针对肿瘤细胞周期的时相靶向制剂的研究

Nowdays, the majority of conventional chemotherapeutic agents are cell-cycle specific, acting preferentially on specific phase of the cell cycle. Deliverying these drugs to the desired phase of the tumor cells has been the key to enhance efficacy and/or reduced toxicity for cell cycle-specific anticancer drugs. However, previous drug delivery system was not designed based on the cell cycle. In this project, the duramycin (was coupled with PEG-PDLLA to form Dur-PEG-PDLLA) was used as a targeting ligand to design a cell cycle-specific drug delivery system. Duramycin was usually as a probe for studying the phosphatidylethanolamine (PE), while that PE localized in the cell’s inner membrane leaflet and exposed on the cell surface of the cleavage furrow during late cytokinesis. At the same time, the pH-responsive polymeric micelles of PEG-P(His) was used with Dur-PEG-PDLLA to form mixed micelles. With this novel drug delivery system, the micelles were gathered in the tumor tissue based on the Enhanced Permeation and Retention effect, then the PEG-P(His) disassembled in the acidic environment and, the Dur-PEG-PDLLA were exposed. Under the binding of duramycin and PE, the miclles gathered in the M phased tumor cell. This project will focus on the study of formation mechanism of the mixed micelles, the characteristics of drug loading, drug release and the cell cycle-specific behavior of this novel drug delivey system. This project will provide a novel method and idea for building highly efficient, low toxicity and cell cycle-specific tumor-targeting drug delivery system. This drug delivery system has great potential for applications in clinical practice.

鉴于很多化疗药物为细胞周期特异性药物，将其高效、靶向导入目标时相的肿瘤细胞是增效减毒的关键。本课题首次提出针对肿瘤细胞周期的时相靶向载体设计思路，该载体以磷脂酰乙醇胺（PE）探针Duramycin为靶向配体修饰PEG化聚乳酸（PEG-PDLLA）, 并与pH敏感的PEG化聚组氨酸（PEG-P(His)）组成混合胶束，构建以肿瘤细胞M时相外化PE及其pH偏酸性的肿瘤组织为标的的细胞周期特异性靶向载体。该载体借助EPR效应聚集于肿瘤组织后，pH敏感的PEG-P(His)胶束发生解聚合，暴露出Dur-PEG-PDLLA，利用Duramycin特异性结合至M期肿瘤细胞膜外化的PE，携带胶束聚集于M期肿瘤细胞而实现化疗药的增效减毒。本项目重点围绕混合胶束形成机制，载药和释药特性，肿瘤细胞周期特异性等展开系统性研究，为研究高效、低毒、肿瘤细胞靶向新型载体及技术提供新思路和新方法。

聚合物胶束是解决抗肿瘤药物靶向输送的重要手段。然而，由于很多化疗药物存在细胞周期特异性，聚合物胶束在应用时缺乏细胞周期特异性。本课题采用细胞周期特异性配体duramycin（Dur）修饰聚乙二醇-聚乳酸（PEG-PDLLA）（Dur-PEG-PDLLA），并针对肿瘤低pH环境，与具pH敏感性聚乙二醇-聚组氨酸（PEG-PHIS）成功构建混合胶束（Dur-PEG-PDLLA/PEG-PHIS）。该混合胶束临界相对胶束浓度（CMC）仅为0.09 μg/ml。该胶束具有粒径均一（188.6± 5.3 nm），载药量高（14.7± 0.3%），包封率高（89.1± 2.5%）的优点。体外pH敏感性研究表明，胶束粒径随pH发生变化，体外pH 5.0时释放速度提高约2倍。该项目以未修饰的胶束为对照，考察了胶束的摄取，流式细胞仪及激光共聚焦结果表明，混合胶束在G2/M期HeLa细胞的摄取显著高于G1、S期及正常组细胞，并且发现纳米粒有细胞分裂沟聚集现象，验证了该混合胶束的细胞周期特异性靶向效果。 MTT研究表明，空白混合胶束在200 μg /mL没有发现明细细胞毒性，而市售Taxol空白溶剂组细胞存活率只有53.9%，包载紫杉醇的混合胶束显示出更低的IC50（0.604± 0.016 µg/mL)。大鼠尾静脉注射紫杉醇Dur-PEG-PDLLA/PEG-PHIS混合胶束与Taxol相比，消除半衰期从Taxol的0.866± 0.016 h提高到4.144± 0.548 h，表明紫杉醇混合胶束延长了药物在体内的循环时间。小动物活体成像初步研究了胶束在小鼠体内的分布情况，与Taxol相比，胶束在肿瘤部位有明显蓄积，且在8 h 达到了峰值，而Taxol无此现象，说明紫杉醇混合胶束具有明显的靶向作用。本项目研究结果表明，所构建的细胞周期特异性混合胶束在抗肿瘤药物传递领域具有优越的应用前景。