Dos Santos Nancy, Cox Kelly A, McKenzie Cheryl A, van Baarda Floris, Gallagher Ryan C, Karlsson Goran, Edwards Katarina, Mayer Lawrence D, Allen Christine, Bally Marcel B
Department of Advanced Therapeutics, British Columbia Cancer Agency, BC Cancer Research Centre, 600 West 10th Avenue, Vancouver, BC, Canada V5Z 4E6.
Biochim Biophys Acta. 2004 Feb 10;1661(1):47-60. doi: 10.1016/j.bbamem.2003.11.016.
Application of cholesterol-free liposomes as carriers for anticancer drugs is hampered, in part, because of standard pH gradient based loading methods that rely on incubation temperatures above the phase transition temperature (Tc) of the bulk phospholipid to promote drug loading. In the absence of cholesterol, liposome permeability is enhanced at these temperatures which, in turn, can result in the collapse of the pH gradient and/or unstable loading. Doxorubicin loading studies, for example, indicate that the drug could not be loaded efficiently into cholesterol-free DSPC liposomes. We demonstrated that this problem could be circumvented by the addition of ethanol as a permeability enhancer. Doxorubicin loading rates in cholesterol-free DSPC liposomes were 6.6-fold higher in the presence of ethanol. In addition, greater than 90% of the added doxorubicin was encapsulated within 2 h at 37 degrees C, an efficiency that was 2.3-fold greater than that observed in the absence of ethanol. Optimal ethanol concentrations ranged from 10% to 15% (v/v) and these concentrations did not significantly affect liposome size, retention of an aqueous trap marker (lactose) or, most importantly, the stability of the imposed pH gradient. Cryo-transmission electron micrographs of liposomes exposed to increasing concentrations of ethanol indicated that at 30% (v/v) perturbations to the lipid bilayer were present as evidenced by the appearance of open liposomes and bilayer sheets. Ethanol-induced increased drug loading was temperature-, lipid composition- and lipid concentration-dependent. Collectively, these results suggest that ethanol addition to preformed liposomes is an effective method to achieve efficient pH gradient-dependent loading of cholesterol-free liposomes at temperatures below the Tc of the bulk phospholipid.
无胆固醇脂质体作为抗癌药物载体的应用受到了一定阻碍，部分原因是基于标准pH梯度的加载方法，该方法依赖于高于主体磷脂相变温度（Tc）的孵育温度来促进药物加载。在没有胆固醇的情况下，这些温度下脂质体的通透性会增强，这反过来又可能导致pH梯度的崩溃和/或加载不稳定。例如，阿霉素加载研究表明，该药物无法有效地加载到无胆固醇的二硬脂酰磷脂酰胆碱（DSPC）脂质体中。我们证明，通过添加乙醇作为通透性增强剂可以规避这个问题。在乙醇存在下，无胆固醇DSPC脂质体中的阿霉素加载率提高了6.6倍。此外，在37℃下2小时内，超过90%添加的阿霉素被包封，该效率比在没有乙醇的情况下观察到的高2.3倍。最佳乙醇浓度范围为10%至15%（v/v），这些浓度不会显著影响脂质体大小、水相捕获标记物（乳糖）的保留，或者最重要的是，所施加pH梯度的稳定性。暴露于不同浓度乙醇的脂质体的低温透射电子显微镜图像表明，在30%（v/v）时，脂质双层出现了扰动，表现为开放脂质体和双层片层的出现。乙醇诱导的药物加载增加与温度、脂质组成和脂质浓度有关。总的来说，这些结果表明，向预先形成的脂质体中添加乙醇是一种在低于主体磷脂Tc的温度下实现无胆固醇脂质体高效pH梯度依赖性加载的有效方法。
