乏氧响应型抗肿瘤诊疗前药的设计、合成及评价

发布时间：2018-07-31 09:35

【摘要】：化疗是治疗癌症的重要手段之一,但现有抗癌药物一般具有较大的毒副作用,为了减小其副作用,大量新颖的前药被开发出来。诊断治疗型前药,兼备了诊断与治疗的功能,实现了药物的可控释放,并对活性药物的释放进行检测,及时反馈了治疗效果。有机小分子诊疗前药具有结构简单、便于修饰、生物相容性好等特点,成为研究的热点。大多数肿瘤是实体瘤,乏氧是实体瘤的重要特征之一,导致肿瘤对化疗药物的不敏感并产生耐药性。然而,乏氧也为肿瘤的治疗提供了思路,即用乏氧响应基团修饰活性药物,使其只在肿瘤的乏氧区域被激活,释放活性药物,达到选择性治疗的效果。氟脲苷(FDU)是一种抗代谢类抗肿瘤药物,用于临床治疗胃癌、乳腺癌等,但其引起的毒副作用(如恶心、呕吐等)限制了其应用。为降低FDU在肿瘤治疗过程中产生的毒副作用,并在线检测药物在体内的分布和积蓄情况,本文以硝基苄基为乏氧响应基团,以2-羧基-2’-羟基联苯(BCM)为荧光染料前体,通过共价键与活性药物FDU进行偶联,设计合成了乏氧响应型诊疗前药FDU-BCM-NO_2。采用NMR、MS和IR等手段对前药和中间体进行表征;采用HPLC法对前药的还原组分进行测定,探讨其释药机制;采用荧光光谱法测定前药与Na2S2O4孵育不同时间后的荧光强度,探讨FDU释放量随时间的变化;采用MTT法测试了FDU和FDU-BCM-NO_2对MCF-7、MGC-803肿瘤细胞和BRL-3A正常肝细胞的细胞毒性,采用流式细胞分析法测试FDU-BCM-NO_2诱导肿瘤细胞凋亡的情况,评价了FDU-BCM-NO_2的体外抗肿瘤效果;采用荧光成像法对FDU-BCM-NO_2孵育的乏氧肿瘤细胞及肿瘤球成像,评价了FDU-BCM-NO_2对乏氧的诊断和对FDU的示踪效果。结果表明:乏氧条件下,FDU-BCM-NO_2在被还原的同时释放出活性药物FDU和荧光分子BCM,BCM释放量随乏氧孵育时间的延长而增加,可利用BCM的荧光信号监测FDU的释放。在常氧条件下,FDU分别孵育MCF-7乳腺癌细胞、MGC-803胃癌细胞和BRL-3A肝细胞48 h后,细胞存活率分别为41.43%、41.70%和48.09%,而FDU-BCM-NO_2孵育的三种细胞存活率则分别为91.09%、91.22%和99.08%;在乏氧条件下,FDU孵育的MCF-7和MGC-803的细胞存活率则分别为22.57%和41.70%,而采用FDU-BCM-NO_2孵育后,细胞存活率则分别44.76%和51.27%,表明FDU对正常细胞和肿瘤细胞均有抑制作用,而FDU-BCM-NO_2具有乏氧选择性,只对乏氧肿瘤细胞具有抑制作用,对正常细胞则不产生毒性。在乏氧条件下,FDU-BCM-NO_2孵育的MGC-803细胞,52.21%的细胞发生凋亡,表明FDU-BCM-NO_2以凋亡的方式抑制肿瘤细胞增殖。FDU-BCM-NO_2在乏氧条件下释放出的BCM具有双光子激发特性,其荧光信号可用于肿瘤细胞及肿瘤球乏氧程度和药物释放的检测。FDU-BCM-NO_2具有诊断、治疗的双重功能,利用实体瘤的乏氧特性可实现FDU的可控释放,避免了其对正常细胞的毒副作用,为实现肿瘤的个体化治疗提供了新的思路。
[Abstract]:Chemotherapy is one of the important means for the treatment of cancer, but the existing anticancer drugs generally have large toxic and side effects. In order to reduce their side effects, a large number of novel prodrugs have been developed. The diagnostic and therapeutic prodrugs have both the function of diagnosis and treatment, the controlled release of drugs, the detection of the release of active drugs, and timely feedback. The effect of treatment. The drug has the characteristics of simple structure, easy to modify and good biocompatibility, and it has become a hot spot. Most tumors are solid tumors. Hypoxia is one of the important characteristics of solid tumor, which causes the tumor to be insensitive to chemotherapy drugs and produces resistance. However, hypoxia also provides thought for the treatment of tumor. FDU is an antitumor antitumor drug used in the clinical treatment of gastric cancer and breast cancer, but its toxic and side effects (such as nausea, vomiting, etc.) restrict its application. In order to reduce the toxic and side effects of FDU in the treatment of tumor, and to detect the distribution and accumulation of drugs in vivo, this paper uses nitrobenzyl as the hypoxia response group and 2- carboxyl -2 'hydroxybiphenyl (BCM) as the precursor of the fluorescent dye, and coupling the covalent bond with the active drug FDU, and designs and syntheses the anoxy responsive diagnosis and treatment drug FD U-BCM-NO_2. was used to characterize the prodrugs and intermediates by means of NMR, MS and IR, and the HPLC method was used to determine the reductive components of the precursor. The fluorescence intensity of the precursor and Na2S2O4 at different time was measured by the fluorescence spectrum method, and the changes of the release amount of FDU were discussed with the time. FDU and FDU-BCM-NO_2 were tested by MTT method. For the cytotoxicity of MCF-7, MGC-803 tumor cells and BRL-3A normal liver cells, the flow cytometry was used to test the apoptosis of the tumor cells induced by FDU-BCM-NO_2, and the anti tumor effect of FDU-BCM-NO_2 in vitro was evaluated. The hypoxic tumor cells and tumor spheres incubated with FDU-BCM-NO_2 were detected by fluorescence imaging, and the FDU-BCM-NO_2 pair was evaluated. The diagnosis of hypoxia and the tracer effect on FDU showed that, under the hypoxic condition, the active drug FDU and the fluorescent molecule BCM were released while the FDU-BCM-NO_2 was reduced. The release of BCM increased with the prolonged incubation time of hypoxia, and the release of FDU could be monitored by the fluorescence signal of BCM. FDU incubated MCF-7 breast cancer cells in normal oxygen condition, M, respectively, M. The survival rates of GC-803 gastric cancer cells and BRL-3A hepatocytes were 41.43%, 41.70% and 48.09% respectively after 48 h, while the survival rates of three cells incubated in FDU-BCM-NO_2 were 91.09%, 91.22% and 99.08%, respectively, and the survival rates of MCF-7 and MGC-803 incubated by FDU were 22.57% and 41.70%, while FDU-BCM-NO_2 incubation was fine. The cell survival rate was 44.76% and 51.27%, respectively, indicating that FDU had inhibitory effect on normal and tumor cells, while FDU-BCM-NO_2 had hypoxia selectivity, only inhibited hypoxia and did not produce toxicity to normal cells. In hypoxic conditions, FDU-BCM-NO_2 incubated in MGC-803 cells, 52.21% cells were apoptotic, indicating FD U-BCM-NO_2 inhibits the proliferation of tumor cells in the manner of apoptosis, and the release of.FDU-BCM-NO_2 under hypoxic conditions has two photon excitation characteristics. The fluorescence signal can be used to detect the hypoxic and drug release of tumor cells and tumor spheres and to detect the release of drugs. The dual function of.FDU-BCM-NO_2 can be used to realize FD with the hypoxic characteristic of solid tumor. The controlled release of U has avoided its toxic and side effects on normal cells, and provided a new way of thinking for individual treatment of tumors.
【学位授予单位】：河北大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914;R96

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