In-silico Molecular Interaction of Short Synthetic Lipopeptide/Importin-alpha and In-vitro Evaluation of Transgene Expression Mediated by Liposome-Based Gene Carrier

Background: Lipopeptide-based gene carriers have shown low cytotoxicity, are capable of cell membrane penetration, are easy to manufacture and therefore are great potential candidates for gene delivery applications. Objectives: This study aims to explore a range of short synthetic lipopeptides, (Lau: Lauryl; Pal: Palmitoyl) consisting of an alkyl chain, one cysteine (C), 1 to 2 histidine (H), and lysine (K) residues by performing in-silico molecular interaction and in-vitro evaluation. Methods: The molecular interactions between the lipopeptides and Importin-alpha receptor were performed using AutoDock Vina and Amber14. The lipopeptide/DNA complexes were evaluated in-vitro for their interactions, particle size, zeta potential and transgene expression. Transfection efficiency of the lipopeptides and Pal-CKKI III-derived liposome was carried out based on luciferase transgene expression. Results: The in-silico interaction showed that Lau-CKKH and Pal-CKKHH hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, nuclear localization sequence (-5.4 kcal/mol). The short lipopeptides were able to condense DNA molecules and efficiently form compacted nanoparticies. Based on the in-vitro evaluation on COS-7, Pal-CKKHH was found to be the best transfection agent amongst the lipopeptides. Its transfection efficiency (ng Luc/mg total protein) increased up to similar to 3-fold higher (1163 +/- 55) as it was formulated with helper lipid DOPE (1:2). The lipopeptide-based liposome (Pal-CKKHH: DOPE=1:2) also facilitated luciferase transgene expression on human embryonic kidney cells (293T) and human cervical adenocarcinoma cells (HeLa) with transfection efficiency 1779 +/- 52 and 260 +/- 22, respectively. Conclusion: Our study for the first time has shown that the fully synthesized short lipopeptide PalCKKHH is able to interact firmly with the Importin-alpha. The lipopeptide is able to condense DNA molecules efficiently, facilitate transgene expression, expedite the nuclear uptake process, and hence has the characteristics of a potential transfection agent.