Angelova M I, Hristova N, Tsoneva I
Institute of Biophysics, Bulgarian Academy of Sciences, Sofia, Bulgaria.
Eur Biophys J. 1999;28(2):142-50. doi: 10.1007/s002490050193.
We suggest a novel approach for direct optical microscopy observation of DNA interaction with the bilayers of giant cationic liposomes. Giant unilamellar vesicles, about 100 microns in diameter, made of phosphatidyl-cholines and up to 33 mol% of the natural bioactive cationic amphiphile sphingosine, were obtained by electroformation. "Short" DNAs (oligonucleotide 21 b and calf thymus 250 bp) were locally injected by micropipette to a part of the giant unilamellar vesicle (GUV) membrane. DNAs were injected native, as well as marked with a fluorescent dye. The resulting membrane topology transformations were monitored in phase contrast, while DNA distribution was followed in fluorescence. We observed DNA-induced endocytosis due to the DNA/lipid membrane local interactions and complex formation. A characteristic minimum concentration (Cendo) of D-erythrosphingosine (Sph+) in the GUV membrane was necessary for the endocytic phenomenon to occur. Below Cendo, only lateral adhesions between neighboring vesicles were observed upon DNA local addition. Cendo depends on the type of zwitterionic (phosphocholine) lipid used, being about 10 mol% for DPhPC/Sph+ GUVs and about 20 mol% for SOPC/Sph+ or eggPC/Sph+ GUVs. The characteristic sizes and shapes of the resulting endosomes depend on the kind of DNA, and initial GUV membrane tension. When the fluorescent DNA marker dye was injected after the DNA/lipid local interaction and complex formation, no fluorescence was detected. This observation could be explained if one assumes that the DNA is protected by lipids in the DNA/lipid complex, thereby inaccessible for the dye molecules. We suggest a possible mechanism for DNA/lipid membrane interaction involving DNA encapsulation within an inverted micelle included in the lipid membrane. Our model observations could help in understanding events associated with the interaction of DNA with biological membranes, as well as cationic liposomes/DNA complex formation in gene transfer processes.
我们提出了一种用于直接光学显微镜观察DNA与巨型阳离子脂质体双层相互作用的新方法。通过电形成法获得了由磷脂酰胆碱和高达33摩尔%的天然生物活性阳离子两亲性鞘氨醇制成的直径约100微米的巨型单层囊泡。用微量移液器将“短”DNA（21个碱基的寡核苷酸和250碱基对的小牛胸腺DNA）局部注射到巨型单层囊泡（GUV）膜的一部分。DNA以天然形式注射，也用荧光染料标记。通过相差显微镜监测由此产生的膜拓扑结构转变，同时通过荧光追踪DNA分布。我们观察到由于DNA/脂质膜局部相互作用和复合物形成导致的DNA诱导的内吞作用。GUV膜中D-赤藓糖鞘氨醇（Sph+）的特征性最低浓度（Cendo）是内吞现象发生所必需的。低于Cendo时，在局部添加DNA后仅观察到相邻囊泡之间的侧向粘连。Cendo取决于所用两性离子（磷脂酰胆碱）脂质的类型，对于DPhPC/Sph+ GUV约为10摩尔%，对于SOPC/Sph+或eggPC/Sph+ GUV约为20摩尔%。所得内体的特征尺寸和形状取决于DNA的种类以及初始GUV膜张力。当在DNA/脂质局部相互作用和复合物形成后注射荧光DNA标记染料时，未检测到荧光。如果假设DNA在DNA/脂质复合物中被脂质保护，从而染料分子无法接近，那么这一观察结果可以得到解释。我们提出了一种DNA/脂质膜相互作用的可能机制，涉及DNA被包裹在脂质膜中的反相胶束内。我们的模型观察结果有助于理解与DNA与生物膜相互作用相关的事件，以及基因转移过程中阳离子脂质体/DNA复合物的形成。
