Three-dimensional imaging of lipid gene-carriers: Membrane charge density controls universal transfection behavior in lamellar cationic liposome-DNA complexes
Three-dimensional imaging of lipid gene-carriers: Membrane charge density controls universal transfection behavior in lamellar cationic liposome-DNA complexes
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DOI：
10.1016/s0006-3495(03)70055-1
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发表时间：
2003-05-01
影响因子：
3.4
通讯作者：
Safinya, CR
中科院分区：
文献类型：
--
作者：
Lin, AJ;Slack, NL;Safinya, CR
阳离子脂质体;（CLs）作为基因载体（载体）在非病毒基因传递的临床应用中被广泛使用，尽管转染效率（TE）低得令人无法接受。我们展示了三维激光扫描共聚焦显微镜研究，揭示了层状l - α (C)和倒六边形H-II(C)纳米结构的CL-DNA复合物与小鼠成纤维细胞之间明显的相互作用。细胞中L复合物的共聚焦图像确定了两种制度。对于低膜电荷密度（sigma(M)）， DNA仍然被困在cl载体中。相比之下，高sigma(M)时，在细胞质中观察到释放的DNA，表明从核内体中逃逸；通过融合。值得注意的是，萤火虫荧光素酶报告基因在高度复杂的l - α (C)-哺乳动物细胞系统中的研究揭示了一种意想不到的简单性，在恒定的阳离子与阴离子电荷比下，一价和多价阳离子脂质的TE数据；作为σ (M)的函数合并成一条曲线，将其确定为关键的通用参数。l - α (C)复合物转染的通用曲线在大约40年的时间里呈指数增长，随着sigma(M)的增加而低于最佳电荷密度（sigma(M)*），并在sigma(M) > sigma(M)*达到饱和，其值与H-II(C)复合物的高转染效率相媲美。相反，H-II复合物的转染效率(C)与sigma(M)无关。对于l - α (C)复合物，TE对sigma(M)的指数依赖性表明，存在一个阻止内体融合的动力学屏障，其中sigma(M)的增加降低了屏障。在饱和TE状态下，对于l - α (C)复合物和H-II(C)，共聚焦显微镜显示脂质和DNA的解离。然而，脂质释放的DNA被观察到处于凝聚状态，很可能是与来自细胞质的带相反电荷的大离子凝聚剂，这仍有待鉴定。观察到的大部分浓缩DNA可能是转录无活性的，并且可能决定阳离子脂质基因载体转染的当前限制因素。
Cationic liposomes; (CLs) are used worldwide as gene vectors (carriers) in nonviral clinical applications of gene delivery, albeit with unacceptably low transfection efficiencies (TE). We present three-dimensional laser scanning confocal microscopy studies revealing distinct interactions between CL-DNA complexes, for both lamellar L-alpha(C) and inverted hexagonal H-II(C) nanostructures, and mouse fibroblast cells. Confocal images of L complexes in cells identified two regimes. For low membrane charge density (sigma(M)), DNA remained trapped in CL-vectors. By contrast, for high sigma(M), released DNA was observed in the cytoplasm, indicative of escape from endosomes; through fusion. Remarkably, firefly luciferase reporter gene studies in the highly complex L-alpha(C)-mammalian cell system revealed an unexpected simplicity where, at a constant cationic to anionic charge ratio, TE data for univalent and multivalent cationic lipids; merged into a single curve as a function of sigma(M), identifying it as a key universal parameter. The universal curve for transfection by L-alpha(C) complexes climbs exponentially over approximate to four decades with increasing sigma(M) below an optimal charge density (sigma(M)*), and saturates for sigma(M) > sigma(M)* at a value rivaling the high transfection efficiency of H-II(C), complexes. In contrast, the transfection efficiency of H-II(C), complexes is independent of sigma(M). The exponential dependence of TE on sigma(M) for L-alpha(C) complexes, suggests the existence of a kinetic barrier against endosomal fusion, where an increase in sigma(M) lowers the barrier. In the saturated TE regime, for both L-alpha(C) complexes and H-II(C), confocal microscopy reveals the dissociation of lipid and DNA. However, the lipid-released DNA is observed to be in a condensed state, most likely with oppositely charged macro-ion condensing agents from the cytoplasm, which remain to be identified. Much of the observed bulk of condensed DNA may be transcriptionally inactive and may determine the current limiting factor to transfection by cationic lipid gene vectors.