptide / cholesterol / DOPE formula was prepared by ethanol injection technique with a flow rate of cholesterol / DOPE / ethanol solution of 300-500 µL / minute. The lipopeptide / cholesterol / DOPE transfection agent formula was prepared by heating at a temperature of 45-50oC using a magnetic stirrer at a speed of 200 rpm for 45 minutes. The lipopeptide / cholesterol / DOPE formula colloid was then sonicated (water bath sonication) for 45 minutes or until the colloid appeared transparent, and then filtered with a sterile membrane measuring 0.22 µm.The main objective of this invention is to overcome the cytotoxicity of the cationic lipopeptide transfection agent C18:2-CHS-PKKKRKV while maintaining the ability to deliver genetic material to HEK-293T cells. The test results prove that the lipopeptide / cholesterol / DOPE formula is able to protect DNA from DnaseI enzymatic degradation, form nanoparticles (~300-400 nm), significantly increase the viability of test cells (20-30%) and still have the ability to deliver genes to HEK-293T cells. Overall, the lipopeptide / cholesterol / DOPE formula can be used as a safer transfection agent in HEK-293T mammalian cells compared to the C18:2-CHS-PKKKRKV lipopeptide without cholesterol and DOPE.