肿瘤微环境响应的药物连环递送系统的构建及评价

Tumor acidity (tumor intracellular and extracellular acidity) and elevated glutathione levels are characteristic features in the tumor microenvironment. The purpose of this application is to construct an extracellular responsive and intracellular triggered consecutive drug delivery system by designing redox-sensitive prodrug delivered with an acidity-responsive carrier. To construct the tumor microenvironment responsive and consecutive drug delivery system, cisplatin was selected as a model drug. Based on the former research work, Pt (II) was oxidized into less toxic Pt (IV) prodrug, coupled with acidity-sensitive polyhistidine peptides for cell-penetrating functions, and sequently conjugated with modified γ-polyglumatic acid. The nanoconjugates showed a micellar conformation under neutral conditions and the negatively charged polyglumatic acid shell of micelles extended the blood circulation time. Exposed into the tumor extracellular acidity, the micellar conformation disassembled rapidly and stretched into linear polymer compounds. After the transitions of nanoconjugates, polyhistidine induced cell-penetrating uptake and endosomal escape. The configuration reversions of Pt(IV) were demonstrated in the presence of reduction conditions. The low reactive Pt(IV) structure inverted into highly active Pt (II) structure and then exerted cytotoxicity for tumor targeted therapy. In this study, the tumor targeting and safety of the consecutive drug delivery system were examined through cell tests, in vivo antitumor activities and pharmacokinetics studies. We hope that the tumor microenvironment responsive and consecutive drug delivery system can provide the foundation for further research on targeted tumor therapy.

肿瘤微环境包含酸性（内、外环境均酸性）和还原性，本申请通过设计还原敏感型前药，利用酸性敏感的载体递送，达到对肿瘤“胞外响应，胞内触发”式连环靶向。以顺铂为模型药物，将Pt(II)氧化成细胞毒性低的Pt(IV)前药，通过与酸敏感的聚组氨酸结合，并与改性聚谷氨酸配位键合载药，构建肿瘤微环境响应的连环机制递送系统。本课题的连环递送系统由于选择的聚组氨酸的亲脂性及聚谷氨酸的亲水性，在中性条件下以胶束形式存在，带负电性的聚谷氨酸胶束外壳，延长了体内循环时间，在肿瘤组织酸性环境下，胶束伸展为链状高分子复合物，聚组氨酸介导穿透肿瘤细胞膜并引发溶酶体逃逸，Pt(IV)在肿瘤细胞内经还原作用发生构型翻转，由低活性八面体Pt(IV)翻转为高活性平面四边形Pt(II)，继而发挥细胞毒性，实现靶向治疗。本课题将通过多种体内外研究手段，研究该连环递送系统的靶向性和安全性，为进一步研究肿瘤靶向治疗提供基础。

本研究基于肿瘤弱酸和还原性微环境，设计了一种多重响应的药物连环递送系统，旨在避免单一靶向机制靶向效率低的缺点，实现高度肿瘤靶向性，提高疗效，降低毒副作用。首先对γ-聚谷氨酸（γ-PGA）的侧链羧基进行氨基修饰，得到γ-PGA-NH2，再将具穿膜作用的两亲性短链聚组氨酸（PHP）连接在γ-PGA-NH2上，得到两亲性接枝共聚物γ-PGA-PHP，以此聚合物作为载体基础，构建聚合物胶束递药系统。选择顺铂为模型药物，先将其氧化为四价铂，再用另一化疗药物去甲斑蝥素（DMC）对四价铂进行修饰，得到DMC-Pt(IV)-DMC“夹心铂”小分子，将“夹心铂”小分子连接在γ-PGA-PHP上，得到最终制剂γ-PGA-PHP-Pt(DMC)；选择琥珀酸酐（SA）对四价铂进行修饰得到SA-Pt(IV)-SA，最终得到对照制剂γ-PGA-PHP-Pt(SA)。体外实验表明，制剂具有一定pH敏感性和还原敏感性。通过调节pH可实现制剂在球形和链状结构的可逆转化；制剂在含还原剂条件下释放快于不含还原剂的条件，表明还原条件可以促进药物的释放。体外细胞实验发现，在不同pH条件下，γ-PGA-PHP-Pt(DMC)的细胞毒性均强于对照制剂γ-PGA-PHP-Pt(SA)，两种制剂在pH6.5条件下的毒性均强于pH7.4条件下的毒性，且γ-PGA-PHP-Pt(DMC)在pH6.5条件下的细胞摄取量要明显高于pH7.4组，进一步表明制剂具有一定的pH敏感性；摄取途径研究表明γ-PGA-PHP-Pt(DMC)的内化是能量依赖的，且由网格蛋白和小窝蛋白介导；胞内转运结果发现PEP(DMC-Pt(IV)-DMC)可以通过PHP质子化发生溶酶体逃逸进入胞质。体内分布实验表明，24h内制剂组小鼠肿瘤组织内Pt含量不断升高，显著高于游离药物顺铂组。药效实验表明制剂组PEP(DMC-Pt(IV)-DMC)可显著抑制肿瘤的生长，延长荷瘤鼠的生存期，且对正常组织无明显毒副作用。本研究表明γ-PGA-PHP递药系统具有良好的靶向作用和抗肿瘤作用，具有很好的应用前景。