Cholesterol Sequestration from Caveolae/Lipid Rafts Enhances Cationic Liposome-Mediated Nucleic Acid Delivery into Endothelial Cells

Maddila, SC; Voshavar, C; Arjunan, P; Chowath, RP; Rachamalla, HKR; Balakrishnan, B; Balasubramanian, P; Banerjee, R; Marepally, S

Abstract

Delivering nucleic acids into the endothelium has great potential in treating vascular diseases. However, endothelial cells, which line the vasculature, are considered as sensitive in nature and hard to transfect. Low transfection efficacies in endothelial cells limit their potential therapeutic applications. Towards improving the transfection efficiency, we made an effort to understand the internalization of lipoplexes into the cells, which is the first and most critical step in nucleic acid transfections. In this study, we demonstrated that the transient modulation of caveolae/lipid rafts mediated endocytosis with the cholesterol-sequestrating agents, nystatin, filipin III, and siRNA against Cav-1, which significantly increased the transfection properties of cationic lipid-(2-hydroxy-N-methyl-N,N-bis(2-tetradecanamidoethyl)ethanaminium chloride), namely, amide liposomes in combination with 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (AD Liposomes) in liver sinusoidal endothelial cells (SK-Hep1). In particular, nystatin was found to be highly effective with 2-3-fold enhanced transfection efficacy when compared with amide liposomes in combination with Cholesterol (AC), by switching lipoplex internalization predominantly through clathrin-mediated endocytosis and macropinocytosis.

Keywords: cationic lipids; clathrin-mediated endocytosis; caveolae-mediated endocytosis; macropinocytosis; transfection; endothelial cells

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Liposomes for DNA/RNA Delivery

Cholesterol sequestration can enhance cationic liposome-mediated nucleic acid delivery. Research in this area is about using liposomes as carriers to deliver nucleic acids (such as DNA or RNA) into cells for purposes such as gene therapy or gene editing. Cationic liposomes are positively charged liposomes that can form complexes with negatively charged nucleic acids to improve the adsorption and endocytosis of nucleic acids on the cell membrane. Cholesterol is a common component of liposomes and plays an important role in the structure and function of cell membranes. Studies have shown that introducing cholesterol into cationic liposomes can enhance their interaction with cell membranes and improve nucleic acid delivery efficiency. This may be because cholesterol can increase the binding force of liposomes to cell membranes, promote the endocytosis of the complex, and help stabilize the liposome structure. Therefore, cholesterol sequestration enhances the efficiency and stability of cationic liposome-mediated nucleic acid delivery, providing an important theoretical and practical basis for research and applications in the field of gene delivery.