Zhang W, van Winden E C, Bouwstra J A, Crommelin D J
Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, 3508 TB, The Netherlands.
Cryobiology. 1997 Nov;35(3):277-89. doi: 10.1006/cryo.1997.2050.
Until now, studies on the protection of liposomes against freeze-drying damage have mainly focused on the bilayer integrity during the freezing or drying step of this process. Here, we investigated the bilayer permeability of freeze-dried, lyoprotected liposomes to a nonbilayer interacting compound after rehydration, by monitoring the leak-in kinetics of externally added carboxyfluorescein (CF). The results showed that freeze-drying and rehydration of DPPC:DPPG 10:1 liposomes with sucrose in- and outside the vesicles caused a temporary increase in the bilayer permeability for CF, which leveled off after approximately 20 h. The amount of CF/mol phospholipid which leaked into the vesicles increased with vesicle size (range 0.1-1 micro m) / lamellarity. Reduction of the number of bilayers in 1-1 micro m) vesicles enhanced the permeability to CF after freeze-drying and rehydration. The presence of CHOL decreased CF leak-in rates into 1 micro m MLVs consisting of DPPC:DPPG 10:1, but not into 0.1-micro m unilamellar vesicles. In the absence of sucrose similar leak-in profiles as a function of time were found after rehydration, suggesting that repacking processes of the bilayer were responsible for the enhanced permeability after freeze-drying and dehydration both with and without sucrose. The effect of size and lamellarity on the CF leak-in correlated with the retention of encapsulated CF after freeze-drying and rehydration, but no correlation was found with the effect of lipid composition. Both small (0.1 micro m) lyoprotected liposomes made of DPPC:DPPG 10:1 and DPPC:DPPG:CHOL 10:1:4 were highly permeable during the rehydration step itself. The results indicate that, despite the presence of the lyoprotectant, "repacking" of the bilayer components takes place both during and after rehydration. This eventually leads to regaining of its barrier function.
到目前为止，关于脂质体抗冻干损伤保护作用的研究主要集中在该过程冷冻或干燥步骤中的双层完整性。在此，我们通过监测外部添加的羧基荧光素（CF）的渗漏动力学，研究了冻干、冻干保护脂质体复水后对非双层相互作用化合物的双层渗透性。结果表明，在囊泡内外均含有蔗糖的情况下，DPPC:DPPG 10:（此处原文有误，应为1）脂质体的冻干和复水导致CF的双层渗透性暂时增加，约20小时后趋于平稳。渗漏到囊泡中的CF/摩尔磷脂量随囊泡大小（范围为0.1 - 1微米）/层数增加。1 - 1微米囊泡中双层数量的减少增强了冻干和复水后对CF的渗透性。胆固醇（CHOL）的存在降低了CF渗漏到由DPPC:DPPG 10:（此处原文有误，应为1）组成的1微米多层大泡中的速率，但对0.1微米单层囊泡则没有影响。在没有蔗糖的情况下，复水后发现了类似的随时间变化的渗漏曲线，这表明双层的重新排列过程是冻干和脱水后（无论有无蔗糖）渗透性增强的原因。大小和层数对CF渗漏的影响与冻干和复水后包封CF的保留情况相关，但与脂质组成的影响无关。由DPPC:DPPG 10:（此处原文有误，应为1）和DPPC:DPPG:CHOL 10:（此处原文有误，应为1）:4制成的小（0.1微米）冻干保护脂质体在复水步骤本身就具有高渗透性。结果表明，尽管存在冻干保护剂，但双层成分在复水期间和复水后都会发生“重新排列”。这最终导致其屏障功能的恢复。
