带有QS21(ALFQ)的陆军脂质体制剂,疫苗佐剂的制备,包含含有饱和磷脂的脂质体,与55摩尔%的胆固醇相对于磷脂,和两种佐剂,单磷酰脂质A(MPLA)和QS21皂苷。ALFQ的一个独特特征是形成直径>1.0µm的巨大单层囊泡(GUV),由于在将QS21添加到含有MPLA和相对于总磷脂55mol%的胆固醇的纳米脂质体中时引发了显着的融合事件。这导致ALFQ颗粒的多分散尺寸分布,直径范围从~50nm到~30,000nm。这项工作的目的是深入了解导致QS21诱导的GUV的纳米囊泡的独特融合反应。通过比较从ALFQ纯化的囊泡的脂质组成和结构来探测该融合反应,>1µm(即,GUV)和直径<1µm的较小囊泡。这里,我们证明了差速离心后,胆固醇,MPLA,脂质体磷脂双层中的QS21主要存在于GUV中(在颗粒中)。大概,这是通过从纳米尺寸到微米尺寸颗粒的过渡过程中的快速横向扩散而发生的。而脂质体磷脂在颗粒和上清液中的重量回收率分别为44%和36%,分别,胆固醇的重量百分比更高(〜88%),MPLA(94%),和QS21(96%)在含有GUV的颗粒中回收,和≤10%的这些单独的脂质体成分在上清液中回收。尽管ALFQ具有多分散性,大部分的胆固醇,几乎所有的佐剂分子,存在于GUV中。我们假设QS21与胆固醇的结合导致了新的结构纳米结构域,随后在脂质双层中的交织体偶联可能引发了融合过程,导致创建GUV。然而,MPLA和QS21的极地区域一起有十个糖的“糖草坪”,其亲水性可能为GUV中MPLA和QS21的快速横向扩散和浓缩提供了驱动力。
Army Liposome Formulation with QS21 (ALFQ), a vaccine adjuvant preparation, comprises liposomes containing saturated phospholipids, with 55 mol% cholesterol relative to the phospholipids, and two adjuvants, monophosphoryl lipid A (MPLA) and QS21 saponin. A unique feature of ALFQ is the formation of giant unilamellar vesicles (GUVs) having diameters >1.0 µm, due to a remarkable fusion event initiated during the addition of QS21 to nanoliposomes containing MPLA and 55 mol% cholesterol relative to the total phospholipids. This results in a polydisperse size distribution of ALFQ particles, with diameters ranging from ~50 nm to ~30,000 nm. The purpose of this work was to gain insights into the unique fusion reaction of nanovesicles leading to GUVs induced by QS21. This fusion reaction was probed by comparing the lipid compositions and structures of vesicles purified from ALFQ, which were >1 µm (i.e., GUVs) and the smaller vesicles with diameter <1 µm. Here, we demonstrate that after differential centrifugation, cholesterol, MPLA, and QS21 in the liposomal phospholipid bilayers were present mainly in GUVs (in the pellet). Presumably, this occurred by rapid lateral diffusion during the transition from nanosize to microsize particles. While liposomal phospholipid recoveries by weight in the pellet and supernatant were 44% and 36%, respectively, higher percentages by weight of the cholesterol (~88%), MPLA (94%), and QS21 (96%) were recovered in the pellet containing GUVs, and ≤10% of these individual liposomal constituents were recovered in the supernatant. Despite the polydispersity of ALFQ, most of the cholesterol, and almost all of the adjuvant molecules, were present in the GUVs. We hypothesize that the binding of QS21 to cholesterol caused new structural nanodomains, and subsequent interleaflet coupling in the lipid bilayer might have initiated the fusion process, leading to creation of GUVs. However, the polar regions of MPLA and QS21 together have a \"sugar lawn\" of ten sugars, the hydrophilicity of which might have provided a driving force for rapid lateral diffusion and concentration of the MPLA and QS21 in the GUVs.