“高渗序贯-均质逆转”治疗肿瘤耐药

The combined treatment of drug-resistant tumor with chemotherapeutic drug and drug-resistance gene targeted siRNA is now hindered by a mild tumor-targeted penetration and a poor drug-release control. This means that a uniformed drug-resistance reversal is not achieved. To make matters worse, the chemotherapeutic drug payload is always released before the drug resistance phenotype has been silenced by siRNA. Herein, a graphene-based co-delivery system (IPHG) with iRGD modification and calcium phosphate mineralization strategy was designed with function of tumor homing and penetration, as well as a spatiotemporally controlled sequential drug release. Resultantly, IPHG is to achieve an effective drug co-encapsulation, tumor homing and penetration, and a reasonable dual-drug release kinetics that chemotherapeutic drug is burst released after the drug resistance phenotype has been silenced by siRNA for a maximized synergistic anti-tumor effect. In this project, the effect of carrier structure and mineralization technology on nano-morphology, drug co-loading ability, intracellular controlled sequential drug release kinetics of IPHG will be systematically studied. Besides, an in-vitro and in-vivo evaluation system based on a spatiotemporally controlled manner for drug resistance reversal will be established. It means the aggregation degree of the chemical drug on target sites and its subsequent anti-tumor efficiency under the different chemical drug release rate in the silencing duration of siRNA will be studied deeply. Finally, the drug micro-distribution and the reversal degree of drug resistance in different domains within the same tumor will be detected to demonstrate the effective and homogeneous reversal effect of drug resistance by IPHG.

耐药肿瘤联合治疗的共递系统存在化药在耐药逆转剂起效前释放而被细胞外排，及肿瘤靶向渗透能力差、耐药逆转不均一的问题。本项目联合具肿瘤归巢及穿透能力的iRGD肽修饰和新型氧化石墨烯矿化策略，构建具肿瘤靶向渗透及胞内触发式序贯异药释放功能的共递系统，实现异药有效共载、肿瘤靶向促渗、耐药逆转先行而化药时控断后，通过调控双药释放动力学达到协同药效最大化。本项目将系统考察载体结构及矿化工艺对纳米形态、药物共载、胞内序贯释药能力的影响，探讨释药机制并尝试建立体内外肿瘤耐药逆转“时控”评价体系。从耐药逆转剂的生效“时间窗”及化药在“时间窗”内释放程度为切入点，观察胞内化药对靶点的聚集度、对耐药肿瘤抑制效果的影响，并通过体系于肿瘤的微分布及治疗时肿瘤不同部位耐药水平的逆转程度等，多层次考察该体系序贯深入、高效均质的耐药逆转效果，为实现针对耐药肿瘤的优化逆转提供一种全新的设计思路和方法。

耐药肿瘤联合治疗体系存在化药无法在耐药逆转策略的有效时间窗内释放，及肿瘤靶向渗透能力差、耐药逆转不均一的问题。本项目以具肿瘤归巢及穿透能力的iRGD肽修饰具光热转化能力的氧化石墨烯，构建肿瘤靶向渗透型石墨烯纳米片（IPHG），有效荷载模型药物阿霉素（IPHG-DOX），载药量高达64.5%。本项目首先考察验证得IPHG-DOX理化性质合理（117.1 nm，-19.36 mV）、光热性能良好、生理稳定性高、酸触发释药能力显著（72 h释放60%）、且光热可进一步加速药物释放（36 h释放80%）；以小鼠乳腺癌4T1、人脐静脉内皮细胞HUVEC、人乳腺癌MCF-7及耐药株MCF-7/ADR为细胞模型，系统考察了IPHG-DOX的入胞机制及胞内转运行为，结果表明iRGD和HA能特异性与各自受体结合介导主动胞吞，且IPHG-DOX的光热性能可触发溶酶体逃逸、瞬时沉默耐药蛋白P-gp、同时加速药物释放，共同介导药物于靶标的快速递送；4T1肿瘤球及体内靶向分布实验结果验证了iRGD介导的跨内皮蓄积及瘤内持续促渗；4T1模型体内外药效学结果表明瘤内高渗型IPHG-DOX可基于“光热—化疗”联合抗肿瘤，并可通过整合素靶向和光热介导的联合骨架重构干扰实现高效抗迁。更为关键的，本项目继续以MCF-7/ADR为模型考察了低温热疗逆转多药耐药的有效性、时效关系及其相关机制，结果表明瞬时低温热疗可引发耐药株的热应激效应，加速泛素-蛋白酶体系统（UPS）降解异常的蛋白质，在3 h内即可快速、显著下降P-gp的胞内蛋白含量，但由于热应激并未损伤细胞的正常功能，在低温热疗给与后12 h内，胞内P-gp表达快速反弹，即低温热疗的耐药逆转有效时间窗为3-12 h；在MCF-7/ADR模型验证了时间窗内低温热疗可有效增加化药的靶标聚集度、增强耐药逆转效果；并且通过MCF-7/ADR荷瘤裸鼠模型瘤内活检P-gp蛋白检测，验证瘤内高渗型IPHG-DOX可实现“均质”逆转多药耐药。本项目成功建立了体内外肿瘤耐药逆转“时控”及“空间多维”评价体系，为实现针对耐药肿瘤的优化逆转提供了全新的思路和方法，研究结果提示基于低温热疗的耐药逆转策略须配合药物在给与瞬时热疗后的及时释放，才可最大化耐药肿瘤的抑制率。