Pham Hoang L, Ross Benjamin P, McGeary Ross P, Shaw P Nicholas, Hewavitharana Amitha K, Davies Nigel M
School of Pharmacy, The University of Queensland, Brisbane, Australia.
Curr Drug Deliv. 2006 Oct;3(4):389-97. doi: 10.2174/156720106778559092.
ISCOMs have received much attention as vaccine adjuvants due to their immunostimulatory effects. They are colloidal particles typically comprised of phospholipids, cholesterol and Quil A, a crude mixture of saponins extracted from the bark of Quillaja saponaria Molina. We have previously shown that ISCOMs can be prepared by ether injection wherein an ether solution of phospholipids and cholesterol in a mass ratio of 5:2 is injected into a solution of Quil A at a mass ratio of 7 lipids: 3 Quil A. The aim of this study was firstly to isolate and characterise discrete fractions of Quil A and secondly to investigate which of these fractions were able to form ISCOMs by the method of ether injection. Six fractions of Quil A were isolated by semi-preparative reverse phase high performance liquid chromatography (RP-HPLC) and characterised by analytical HPLC, liquid chromatography tandem mass spectrometry (LC-MS) and the qualitative Liebermann-Burchard and Molisch tests for triterpenoids and carbohydrates respectively. ISCOMs were subsequently prepared from the isolated fractions by the method of ether injection and the resulting preparations characterized by photon correlation spectroscopy (PCS) and negative stain transmission electron microscopy (TEM). The molecular weights of the major compounds in the fractions ranged from approximately 1200 to approximately 2300 Da; all fractions tested positive for triterpenoids and saccharides and four of the fractions were identified as QS-7, QS-17, QS-18 and QS-21 by analysis (LC-MS and analytical HPLC). Injection of ether solutions of lipids into aqueous solutions of QS-17, QS-18 or QS-21 all resulted in homogeneous ISCOM dispersions. The combination of lipids and QS-7 by ether injection produced lamellae and liposomes as the prominent structures and a minor amount of ISCOMs. The remaining two hydrophilic, low molecular weight fractions of Quil A did not produce ISCOMs, instead liposomes and helical structures predominated in the samples.
免疫刺激复合物（ISCOMs）因其免疫刺激作用作为疫苗佐剂受到了广泛关注。它们是胶体颗粒，通常由磷脂、胆固醇和皂树素A（Quil A）组成，皂树素A是从皂树（Quillaja saponaria Molina）树皮中提取的皂苷粗混合物。我们之前已经表明，免疫刺激复合物可以通过乙醚注入法制备，即将质量比为5:2的磷脂和胆固醇的乙醚溶液以脂质:皂树素A质量比为7:3注入皂树素A溶液中。本研究的目的首先是分离和表征皂树素A的离散级分，其次是研究这些级分中哪些能够通过乙醚注入法形成免疫刺激复合物。通过半制备反相高效液相色谱（RP - HPLC）分离出六个皂树素A级分，并分别通过分析型HPLC、液相色谱串联质谱（LC - MS）以及分别针对三萜类化合物和碳水化合物的定性Liebermann - Burchard和Molisch试验进行表征。随后通过乙醚注入法从分离出的级分中制备免疫刺激复合物，并通过光子相关光谱（PCS）和负染色透射电子显微镜（TEM）对所得制剂进行表征。级分中主要化合物的分子量范围约为1200至约2300 Da；所有级分的三萜类化合物和糖类检测均呈阳性，并且通过分析（LC - MS和分析型HPLC）确定其中四个级分为QS - 7、QS - 17、QS - 18和QS - 21。将脂质的乙醚溶液注入QS - 17、QS - 18或QS - 21的水溶液中均产生均匀的免疫刺激复合物分散体。通过乙醚注入法将脂质与QS - 7组合产生了以片层和脂质体为主要结构以及少量免疫刺激复合物的产物。皂树素A的其余两个亲水性低分子量级分未产生免疫刺激复合物，相反，样品中脂质体和螺旋结构占主导。
