Lesieur S, Grabielle-Madelmont C, Paternostre M T, Ollivon M
Equipe Physicochimie des Systémes Polyphasés, CNRS URA 1218, Université Paris Sud, Châtenay-Malabry, France.
Anal Biochem. 1991 Feb 1;192(2):334-43. doi: 10.1016/0003-2697(91)90545-5.
Vesicles of egg phosphatidylcholine (EPC) and phosphatidic acid (EPA) were prepared by reverse-phase evaporation (REV) followed either by sequential extrusion through polycarbonate membranes with pore diameters of 0.8, 0.4, 0.2, 0.1, and 0.05 micron or by filtration through 0.8-micron cellulosic or 0.22-micron polyvinylidene fluoride (PVF) membranes. The resulting vesicles ranging from 130 to 640 nm in mean diameter (REVs) were characterized by high-performance liquid chromatography (HPLC) using a TSK G6000 PW gel exclusion column. The efficiency of this technique to determine vesicle size parameters was studied by the analysis of the chromatograms in combination with dynamic light scattering (DLS) determination of the mean diameters (MD) of the fractionated vesicles in the region of the elution profile maxima. The HPLC TSK G6000 PW gel exclusion provides a reproducible and fast method of size characterization for lipid vesicles having MD up to 1 micron, the best selectivity being obtained in the 20- to 500-nm MD range. HPLC analysis of REV's demonstrates that: (i) both the average size and polydispersity of the vesicles decrease with decreasing pore size of the membranes, cellulosic or PVF "tortuous" ones being less efficient than "straight bores" polycarbonate ones; (ii) mixed EPC/EPA REVs sequentially extruded down through 0.2-micron polycarbonate membranes are highly deformable without rupture of the bilayer; and (iii) the mean size of extruded REV's is stable for at least 1 week. The role of EPA on the size stability of mixed EPC/EPA vesicles was studied by coupling HPLC gel exclusion and turbidity analysis of pure EPC and EPC/EPA (mole ratio: 91/9) sonicated small unilamellar vesicles as a function of time. The apparent size variation of EPC vesicles observed over a week, is mainly due to their aggregation which is significantly reduced by the introduction of a small amount of EPA in the vesicle membrane.
通过反相蒸发（REV）制备鸡蛋磷脂酰胆碱（EPC）和磷脂酸（EPA）囊泡，随后依次通过孔径为0.8、0.4、0.2、0.1和0.05微米的聚碳酸酯膜挤压，或通过0.8微米纤维素或0.22微米聚偏二氟乙烯（PVF）膜过滤。使用TSK G6000 PW凝胶排阻柱通过高效液相色谱（HPLC）对所得平均直径在130至640纳米之间的囊泡（REV）进行表征。通过结合洗脱图谱最大值区域中分级分离囊泡的平均直径（MD）的动态光散射（DLS）测定来分析色谱图，研究该技术测定囊泡尺寸参数的效率。HPLC TSK G6000 PW凝胶排阻为平均直径高达1微米的脂质囊泡提供了一种可重现且快速的尺寸表征方法，在20至500纳米的平均直径范围内获得最佳选择性。对REV的HPLC分析表明：（i）随着膜孔径减小，囊泡的平均尺寸和多分散性均降低，纤维素或PVF“曲折”膜的效率低于“直孔”聚碳酸酯膜；（ii）依次通过0.2微米聚碳酸酯膜向下挤压的混合EPC/EPA REV具有高度可变形性，双层不破裂；（iii）挤压后的REV平均尺寸至少1周保持稳定。通过将HPLC凝胶排阻与纯EPC和EPC/EPA（摩尔比：91/9）超声处理的小单层囊泡的浊度分析作为时间的函数进行耦合，研究了EPA对混合EPC/EPA囊泡尺寸稳定性的作用。在一周内观察到的EPC囊泡的表观尺寸变化主要是由于它们的聚集，而在囊泡膜中引入少量EPA可显著减少这种聚集。
