Corley P, Loughrey H C
Department of Biochemistry, University College Galway, Ireland.
Biochim Biophys Acta. 1994 Oct 12;1195(1):149-56. doi: 10.1016/0005-2736(94)90021-3.
We describe an 'in vitro' assay which allows rapid quantification of the binding of biotinated-vesicles to streptavidin immobilised on microtitre plates by estimating levels of a liposome encapsulated fluorescent molecule, rhodamine 123. It is shown that optimal vesicle binding to streptavidin occurs when a six carbon biotin spacer arm derivative of distearoylphosphatidylethanolamine (biotin-X-DSPE) is incorporated in liposomes. This alleviates steric hindrance arising due to the inclusion of small amounts of large bulky amphiphiles such as monosialoganglioside (GM1, 5 mol%) in vesicles. In contrast the ability of liposomes containing poly(ethylene glycol) derivatives of DSPE (PEG2000-DSPE, 5 mol%) to bind streptavidin was only marginally better when biotin-X-DSPE was substituted for biotin-DSPE in vesicles. It is further shown that amounts of biotinated-vesicles bound to streptavidin were minimally influenced by the fluidity of the liposome preparation when assayed at 4 degrees C. However, at elevated temperatures (37 degrees C) lipid estimates as determined by vesicle entrapped rhodamine 123 were low due to leakage of this marker from vesicles. This was shown by comparing amounts of biotinated-liposomes bound to streptavidin coated plates using the lipid marker [3H]cholesteryl hexadecyl ether to estimates determined by vesicle entrapped rhodamine 123. The 'in vitro' assay protocol described here is a general method applicable in the optimisation of other targeting protocols. In conclusion our work suggests that liposomes containing GM1 and the spacer arm derivative biotin-X-DSPE bind optimally to immobilised streptavidin which should aid in the use of biotinated-liposomes in 'in vivo' targeted delivery applications.
我们描述了一种“体外”测定法，该方法通过估计脂质体包裹的荧光分子罗丹明123的水平，能够快速定量生物素化囊泡与固定在微量滴定板上的链霉亲和素的结合。结果表明，当二硬脂酰磷脂酰乙醇胺的六碳生物素间隔臂衍生物（生物素-X-二硬脂酰磷脂酰乙醇胺）掺入脂质体时，囊泡与链霉亲和素的结合最为理想。这减轻了由于在囊泡中包含少量大体积两亲物（如单唾液酸神经节苷脂（GM1，5摩尔%））而产生的空间位阻。相比之下，当在囊泡中用生物素-X-二硬脂酰磷脂酰乙醇胺替代生物素-二硬脂酰磷脂酰乙醇胺时，含有二硬脂酰磷脂酰乙醇胺的聚乙二醇衍生物（PEG2000-二硬脂酰磷脂酰乙醇胺，5摩尔%）的脂质体与链霉亲和素结合的能力仅略好一些。进一步表明，在4℃下测定时，与链霉亲和素结合的生物素化囊泡的量受脂质体制剂流动性的影响最小。然而，在较高温度（37℃）下，由于该标记物从囊泡中泄漏，由囊泡包裹的罗丹明123测定的脂质估计值较低。通过比较使用脂质标记物[3H]胆固醇十六烷基醚与由囊泡包裹的罗丹明123测定的估计值，来确定与链霉亲和素包被板结合的生物素化脂质体的量，从而证明了这一点。这里描述的“体外”测定方案是一种通用方法，适用于优化其他靶向方案。总之，我们的工作表明，含有GM1和间隔臂衍生物生物素-X-二硬脂酰磷脂酰乙醇胺的脂质体与固定的链霉亲和素的结合最为理想，这将有助于生物素化脂质体在“体内”靶向递送应用中的使用。
