Liposome co-encapsulation of anti-cancer agents for pharmacological optimization of nanomedicine-based combination chemotherapy.
Liposome co-encapsulation of anti-cancer agents for pharmacological optimization of nanomedicine-based combination chemotherapy.
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DOI：
10.20517/cdr.2020.87
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发表时间：
2021
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目的：聚乙二醇化脂质体中抗癌药物的共包封可以利用脂质体制剂循环时间长、被动靶向和毒性低的优点，为最大限度地提高联合药物治疗的疗效提供有效的工具。方法：我们开发了几种脂质体制剂的共包封药物使用不同排列的三种活性剂：阿霉素（Dox），丝裂霉素- c脂质前药（MLP）和阿仑膦酸钠（Ald）。Dox和MLP以单药脂质体形式提供：聚乙二醇化脂质体Dox （PLD, Doxil®），临床批准，聚乙二醇化脂质体MLP （PL-MLP, Promitil®），处于1-2期临床试验。我们之前的研究表明，在免疫能力强的动物模型中，Dox和Ald在聚乙二醇化脂质体（PLAD）中共包被产生了一种具有有价值的免疫药理学特性和比PLD更优越的抗肿瘤特性的制剂。在PLAD和PL-MLP平台的基础上，我们开发了一种新的聚乙二醇化脂质体制剂，将Dox和MLP共包埋（PLAD-MLP），前者通过阿仑膦酸铵的远程加载定位在脂质体水相中，后者被动加载到脂质体脂质双分子层中。为了进行比较，还测试了一种共包埋MLP和Dox的替代配方，其中用硫酸铵取代Ald铵（PLD-MLP）。结果：PLAD-MLP对Dox和MLP的负载效率接近100%，平均囊泡直径为110 nm。PLAD-MLP的低温透射电镜（cro - tem）显示圆形囊泡，囊泡内有dox -阿仑膦酸沉淀。用还原剂二硫苏糖醇对PLAD-MLP进行了体外MLP活化试验，发现PLAD-MLP对硫溶活化的敏感性明显低于PL-MLP。随着MLP的硫溶活化，很大一部分被封装的Dox从脂质体中释放出来。PLAD-MLP在人血浆中体外培养后稳定，药物保留率接近100%。在小鼠药代动力学研究中，与以PL-MLP形式递送的MLP相比，PLAD-MLP延长了MLP在循环中的半衰期。此外，与PL-MLP相比，PLAD-MLP在组织中的水平更高，这表明PLAD-MLP减缓了前药MLP向MMC的裂解，从而导致更持续和更长的暴露时间。PLAD-MLP中Dox的循环半衰期与PLD中的Dox半衰期相似。共包膜药物的组织分布模式相似，尽管Dox水平普遍高于MLP，正如MLP裂解其活性代谢物MMC所预期的那样。在小鼠肿瘤模型中，PLAD-MLP的治疗活性优于PL-MLP和PLD，且具有方便的安全剂量窗。无ald制剂PLD-MLP与PLAD-MLP具有相似的药代动力学特性，但其治疗活性较低。结论：PLAD-MLP是一种新型的多药脂质体制剂，具有良好的药理作用和较强的抗肿瘤活性，是一种很有前景的肿瘤联合化疗的治疗工具。
Aim: Co-encapsulation of anti-cancer agents in pegylated liposomes may provide an effective tool to maximize efficacy of combined drug therapy by taking advantage of the long circulation time, passive targeting, and reduced toxicity of liposome formulations. Methods: We have developed several liposome formulations of co-encapsulated drugs using various permutations of three active agents: doxorubicin (Dox), mitomycin-C lipidic prodrug (MLP), and alendronate (Ald). Dox and MLP are available in single drug liposomal formulations: pegylated liposomal Dox (PLD, Doxil®), clinically approved, and pegylated liposomal MLP (PL-MLP, Promitil®), in phase 1-2 clinical testing. We have previously shown that co-encapsulation of Dox and Ald in pegylated liposomes (PLAD) results in a formulation with valuable immuno-pharmacologic properties and superior antitumor properties over PLD in immunocompetent animal models. Building on the PLAD and PL-MLP platforms, we developed a new pegylated liposomal formulation of co-entrapped Dox and MLP (PLAD-MLP), with the former localized in the liposome water phase via remote loading with an ammonium alendronate and the latter passively loaded into the liposome lipid bilayer. An alternative formulation of co-entrapped MLP and Dox in which ammonium Ald was replaced with ammonium sulfate (PLD-MLP) was also tested for comparative purposes. Results: PLAD-MLP displays high loading efficiency of Dox and MLP nearing 100%, and a mean vesicle diameter of 110 nm. Cryo-transmission electron microscopy (cryo-TEM) of PLAD-MLP reveals round vesicles with an intra-vesicle Dox-alendronate precipitate. PLAD-MLP was tested in an in vitro MLP activation assay with the reducing agent dithiothreitol and found to be significantly less susceptible to thiolytic activation than PL-MLP. Alongside thiolytic activation of MLP, a significant fraction of encapsulated Dox was released from liposomes. PLAD-MLP is stable upon in vitro incubation in human plasma with nearly 100% drug retention. In mouse pharmacokinetic studies, PLAD-MLP extended MLP half-life in circulation when compared to that of MLP delivered as PL-MLP. In addition, the MLP levels in tissues were greater than those obtained with PL-MLP, indicating that PLAD-MLP slows down the cleavage of the prodrug MLP to MMC, thus resulting in a more sustained and prolonged exposure. The circulation half-life of Dox in PLAD-MLP was similar to the PLD Dox half-life. The pattern of tissue distribution was similar for the co-encapsulated drugs, although Dox levels were generally higher than those of MLP, as expected from cleavage of MLP to its active metabolite MMC. In mouse tumor models, the therapeutic activity of PLAD-MLP was superior to PL-MLP and PLD with a convenient safety dose window. The Ald-free formulation, PLD-MLP, displayed similar pharmacokinetic properties to PLAD-MLP, but its therapeutic activity was lower. Conclusion: PLAD-MLP is a novel multi-drug liposome formulation with attractive pharmacological properties and powerful antitumor activity and is a promising therapeutic tool for combination cancer chemotherapy.