基于代谢机制靶向红细胞内疟原虫的青蒿素类药物纳米递药系统研究

Our previous studies demonstrated that artemisinin antimalarials exihibit auto-induction metabolism resulting remarkable decrease in plasma concentrations after consecutive oral administration, which might result in related recrudescence, susceptibility and drug-drug interactions. However the autoinduction metabolism was not found after intravenous administrations. In this project, based on the metabolic characteristics of this kind of drugs and the unique metabolic pathways of plasmodium in infected-erythrocytic, the targeted nano intra-erythrocytic drug dilivery system was constructed by using lipid nanoparticle as carrier. Intravenous administration route is adapted for refraining from auto-induction metabolism and PEG modified nanoparticle was designed for extending its circulation time in the blood stream. The choline-derivate-modified artemisinins nanoparticles with suitable particle size can be specifically recognized by Plasmodium-infected erythrocytes, by which the modified artemisinins nanoparticles is expected to be targeting to the plasmodium in infected-erythrocytes. Techniques such as transmission electron microscope are utilized for characterization of nanoparticles. Confocal laser scan microscopy and flow cytometry are adapted for assessment of in vitro targeting potency of artemisinins nanoparticles based on their antimalarial activity, biodistribution and erythrocytes uptakes. The in vivo antimalarial efficacies, pharmacokinetics, sustained release feature and targeting bio-distribution of the artemisinins lipid nanoperticles are investigated by using Plasmodium berghei infected mice. The results will be significant to improving antimalarial activity, reducing recrudescence, and preventing drug resistance as well as drug-drug interactions related to artemisinin antimalarials, and can provide inspirations for designing antimalarial targeted drug delivery system.

前期研究表明，青蒿素类抗疟药连续口服给药后存在自身诱导代谢现象、导致血药浓度显著降低，与其愈后复燃、敏感性及药物相互作用直接相关；而静脉给药无自身诱导代谢问题。本课题根据该类药物代谢特征及其作用靶点红内期疟原虫的磷脂等特异性代谢途径，以脂质纳米粒为载体，构建其静脉纳米靶向细胞内递药系统。通过静脉给药避免其自身诱导代谢，以PEG修饰纳米粒延长血中循环时间，采用胆碱衍生物修饰和粒度控制使其选择性经感染红细胞膜上特异性的胆碱转运载体和新渗透性通道摄取，实现对红细胞内疟原虫的靶向性；用透射电镜等进行表征；采用激光共聚焦、流式细胞等技术，从抗疟活性、生物分布、红细胞摄取等方面评价其对红细胞内疟原虫的靶向性；采用伯氏疟原虫感染小鼠考察其体内抗疟活性、药动学及靶向性分布。研究结果对提高该类药物抗疟活性、降低毒性、降低复燃、耐药及药物相互作用风险有直接的科学意义，为抗疟药的靶向给药系统提供新思路。

疟原虫感染红细胞膜上会特异性表达胆碱转运体（ECC）和新渗透性通道（NPP）。该项目构建了胆碱-PEG双修饰脂质纳米粒，通过PEG修饰实现长循环，通过胆碱修饰和控制粒度<80nm实现ECC和NPP介导的靶向感染红细胞的目的。.（1）分别采用混料比率设计-遗传算法和星点设计-效应面法优化了胆碱-PEG双修饰青蒿素/蒿甲醚脂质纳米粒（CD-PEG-ART-NLC和CD-PEG-ARM-NLC）的处方，其粒度、zeta电位、包封率分别为63和64nm、-25.5 和-25.9 mv、88%和87%，无溶血性，缓释效果良好。（2）采用鼠疟模型考察了纳米粒药效学，对疟原虫抑制率次序为CD-PEG-ART-NLC>PEG-ART-NLC>ART-NLC>ART，CD-PEG-ART-NLC组的肝脾损伤降低、生存时间延长；CD-PEG-ARM-NLC组药效学结果与之类似。（3）采用液质联用技术考察了大鼠尾静脉注射青蒿素/蒿甲醚脂质纳米粒药动学，PEG-ART-NLC的AUC和MRT（6869 h·ng·mL-1和10.26 h）明显>ART-NLC（3659 h·ng·mL-1和4.42 h）（P<0.01）；PEG-ARM-NLC中蒿甲醚和代谢物双氢青蒿素的MRT（分别为6.63和6.51 h）均明显>ARM-NLC（3.85和3.51h），AUC（1442和1142 h·ng·mL-1）显著>ARM-NLC（927和704 h·ng·mL-1）（P<0.01）。（4）采用3D7疟原虫株和BY265疟鼠红细胞培养体系，考察了香豆素6（C6）标记纳米粒的靶向性，感染红细胞内CD-PEG-C6-NLC组荧光强度明显高于其它组；加入NPP抑制剂可明显减少感染红细胞对C6-NLC和PEG-C6-NLC的摄取，但对CD-PEG-C6-NLC影响不大；同时加入NPP和ECC抑制剂可基本阻断感染红细胞对CD-PEG-C6-NLC的摄取。CD-PEG双修饰纳米粒达到了抗吞噬、长循环、靶向感染红细胞的目标，抗疟活性良好,确证了PEG修饰的抗吞噬作用,ECC和NPP可以作为抗疟药靶向递药的靶点。以上结果可为抗疟药靶向递药系统设计提供科学依据和新策略。