P-selectin targeted cationic liposomes efficiently deliver siRNA to endothelial cells

Introduction. P-selectin is specifically expressed by endothelial cells (EC) in inflammatory pathologies (such as atherosclerosis) and therefore a potential target for nanotherapy. The aim of this study was to obtain... [ view full abstract ]

Introduction. P-selectin is specifically expressed by endothelial cells (EC) in inflammatory pathologies (such as atherosclerosis) and therefore a potential target for nanotherapy. The aim of this study was to obtain P-selectin targeted cationic liposomes to function as efficient vectors for siRNA delivery to EC.

Methods. A peptide with high affinity for P-selectin was coupled to the surface of PEGylated liposomes containing the cationic lipid 2-{3-[Bis-(3-amino-propyl)-amino]-propylamino}-N-ditetradecyl carbamoyl methyl-acetamide (DMAPAP) combined with 1,2-Dioleoyl-sn-glycero- 3-phosphoethanolamine (DOPE). The lipoplexes obtained after complexation of cationic liposomes (P-sel_Lipo) with siRNA were characterized for size (DLS) and the amount of peptide coupled to the surface (HPLC). The cytotoxicity studies were performed by MTT assay after exposing bEnd.3 endothelial cells to various charge ratio (+/-) of lipoplexes P-sel_Lipo/siRNA for 48 hours. The binding and internalization of fluorescently labeled P-sel_Lipo/siRNA lipoplexes were determined in static and dynamic conditions, using bEnd.3 cells (that constitutively express P-selectin) by flow cytometry and fluorescence microscopy. As control, scrambled peptide coupled liposomes complexed with siRNA (Scram_Lipo/siRNA) was used.

Results and Discussions. 1) the size of lipoplexes was around 300 nm; 2) the cellular viability was not significantly affected for charge ratios (+/-) up to 4 and a siRNA concentration of 20 nM; 3) under dynamic conditions, the binding of P-sel_Lipo/siRNA to EC was higher in comparison to Scram_Lipo/siRNA, suggesting a specific adhesion; 4) at 10 minutes of static incubation, the internalization of P-sel_Lipo/siRNA was significantly higher as compared to Scram_Lipo/siRNA; 5) P-selectin targeted lipoplexes deliver intracellularly siRNA with higher efficiency than commercial transfection vectors.

Conclusion. P-selectin-targeted lipoplexes bind specifically and efficiently deliver siRNA to P-selectin expressing endothelial cells.

Acknowledgements. The work was supported by UEFISCDI: PN-II-RU-TE-2014-4-1837 project.