Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters
Filter-extruded liposomes revisited: a study into size distributions and morphologies in relation to lipid-composition and process parameters
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DOI：
10.3109/08982104.2015.1022556
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发表时间：
2016-01-02
影响因子：
4.4
通讯作者：
Brandl, Martin
中科院分区：
文献类型：
--
作者：
Hinna, Askell;Steiniger, Frank;Brandl, Martin
过滤挤出是一种广泛使用的技术，用于缩小磷脂囊泡的尺寸。为了详细了解由卵磷脂酰胆碱（具有不同比例的胆固醇）组成的过滤挤出囊泡的尺寸和尺寸分布-与挤出参数（孔径、过滤通道的数量和流速）的关系，结合多角度激光散射的流场流分级（AF 4-MALLS，Wyatt Technology Corp.，圣巴巴拉，CA）。通过AF 4-MALLS确定的脂质体尺寸分布与动态光散射进行了比较，并与低温透射电子显微镜和P-31-NMR层状分析相关。发现脂质体的平均尺寸和尺寸分布的宽度随着通过较小孔径过滤器的顺序挤出而减小，在通过400 nm孔径过滤器的重复挤出后，从约70- 415 nm的尺寸范围开始，最终在通过30 nm孔径过滤器的挤出后，以约30 - 85 nm的尺寸范围结束。而对于小孔径（50 nm），增加流速导致更小的囊泡，流速对平均囊泡尺寸没有显着影响，与较大的孔。胆固醇在增加摩尔分数高达0.45产生更大的囊泡（在相同的工艺条件下）。对于胆固醇摩尔分数为0.5与小过滤器孔径的组合，观察到双峰尺寸分布，表明胆固醇微晶。最后，提出了一种方案，以制备具有相当窄的尺寸分布的大（约300 nm）脂质体，该方案基于在限定的流速下的过滤器挤出结合冷冻/解冻循环和台式离心。
Filter-extrusion is a widely used technique for down-sizing of phospholipid vesicles. In order to gain a detailed insight into size and size distributions of filter-extruded vesicles composed of egg phosphatidyl-choline (with varying fractions of cholesterol) - in relation to extrusion-parameters (pore-size, number of filter passages, and flow-rate), flow field-flow fractionation in conjunction with multi-angle laser light scattering (AF4-MALLS, Wyatt Technology Corp., Santa Barbara, CA) was employed. Liposome size-distributions determined by AF4-MALLS were compared with those of dynamic light scattering and correlated with cryo-transmission electron microscopy and P-31-NMR-analysis of lamellarity. Both the mean size of liposome and the width of size distribution were found to decrease with sequential extrusion through smaller pore size filters, starting at a size range of approximate to 70-415nm upon repeated extrusion through 400nm pore-filters, eventually ending with a size range from approximate to 30 to 85nm upon extrusion through 30nm pore size filters. While for small pores sizes (50nm), increased flow rates resulted in smaller vesicles, no significant influence of flow rate on mean vesicle size was seen with larger pores. Cholesterol at increasing mol fractions up to 0.45 yielded bigger vesicles (at identical process conditions). For a cholesterol mol fraction of 0.5 in combination with small filter pore size, a bimodal size distribution was seen indicating cholesterol micro-crystallites. Finally, a protocol is suggested to prepare large (approximate to 300nm) liposomes with rather narrow size distribution, based on the filter extrusion at defined flow-rates in combination with freeze-/thaw-cycling and bench-top centrifugation.