Li X, Hirsh D J, Cabral-Lilly D, Zirkel A, Gruner S M, Janoff A S, Perkins W R
The Liposome Company, Inc., One Research Way, Princeton, NJ 08540, USA.
Biochim Biophys Acta. 1998 Dec 9;1415(1):23-40. doi: 10.1016/s0005-2736(98)00175-8.
We have examined doxorubicin's (DOX) physical state in solution and inside EPC/cholesterol liposomes that were loaded via a transmembrane pH gradient. Using cryogenic electron microscopy (cryo-EM) we noted that DOX loaded to 200-300 mM internal concentrations in citrate containing liposomes formed linear, curved, and circular bundles of fibers with no significant interaction/perturbation of the vesicle membrane. The individual DOX fibers are putatively comprised of stacked DOX molecules. From end-on views of bundles of fibers it appeared that they are aligned longitudinally in a hexagonal array with a separation between fibers of approx. 3-3.5 nm. Two distinct small angle X-ray diffraction patterns (oblique and simple hexagonal) were observed for DOX-citrate fiber aggregates that had been concentrated from solution at either pH 4 or 5. The doxorubicin fibers were also present in citrate liposomes loaded with only one-tenth the amount of doxorubicin used above (approx. 20 mM internal DOX concentration) indicating that the threshold concentration at which these structures form is relatively low. In fact, from cryo-EM and circular dichroism spectra, we estimate that the DOX-citrate fiber bundles can account for the vast majority (>99%) of DOX loaded via a pH gradient into citrate buffered liposomes. DOX loaded into liposomes containing lactobionic acid (LBA), a monoanionic buffer to control the internal pH, remained disaggregated at internal DOX concentrations of approx. 20 mM but formed uncondensed fibers (no bundles) when the internal DOX concentration was approx. 200 mM. This finding suggests that in the citrate containing liposomes the citrate multianion electrostatically bridged adjacent fibers to form the observed bundles. 13C-NMR measurements of [1,5-13C]citrate inside liposomes suggested that citrate 'bound' to the DOX complex and 'free' citrate rapidly exchange indicating that the citrate-DOX interaction is quite dynamic. DOX release into buffer was relatively slow (<4% at 1 h) from liposomes containing DOX fibers (in citrate loaded to a low or high DOX concentration or in LBA liposomes loaded to a high internal DOX concentration). LBA containing liposomes loaded with disaggregated DOX, where the internal DOX concentration was only approx. 20 mM, experienced an osmotic stress induced vesicle rupture with as much as 18% DOX leakage in less than 10 min. The possible implications for this in vivo are discussed.
我们研究了阿霉素（DOX）在溶液中以及通过跨膜pH梯度加载到EPC/胆固醇脂质体内部后的物理状态。使用低温电子显微镜（cryo-EM），我们注意到在含柠檬酸盐的脂质体中加载到200 - 300 mM内部浓度的DOX形成了线性、弯曲和圆形的纤维束，对囊泡膜没有明显的相互作用/扰动。单个DOX纤维据推测由堆叠的DOX分子组成。从纤维束的端视图来看，它们似乎以六边形阵列纵向排列，纤维之间的间距约为3 - 3.5 nm。对于在pH 4或5从溶液中浓缩得到的DOX - 柠檬酸盐纤维聚集体，观察到两种不同的小角X射线衍射图案（斜方和简单六方）。在仅加载上述用量十分之一的阿霉素（内部DOX浓度约为20 mM）的柠檬酸盐脂质体中也存在阿霉素纤维，这表明形成这些结构的阈值浓度相对较低。事实上，通过低温电子显微镜和圆二色光谱，我们估计DOX - 柠檬酸盐纤维束可以占通过pH梯度加载到柠檬酸盐缓冲脂质体中的DOX的绝大多数（>99%）。加载到含有乳糖酸（LBA）的脂质体中的DOX，LBA是一种用于控制内部pH的单阴离子缓冲剂，在内部DOX浓度约为20 mM时仍保持分散状态，但当内部DOX浓度约为200 mM时形成未凝聚的纤维（无纤维束）。这一发现表明，在含柠檬酸盐的脂质体中，柠檬酸盐多阴离子通过静电作用桥接相邻纤维形成观察到的纤维束。脂质体内[1,5 - 13C]柠檬酸盐的13C - NMR测量表明，柠檬酸盐“结合”到DOX复合物上，“游离”柠檬酸盐快速交换，这表明柠檬酸盐 - DOX相互作用相当动态。从含有DOX纤维的脂质体（在低或高DOX浓度的柠檬酸盐中加载或在高内部DOX浓度的LBA脂质体中加载）中，DOX释放到缓冲液中的速度相对较慢（1小时时<4%）。加载有分散DOX的含LBA脂质体，其内部DOX浓度仅约为20 mM，在不到10分钟内经历渗透应激诱导的囊泡破裂，DOX泄漏高达18%。讨论了这在体内可能产生的影响。
