SEPARATION OF LARGE UNILAMELLAR LIPOSOMES FROM BLOOD COMPONENTS BY A SPIN COLUMN PROCEDURE - TOWARDS IDENTIFYING PLASMA-PROTEINS WHICH MEDIATE LIPOSOME CLEARANCE INVIVO
SEPARATION OF LARGE UNILAMELLAR LIPOSOMES FROM BLOOD COMPONENTS BY A SPIN COLUMN PROCEDURE - TOWARDS IDENTIFYING PLASMA-PROTEINS WHICH MEDIATE LIPOSOME CLEARANCE INVIVO
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DOI：
10.1016/0005-2736(91)90167-7
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发表时间：
1991-11-18
期刊：
影响因子：
--
通讯作者：
CULLIS, PR
中科院分区：
文献类型：
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作者：
CHONN, A;SEMPLE, SC;CULLIS, PR
为了便于从血液成分中分离脂质体，我们开发了一种简单快速的方法，结合色谱法和离心法。 该“旋转柱”程序用于从与人血清的孵育混合物中或从静脉内施用脂质体后的CD 1小鼠的血液中分离脂质体。 该程序的优点是处理时间快（通常为几分钟），使得分离程序可以在不存在可能影响蛋白质/脂质体相互作用的螯合剂或其他凝血抑制剂的情况下进行。 此外，可以同时分析多个样品，并处理少量样品。 此外，我们表明，这种旋转柱程序可用于分离大的单层囊泡平均直径为100 nm的脂蛋白和血浆蛋白。 该旋转柱程序在研究蛋白质/脂质体相互作用中的适用性通过用人血清孵育后使用C3竞争性ELISA测定法定量每个脂质体结合的人补体组分C3的量来证明。 通过常规SDS-聚丙烯酰胺凝胶电泳进一步分析与回收的脂质体相关的蛋白质。 我们表明，卵磷脂/胆固醇（55：45，mol/mol）或卵磷脂/胆固醇/二油酰磷脂酰丝氨酸（35：45：20，mol/mol）脂质体从CD 1小鼠的循环中分离的管理分钟内有不同的，复杂的相关蛋白质的配置文件。 通过使用旋转柱方法分离循环的大单层脂质体并表征与其膜相关的蛋白质，这种蛋白质指纹法将加快鉴定影响脂质体稳定性和体内清除的蛋白质相互作用。
In order to facilitate the isolation of liposomes from blood components, we have developed a simple and rapid procedure combining chromatographic and centrifugal methods. This 'spin column' procedure was used to isolate liposomes from incubation mixtures with human serum or from the blood of CD1 mice after intravenous administration of liposomes. An advantage of this procedure is that processing times are fast (typically minutes) such that the isolation procedure can be done in the absence of chelators or other coagulation inhibitors which may affect protein/liposome interactions. Furthermore, several samples can be analyzed together and small sample volumes can be processed. In addition, we show that this spin column procedure can be employed to isolate large unilamellar vesicles averaging 100 nm in diameter from lipoproteins and plasma proteins. The applicability of this spin column procedure in studying protein/liposome interactions is demonstrated by quantitating the amount of human complement component C3 bound per liposome using a C3 competitive ELISA assay after incubation with human serum. The proteins associated with the recovered liposomes were further analyzed by conventional SDS-polyacrylamide gel electrophoresis. We show that egg phosphatidylcholine/cholesterol (55:45, mol/mol) or egg phosphatidylcholine/cholesterol/dioleoylphosphatidylserine (35:45:20, mol/mol) liposomes isolated from the circulation of CD1 mice within minutes of administration have distinct, complex profiles of associated proteins. By isolating circulating large unilamellar liposomes using the spin column method and characterizing the proteins associated with their membranes, this protein fingerprinting approach will expedite identifying protein interactions which affect liposome stability and clearance in vivo.