肿瘤微环境响应复合纳米囊的构建及其肿瘤深部渗透的研究

Nanoparticles can only concentrate on the peripheral tissue but cannot penetrate deep into the tumor. The most of the uptaken nanoparticles cannot release drug timely and impede the drug from penetrating into the target organelle. As a result, the anti-tumor effect of nanoparticles will be greatly decreased. It is widely concerned that how to improve the deep penetration of nanoparticles. This project intends to design a composite, large-size and pH-responsive nano-capsules, which were prepared from lentinan (LNT)-polyglutamic acid (PGA) as outer capsule. They also contain several small-size TAT peptide-modified redox-responsive micelles. The drug delivery system achieves the aim that not only micelle-release will be controlled by acidic tumor environment, but also drug-release will be regulated by intracellular reductive environment. The final result is that the anti-tumor drug can deeply penetrate in tumor. The effects of molecular weight and grafting ratio of PGA and LNT on pH responsiveness and stability of nano-capsules will be studied to optimize the carrier. By the means of multicellular tumor spheroids model, the levels of tumor penetration and intracellular drug release of TAT-modified redox-responsive micelles will be investigated. And the relationship between deep penetration of drug and tumor inhibition will be explored. The environmental responsiveness, deep penetration, pharmacokinetics and pharmacodynamics of the composite nano-capsules will be systematically investigated. This project is intended to promote drug deep penetrating in tumor, and provide a new method to improve the efficacy of anti-tumor drugs.

纳米制剂到达肿瘤组织后，往往只能聚集在外围组织而无法渗透进肿瘤深部；而摄取进肿瘤细胞的制剂，常因不能及时释药而阻碍药物渗透到靶细胞器发挥疗效，以上两点大大降低了纳米制剂的抗肿瘤效果。如何提高纳米制剂的肿瘤深部渗透性受到人们的广泛关注。本项目拟构建复合纳米囊体系，外囊由大粒径pH响应香菇多糖（LNT）-聚谷氨酸（PGA）组成，囊内包裹多个小粒径TAT肽化还原响应胶束。该体系可实现肿瘤微酸环境激发胶束释放和胞内还原环境激发药物释放，以促进药物在肿瘤中的深部渗透。本项目通过研究PGA和LNT分子量及接枝率对纳米囊pH敏感性和稳定性的影响来优化载体，通过肿瘤多细胞球模型考察TAT肽化还原响应胶束的肿瘤组织渗透性和胞内药物释放性，探索药物深部渗透与肿瘤抑制之间的关系，系统评价复合纳米囊的环境响应性、药物渗透性、药动学和药效学特性。本项目通过促进药物在肿瘤组织的深部渗透，为提高抗肿瘤药疗效提供新方法。

PEG化纳米制剂到达肿瘤组织后，PEG化胶束能够阻碍药物在肿瘤细胞内的释放，使药物难以发挥最佳疗效，PEG水化层带来的弊端引起了研究人员的广泛关注。本研究设计并合成出还原敏感Y型两亲性嵌段共聚物--聚乙二醇2000二硫二硬脂酸甘油酯（P2kSSGCDS），通过还原敏感键--二硫键，将亲水基团PEG和疏水基团二硬脂酸甘油酯连接。合成的载体在水溶液中可自组装成胶束，进入肿瘤细胞后，受还原环境刺激，二硫键发生断裂使胶束结构破坏，从而实现药物的快速释放。以阿霉素（DOX）为模型药物，制备载DOX胶束（P2kSSGCDS- DOX），胶束粒径较小，约21nm；体外释放研究显示，P2kSSGCDS- DOX在非还原条件下，DOX释放较少，但在还原条件下（10 mM DTT），DOX释放显著增加。进一步考察了P2kSSGCDS- DOX的抗肿瘤效果，与非还原胶束P2kGCDS- DOX相比，P2kSSGCDS- DOX对MCF7细胞48h的IC50值更低，为0.38 µg/mL；P2kSSGCDS- DOX制剂中DOX更容易进入细胞核发挥抗肿瘤疗效；P2kSSGCDS- DOX 24h促细胞凋亡值更高，为49.36%；P2kSSGCDS- DOX抑制荷瘤小鼠实体瘤体积的效果更好，体重降低更少。综上所述，与非还原响应胶束相比，还原响应胶束可进一步促进药物在肿瘤细胞内的释放，提高抗肿瘤药物的药效，为抗肿瘤药物的纳米制剂涉及提供参考。本研究原计划在P2kSSGCDS- DOX的基础上包裹聚合物多糖，实现肿瘤深层渗透的目的，但由于原计划设计的载体--聚谷氨酸修饰的香菇多糖（LNT-PGA）和穿膜肽TAT修饰的P2kSSGCDS合成失败，因此转换思路，制备聚合物多糖包裹的纳米硒复合纳米粒T70-SeNPs，考察其抗腹水癌的疗效，并进一步制备了双载药递药系统--顺铂及阿霉素双载药脂质体，考察其对腹水癌的抗肿瘤效果。研究结果显示，T70-SeNPs对HepG2细胞的抑制作用最强，IC50值为9.074 μg/mL；T70-SeNPs通过诱导HepG2细胞凋亡、细胞线粒体膜电位下降以及活性氧水平上升来发挥抗肿瘤机制。顺铂及阿霉素双载药脂质体与单一抗肿瘤药相比可显著提高药物对细胞的毒性和细胞凋亡。综上所述，T70-SeNPs及双载药脂质体具有较强的抗肿瘤活性，为临床应用提供参考。