基于增强巨胞饮的共载化疗药物和siRNA仿生纳米载体的构建及其通过协同致死效应靶向治疗复发脑胶质瘤的研究

Glioma stem cell subpopulation are crucial for the recurrence of glioma. However, in the absence of effective carriers of overcoming several obstacles, the application for delivery drugs to this subpopulation has lagged behind. It is because that we face several key challenges: The blood-brain barrier prevents most drug in the blood from entering the brain. It is difficult to make the drugs concentrate into the subpopulation of glioma stem cells in the tumor region. Glioma stem cells are composed of complex gene network and heterogenetic, which results in that most drugs cannot meet the demand to regulate the disorder. Therefore, we propose the hypothesis that a forward feedback loop could be established to induce the heterogeneity drug-resistant tumor cells to uptake the nanocarrier which can cross the blood brain barrier much more efficiently through the enhanced macropinocytosis pathway. Meanwhile, the chemotherapy drugs and siRNA can be co-delivered in the core of nanocarrier, which will produce the synthetic lethal effect to kill the glioma stem cell. .In this study we would like to systematically investigate these questions: 1. The basic nanocarrier with several properties should be synthesized. It can cross the blood-brain barrier, co-deliver the chemotherapy drugs and siRNA with a core and release the drug into cell plasma in a safe manner. 2. The basic nanocarrier should be further modified by α-helix peptide. It can enhance the macropinocytosis pathway as proposed. 3. The proper chemotherapy drugs and siRNA should be selected to produce the synthetic lethal effect. The glioma stem cell subpopulation should uptake the nanocarrier in a high performance and should be effectively inhibited as proposed. Utilizing the response to these questions would help us to construct a new nanocarrier to induce the recurrent tumor cells to 'drink' the poisons by enhanced macropinocytosis mechanism. There will be a breakthrough with this method, compared with the simple ligand binding to the high expression receptor method, which will provide a novel therapeutic method by synthetic lethal effect through enhanced drug co-delivery nanocarrier.

胶质瘤干细胞是肿瘤复发的关键细胞亚群，对其进行高效的靶向致死有望提高复发患者的生存期。然而目前由于血脑屏障对药物入脑阻碍、药物难以有效分布于肿瘤内部且富集于该细胞亚群以及传统药物难以调控胶质瘤干细胞凋亡等原因而缺少有效的治疗策略。因此，我们提出利用其自身高表达的巨胞饮通路并予以进一步增强使可过血脑屏障的仿生纳米载体能高效靶向富集于胶质瘤干细胞，并通过核心共载化疗药物和siRNA的方法产生协同致死效应抑制复发胶质瘤。本课题拟开展如下工作：1.构建安全可通过血脑屏障、核心稳定共载两种药物且能够在靶细胞胞浆释药的仿生纳米载体；2.靶向激活CXCR4增强其巨胞饮通路高效摄取载药纳米粒；3.共载合适的化疗药物及siRNA药物产生协同致死效应。通过本项目的研究，构建一种新的纳米递药载体，诱导胶质瘤干细胞“智能高效吞饮”含药物的纳米载体，突破现有单一靶向模式，通过产生协同致死效应综合治疗复发胶质瘤。

靶向调控胶质瘤起始细胞亚群有助于提高胶质母细胞瘤患者的预后，但需克服跨血脑屏障进行药物递送、对实体肿瘤内部该亚群实现有效富集、针对多靶点协同调控治疗等一系列的困难和挑战。本项目在前期发现的肿瘤细胞中Ras通路激活的、依赖于巨胞饮的脂蛋白纳米载体摄取的基础上，提出：通过激活受体介导的增强巨胞饮机制，使纳米药物高效地被胶质瘤起始细胞“主动”摄取，以达到对该细胞亚群靶向富集的目的。为证实该靶向策略的效果，本项目在符合伦理和知情同意的基础上，提取了患者来源的胶质瘤起始细胞亚群，构建了原位人源胶质瘤小鼠模型。由于CXCR4受体满足以下几种特点：一、在胶质瘤起始细胞亚群中高度表达；二、能够激活下游的巨胞饮通路；三、通过数据库筛选及实验发现CXCR4蛋白表达的水平在胶质瘤组织中与胶质瘤起始细胞亚群的空间分布密切相关，故而选择CXCR4受体作为利用受体介导的增强巨胞饮机制靶向胶质瘤起始细胞亚群的关键靶向受体。本项目筛选了三条能够激动CXCR4受体及其下游通路的靶向肽，并通过α-螺旋肽修饰至脂蛋白纳米载体表面，通过体内外实验评价分析这三条多肽的靶向效果，发现38个氨基酸组成的FH38靶向肽在体内外对胶质瘤起始细胞亚群有显著的增强靶向富集能力，较未修饰靶向肽的脂蛋白纳米载体能够提升3-5倍。利用磷酸钙核心载带microRNA-34a核苷酸药物，表面包裹并修饰磷脂、ApoE脂蛋白和靶向肽，能够实现跨血脑屏障、靶向胶质瘤组织的高效核苷酸药物递送，并利用受体激活的巨胞饮机制被胶质瘤起始细胞亚群所高效摄取，利用磷酸钙核心实现溶酶体逃逸释放药物，通过microRNA-34a基因药物抑制胶质瘤起始细胞的SOX2蛋白和Notch1蛋白的表达，在较低剂量下有效地抑制了胶质瘤起始细胞的自我更新能力和耐药性，降低化疗药物替莫唑胺的治疗剂量并提高其治疗效果，有效延长原位荷瘤小鼠模型的生存期，且具有良好的生物相容性，能够有效克服上述的一系列困难和挑战。本项目进一步利用原位胰腺癌模型进行了利用受体增强巨胞饮的机制提高靶向治疗效果的初步普适性验证，同样发现该药物制剂能够显著延长原位胰腺癌小鼠模型的生存期。因此，本项目为利用受体激活巨胞饮作用靶向肿瘤起始细胞亚群的主动靶向纳米递药策略上提供了关键实验依据。