Gordon Sean Patrick, Berezhna Svitlana, Scherfeld Dag, Kahya Nicoletta, Schwille Petra
International Max Planck Research School for Molecular Biology, University of Goettingen, Goettingen, Germany.
Biophys J. 2005 Jan;88(1):305-16. doi: 10.1529/biophysj.104.043133. Epub 2004 Oct 29.
Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides (CL-ODN) are promising delivery systems for antisense therapy. ODN release from the complexes is an essential step for inhibiting activity of antisense drugs. We applied fluorescence correlation spectroscopy and confocal laser scanning microscopy to monitor CL-ODN complex interaction with membrane lipids leading to ODN release. To model cellular membranes we used giant unilamellar vesicles and investigated the transport of Cy-5-labeled ODNs across DiO-labeled membranes. For the first time, we directly observed that ODN molecules are transferred across the lipid bilayers and are kept inside the giant unilamellar vesicles after release from the carriers. ODN dissociation from the carrier was assessed by comparing diffusion constants of CL-ODN complexes and ODNs before complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence correlation spectroscopy curves for CL-ODN complexes were fitted with two components, which both have significantly slower diffusion in the range of D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is dissociated from cationic lipids. In contrast to earlier studies, we report that phosphatidylethanolamine can trigger ODN release from the carrier in the full absence of anionic phosphatidylserine in the target membrane and that phosphatidylethanolamine-mediated release is as extensive as in the case of phosphatidylserine. The presented methodology provides an effective tool for probing a delivery potential of newly created lipid formulations of CL-ODN complexes for optimal design of carriers.
阳离子脂质体与单链寡脱氧核苷酸（CL - ODN）形成的复合物是用于反义治疗的有前景的递送系统。复合物中ODN的释放是抑制反义药物活性的关键步骤。我们应用荧光相关光谱和共聚焦激光扫描显微镜来监测CL - ODN复合物与膜脂的相互作用，这会导致ODN释放。为了模拟细胞膜，我们使用了巨型单层囊泡，并研究了Cy - 5标记的ODN跨DiO标记膜的转运。首次，我们直接观察到ODN分子穿过脂质双层，并在从载体释放后保留在巨型单层囊泡内部。通过比较CL - ODN复合物和ODN在复合前和释放后的扩散常数来评估ODN与载体的解离。自由扩散的Cy - 5标记的ODN（16个核苷酸）的扩散常数D(ODN) = 1.3 ± 0.1×10⁻⁶ cm²/s。CL - ODN复合物的荧光相关光谱曲线用两个成分拟合，这两个成分在D(CL - ODN) = 约1.5×10⁻⁸ cm²/s范围内的扩散都明显较慢。释放的ODN的平均扩散常数D = 1.1 ± 0.2×10⁻⁶ cm²/s，这表明ODN已从阳离子脂质中解离。与早期研究不同，我们报道磷脂酰乙醇胺可以在靶膜中完全不存在阴离子磷脂酰丝氨酸的情况下触发ODN从载体释放，并且磷脂酰乙醇胺介导的释放与磷脂酰丝氨酸的情况一样广泛。所提出的方法为探究新创建的CL - ODN复合物脂质制剂的递送潜力以进行载体的优化设计提供了一种有效工具。
