Grupo de Química Coloidal y Supramolecular, Departamento de Química Física I, Facultad de Ciencias Químicas, Universidad Complutense de Madrid , 28040 Madrid, Spain.
ACS Appl Mater Interfaces. 2016 Aug 31;8(34):22113-26. doi: 10.1021/acsami.6b08823. Epub 2016 Aug 18.
The use of small interfering RNAs (siRNAs) to silence specific genes is one of the most promising approaches in gene therapy, but it requires efficient nanovectors for successful cellular delivery. Recently, we reported liposomal gene carriers derived from a gemini cationic lipid (GCL) of the 1,2-bis(hexadecyl dimethyl imidazolium) oligo-oxyethylene series ((C16Im)2(C2H4O)nC2H4 with n = 1, 2, or 3) and 1,2-dioleyol phosphatidylethanolamine as highly efficient cytofectins for pDNA. On the basis of the satisfactory outcomes of the previous study, the present work focuses on the utility of coliposomes of these gemini lipids with the biocompatible neutral lipid mono oleoyl glycerol (MOG) as highly potent vectors for siRNA cellular transport in the presence of serum. The (C16Im)2(C2H4O)nC2H4/MOG-siRNA lipoplexes were characterized through (i) a physicochemical study (zeta potential, cryo-transmission electron microscopy, small-angle X-ray scattering, and fluorescence anisotropy) to establish the relationship between size, structure, fluidity, and the interaction between siRNA and the GCL/MOG gene vectors and (ii) a biological analysis (flow cytometry, fluorescence microscopy, and cell viability) to report the anti-GFP siRNA transfections in HEK 293T, HeLa, and H1299 cancer cell lines. The in vitro biological analysis confirms the cellular uptake and indicates that a short spacer, a very low molar fraction of GCL in the mixed lipid, and a moderate effective charge ratio of the lipoplex yielded maximum silencing efficacy. At these experimental conditions, the siRNA used in this work is compacted by the GCL/MOG nanovectors by forming two cubic structures (Ia3d and Pm3n) that are correlated with excellent silencing activity. These liposomal nanocarriers possess high silencing activity with a negligible cytotoxicity, which strongly supports their practical use for in vivo knockdown studies.
使用小干扰 RNA（siRNA）沉默特定基因是基因治疗中最有前途的方法之一，但它需要高效的纳米载体才能成功进行细胞递送。最近，我们报道了源自双子阳离子脂质（GCL）的脂质体基因载体，该 GCL 来自 1,2-双（十六烷基二甲基咪唑啉）聚氧乙烯系列（（C16Im）2（C2H4O）nC2H4，其中 n = 1、2 或 3）和 1,2-二油酰基磷脂酰乙醇胺，作为 pDNA 的高效细胞转染剂。在前一项研究令人满意的结果基础上，本工作重点研究了这些双子脂质与生物相容性中性脂质单油酰基甘油（MOG）的共脂质体作为在血清存在下 siRNA 细胞转运的高效载体的用途。（C16Im）2（C2H4O）nC2H4 / MOG-siRNA 脂质体通过（i）物理化学研究（Zeta 电位、冷冻传输电子显微镜、小角 X 射线散射和荧光各向异性）进行表征，以建立大小、结构、流动性和 siRNA 与 GCL / MOG 基因载体之间的相互作用之间的关系，以及（ii）生物学分析（流式细胞术、荧光显微镜和细胞活力），以报告在 HEK 293T、HeLa 和 H1299 癌细胞系中的抗 GFP siRNA 转染。体外生物学分析证实了细胞摄取，并表明短间隔、混合脂质中 GCL 的非常低的摩尔分数和脂质体的中等有效电荷比产生最大的沉默效果。在这些实验条件下，本工作中使用的 siRNA 由 GCL / MOG 纳米载体压缩，通过形成两种立方结构（Ia3d 和 Pm3n）而得到压缩，这与优异的沉默活性相关。这些脂质体纳米载体具有高沉默活性和可忽略的细胞毒性，这强烈支持它们在体内敲低研究中的实际应用。
