“配体-超声-转运体”级联介导MTX滋养细胞精准给药及机制研究

Methotrexate (MTX) is the main and irreplaceable therapeutic for the trophoblast-related diseases. However, due to several intrinsic drawbacks such as the low therapeutic efficiency and severe side effects, caused by the non-targeted distribution of the drugs and the low transport efficiency for the trophoblast cells, MTX faces the probability of treating failure. In order to overcome the above challenges and achieve desirable therapeutic benefits, a "ligand-ultrasound-transporter" gradually mediated strategy has been designed in our project. Firstly, the structure of the methotrexate will be coupled with L-valine-L-valine, the substrate of the oligopeptide transporters which is overexpressed on gestational trophoblast cells. Secondly, the coupled drugs will be encapsulated into PLGA nanobubbles and the nano-formulation will be further modified by ligands for targeting delivery to the trophoblast-related disease sites. Thirdly, the coupled drugs will be released from the nanobubbles under stimulation of low energy ultrasound and transported into cells by the oligopeptide transporter mediation. Finally, in the intracellular acid environment, the coupled drugs will be disassembled into free methotrexate to achieve precise drug delivery. In addition, this project will demonstrate the transport and therapeutic mechanism through animal, cell and molecular levels and might provide a new remedy or therapeutic schedule based on trophoblast membrane transporters.

甲氨蝶呤（MTX）治疗/辅助治疗滋养细胞相关疾病的地位不可替代，但一直存在低效、高毒的致命缺陷。究其根本是MTX给药后在病灶部位的富集不足以及滋养细胞对其转运效率的低下。本项目基于多年来积累的PLGA载体构建经验，创新性地结合细胞膜转运体研究成果，借助低能量超声技术，设计了一种“配体-超声-转运体”级联介导策略：首先将MTX进行结构改造，与靶细胞（妊娠滋养细胞）膜上高表达的寡肽转运体的底物——L-缬氨酸-L-缬氨酸进行偶联，将偶联药物制备成PLGA纳米泡并修饰配体后靶向滋养细胞病灶部位，在低能量超声刺激下释出偶联药物，再经寡肽转运体介导入胞，并在胞内酸性环境中游离出MTX，实现滋养细胞的精准给药，降低对育龄妇女机体毒副作用。本项目还将从动物-细胞-分子三个水平阐述药物的转运及作用机制。本项目不仅为滋养细胞相关疾病的精准治疗提供新策略，而且有望为滋养细胞膜转运体参与疾病的治疗开辟全新的方向。

滋养细胞相关疾病显著影响女性的生殖需求与生命健康。甲氨蝶呤（MTX）治疗/辅助治疗滋养细胞相关疾病的地位不可替代，但一直存在低效、高毒的缺陷。MTX给药后在病灶部位富集不足以及滋养细胞对其转运效率低下是MTX治疗缺陷的主要原因。在本项目的资助下，课题组研究发现：除了寡肽转运体，细胞膜核苷转运蛋白（ENT1）在妊娠滋养细胞肿瘤细胞膜中也高度表达，因此构建了ENT1底物（阿糖胞苷，Cy）接枝的脂质体（Cy-Lipo@MTX）用于靶向递送MTX到妊娠滋养细胞肿瘤细胞中。Cy-磷脂接枝物不但可以实现与ENT1的特异性结合，而且能够与MTX协同发挥抗肿瘤的作用。在Cy-Lipo@MTX内吞的过程中，ENT1与内吞小泡一起进入细胞质，随后细胞膜上的ENT1水平通过内吞转运蛋白再循环和mRNA的从头合成两条途径恢复。药物动力学与组织分布研究表明Cy-Lipo@MTX具有长循环的特征并且能够将MTX靶向递送至妊娠滋养细胞肿瘤。最终纳米脂质体发挥出优越的滋养细胞肿瘤治疗作用并没有产生明显的机体副作用。总的来说，课题组从动物-细胞-分子三个水平阐明了转运体介导的纳米药物的入胞转运机制与抗肿瘤作用机制。除此之外，课题组还制备并表征了PLGA纳米泡，以期后期利用超声进一步提高转运体介导的药物递送效率。本项目的实施为滋养细胞相关疾病的精准治疗提供了新策略。