肿瘤微环境响应性纳米胶束的制备及其用于“抗癌前药—酶”的联合输送研究

发布时间：2018-07-25 21:09

【摘要】：癌症已经成为世界人口死亡的主要原因之一。目前,癌症的主要治疗方法有化学疗法、光热疗法和辐射疗法等等,已经得到了广泛的研究。近年来,“酶-前药”疗法(“enzyme-prodrug”therapy,EPT)作为一种新的癌症治疗策略,已展现出很好的癌症治疗效果,并得到了大量的临床验证。EPT是一种两步疗法:首先,使用靶向策略将前药活化酶传输到肿瘤部位;其次,将无毒的前药静脉注射后通过血液循环传递到肿瘤组织,无毒的抗肿瘤前药就会在前药活化酶的催化下产生活性抗肿瘤药物。虽然“酶-抗肿瘤前药”疗法为人们展示了广阔的应用前景,给肿瘤治疗带来了一个新思路,但仍有许多关键性问题亟待解决:“酶-前药”疗法需要两步治疗,这就增加了病人的痛苦与治疗成本,并且第二次用药的时间也不易把握(对于抗体导向“酶-前药”疗法,首先将抗体-酶偶联物静脉注入体内,借助抗体识别肿瘤表面抗原的特性,使酶被带到肿瘤靶位,然后当抗体-酶偶联物从血液中清除后才能静脉注入无抗癌活性或低活性的前药,不然滞留在体内正常组织器官的酶就会催化前药,进而对正常组织造成损伤,因此对于第二次用药的时间就不易把握)。“酶-前药”疗法中前药的用药剂量比较大,这是由于药物在病变部位只有达到一定浓度才能发挥治疗作用,而药物在体内传输过程中会有滞留等现象,给药剂量太小就会造成到达病变组织药物浓度不够,但用药剂量太大则会造成药物的蓄积,可能对机体造成一定的毒副作用,并且增加了治疗成本。如果酶和前药被同时地输送到肿瘤细胞,由两步疗法所造成的缺陷就会被极大地避免。所以,发展一种有效的能够同时传输酶和前药的载体是极其重要的。辣根过氧化物酶-吲哚乙酸(IAA-HRP)体系是近年来研究比较多的一种“酶-前药”系统。IAA作为一种植物激素,被HRP氧化后会产生包括活性氧自由基(ROS),如吲哚基、3-甲基吲哚基以及过氧化基在内的自由基。其可以通过引起细胞脂质过氧化引起细胞膜结构改变、DNA损伤、降低Bcl-2活性以及激活凋亡相关基因,导致细胞内损伤和凋亡。然而,相同浓度IAA对正常细胞生长的影响并不显著,IAA也不易被哺乳动物的过氧化物酶氧化。酯键是前药设计中一种非常普遍的键接方式,其可在人体酯酶的作用下发生水解。吲哚乙酸乙酯(EIA)是一种IAA的衍生物,当其被传输到肿瘤组织后,在酯酶及肿瘤部位低的pH作用下发生水解,产生前药IAA。由两亲性大分子通过自组装形成的聚合物胶束,在传递抗肿瘤药物、siRNA以及其它治疗剂方面有很大的潜力。纳米胶束可以通过EPR效应优先进入到肿瘤组织,大大减少了化疗的副作用,同时,胶束有较长的血液循环时间,可提高药物的利用率。此外,聚合物胶束有对于肿瘤微环境的可设计性。例如,肿瘤组织(pH 6.5-6.8)、核内体(pH 5.5-6.5)和溶酶体(pH 4.5-5.5)是微酸性性环境,不同于血液和正常组织(pH 7.4)。因此,pH敏感型胶束可以实现肿瘤部位药物的智能可控释放。此外,肿瘤部位细胞内还原性谷胱甘肽(GSH)的浓度是细胞外GSH浓度的100-1000倍。含二硫键链的还原敏感型聚合物胶束在肿瘤部位会发生解体,胶束载体被迅速破坏,可实现药物在肿瘤部位可控释放。因此,聚合物胶束可以作为理想的载体来实现酶和药物的联合输送。本论文主要通过设计不同肿瘤微环境敏感型的聚合物胶束,来实现酶和前药的联合输送,从而避免了两步“酶-前药”疗法的缺陷。具体内容包括:(1)设计合成了还原敏感型三嵌段共聚物mPEG-PAsp(AED)-CA,自组装形成纳米胶束,共负载了HRP和EIA。PEG为胶束的外壳层,HRP通过静电作用于PAsp(AED)段的氨基上,EIA通过疏水作用包覆于胶束核层内。当胶束到达肿瘤部位后,聚合物胶束PAsp(AED)段上的二硫键在还原型谷胱甘肽的作用下断裂,胶束结构被破坏,HRP和EIA得到释放。EIA水解产生IAA,IAA被HRP激活将产生大量的ROS,杀死肿瘤细胞。通过透射电镜(TEM)测得负载HRP和EIA的胶束尺寸大约为40 nm,细胞存活率测试结果表明HRP/EIA胶束对人体肺腺癌细胞有很强的杀伤力,证明一步“酶-前药”疗法具有很大的潜力。(2)设计合成了pH敏感型三嵌段共聚物mPEG-PAsp(THA)-AcMal7,自组装形成纳米胶束,共负载了HRP和EIA。PEG为胶束外壳,PAsp(THA)段可静电吸附HRP,形成胶束次外层,AcMal7为pH敏感型疏水链段,用于形成疏水型的胶束内核来包覆EIA。当聚合物胶束到达肿瘤部位后,在肿瘤低的pH作用下,AcMal7脱保护由疏水变为亲水,此时,胶束结构被破坏,EIA和HRP得到释放,进而产生大量ROS,杀死肿瘤细胞。通过TEM对胶束形态进行了表征,通过MTT对细胞活率进行了测试,结果表明所制备pH敏感型聚合物胶束在酶前药一步疗法中具有很大的优势,与上一个工作相比,避免了氨基还原性对自由基的消耗,提高了治疗效率。
[Abstract]:Cancer has become one of the major causes of death in the world. At present, the main treatments for cancer are chemotherapy, photothermal therapy and radiation therapy, and so on. It has been widely studied. In recent years, "enzyme-prodrug" ("enzyme" therapy, EPT) as a new cancer treatment strategy, has shown good cancer. .EPT is a two step therapy: first, using the target strategy to transfer the prodrug activator to the tumor site; secondly, the nontoxic prodrug is injected through the blood circulation to the tumor tissue, and the nontoxic antitumor prodrug will produce a living anti swelling under the catalysis of the precursor activator. Tumor drugs. Although the "enzyme antiantineoantineoantineodrug" therapy has shown a broad prospect of application and brings a new idea to the treatment of cancer, there are still many key problems to be solved: "enzyme prodrug" therapy requires two steps of treatment, which increases the patient's pain and treatment cost, and the time of the second medication is not easy to take. For the antibody directed enzyme - prodrug therapy, the antibody enzyme coupling is injected into the body and the antibody identification of the tumor surface antigen is used to identify the tumor surface antigen, so that the enzyme is taken to the tumor target and then the antibody enzyme coupling is removed from the blood until the anticancer or low activity prodrug is injected into the body, otherwise the body is detained in the body. The enzymes that organize the organs will catalyze the prodrugs and then cause damage to the normal tissue, so the time for the second drug use is not easy to grasp. The dosage of the drug in the "enzyme pre drug" therapy is relatively large, because the drug can be used only to reach a certain concentration in the lesion, and the drug will be transmitted in the body. If the dosage is too small, the drug concentration is too small to cause the drug concentration to reach the lesion, but the dosage is too large will cause the accumulation of drugs, may cause certain toxic side effects to the body, and increase the cost of treatment. If the enzyme and the prodrug are transported to the tumor cells at the same time, the defects caused by the two step therapy will be Therefore, it is very important to develop an effective carrier that can transmit enzymes and prodrugs at the same time. The horseradish peroxidase indoloacetic acid (IAA-HRP) system is a kind of "enzyme - prodrug" system.IAA as a plant hormone, which is oxidized by HRP to include reactive oxygen free radicals (ROS), such as The free radicals, such as indolyl, 3- methyl indolyl and peroxidation, can cause cell membrane structure change, DNA damage, Bcl-2 activity and activation of apoptosis related genes by causing lipid peroxidation in cells, causing intracellular damage and apoptosis. However, the effect of the same concentration of IAA on normal cell growth is not significant, and IAA does not Peroxidase is easily oxidized by mammalian peroxidase. Ester bond is a very common bonding method in the design of prodrug. It can be hydrolyzed under the action of human esterase. EIA is a derivative of IAA. When it is transmitted to the tumor tissue, it is hydrolyzed under the action of esterase and the low pH of the tumor, producing the prodrug IAA. The polymer micelles formed by two Pro macromolecules through self-assembly have great potential in the delivery of antitumor drugs, siRNA and other therapeutic agents. Nanomicelles can first enter the tumor tissue through the EPR effect, greatly reducing the side effects of chemotherapy, while the micelle has a long blood circulation time, which can improve the benefit of the drug. In addition, polymer micelles have a design for tumor microenvironment. For example, the tumor tissue (pH 6.5-6.8), the pH 5.5-6.5 (pH 5.5-6.5) and the lysosome (pH 4.5-5.5) are microacid environments, different from the blood and normal tissues (pH 7.4). Therefore, the pH sensitive adhesive bundles can realize the intelligent controlled release of the tumor site drugs. In addition, the tumor part The concentration of reduced glutathione (GSH) in the cell is 100-1000 times that of the extracellular GSH. The reduction sensitive polymer micelles containing two sulfur bonds are disintegrated at the tumor site, and the micellar carrier is rapidly destroyed and the drug can be controlled to release in the tumor site. Therefore, the polymer micelle can be used as an ideal carrier to realize the enzyme and the enzyme. Combined transport of drugs. In this paper, the polymer micelles of different tumor microenvironment sensitive types were designed to combine the enzyme with the precursor, thus avoiding the defects of the two step "enzyme prodrug" therapy. The specific contents include: (1) the design and synthesis of the reduction sensitive three block copolymer mPEG-PAsp (AED) -CA and the self-assembled formation of nanoscale Micelles, CO loaded with HRP and EIA.PEG as the shell layer of the micelle, HRP by electrostatic action on the amino group of the PAsp (AED) section, EIA is coated in the nuclear layer of the micelle by hydrophobicity. When the micelle reaches the tumor site, the two sulfur bond on the PAsp (AED) segment of the polymer micelle breaks under the effect of the cystamine in the prototype Valley, the micelle structure is destroyed, HRP and EIA obtained. By releasing.EIA hydrolysis to produce IAA and IAA being activated by HRP, a large number of ROS will be produced to kill tumor cells. The micellar size of the loaded HRP and EIA is estimated to be about 40 nm by transmission electron microscopy (TEM). The cell viability test results show that the HRP/EIA micelle has a strong killing force on human lung adenocarcinoma cells, proving that one step "enzyme prodrug" therapy is very large. (2) (2) the design and synthesis of pH sensitive three block copolymer mPEG-PAsp (THA) -AcMal7, self-assembled to form nano micelle, CO loaded with HRP and EIA.PEG as micellar shell, PAsp (THA) segment can be electrostatic adsorbed HRP, form a micellar secondary layer, AcMal7 for pH sensitive hydrophobic chain section, to form a hydrophobic micellar core to encapsulate EIA. when polymer glue After the beam reaches the tumor site, the AcMal7 deprotection is changed from hydrophobicity to hydrophilic under the low pH action of the tumor. At this time, the micelle structure is destroyed, EIA and HRP are released, and a large number of ROS are produced and the tumor cells are killed. The micelle morphology is characterized by TEM and the cell viability is tested by MTT. The results show that the pH sensitive polymerization is prepared. The micelle has a great advantage in the one step therapy of enzyme predrug. Compared with the previous work, it avoids the consumption of amino reducibility to free radicals and improves the efficiency of treatment.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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