肝素纳米载药系统跨血脑屏障运载阿霉素和cRGD靶向杀伤胶质瘤细胞和血管的研究

The presence of the blood-brain barrier (BBB) and incapable of efficiently blocking the blood flow in glioma are considered as leading causes of poor glioma chemotherapy effects by current studies. Therefore, the development of novel drug delivery systems carrying chemotherapeutic and anti-angiogenesis drugs across the BBB for glioma treatment might be a promising way to enhance the efficiency of glioma chemotherapy. In previous work, we reported heparin-based nanoparticle delivery system could re-self-assemble in blood-mimic condition, leading to a smaller size helps to cross the BBB. In this study, we aim to apply the re-self-assembly heparin-based nanoparticles to load cRGD targeting peptides with dual blocking blood supply, and then fabricate nanoparticle drug complex can across the BBB and accurately rapid release drugs in glioma, through conjugate doxorubicin to heparin with PH-sensitive hydrazine bond and reduction-sensitive disulfide bond for targeting to the reducing acidic microenvironment of glioma. Afterwards, detect the biological activity of the nanoparticle complex in vitro, and investigate the in vivo efficacy of the complex in intracranial xenograft glioma and glioma surgical resection animal models. Finally, further study the mechanism of re-self-assembly in blood condition and anti-glioma effect of the complex. This study is hopeful for improving glioma chemotherapy efficacy and enlarging kinds of glioma drugs, the research method might provide new ideas for the development of drugs target to glioma.

目前研究认为血脑屏障的存在及不能有效阻断肿瘤血供是导致胶质瘤化疗疗效不佳的重要因素，因此，研发可携带化疗药物和抗血管生成药物跨越血脑屏障的新型载药系统可能是提高胶质瘤化疗疗效的有效途径。我们前期研究中报道了肝素纳米载药系统在血液模拟环境中发生重新自组装导致粒径变小，从而有利于其跨血脑屏障的特性。本研究拟应用可重新自组装的肝素纳米载药系统，负载具有双重阻断血供作用的cRGD导向肽，并针对胶质瘤微环境呈弱酸性和还原性的特点，设计PH敏感的腙键和还原敏感的二硫键，将阿霉素偶联到肝素上，制备能跨血脑屏障在胶质瘤组织中精确快速释放药物的纳米药物复合体，体外检测该复合体的生物活性，并在原位胶质瘤和原位胶质瘤手术切除后动物模型上评估其在体疗效，同时探讨该复合体在血液环境中重新自组装及杀伤肿瘤的作用机制。本研究有望提高胶质瘤化疗疗效、丰富胶质瘤治疗药品种类，该研究方法可能为靶向胶质瘤的药物研发提供新思路。

纳米颗粒的细胞内在化、传递效率和治疗效果因肿瘤类型的微环境复杂性而异。调整它们的物理化学性质，如表面性质和尺寸，对于处理这种复杂性具有重要的潜力。本文中，我们通过简单改变反应介质或反应物比例，制备了四种ph敏感的阿霉素纳米颗粒(DOX-D1、DOX-D2、DOX-W1和DOX-W2)。DOX-D1和DOX-D2表现出相似的表面特性(表面包覆和靶向配体含量)，且尺寸不同，而DOX-W表现出相似的表面特性和尺寸。在模拟血液条件下，它们可以自组装成更小的颗粒，大小顺序为:DOX-D1> DOX-D2≈DOX-W ，且DOX-W比DOX-D具有更高的靶向配体含量。因此，由于DOX-D1、DOX-D2和DOX-W1的物理化学性质不同，我们进一步研究了它们的体外生物活性和肿瘤微环境反应。与DOX-D1和DOXD2相比，DOX-W1具有更高的细胞摄取，更强的抗增殖能力，这主要归功于其更小的尺寸和更高的靶向部分含量。尽管DOX-W1和DOX-D2的尺寸相似，但DOX-D2表现出与表面涂层相关的更大的体外血脑屏障(BBB)通透性。有趣的是，尺寸和表面性质合适的DOX-D1可以有效绕过血脑屏障并传递到颅内胶质瘤;相比之下，DOX-W1在皮下肿瘤(胶质瘤和乳腺癌)中具有良好的靶向效果。因此，DOX-D1在治疗颅内胶质瘤方面具有优势，而DOX-W1对皮下肿瘤具有强大的杀伤能力。我们的工作表明，调整纳米粒子的多种物理化学性质可以在解决肿瘤微环境复杂性方面发挥重要作用。