Yadav Mange R, Kumar Mukesh, Murumkar Prashant R, Hazari Puja P, Mishra Anil K
Faculty of Pharmacy, Kalabhavan Campus, The Maharaja Sayajirao University of Baroda, Vadodara, 390 001 Gujarat State, India.
Division of Radiopharmaceuticals, Institute of Nuclear Medicine & Allied Sciences (INMAS), Lucknow Road, Timarpur, 110 054 Delhi, India.
ACS Omega. 2018 Sep 30;3(9):11802-11816. doi: 10.1021/acsomega.8b01014. Epub 2018 Sep 24.
Some quaternary gemini amphiphiles (GAs) were synthesized as nonviral gene delivery carriers. The critical miceller concentration values of these amphiphiles are indicative of their superior surface-active properties. All of the synthesized GAs, alone or along with lipids like cholesterol and/or dioleoylphosphatidyl ethanolamine (DOPE), were formulated as liposomes. Formulations of GAs with DOPE showed average particle diameters of 326-400 nm with positive ζ-potential (30.1-46.4 mV). The lipoplexes of theses formulations showed complete pDNA retention at the base at a N/P ratio higher than 1.0 in gel retardation study. The GAs were effective in condensing pDNA into a ψ-phase, as indicated by circular dichroism study, and provided complete protection of the pDNA against the enzyme DNase at a N/P ratio more than 1. In vitro cell line studies showed that GA liposomal formulations caused β-gal expression and offered a higher transfection efficiency than that of liposomes prepared with the help of -[1-(2,3-dioleoyloxy)propyl]-,,-trimethylammonium methyl-sulfate (DOTAP)/DOPE and dicyclocarbodiimide (DCC)/DOPE but comparable to those of Lipofectamine 2000 in A549 and HeLa cell lines. Modulation of head group polarity significantly affected the transfection efficacy of GAs. The cell viabilities of almost all of the formulations were comparable to those of the standards (DCC/DOPE and DOTAP/DOPE liposomes). Incorporation of cholesterol [GA/DOPE/cholesterol in the ratio of 1:1:1] further improved the serum compatibility of the formulations and improved the transfection efficacy when evaluated in A549 and HeLa cell lines. Fluorescence-assisted cell sorting studies showed comparable number of transfected cells to Lipofectamine 2000 in the HeLa cell line. Intracellular trafficking studies using confocal microscopy indicated transfection of the HeLa cells with the reporter gene within 30 min of lipoplex treatment. γ-Scintigraphy using Tc-labeled lipoplexes showed higher concentrations of the lipoplexes in vital tissues like liver, spleen, lungs, and kidneys.
合成了一些季铵盐型双子两亲分子（GAs）作为非病毒基因传递载体。这些两亲分子的临界胶束浓度值表明了它们优异的表面活性性质。所有合成的GAs，单独或与胆固醇和/或二油酰磷脂酰乙醇胺（DOPE）等脂质一起，被制备成脂质体。GAs与DOPE的制剂显示平均粒径为326 - 400 nm，ζ电位为正（30.1 - 46.4 mV）。在凝胶阻滞研究中，这些制剂的脂质体复合物在N/P比高于1.0时在底部显示出完全的pDNA保留。圆二色性研究表明，GAs能有效地将pDNA浓缩成ψ相，并且在N/P比大于1时能为pDNA提供完全的抗DNase酶保护。体外细胞系研究表明，GA脂质体制剂能引起β - 半乳糖苷酶表达，并且与在 - [1 - (2,3 - 二油酰氧基)丙基] - ，， - 三甲基硫酸甲酯铵（DOTAP）/DOPE和二环己基碳二亚胺（DCC）/DOPE帮助下制备的脂质体相比，具有更高的转染效率，但在A549和HeLa细胞系中与Lipofectamine 2000相当。头基极性的调节显著影响GAs的转染效果。几乎所有制剂的细胞活力都与标准品（DCC/DOPE和DOTAP/DOPE脂质体）相当。加入胆固醇[GA/DOPE/胆固醇比例为1:1:1]进一步提高了制剂的血清相容性，并在A549和HeLa细胞系中评估时提高了转染效果。荧光辅助细胞分选研究表明，在HeLa细胞系中转染细胞的数量与Lipofectamine 2000相当。使用共聚焦显微镜的细胞内运输研究表明，脂质体复合物处理后30分钟内HeLa细胞中的报告基因发生了转染。使用锝标记的脂质体复合物进行的γ闪烁显像显示，脂质体复合物在肝脏、脾脏、肺和肾脏等重要组织中的浓度更高。
