Patil Siddhesh D, Rhodes David G, Burgess Diane J
Department of Pharmaceutical Sciences, University of Connecticut, Storrs, CT 06269, USA.
Biochim Biophys Acta. 2005 Jun 1;1711(1):1-11. doi: 10.1016/j.bbamem.2005.03.004. Epub 2005 Mar 31.
Transfection efficiency of liposomal gene delivery vectors depends on an optimal balance in the electro-chemical and structural properties of the transfection-capable complexes. We have recently reported a novel anionic lipoplex DNA delivery system composed of a ternary complex of endogenous occurring non-toxic anionic lipids, physiological Ca2+ cations, and plasmid DNA encoding a gene of interest with high transfection efficiency and low toxicity. In this work, we investigate the electro-chemical and structural properties anionic lipoplexes and compare them with those of Ca2+-DNA complexes. Biophysical characterization is used to explain the transfection efficiency of anionic lipoplexes in mammalian CHO-K1 cells. Circular dichroism and fluorescence spectroscopy showed that the plasmid DNA underwent conformational transition from native B-DNA to Z-DNA due to compaction and condensation upon Ca2+-mediated complexation with anionic liposomes. Zeta potential measurements and gel electrophoresis studies demonstrated that Ca2+ interaction with plasmid DNA during the formation of lipoplexes also led to increased association of supercoiled plasmid DNA with the lipoplexes, leading to charge neutralization which is expected to facilitate transfection. However, even 10-fold higher concentrations of Ca2+ alone (in the absence of the anionic liposomes) were unable to induce these changes in plasmid DNA molecules. A model explaining the possible mechanism of anionic lipoplex formation and the correlation of high transfection efficiency to biophysical properties was proposed. These studies confirm the utility of biophysical studies to identify optimal formulation conditions to design efficient liposomal gene delivery vectors.
脂质体基因递送载体的转染效率取决于具有转染能力的复合物在电化学和结构性质上的最佳平衡。我们最近报道了一种新型阴离子脂质体DNA递送系统，它由内源性无毒阴离子脂质、生理Ca2+阳离子和编码感兴趣基因的质粒DNA组成的三元复合物构成，具有高转染效率和低毒性。在这项工作中，我们研究了阴离子脂质体复合物的电化学和结构性质，并将它们与Ca2+-DNA复合物的性质进行比较。生物物理表征用于解释阴离子脂质体复合物在哺乳动物CHO-K1细胞中的转染效率。圆二色性和荧光光谱表明，由于Ca2+介导的与阴离子脂质体络合时的压缩和凝聚，质粒DNA经历了从天然B-DNA到Z-DNA的构象转变。zeta电位测量和凝胶电泳研究表明，脂质体形成过程中Ca2+与质粒DNA的相互作用也导致超螺旋质粒DNA与脂质体的结合增加，导致电荷中和，这有望促进转染。然而，即使单独使用高10倍浓度的Ca2+（在没有阴离子脂质体的情况下）也无法诱导质粒DNA分子发生这些变化。提出了一个解释阴离子脂质体复合物形成的可能机制以及高转染效率与生物物理性质相关性的模型。这些研究证实了生物物理研究在确定设计高效脂质体基因递送载体的最佳配方条件方面的实用性。
