Departments of ‡Organic Chemistry and ⊥Molecular Reproduction Development and Genetics, Indian Institute of Science , 560012 Bangalore, India.
Biomacromolecules. 2013 Nov 11;14(11):3951-63. doi: 10.1021/bm401079h. Epub 2013 Oct 15.
Lipoplex-type nanoaggregates prepared from pEGFP-C3 plasmid DNA (pDNA) and mixed liposomes, with a gemini cationic lipid (CL) [1,2-bis(hexadecyl imidazolium) alkanes], referred as (C16Im)2Cn (where Cn is the alkane spacer length, n = 2, 3, 5, or 12, between the imidazolium heads) and DOPE zwitterionic lipid, have been analyzed by zeta potential, gel electrophoresis, SAXS, cryo-TEM, fluorescence anisotropy, transfection efficiency, fluorescence confocal microscopy, and cell viability/cytotoxicity experiments to establish a structure-biological activity relationship. The study, carried out at several mixed liposome compositions, α, and effective charge ratios, ρeff, of the lipoplex, demonstrates that the transfection of pDNA using CLs initially requires the determination of the effective charge of both. The electrochemical study confirms that CLs with a delocalizable positive charge in their headgroups yield an effective positive charge that is 90% of their expected nominal one, while pDNA is compacted yielding an effective negative charge which is only 10-25% than that of the linear DNA. SAXS diffractograms show that lipoplexes formed by CLs with shorter spacer (n = 2, 3, or 5) present three lamellar structures, two of them in coexistence, while those formed by CL with longest spacer (n = 12) present two additional inverted hexagonal structures. Cryo-TEM micrographs show nanoaggregates with two multilamellar structures, a cluster-type (at low α value) and a fingerprint-type, that coexist with the cluster-type at moderate α composition. The optimized transfection efficiency (TE) of pDNA, in HEK293T, HeLa, and H1299 cells was higher using lipoplexes containing gemini CLs with shorter spacers at low α value. Each lipid formulation did not show any significant levels of toxicity, the reported lipoplexes being adequate DNA vectors for gene therapy and considerably better than both Lipofectamine 2000 and CLs of the 1,2-bis(hexadecyl ammnoniun) alkane series, recently reported.
由 pEGFP-C3 质粒 DNA (pDNA) 和混合脂质体制备的脂质体型纳米聚集体，其中包含一种双子阳离子脂质（CL）[1,2-双（十六烷基咪唑）烷]，称为（C16Im）2Cn（其中 Cn 是咪唑头之间的烷烃间隔长度，n = 2、3、5 或 12）和 DOPE 两性离子脂质，已经通过 zeta 电位、凝胶电泳、小角 X 射线散射（SAXS）、冷冻透射电子显微镜（cryo-TEM）、荧光各向异性、转染效率、荧光共聚焦显微镜和细胞活力/细胞毒性实验进行了分析，以建立结构-生物学活性关系。该研究在几种混合脂质体组成、α 和脂质体有效电荷比 ρeff 下进行，表明使用 CL 进行 pDNA 的转染最初需要确定两者的有效电荷。电化学研究证实，头部具有可离域正电荷的 CL 产生的有效正电荷是其预期标称值的 90%，而 pDNA 被压缩，产生的有效负电荷仅比线性 DNA 高 10-25%。SAXS 衍射图谱显示，具有较短间隔基（n = 2、3 或 5）的 CL 形成的脂质体呈现出三种层状结构，其中两种共存，而具有最长间隔基（n = 12）的 CL 形成的脂质体呈现出两种额外的反向六方结构。冷冻透射电子显微镜照片显示，纳米聚集体具有两种双层结构，一种是簇状（在低 α 值下）和指纹状，它们在中等 α 组成下共存。在 HEK293T、HeLa 和 H1299 细胞中，pDNA 的最佳转染效率（TE）使用在低 α 值下含有较短间隔基的双子 CL 的脂质体更高。每种脂质配方均未显示出任何明显的毒性水平，所报道的脂质体是用于基因治疗的合适 DNA 载体，并且明显优于最近报道的 Lipofectamine 2000 和 1,2-双（十六烷基氨）烷系列的 CL。
