School of Pharmacy, Bandung Institute of Technology, Bandung 40132, Indonesia.
School of Biomedical Sciences, Faculty of Science, Charles Sturt University, Wagga-Wagga, New South Wales 2678, Australia.
Curr Gene Ther. 2020;20(5):383-394. doi: 10.2174/1566523220666201005104224.
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.
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.
The molecular interactions between the lipopeptides and Importin-α 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-CKKHH-derived liposome was carried out based on luciferase transgene expression.
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 nanoparticles. 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 ~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.
Our study for the first time has shown that the fully synthesized short lipopeptide Pal- CKKHH is able to interact firmly with the Importin-α. 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.
基于脂肽的基因载体显示出低细胞毒性，能够穿透细胞膜，易于制造，因此具有成为基因传递应用的潜在候选物的巨大潜力。
本研究旨在探索一系列短合成脂肽（Lau：月桂基；Pal：棕榈酰基），由烷基链、一个半胱氨酸（C）、1 至 2 个组氨酸（H）和赖氨酸（K）残基组成，通过进行计算机分子相互作用和体外评估。
使用 AutoDock Vina 和 Amber14 进行脂肽与 Importin-α 受体之间的分子相互作用。在体外评估脂肽/DNA 复合物的相互作用、粒径、ζ 电位和转基因表达。根据荧光素酶转基因表达，进行脂肽和 Pal-CKKHH 衍生脂质体的转染效率。
计算机模拟相互作用表明，Lau-CKKH 和 Pal-CKKHH 假设加速核摄取。与天然配体（核定位序列为-5.4 kcal/mol）相比，两种脂肽的结合能都较低（分别为-6.3 kcal/mol 和-6.2 kcal/mol）。短脂肽能够使 DNA 分子凝聚，并有效地形成紧密的纳米颗粒。基于对 COS-7 的体外评估，发现 Pal-CKKHH 是脂肽中最好的转染剂。其转染效率（ng Luc/mg 总蛋白）增加了约 3 倍（1163+55），因为它与辅助脂质 DOPE（1:2）一起配制。基于 Pal-CKKHH：DOPE=1:2 的脂肽脂质体也促进了人胚肾细胞（293T）和人宫颈癌细胞（HeLa）的荧光素酶转基因表达，转染效率分别为 1779+52 和 260+22。
我们的研究首次表明，完全合成的短脂肽 Pal-CKKHH 能够与 Importin-α 牢固地相互作用。该脂肽能够有效地使 DNA 分子凝聚，促进转基因表达，加速核摄取过程，因此具有成为潜在转染剂的特征。
