具有GLUT1主动转运、葡萄糖和RGD肽双重肿瘤识别的脑靶向脂质体的制备及其在耐药脑肿瘤的应用研究

In recent years, the incidence of brain tumors has been increasing, which greatly affects human health. Chemotherapeutic drugs have limited brain targeting ability, poor tumor selectivity and easy been excluded by tumor cells, resulting in unsatisfactory therapeutic effect. In our previous study, it was found that the glucose-modified liposome had good brain targeting ability. Based on this, we propose the theory of the preparation and application of brain-targeted liposome with active transport by GLUT1, dual cancer recognition by glucose and RGD peptide in drug-resistant brain tumors for the first time. After designing and synthesizing series liposome ligand, glucose-RGD-cholesterol conjugate, the paclitaxel and verapamil co-loaded liposome modified with the ligand will be prepared. The exposed glucose residues could recognize GLUT1, which would transport the liposome across the BBB into brain to improve the brain targeting ability. Subsequently, The glucose and RGD peptide residues can identify the transporter and receptor on the surface of tumor cells, and the liposome was targeted delivered into the tumor cells and released paclitaxel and verapamil. The verapamil will be bind with P-glycoprotein to prevent paclitaxel from being reversibly transported out of the tumor cells, playing an effective treatment of drug-resistant brain tumors. The study of targeting of liposome and activity of liposome in vitro and in vivo can suppose new ideas and methods of the treatment of drug-resistant brain tumors and other brain diseases, with high academic and applied value.

近年来，脑肿瘤的发病率逐渐增加，严重影响人类健康。目前化疗药物的脑靶向能力有限、肿瘤选择性差且易被肿瘤细胞外排，导致其治疗效果不理想。申请者前期研究中发现葡萄糖修饰的脂质体具有很好的脑靶向性。基于此，本项目首次提出“具有GLUT1主动转运、葡萄糖和RGD肽双重肿瘤识别的脑靶向脂质体的制备及其在耐药脑肿瘤的应用研究”的设想，设计并拟合成系列葡萄糖-RGD肽-胆甾偶联物，以此为配体，制备包封紫杉醇和维拉帕米脂质体。暴露在脂质体表面的葡萄糖残基可识别GLUT1转运蛋白，透过血脑屏障进入脑中；随后利用葡萄糖和RGD肽识别相应的转运体和受体，将脂质体靶向传递到肿瘤细胞释放原药；且维拉帕米与P-gp蛋白结合，避免药物再次被逆转运出肿瘤细胞，进而对耐药脑肿瘤起到有效的治疗。本项目设计并对比研究该类脂质体的体内外靶向性及活性，为耐药脑肿瘤及其他脑部疾病的治疗创立新思路和新方法，具极高的学术和应用价值。

在国内外研究基础上，首次提出“具有GLUT1主动转运、葡萄糖和RGD肽双重肿瘤识别的脑靶向脂质体的制备及其在耐药脑肿瘤的应用研究”的设想，设计并合成葡萄糖-RGD肽-胆甾偶联物A，以此为配体，制备包封紫杉醇和P-gp抑制剂维拉帕米的脂质体。该脂质体具有以下特点：（1）配体中胆甾部分嵌入到双分子层中，而葡萄糖和RGD肽残基暴露在脂质体的表面，起到PEG化作用，增加其在体循环中的稳定性。（2）当脂质到达血脑屏障时，暴露在脂质体表面的葡萄糖残基识别GLUT1，可使得脂质体透过血脑屏障进入脑中，有效的提高脂质体的脑靶向性。（3）葡萄糖和RGD肽残基分别与肿瘤细胞表面GLUT1和αvβ3结合，将脂质体靶向到肿瘤细胞释放原药，进而对脑肿瘤起到有效的治疗。（4）维拉帕米与P-gp蛋白结合，避免了药物被外排出肿瘤细胞，从而增强耐药脑肿瘤的治疗效果。体外细胞毒性和凋亡实验表明：葡萄糖-RGD肽-胆甾偶联物修饰后的脂质体LipA显示出良好的抑制活性和缓释效果，有较强的诱导凋亡和坏死能力。体外细胞摄取实验进一步验证了设计思路：葡萄糖和RGD肽的修饰均可促进C6/adrMDR细胞对脂质体的摄取，双重修饰的脂质体可进一步提高对C6/adrMDR细胞的靶向能力，维拉帕米与P-gp蛋白结合，避免了药物被外排出肿瘤细胞。摄取机制实验表明C6/adrMDR对脂质体LipA的摄取具有能量依赖性的、GLUT1和αvβ3受体主动转运的、通过多种入胞途径综合介导实现的。在动物模型中，给药4 h后，脑部DiD荧光最强，而给药24 h后，荧光显著降低，脂质体在体内被大量代谢，仅载有维拉帕米的脂质体还有少量残留。脂质体LipA组中位生存期相比于生理盐水组延长135%，表现出最佳的抗耐药脑肿瘤活性。总的来说，在葡萄糖和RGD肽的共同作用下，增加了脂质体对肿瘤细胞的选择性，促进肿瘤细胞对药物的摄取，降低药物对正常细胞的毒性；维拉帕米与P-gp蛋白结合，避免了药物被外排出肿瘤细胞，增强耐药脑肿瘤的治疗效果；为新型耐药脑肿瘤靶向药物的研制提供新思路和新方法，并将该方法拓展于其他脑部疾病的治疗。