【作者】 王静；

【导师】 杨红；

【作者基本信息】 电子科技大学 ， 生物化学与分子生物学， 2021， 硕士

【摘要】 化疗是肿瘤临床治疗的重要方式,但化疗存在着对肿瘤选择性差、副作用严重等不足。随着纳米药物递送系统(Drug delivery systems,DDS)的快速发展,搭载化疗药物的DDS能在循环系统中保护化疗药物,并且可以将药物被动靶向到肿瘤部位,显著提高药物的生物利用度和抗肿瘤治疗的效果。但DDS也面临着难以向肿瘤深处渗透,细胞摄取困难以及药物释放不完全等弊端,削弱了DDS抗肿瘤效果。目前,开发响应于肿瘤微环境释放药物的DDS能有效解决以上阻碍,这类DDS在循环过程中结构稳定,保护药物不被漏释,当它们到达肿瘤微环境,随着pH,ROS,酶等浓度变化,DDS就会通过改变自身电荷、结构、大小等来增强其肿瘤深层渗透,细胞内吞,并提高药物释放量。此外,这类响应于肿瘤微环境的DDS能区分肿瘤组织和正常组织,降低药物对正常组织的毒副作用,改善整体治疗效果。基于上述分析,本课题通过薄膜水化法制备了一种pH/ROS级联响应性纳米前药递送系统PPHI@B/L,由具有pH响应性腙键的PEG-Hyd-PCL和PEI-PCL两种单体形成杂化胶束(PPHI),同时搭载ROS产生剂β-Lapachone(Lap)和ROS响应性前药(BDOX)。通过TEM、DLS、H-NMR、TOF-MS等证明了PPHI@B/L的成功合成,粒径在100 nm左右,PDI在0.2左右。共聚焦和流式细胞术证明了PPHI@B/L中的腙键响应于肿瘤弱酸微环境而断裂,使得粒径缩小,增加了药物的深层渗透和细胞摄取。通过ROS探针DCFH-DA验证了PPHI@B/L中的Lap可以在肿瘤细胞内过量NQO1诱导下产生ROS,实验组的ROS水平是空白组的3.9倍。此外,由于Lap产生ROS的过程会消耗ATP,实验组细胞内ATP含量比空白组降低了8.9倍,有效抑制了ATP依赖性的P-gp蛋白活性,进而克服肿瘤细胞多药耐药。通过Annexin V-FITC/PI、Calcein-AM/PI以及CCK-8等实验证明PPHI@B/L可以有效杀死多药耐药肿瘤细胞,PPHI@B/L组对耐药肿瘤细胞的杀伤效果是DOX组的5.6倍,还可以区分正常细胞与肿瘤细胞,降低毒副作用,改善治疗效果。通过小鼠活体荧光成像证实了PPHI@Di R可有效延长药物在的体内循环时间,并可被动靶向累积到肿瘤部位。小鼠皮下肿瘤模型证实PPHI@B/L与其它对照组相比有最好的抗肿瘤效果,且具有良好的生物安全性。裸鼠MCF-7 ADR模型证实PPHI@B/L组与DOX组相比可以抑制多药耐药肿瘤的生长,提高治疗效果。总之,本课题为开发具有肿瘤高选择性和克服肿瘤多药耐药的前药递送系统提供了新的策略,该纳米前药递送系统具有良好的应用前景。更多还原

【Abstract】 Chemotherapy is the main method of clinical cancer treatment which has some shortcomings such as poor tumor selectivity and serious side effects.Drug delivery systems(DDS)has been widely explored for which can protect drugs from phagocytosis and degradation in the circulatory system,prolong the blood circulation time,and significantly improve the targeting efficiency of tumors,thus significantly improving the bioavailability of drugs and the efficacy of anti-tumor therapy.However,DDS also face the challenges of low drug penetration,poor cell uptake and incomplete drug release,which limit the application of DDS in anti-tumor therapy.The emerging stimulusresponsive DDS will remain "invisible" during circulation to avoid being cleared.Once these kinds of DDS are exposed to pH,ROS,enzyme variation,they will change their charges,structure or size for deeper tumor penetration,more cell endocytosis,and higher drug release.In addition,stimulus-responsive DDS can distinguish tumor tissue from healthy tissue by responding to tumor microenvironment so as to reduce the toxic and side effects.Firstly,we fabricated a pH/ROS cascade responsive prodrug nanomicells(denoted as PPHI@B/L)by utilizing two amphiphilic diblock copolymers(PEG-Hyd-PCL,PEIPCL)with the loading of ROS-generation agent β-Lapachone and ROS responsive prodrug(BDOX).The successful synthesis of PPHI@B/L was proved by TEM,DLS,HNMR,TOF-MS and other characterization technique.The results of TEM and DLS verified that the particle size of PPHI@B/L could be reduced through the pH-responsive cleavage of hydrazone bonds under the weak acidic conditions(pH=6.8).The results of HPLC verified that the BDOX could release the therapeutic drug DOX through ROS response activation in the presence of ROS which was much less than the ROS content of tumor cells.Moreover,it is proved that the empty vector(PPHI)has good blood compatibility and biosafety by co-incubating with blood cells and many kinds of cells.Secondly,the effect of PPHI@B/L on overcoming multidrug resistance and antitumor therapy was explored in vitro.Confocal and flow cytometry verified that the reduction of PPHI@B/L particle size caused by pH response could increase the deep infiltration of tumor and expand cell uptake.ROS probe DCFH-DA verified that Lap in PPHI@B/L could generate effective ROS under the induction of excessive NQO1 in tumor cells.And Lap also consumed ATP and inhibited ATP-dependent P-gp protein in the process of genarating ROS,which overcame the multidrug resistance of tumor cells.Subsequently,the mechanism of overcoming multidrug resistance of PPHI@B/L was explored by examining intracellular ATP level and observing by confocal microscope.In addition,it was proved by Annexin V-FITC/PI,Calcein AM/PI,and CCK-8 that PPHI@B/L could effectively overcome multidrug resistance,distinguish normal cells from tumor cells,reduce the toxicity and side effects and improve the therapeutic effect.Finally,the effect of PPHI@B/L on overcoming multidrug resistance and anti-tumor therapy was explored in vivo.PPHI@Di R,carrying fluorescent molecule Di R was synthesized and verified by in vivo fluorescence imaging in mice that PPHI@Di R could effectively prolong the circulation time in vivo and accumulate passively to the tumor site,and that PPHI@B/L had the best anti-swelling effect and good biosafety compared with other control groups by BALB/c 4T1 cell tumor model.Through the MCF-7ADR model of BALB/c nude mice,it was proved that PPHI@B/L could inhibit multidrug resistance and improve the therapeutic effect compared with DOX group.Therefore,PPHI@B/L is a promising nanoplatform for overcoming tumor multidrug resistance and pathological barriers to enhance drug delivery.更多还原