用于脑胶质瘤治疗的逆序D型缓激肽与聚合物胶束联合给药系统的研究

Due to the two special barriers of brain: blood-brain barrier (BBB) and blood-brain tumor barrier (BTB), the antitumor agents cannot through the hindrance into brain tumor tissues successfully to achieve the effective therapy. In order to decrease the damage of normal brain tissue, we need to look for a targeted strategy to deliver the antitumor agents to brain tumor by openning the BTB selectively without affecting the permeability of BBB. In previous studies, we demonstrated that co-administration of retro-inverso Bradykinin (RI-BK) can enhance the accumulation of drug loaded polymeric micelles in brain tumor and significantly prolonged the median survival time. Based on our studies, we plan to research the application of RI-BK in the treatment of brain tumor exhaustively from the following aspects: firstly, the affinity between RI-BK and bradykinin B2 receptor; secondly, the different respond between BBB and BTB in vitro after administration of RI-BK; thirdly, whether co-administration of RI-BK will influence the transport action of polymeric micelles in in vitro BBB and BTB model and the distribution of polymeric micelles in brain tumor and normal brain; fourthly, the influence of dosage, administration and particle size of polymeric micelles for the distribution of antitumor agents; finally, evaluation of the antitumor effect and toxicity on the drug delivery system that co-administration of RI-BK and polymeric micelles. This study will make contributions to finding a potential strategy for enhancing the efficacy of antitumor agents in theory and practice, to treat the brain tumor and decrease the damage of normal brain simultaneously.

血脑屏障（BBB）和血脑肿瘤屏障(BTB)的存在阻碍了药物对脑胶质瘤的有效治疗。如何在不影响BBB的前提下，选择性开放BTB，将药物靶向递送到脑胶质瘤组织？我们前期工作证明逆序D型缓激肽与聚合物胶束联合给药可增加药物在脑内蓄积，并显著延长原位脑胶质瘤模型动物的中位生存期。本申请项目将进一步研究逆序D型缓激肽与BTB上激肽受体B2的亲和性及其对体外BTB和BBB模型的开放效应是否有所不同、与逆序D型缓激肽联合给药后聚合物胶束在体外BTB和BBB模型上的转运行为是否有所差异、相对剂量、间隔时间和胶束粒径等因素是否影响聚合物胶束在脑肿瘤和正常脑组织的分布、以及评价逆序D型缓激肽与聚合物胶束联合给系统抗原位脑胶质瘤效果和初步生物安全性。这些研究将对寻找一种降低正常脑组织损伤的同时，有效提高药物抗脑胶质瘤作用的治疗策略具有理论和实际意义。

本项目针对脑胶质瘤治疗中存在的血脑屏障（BBB）和血脑肿瘤屏障（BTB），主要开展了以下研究：设计了一种逆序D型缓激肽（RI-BK）、分别从分子水平、体外BBB和BTB模型，以及原位脑肿瘤动物模型水平探讨了RI-BK在不影响BBB前提下选择性开放BTB的机制；系统研究了RI-BK选择性开放BTB的剂量、时间区间和尺度等影响因素；采用两种纳米递药系统（脂质体和聚合物胶束），开展了与RI-BK联合给药用于脑肿瘤的生物分布和药效学研究。实验结果表明：与缓激肽比较，RI-BK显著提高了与缓激肽受体B2结合活性和生物稳定性；在体外BBB和BTB模型上，RI-BK可减少体外BTB模型紧密连接蛋白ZO-1在细胞间隙的表达，同时解聚肌动蛋白F-actin，增加细胞内应力纤维，而对体外BBB模型相关蛋白分布和结构无影响；在原位脑胶质瘤动物模型上，RI-BK可引起脑肿瘤组织的血管内皮细胞间紧密连接开放，而正常脑组织的血管内皮细胞间的紧密连接呈现贴合状态；在原位脑胶质瘤动物模型上，RI-BK选择性开放BTB的剂量为30μg/kg、时间区间约在15~60 min范围内、尺度在0 ~ 170nm范围之间；RI-BK可促进分子药物或纳米药物在脑肿瘤组织蓄积，对正常脑组织中没有影响；与RI-BK联合给药，均显著延长了化疗药物、脂质体和聚合物胶束的原位脑胶质瘤动物模型的中位生存期。综上所述，鉴于BTB区域缓激肽受体B2显著高于BBB，RI-BK可在不激活BBB区域的缓激肽B2受体的前提下，与BTB区域B2结合，使得细胞释放出二级信号因子，进而上调细胞内吞相关蛋白表达和下调紧密连接相关蛋白表达，从而开放BTB，促进化疗药物和纳米药物在脑肿瘤组织的蓄积，提高对脑肿瘤的治疗作用。. 本项目的研究成果提示，作为一种新型化疗药物、抗体药物和纳米药物的辅助用药，RI-BK为脑胶质瘤的治疗提供了具有临床应用前景的方案和思路。