【作者】 杨捷；

【导师】 游剑；

【作者基本信息】 浙江大学 ， 药学， 2019， 硕士

【摘要】 癌症作为全球公共卫生问题,目前仍是亟待克服的疾病。肿瘤低氧微环境是肿瘤的重要特征之一,它会导致肿瘤增殖,侵袭与转移,对化疗、放疗及光动力治疗等的耐受。化疗药物是治疗肿瘤的最常用及有效的手段,现阶段的化疗药物大多存在耐药问题,主要由肿瘤低氧微环境所导致,此外肿瘤部位药物累积受限也会抑制化疗药物的治疗效果。因此缓解肿瘤低氧以及增加肿瘤部位药物累积是增强化疗效果的重要因素。目前已有许多策略用于肿瘤低氧靶向治疗,如低氧激活的前体药物以及低氧响应的纳米载体,产生氧气或递送氧气的纳米系统等。其中携氧载体可以有效地缓解肿瘤低氧,目前研究的携氧载体主要包括全氟化碳(perfluorocarbon,PFC)、人工红细胞、修饰的血红蛋白等。PFC虽然有很高的氧气溶解度,但是只能通过氧浓度梯度释放并扩散氧气,效率比较低。与之不同,血红蛋白(Hemoglobin,Hb)在高氧条件下可以有效地结合氧气,在低氧条件下的快速释放氧气,效率较高,同时血红蛋白脂质体是较成熟的临床血液替代品。本论文在此基础上,制备了化疗药物阿霉素(Doxorubicin,DOX)与携氧载体血红蛋白共载的脂质体(DOX-Hb-lipo,DHL)。对比不同的处方及制备方法,最终确定采用硫酸铵梯度法,最佳处方为E80磷脂与胆固醇摩尔比2:1。制得的脂质体粒径均一,大小为130 nm左右,具有较好的血清稳定性及放置稳定性。由于血红蛋白有自供氧的特性,DHL可以在肿瘤低氧部位释放氧气,克服肿瘤低氧耐药。通过溶氧仪及哌莫硝唑低氧探针、HIF-1α蛋白表达在体外水平检测脂质体缓解肿瘤低氧的能力,结果表明DHL可以持续释放氧气,有效地改善肿瘤低氧。此外,由于Hb与磷脂之间存在疏水作用,脂质体DHL表面结合有一定的血红蛋白,粒径及透射电镜图均验证了这一点。我们惊喜地发现脂质体表面血红蛋白具有一定地肿瘤靶向作用,采用多种肿瘤细胞及正常细胞系考察摄取情况,同时采用活体分布在体内水平进行进一步验证。结果发现相比于阿霉素脂质体(DOX-lipo,DL),DHL可以更多地被肿瘤细胞摄取,累积到肿瘤部位。根据相关的文献及实验探究,肿瘤细胞倾向于摄取铁,初步推测该靶向作用与血红蛋白中的铁有关。细胞毒性实验及药效学实验均说明DHL可以缓解肿瘤低氧,克服低氧导致的耐药,从而增强化疗药物阿霉素的抗肿瘤效果。药效学实验中,利用HIF-1α、VEGF及哌莫硝唑低氧探针考察肿瘤低氧水平,结果表明DHL也可以有效地在体内水平改善肿瘤低氧。此外,采用活性氧检测试剂盒证明了脂质体显著地增加了在缺氧环境中活性氧(Reactive oxygen spieces,ROS)的产生并促进了 ROS介导的阿霉素细胞毒性作用。综上所述,本研究制备了共载携氧血红蛋白和化疗药物阿霉素的纳米载体,通过脂质体表面血红蛋白的肿瘤靶向作用,增加肿瘤部位药物累积,同时血红蛋白释放氧气,克服低氧导致的肿瘤耐药,从而提高化疗药物的抗肿瘤效果。更多还原

【Abstract】 As a global public health problem,cancer is still a disease that needs to be overcome.Hypoxic microenvironment,one of the critical features of tumors,can lead to tumor proliferation,invasion and metastasis,tolerance to chemotherapy,radiotherapy and photodynamic therapy.Chemotherapy is the most common and effective means of treating tumors.However,owing to hypoxia,tumor presents resistance to most of the current chemotherapy drugs.In addition,the limited drug accumulation in tumor sites also inhibits chemotherapy efficiency.Therefore,alleviating tumor hypoxia and increasing drug accumulation in tumor sites are important factors in enhancing chemotherapy effect.Various strategies for tumor hypoxia have been studied,such as hypoxia-activated prodrugs and hypoxic-responsive nanocarriers,nanosystems that produce oxygen or deliver oxygen.Among them,oxygen-carrying carriers can effectively alleviate tumor hypoxia,including perfluorocarbon(PFC),artificial red blood cells,modified hemoglobin,etc.Although PFC shows high oxygen solubility,it can only release and diffuse oxygen depending on oxygen concentration gradient,which is less efficient.In contrast,hemoglobin(Hb)can effectively bind oxygen under high oxygen conditions,and release oxygen rapidly under hypoxic conditions.In addition,hemoglobin liposome is a mature clinical blood substitute.Therefore,we prepared the liposome(DOX-Hb-lipo,DHL)co-loaded with the chemotherapy drug doxorubicin(DOX)and the oxygen-carrying carrier hemoglobin.The ammonium sulfate gradient method was finally determined and the best prescription was E80 phospholipid:cholesterol = 2:1(molar ratio)after comparing different prescriptions and preparation methods.The fabricated liposome DHL presented a uniform particle size which was about 130 nm,and good stability.Thanks to the self-oxygenation characteristics of hemoglobin,DHL can release oxygen to overcome hypoxia-induced chemoresistance.The ability of DHL to alleviate tumor hypoxia was detected by soluble oxygen analyzer,pimonidazole hypoxic probe and HIF-1α protein expression in vitro.The results showed that DHL can continuously release oxygen and effectively improve tumor hypoxia.Furthermore,due to the hydrophobic interaction between Hb and phospholipids,fractional Hb was bound to the surface of the liposome membrane,which was confirmed by particle size and transmission electron micrograph.Interestingly,we found that Hb on the surface of the liposomes presented a certain tumor targeting effect,which was verified by the uptake study in various tumor cells and normal cell lines and the biodistribution study.The results showed that compared to doxorubicin liposomes(DOX-lipo,DL),DHL can be more internalized by tumor cells and accumulated in tumor site.According to relevant literature and experimental research,tumor cells tend to take up iron and hemoglobin consists of iron,which may contribute to the targeting effect.Cytotoxicity and anti-tumor efficacy in vivo indicated that DHL can alleviate tumor hypoxia and overcome the chemoresistance induced by hypoxia,thereby enhancing the anti-tumor effect of doxorubicin.In anti-tumor efficacy experiments,tumor hypoxia was detected using HIF-1α,VEGF,and pimonidazole hypoxic probes.The results showed that DHL was also effective in improving tumor hypoxia in vivo.In addition,the reactive oxygen species(ROS)detection demonstrated that DHL significantly increased the production of ROS in hypoxic environments and promoted ROS-mediated DOX cytotoxicity.In summary,this study prepared a liposome carrying oxygenated hemoglobin and the chemotherapeutic drug doxorubicin.DHL presented increased drug accumulation in tumor site attributed to the tumor targeting effect of Hb on liposome surface.Besides,Hb could release oxygen to overcome tumor hypoxia-induced chemoresistance,thereby improving the anti-tumor efficacy of chemotherapy drugs.更多还原