超声开放血脑屏障介导智能型纳米粒向脑胶质瘤定剂量比递送双重药物的研究

Recently, cancer stem cells (CSCs) have been considered to be a source for initiation, invasion, metastasis and recurrence of glioma. It has become a new strategy to simultaneously target the CSCs and the differentiated tumor cells by concomitant therapeutics of CSCs-targeting medicines and traditional chemotherapeutics. However, in view of existence of blood brain barriers and the inconsistent pharmacokinetics profiles between the concomitant drugs with different physiochemical characteristics, there are few strategies with high efficiency and ability to ratiometricaly delivery them into glioma in clinical treatment. This project synthesized a new synthetic material, D-α-tocopherol grafted ε-polylysine conjugates, to prepare the cationic nanoparticles, which can encapsulate the hydrophobic CSCs-targeting medicines. Moreover, another synthetic conjugates, dopamine-modified poly-γ-glutamic acid conjugates, as an anionic coating material was deposited on surface of the cationic nanoparticles through pH-sensitive bonds, forming the functional corona with ability to load the hydrophilic drugs. It is easy to construct an intelligent dual-drugs loaded nanoparticle with an optimum ratio of the drug loading through adjusting the formula. Using the technology of ultrasound-mediated opening of blood brain barriers, the ability of the intelligent nanoparticles to deliver the concomitant drugs to giloma was evaluated by cytology, pharmacokinetics, imaging, pharmacology and molecular biology, the multiple therapeutic effects and mechanism of the intelligent dual-drugs loaded nanoparticles for glioma-bearing rat models will be investigated. The strategy in this project may provide an efficient mean to facilitate the ratiometric delivery of the synergetic multi-drugs to glioma.

肿瘤干细胞(CSCs) 是导致脑胶质瘤发生、侵袭、转移和复发的根源。靶向CSCs药物与传统化疗药物以一定剂量比联合治疗，分别杀死CSCs和分化的肿瘤细胞，已成为脑胶质瘤化疗的新思路。但由于血脑屏障的存在，以及不同理化性质的药物间体内药动学差异，目前尚缺乏有效方法将联合药物以最佳剂量比、高效递送至肿瘤病灶。本项目合成维生素E琥珀酸酯-接枝-（ε-聚赖氨酸）为新型材料，构建阳离子纳米粒，实现疏水性药物装载，合成多巴胺-接枝-（γ-聚谷氨酸）为阴离子包被材料，以pH敏感方式对阳离子纳米粒层-层包衣、沉积为多功能亲水外壳，供亲水性药物装载，以最佳剂量比负载，构建双重药物负载智能型纳米粒。超声介导血脑屏障开放促进双重药物负载智能型纳米粒脑内转运，通过细胞学、药动学、影像学、药效学、分子生物学等评价方法，研究其在脑胶质瘤动物模型体内的药物递送性能和作用机制，为联合药物定剂量比递送入脑奠定实践基础。

姜黄素(Cur)杀死脑胶质瘤干细胞，盐酸阿霉素(DOX)则杀死分化的脑瘤细胞，两药联合对脑胶质瘤具有协同作用，但治疗效果依赖于二者剂量配比。而且，由于血脑屏障的存在，以及联合药物间理化性质差异大，尚缺乏有效方法定剂量比递送Cur/DOX 治疗脑胶质瘤。本项目合成了维生素E琥珀酸酯-接枝-(ε-聚赖氨酸) 的新型材料并对其结构进行表征，采用芘荧光探针法测定其临界胶束浓度(CMC)。发现VES-PLL-50聚合物具有较低的CMC值，且能自发组装成超小纳米胶束。后又利用自组装法，制备了包载姜黄素的纳米粒（Cur-NP）并评价了其粒径、形态、载药量、包封率、药物晶型，以及体外稳定性。结果表明Cur-NP粒径为32nm，Zeta电位为19.6mV，载药量达到4.32%，包封率达 82.27%，姜黄素以非晶态分布在胶核内，能保持较长时间粒径与载药稳定。接着，合成了多巴胺修饰的聚谷氨酸包衣材料，在pH8的条件下，通过 Michael或Schiff自发交联对Cur-NP表面包衣修饰，制备出pH敏感性的核-壳纳米粒。经静电吸附方式，DOX装载到包衣纳米粒的包衣层，继续控制装载比例，最终制备出以Cur/DOX=3:1载药的智能纳米粒(PDCP-NP)。该PDCP-NP粒径为160.8nm，Zeta电位为–30.5 mV，呈球状，具有核-壳双层结构。体外贴壁细胞以及C6细胞球实验表明，与溶液剂相比，PDCP-NP能够将Cur/DOX以3:1剂量比递送至细胞内，具有强的肿瘤球穿透能力，能有效地抑制C6细胞球的生长(P<0.05)。原位荷C6瘤大鼠活体示踪实验表明，在UTMD辅助下，PDCP-NP尾静脉给药后，能渗透至脑瘤组织，并且肿瘤组织内Cur/DOX浓度比仍然保持为3:1。体内药效实验表明，与溶液剂相比，UTMD辅助的PDCP-NP脑胶质瘤治疗，显著提高了荷瘤大鼠生存期，抑制肿瘤生长 (P<0.05)。与溶液组相比，PDCP-NP治疗后，脑胶质瘤组织内CD133阳性的脑瘤干细胞明显减少，其比例从5.77%降低至0.95%。 由此可见，本项目创新制备的PDCP-NP不但能实现Cur/DOX的定剂量装载与递送，而且在UTMD短暂开放BBB的辅助下能渗透至脑肿瘤组织，分别杀死CSCs和分化的脑瘤细胞，延长荷瘤大鼠的生存期。本项目为临床复方药物联合高效治疗提供新的思路。