Zheng S, Zheng Y, Beissinger R L, Fresco R
Biomedical Engineering Laboratory, Illinois Institute of Technology, Chicago 60616.
Biochim Biophys Acta. 1994 Dec 30;1196(2):123-30. doi: 10.1016/0005-2736(94)00212-6.
A double emulsion, film dehydration/rehydration approach was developed for encapsulation of hemoglobin (Hb) at high concentration in liposomes. The liposome-encapsulated Hb (LEH) membrane was formulated to contain either phosphatidylinositol (PI) or polyethyleneglycol phosphatidylethanolamine (PEG-PE) along with partially hydrogenated egg-PC, cholesterol, and alpha-tocopherol in a molar ratio of 0.1:1:1:0.02, respectively. The methods introduced in this study followed a multi-step procedure. First, a primary emulsion of Hb in organic solvent containing dissolved lipids was formed. Next, the emulsion was dispersed into an aqueous continuous phase to form a water-in-oil-in-water type double emulsion. Other than the lipids noted above, no surfactants were used in this system. The double emulsion was then converted to LEH by the following steps: evaporating the organic solvent; dehydrating the water to form a dry, thin Hb-lipid film; rehydrating the film in Hb solution to form the LEH; reducing the size of the LEH using 'microfluidization' i.e., high pressure/hydrodynamic shear; and lastly washing the down-sized LEH in buffer. Physico-chemical properties of the model LEH were measured, including oxygen content, encapsulated Hb concentration, oxygen affinity and cooperativity, vesicular size distribution, viscosity, and stability. The suitability of LEH prepared in this manner as a red blood cell substitute was shown using continuous isovolemic exchange transfusion techniques in a small animal model: clearance, efficacy and acute toxicity were evaluated.
开发了一种双乳液、薄膜脱水/再水化方法，用于在脂质体中高浓度封装血红蛋白（Hb）。脂质体封装血红蛋白（LEH）膜的配方中含有磷脂酰肌醇（PI）或聚乙二醇磷脂酰乙醇胺（PEG-PE），以及部分氢化的鸡蛋卵磷脂、胆固醇和α-生育酚，摩尔比分别为0.1:1:1:0.02。本研究中介绍的方法遵循多步骤程序。首先，在含有溶解脂质的有机溶剂中形成Hb的初级乳液。接下来，将乳液分散到水连续相中，形成水包油包水型双乳液。除上述脂质外，该系统未使用表面活性剂。然后通过以下步骤将双乳液转化为LEH：蒸发有机溶剂；使水脱水形成干燥的薄Hb-脂质膜；在Hb溶液中使膜再水化以形成LEH；使用“微流化”即高压/流体动力剪切减小LEH的尺寸；最后在缓冲液中洗涤尺寸减小的LEH。测量了模型LEH的物理化学性质，包括氧含量、封装的Hb浓度、氧亲和力和协同性、囊泡大小分布、粘度和稳定性。在小动物模型中使用连续等容交换输血技术显示了以这种方式制备的LEH作为红细胞替代物的适用性：评估了清除率、疗效和急性毒性。
