Departamento de Quı́mica Fı́sica, Facultad de Ciencias Químicas, Universidad Complutense de Madrid, 28040 Madrid, Spain.
Departamento de Fı́sica de Partı́culas, Facultad de Fı́sicas e Instituto de Investigaciones Sanitarias (IDIS), Universidad de Santiago de Compostela, Campus Vida, E-15782 Santiago de Compostela, Spain.
ACS Appl Mater Interfaces. 2020 Aug 5;12(31):34536-34547. doi: 10.1021/acsami.0c06273. Epub 2020 Jul 26.
Despite the use of small interfering RNAs (siRNAs) as therapeutic agents through the knockdown expression of pathogenic proteins, transportation and delivery of such siRNAs into cells continue to be under investigation. Within nonviral vectors, cationic lipids that include amino acid residues in their structures, and that have already demonstrated their suitability as plasmid DNA nanocarriers, may be also considered as potential siRNA vehicles. A double-chain cationic lipid based on the amino acid arginine mixed with a helper lipid has been the object of this biophysical study. First, ζ-potential measurements and agarose gel electrophoresis experiments confirmed the siRNA compaction, while small-angle X-ray scattering analysis (SAXS) revealed the structural pattern of the lipoplexes. Two bicontinuous cubic phases were found to coexist: the double-gyroid phase () and the double-diamond phase (), with 3 and 3 as crystallographic space groups, respectively; the siRNA is known to be located inside their bicontinuous aqueous channels. Second, studies in HeLa-green fluorescent protein (GFP) and T731-GFP cell lines (modified for GFP overexpression) showed moderate to high gene knockdown levels (determined by flow cytometry and epifluorescence microscopy) with remarkable cell viabilities (CCK-8 assay). Finally, nano-liquid chromatography/mass spectrometry (nanoLC-MS/MS) was used to identify the nature of the proteins adhered to the surface of the lipoplexes after incubation with human serum, simulating their behavior in biological fluids. The abundant presence of lipoproteins and serum albumin in such protein corona, together with the coexistence of the bicontinuous cubic phases, may be behind the remarkable silencing activity of these lipoplexes. The results reported herein show that the use of amino-acid-based cationic lipids mixed with a suitable helper lipid, which have already provided good results as DNA plasmid nanocarriers in cellular transfection processes, may also be a biocompatible option, and so far little investigated, in gene silencing strategies.
尽管通过敲低致病蛋白的表达来使用小干扰 RNA（siRNA）作为治疗剂，但将这些 siRNA 转运并递送到细胞中仍然是研究的重点。在非病毒载体中，包含其结构中氨基酸残基的阳离子脂质，并且已经证明它们适合作为质粒 DNA 纳米载体，也可以被认为是潜在的 siRNA 载体。基于氨基酸精氨酸的双链阳离子脂质与辅助脂质混合已成为这项生物物理研究的对象。首先，ζ-电位测量和琼脂糖凝胶电泳实验证实了 siRNA 的压缩，而小角 X 射线散射分析（SAXS）揭示了脂质体的结构模式。发现两种双连续立方相共存：双回旋相（）和双菱形相（），分别具有 3 和 3 作为晶体空间群；已知 siRNA 位于它们的双连续水通道内。其次，在 HeLa-绿色荧光蛋白（GFP）和 T731-GFP 细胞系（为 GFP 过表达而修饰）中的研究表明，具有中等至高基因敲低水平（通过流式细胞术和荧光显微镜确定）和显著的细胞活力（CCK-8 测定）。最后，纳米液相色谱/质谱联用（nanoLC-MS/MS）用于鉴定与人类血清孵育后附着在脂质体表面的蛋白质的性质，模拟它们在生物流体中的行为。在这种蛋白质冠中，脂蛋白和血清白蛋白的大量存在以及双连续立方相的共存，可能是这些脂质体具有显著沉默活性的原因。本文报道的结果表明，使用基于氨基酸的阳离子脂质与合适的辅助脂质混合，作为细胞转染过程中 DNA 质粒纳米载体已经取得了良好的效果，也可能是一种生物相容性的选择，并且迄今为止在基因沉默策略中研究甚少。
