Nanomalaria Group, Institute for Bioengineering of Catalonia (IBEC), Baldiri Reixac 10-12, ES-08028 Barcelona, Spain; Barcelona Institute for Global Health (ISGlobal, Hospital Clínic-Universitat de Barcelona), Rosselló 149-153, ES-08036 Barcelona, Spain; Nanoscience and Nanotechnology Institute (IN2UB), University of Barcelona, Martí i Franquès 1, ES-08028 Barcelona, Spain.
Tres Cantos Medicines Development Campus, GlaxoSmithKline, Severo Ochoa 2, ES-28760 Tres Cantos, Spain.
J Control Release. 2015 Jul 28;210:217-29. doi: 10.1016/j.jconrel.2015.05.284. Epub 2015 May 23.
One of the most important factors behind resistance evolution in malaria is the failure to deliver sufficiently high amounts of drugs to early stages of Plasmodium-infected red blood cells (pRBCs). Despite having been considered for decades as a promising approach, the delivery of antimalarials encapsulated in immunoliposomes targeted to pRBCs has not progressed towards clinical applications, whereas in vitro assays rarely reach drug efficacy improvements above 10-fold. Here we show that encapsulation efficiencies reaching >96% are achieved for the weak basic drugs chloroquine (CQ) and primaquine using the pH gradient loading method in liposomes containing neutral saturated phospholipids. Targeting antibodies are best conjugated through their primary amino groups, adjusting chemical crosslinker concentration to retain significant antigen recognition. Antigens from non-parasitized RBCs have also been considered as targets for the delivery to the cell of drugs not affecting the erythrocytic metabolism. Using this strategy, we have achieved unprecedented complete nanocarrier targeting to early intraerythrocytic stages of the malaria parasite for which there is a lack of specific extracellular molecular tags. Immunoliposomes studded with monoclonal antibodies raised against the erythrocyte surface protein glycophorin A were capable of targeting 100% RBCs and pRBCs at the low concentration of 0.5μM total lipid in the culture, with >95% of added liposomes retained on cell surfaces. When exposed for only 15min to Plasmodium falciparum in vitro cultures of early stages, free CQ had no significant effect on the viability of the parasite up to 200nM, whereas immunoliposomal 50nM CQ completely arrested its growth. In vivo assays in mice showed that immunoliposomes cleared the pathogen below detectable levels at a CQ dose of 0.5mg/kg, whereas free CQ administered at 1.75mg/kg was, at most, 40-fold less efficient. Our data suggest that this significant improvement is in part due to a prophylactic effect of CQ found by the pathogen in its host cell right at the very moment of invasion.
疟原虫耐药性产生的一个重要原因是未能将足够高剂量的药物递送至早期被感染的红细胞（pRBC）。尽管免疫脂质体包封的抗疟药物靶向 pRBC 已被认为是一种很有前途的方法，但数十年以来，该方法仍未能推进至临床应用，而体外检测很少能提高药物疗效超过 10 倍。在此，我们表明，在含有中性饱和磷脂的脂质体中，使用 pH 梯度加载法可以实现弱碱性药物氯喹（CQ）和伯氨喹的包封效率达到>96%。靶向抗体最好通过其一级氨基基团进行共轭，调整化学交联剂浓度以保留显著的抗原识别。还考虑了来自未感染 RBC 的抗原作为递送至细胞的药物的靶标，这些药物不会影响红细胞代谢。使用这种策略，我们已经实现了对疟原虫早期红细胞内阶段的空前完全的纳米载体靶向，而这些阶段缺乏特异性的细胞外分子标记。用针对红细胞表面蛋白糖蛋白 A 的单克隆抗体缀合的免疫脂质体能够以 0.5μM 总脂质的低浓度靶向 100%的 RBC 和 pRBC，其中>95%的添加脂质体保留在细胞表面上。当在体外仅暴露于早期疟原虫培养物 15 分钟时，游离 CQ 在高达 200nM 的浓度下对寄生虫的活力没有显著影响，而免疫脂质体 50nM CQ 则完全阻止了其生长。在小鼠体内试验中，免疫脂质体在 CQ 剂量为 0.5mg/kg 时将病原体清除至低于检测水平，而以 1.75mg/kg 给予的游离 CQ 的效率最高只有 40 倍。我们的数据表明，这种显著的改善部分归因于 CQ 在寄生虫入侵宿主细胞的那一刻就在其宿主细胞中发现的预防性作用。
