van Winden E C, Zhang W, Crommelin D J
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht Institute for Drug Exploration, Utrecht University, The Netherlands.
Pharm Res. 1997 Sep;14(9):1151-60. doi: 10.1023/a:1012142520912.
In the present study we examined the effect of the freezing protocol on carboxyfluorescein (CF) retention in liposomes after freeze-drying and rehydration.
Liposomes were frozen slowly at 0.5 degree C/min, or quickly by submerging the samples in boiling nitrogen before freeze-drying. The thermal behaviour of the frozen dispersions was analysed by Modulated Temperature Differential Scanning Calorimetry (MTDSC). The dried cakes were analysed by SEM, MTDSC and FTIR. The % encapsulated CF of the (re)hydrated liposomes was determined by fluorimetry after GPC, their vesicle size was measured by the Dynamic Light scattering Technique and their bilayer transition was studied by DSC.
Slow freezing resulted in a markedly higher CF retention after freeze-drying and rehydration as compared to quick freezing. The effect of the freezing rate depended on the lipid composition and was most pronounced for rigid liposomes. The damage caused by quick freezing did not occur after a freezing/thawing cycle. The freezing protocol did not influence the interaction between the phospholipids and the lyoprotectants (sucrose, trehalose or glucose) in the freeze-dried state. However, analysis by DSC of dipalmitoylphosphatidylcholine (DPPC): dipalmitoylphosphatidylglycerol (DPPG) = 10:1 and DPPC liposome dispersions showed that the freezing protocol affected the bilayer melting characteristics of these liposomes after freeze-drying and rehydration.
A proper design of the freezing protocol is essential to achieve optimal stability of rigid liposomes during a freeze-drying and rehydration cycle.
在本研究中，我们检测了冻干和复水后冷冻方案对脂质体中羧基荧光素（CF）保留率的影响。
脂质体以0.5℃/分钟的速度缓慢冷冻，或在冻干前通过将样品浸入沸腾的氮气中快速冷冻。通过调制温度差示扫描量热法（MTDSC）分析冷冻分散体的热行为。通过扫描电子显微镜（SEM）、MTDSC和傅里叶变换红外光谱（FTIR）对干燥后的饼剂进行分析。通过凝胶渗透色谱（GPC）后荧光法测定（复）水脂质体中CF的包封率，通过动态光散射技术测量其囊泡大小，并通过差示扫描量热法（DSC）研究其双层转变。
与快速冷冻相比，缓慢冷冻导致冻干和复水后CF保留率显著更高。冷冻速率的影响取决于脂质组成，对刚性脂质体最为明显。快速冷冻造成的损伤在冻融循环后未出现。冷冻方案在冻干状态下不影响磷脂与冻干保护剂（蔗糖、海藻糖或葡萄糖）之间的相互作用。然而，对二棕榈酰磷脂酰胆碱（DPPC）：二棕榈酰磷脂酰甘油（DPPG）=10:1和DPPC脂质体分散体的DSC分析表明，冷冻方案影响了这些脂质体在冻干和复水后的双层熔化特性。
正确设计冷冻方案对于在冻干和复水循环过程中实现刚性脂质体的最佳稳定性至关重要。
