Purpose

To formulate and characterize nanoparticles from m-7NH-m gemini surfactants, synthesized by a new improved method, for non-invasive gene delivery including optimization of composition for transfection efficiency and corneal penetration.

Methods

A one-pot, solvent-free, DMAP-free method was developed for the synthesis of m-7NH-m (m = 12–18) gemini surfactant series. Lipoplexes (LPXs) and nanoplexes (NPXs) of gemini surfactant-plasmid DNA were formulated with and without DOPE helper lipid, respectively, at various charge ratios and characterized by dynamic light scattering and zeta potential measurements. Transfection efficiency, cellular toxicity, effect of DOPE and gene expression kinetic studies were carried out in A7 astrocytes by flow cytometry and confocal microscopy. Corneal penetration studies of 18-7NH-18 NPXs were carried out using 3D EpiCorneal^® tissue model.

Results

The new synthesis method provides a two-fold improved yield and the production of a pure species of m-7NH-m without DMAP and trimeric m-7N(m)-m surfactants as impurities. Structure and purity was confirmed by ESI-MS, ¹H NMR spectroscopy and surface tension measurements. Particle size of 199.80 ± 1.83 nm ± S.D. and a zeta potential value of +30.18 ± 1.17 mV ± S.D. was obtained for 18-7NH-18 5:1 ratio NPXs showed optimum transfection efficiency (10.97 ± 0.11%) and low toxicity (92.97 ± 0.57% viability) at the 48-h peak expression. Inclusion of DOPE at 1: 0.5 and 1:1 ratios to gemini surfactant reduced transfection efficiency and increased toxicity. Treatment of EpiCorneal^® tissue model showed deep penetration of up to 100 μm with 18-7NH-18 NPXs.

Conclusion

Overall, 18-7NH-18 NPXs are potential gene delivery systems for ophthalmic gene delivery and for further in vivo studies.

中文翻译：

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用阳离子阳离子氨基取代的双子表面活性剂及其纳米复合物：体外转染效率和角膜穿透性的改进合成与表征。

目的

用新型改进方法合成的m-7NH-m双子表面活性剂来配制和表征纳米粒子，用于非侵入性基因传递，包括优化转染效率和角膜渗透的成分。

方法

开发了一种单锅，无溶剂，无DMAP的方法来合成m-7NH-m（m = 12-18）双子表面活性剂系列。双子表面活性剂-质粒DNA的脂质复合物（LPXs）和纳米复合物（NPXs）分别在具有和不具有DOPE辅助脂质的情况下以各种电荷比率配制，并通过动态光散射和ζ电位测量进行表征。通过流式细胞术和共聚焦显微镜对A7星形胶质细胞进行了转染效率，细胞毒性，DOPE的影响和基因表达动力学研究。18-7NH-18 NPXs的角膜穿透研究进行了使用3D EpiCorneal ^®组织模型。

结果

新的合成方法可将产量提高两倍，并且可以生产纯物质m-7NH-m，而没有DMAP和三聚体m-7N（m）-m表面活性剂作为杂质。通过ESI-MS，¹ H NMR光谱和表面张力测量来确认结构和纯度。对于18-7NH-18 5：1比例的NPX，粒径为199.80±1.83 nm±SD，ζ电势值为+30.18±1.17 mV±SD，表现出最佳的转染效率（10.97±0.11％）和低毒性（92.97）在48小时峰值表达时的生存力（±0.57％）。以双子表面活性剂以1：0.5和1：1的比例包含DOPE会降低转染效率并增加毒性。EpiCorneal的治疗^®组织模型显示高达100微米与18-7NH-18 NPXs的深层渗透。

结论

总体而言，18-7NH-18 NPXs是用于眼科基因传递和进一步体内研究的潜在基因传递系统。