Dijkstra J, Ryan J L, Szoka F C
Department of Pharmacy and Pharmaceutical Chemistry, University of California, San Francisco 94143.
J Immunol Methods. 1988 Nov 10;114(1-2):197-205. doi: 10.1016/0022-1759(88)90174-3.
Previous studies on the mechanism of action of lipopolysaccharides (LPS) on macrophages have used wild-type lipopolysaccharide (wt-LPS) containing liposomes. In these studies the endotoxin was incorporated into liposomes by suspending the wt-LPS in the buffer used to rehydrate the lipid. Using this approach (buffer method), we observed that less than 10% of Salmonella minnesota smooth LPS is incorporated into multilamellar vesicles (MLV). If the non-incorporated material is not effectively separated from the liposomal form, erroneous conclusions on the mechanism of action of LPS can be drawn. Prolonged sonication of the wt-LPS-MLV suspension resulted in almost complete incorporation of the LPS into the resulting small unilamellar vesicles (SUV). In order to prepare MLV, we briefly soniated the buffer preparation, dehydrated the resulting smaller vesicles and then rehydrated the mixture (dry method). This procedure resulted in almost complete incorporation of the wt-LPS into MLV. The ability of wt-LPS in MLV prepared by the dry method to activate macrophages or trigger gelation of Limulus amoebocyte lysate was reduced by 100-1000-fold compared to the non-incorporated wt-LPS. This indicates that at least 99% of the wt-LPS is incorporated in MLV made by the dry method.
以往关于脂多糖（LPS）对巨噬细胞作用机制的研究使用了含有野生型脂多糖（wt-LPS）的脂质体。在这些研究中，通过将wt-LPS悬浮在用于使脂质复水的缓冲液中，将内毒素掺入脂质体。使用这种方法（缓冲液法），我们观察到明尼苏达沙门氏菌光滑型LPS中只有不到10%被掺入多层囊泡（MLV）。如果未掺入的物质不能有效地与脂质体形式分离，就可能得出关于LPS作用机制的错误结论。对wt-LPS-MLV悬浮液进行长时间超声处理，可使LPS几乎完全掺入所得的小单层囊泡（SUV）中。为了制备MLV，我们对缓冲液制剂进行了短暂超声处理，将所得较小的囊泡脱水，然后使混合物复水（干燥法）。该程序使wt-LPS几乎完全掺入MLV中。与未掺入的wt-LPS相比，通过干燥法制备的MLV中的wt-LPS激活巨噬细胞或触发鲎变形细胞溶解物凝胶化的能力降低了100 - 1000倍。这表明至少99%的wt-LPS被掺入通过干燥法制备的MLV中。
