【作者】 黄艳；

【导师】 唐汝培；

【作者基本信息】 安徽大学 ， 生物化学与分子生物学， 2018， 硕士

【摘要】 在目前的临床治疗中,化疗仍是针对恶性肿瘤所采用的主要治疗方法之一。但是由于一些抗癌药物存在对肿瘤细胞的选择性较低,在血液中循环的时间较短,对正常组织和器官的毒副作用较大等缺陷,导致其整体的治疗效果不能令人满意。近年来,为了进一步提高抗癌药的治疗效果,很多刺激响应型的聚合物纳米药物载体被广泛的应用于癌症的治疗。这些药物载体能够在血液中稳定存在,并可通过增强渗透与滞留效应(EPR)在肿瘤组织处大量的累积。又由于在肿瘤组织处存在不同于正常组织的pH值、温度、谷胱甘肽(GSH)浓度等生理变化,使得这些具有刺激响应的药物载体能在肿瘤组织处达到靶向药物释放的目的,从而杀死肿瘤细胞。相对于正常组织血液中pH(～7.4),存在于肿瘤组织细胞中弱酸的pH(～5.0-6.0)会使很多pH敏感的药物载体可通过酸敏感的化学键断裂而释放出药物进行癌症的治疗。尽管如此,仅仅依靠这一种酸刺激,很难将药物完全、快速地释放出来,从而使这种药物传递系统在体内治疗时受到了很大的限制。经研究,在肿瘤细胞内还存在着显著的谷胱甘肽浓度差异,在肿瘤细胞中的GSH浓度是正常细胞中两倍以上。因此,通过结合这种GSH浓度的差异和pH值的变化,一些pH和还原双重响应的药物载体得到广泛的发展。通过两种刺激因子的协同作用,这种双重响应的药物载体在癌症治疗方面将有很大的应用潜力。在本论文中,我们通过缩聚的方式合成一种pH和还原双重敏感的聚(原酸酯-二硫键-氨酯)(POEDU)和仅有pH敏感的聚(原酸酯-氨酯)(POEU)(作为对照组)。已知,二硫键是一种还原敏感的化学键,在还原条件下能够快速的断裂。值得注意的是,这里所用的原酸酯键作为一种酸敏感键,具有良好的生物相容性和可降解性,近年来被广泛的应用于药物传递或者基因传递领域。在弱酸环境下,相比较其他酸敏感键,原酸酯键的水解速率较高,能够作出更加灵敏的响应。因此,我们推测这种pH和还原双重响应的纳米药物载体能够在肿瘤位点弱酸和还原的条件下将所载的药物快速的释放出来,从而提高药物治疗效果。所做工作的具体内容分主要为以下几点阐述:(1)pH和还原双重敏感的聚(原酸酯-二硫键-氨酯)(POEDU)和仅有pH敏感的聚(原酸酯-氨酯)(POEU)的合成:这两种聚合物是通过己二醇活性酯分别和新合成的原酸酯二硫键二氨基单体、原酸酯二氨基单体在温和的条件下缩聚反应得到的。通过核磁可以验证两种聚合物的结构都是正确的,通过凝胶渗透色谱测得POEDU和POEU的分子量分别为1.38 × 104和1.29 × 104。(2)POEDU和POEU两种纳米微球的制备:POEDU和POEU两种纳米微球的制备方法为水包油的单层乳液技术。通过动态光衍射仪测得两种纳米微球的粒径均为在200 nm左右,且分布较均一。通过扫描电镜和透射电镜可以观察到两种纳米微球的形貌都为规整的球形结构。除此之外,在体外降解实验发现,POEU纳米微球仅在弱酸环境下有降解行为,而POEDU纳米微球在弱酸和还原环境中都具有明显的时间依赖的降解行为,且在中性条件下较为稳定。(3)两种纳米微球的体外表征:选择阿霉素(DOX)为抗肿瘤模型药物,并在纳米粒子形成的同时将药物高效的负载在其内部,即得到POEDU-DOX和POEU-DOX两种载药纳米微球。从体外药物释放结果可得pH和还原双重敏感的POEDU-DOX在DTT和弱酸环境同时存在的条件下药物释放的最快。通过使用两种癌细胞(人的神经母细胞瘤细胞(SH-SY5Y)和人源的肝癌细胞(HepG2))对两种载药纳米微球在2D层面(单层细胞)和3D层面(多细胞肿瘤球)上评估可以看出,两种载药纳米微球都能够被有效的摄取,并且双重敏感的POEDU-DOX由于其快速的药物释放,具有更强地杀死肿瘤细胞的能力。(4)两种纳米微球的体内表征:用鼠源的肝癌细胞(H22)建立肝癌小鼠模型,并用来评估POEDU-DOX和POEU-DOX在肿瘤组织和各器官中的药物分布的状况和抗肿瘤效果。通过体内药物分布和组织切片可看出,相对于裸药,两种载药纳米微球有效地延长了 DOX在血液中的循环时间,提高肿瘤组织中的DOX浓度,且明显减少裸药对心脏的毒性。体内抗肿瘤结果研究表明双重敏感的POEDU-DOX因其能够更加高效的传递药物从而表现了更好的抗肿瘤效果。综上所述,这种pH和还原双敏感的聚(原酸酯-二硫键-氨酯)共聚物药物载体在抗肿瘤药物传递的领域具有广阔的应用前景。更多还原

【Abstract】 Chemotherapy is still one of the most useful approaches to treat cancers in clinical applications.Unfortunately,the therapeutic efficacy is seriously limited because most anticancer drugs have low-selectivity for tumor cells,short circulation time in the blood stream,and serious side effect in normal tissues and organs.Recently,a lot of tumor stimuli-responsive polymeric nanocarriers have been designed to improve the therapeutic efficacy in anticancer treatment because of the specific accumulation in tumor site via the enhanced permeability and retention(EPR)effect and accelerated drug release triggered by the stimuli factors,such as pH,temperature,and redox conditions.Compared with normal tissues,solid tumors display specific tumor microenvironment,and existence of pH variation between blood vessels(pH～7.4)and intracellular spaces(pH～5.0 to 6.0).It is well-known that most of pH-sensitive polymeric drug carriers can realize drug release resulted from the cleavage of acid labile linkages in the tumor tissues.Nevertheless,the drug release depending on a single acid-responsiveness is still not desirable due to the lower dosages of anticancer drugs than the level of therapeutic window caused by the insufficient intracellular drug release,which greatly restrict therapeutic efficacy in vivo.Thus,a few polymeric drug delivery systems have been developed by the combination of pH and redox stimuli simultaneously.The intracellular concentration of glutathione(GSH)at tumor site is several times higher than that in normal cells.With the synergetic effect of GSH and pH,the dual-stimuli responsive drug carriers may have great potential for efficient chemotherapy with further optimization.In this work,we have successfully synthesized the dual-stimuli-sensitive poly(ortho ester disulfide urethanes)(POEDU)and pH sensitive poly(ortho ester urethanes)(POEU)through a facile polycondensation reaction.As well-known,reduction-responsive linkers such as disulfide would be cleaved by GSH in the cytoplasm of cancer cells.Notably,ortho ester bond as acid-sensitive linkage is chosen since it has shown excellent biocompatibility and been widely applied in biomedical fields including drug/gene delivery.Its hydrolysis rate may improve 1-4 orders of magnitude compare to other acid-labile linkages in response to mildly acidic environments.Hence,We postulate that the pH and redox dual-sensitive drug carriers may obviously enhance the therapeutic efficiency of loaded cargoes through the synergistically intracellular drug release at tumor sites.The main research contents are as follows:(1)Synthesis of two copolymers(POEDU and POEU):POEDU and POEU were obtain by active esters of 1,6-hexanediol(HD)reacted with ortho ester disulfide-based diamine and ortho ester-based diamine under the mild conditions,respectively.Then,the structure and molecular weight of POEDU and POEU were examined by 1H NMR and GPC,respectively.The results showed that POEDU and POEU were successfully synthesized with number molecular weight of 1.38 and 1.29× 104,respectively.(2)Fabrication of two nanospheres:POEDU and POEU were selected for the preparation of nanospheres using an oil-in-water single emulsion technique.The light scattering intensity and particle size of nanospheres were measured using a dynamic light scattering(DLS),and the morphologies of two nanospheres were observed by scanning electronic microscopy(SEM)and transmission electron microscopy(TEM).The results indicated that the size of POEDU and POEU nanospheres were about 200 nm and the morphology is spherical-like.In addition,POEDU nanospheres with pH and redox dual-sensitivity exhibited excellent stability in physiological condition and displayed a time-dependent degradation in DTT and mildly acidic conditions.(3)Nanospheres characterization in vitro:Doxorubicin(DOX)as a model anticancer drug could be loaded into nanospheres with with high drug loading efficiency.Drug release behavior of the DOX-loaded nanospheres(POEDU-DOX and POEU-DOX)in vitro was revealed that the fastest and complete DOX release was observed for POEDU-DOX at pH 5.0 in the presence of 20 mM DTT.In two tumor models(2D monolayer cells,3D multicellular tumor spheroids),the DOX-loaded nanospheres could be preferentially internalized by human neuroblastom cancer cell line(SH-SY5Y)and human hepatic cancer cell line(HepG2),respectively.Importantly,POEDU-DOX nanospheres showed superior antitumor effect,which was because the dual responsive POEDU-DOX could significantly increase the intracellular drug release after a quick simultaneous response to the mild acidic and reductive intracellular environment.(4)Nanospheres characterization in vivo:In vivo anti-tumor effect and drug biodistribution were evaluated in H22 tumor-bearing mice.The drug distribution and histological analysis results demonstrated that both of POEDU-DOX and POEU-DOX showed a improved accumulation in solid tumor,prolonged blood circulation time and reduced cardiac toxicity of DOX,compared to free DOX.Notably,in vivo antitumor results indicated that the POEDU-DOX displayed the best antitumor activity,resulting efficient delivery to the tumor site and sufficient drug release after cellular uptake by tumor cells.In summary,the pH and redox dual-stimuli-sensitive POEDU nanospheres have great potentials as drug carriers in cancer therapy.更多还原