【作者】 张婵；

【导师】 赵良启；

【作者基本信息】 山西大学 ， 微生物学， 2011， 博士

【摘要】 众所周知,癌症是当前威胁人类健康的重大疾病之一,每年仅宫颈癌就会夺去20多万人的生命。在目前癌症的临床治疗中,化学抗癌药物发挥着极为重要的作用,不仅可以杀灭局部肿瘤组织,而且能够清除全身性的癌细胞。然而,这些化疗药物对机体有着强烈的毒副作用,会给患者带来很大的肉体与精神痛苦。因此,迫切需要设计与开发一种新型智能抗癌药物剂型。在新型抗癌药物剂型的研发中,靶向给药系统(TDDS)成为当代智能化药物剂型的热门课题。该系统的突出特点是将治疗药物定向运送到靶区,在显著提高靶区疗效的同时最大限度地减轻药物在非靶区的毒副作用,从而实现高效低毒的治疗策略。构建TDDS主要包括靶向材料、载体材料、抗癌药物及制备技术等要素。靶向材料决定着药剂在体内的运行方向与结合位点,是实现药剂靶向功能的关键。载体材料关系到药物的装载、运输、疗效及药代动力学,需要具备良好的生物相容性、适宜的生物可降解性和相应的加工可行性。抗癌药物赋予TDDS特异的抗癌作用。而制备技术是完成TDDS研发的核心要素,关系到药剂的成型性和生产可行性。通过查阅大量的国内外文献资料,分析比较了各种靶向材料、载体材料和制备技术的特点,设计了本文的研究方案。基于宫颈癌细胞表面叶酸受体(FR)过度表达的特性,选择其相应配体叶酸(FA)作为靶向材料。为了保证药物运输到靶区并延长其作用时间,选用具有良好的理化性质和生物学特性的生物高分子聚酯—羟基丁酸与羟基辛酸共聚物[P(HB-HO)]为载体材料,以阿霉素(DOX)为模型药物,采用W1/O/W2超声乳化法研制了叶酸靶向P(HB-HO)载阿霉素纳米粒,检测了纳米粒的基本特性参数,并进行了其靶向性和抑瘤效果的研究。研制叶酸靶向P(HB-HO)载阿霉素纳米粒的过程分为两个阶段。第一阶段合成具有靶向作用的载体材料[FA-PEG-P(HB-HO)]。根据氨基与羧基缩合形成酰胺键的化学反应原理,采用二步法操作程序,首先将偶联剂聚乙二醇二胺(H2N-PEG-NH2)与叶酸(FA)以酰胺键相连接,合成了FA-PEG-NH2。然后再以FA-PEG-NH2与P(HB-HO)进行酰胺化反应,合成FA-PEG-P(HB-HO),其收率达到86.39%。运用红外光谱(IR)和核磁共振氢谱(1H NMR)对该产物进行了结构表征,结果证实FA-PEG-P(HB-HO)合成取得成功。第二阶段研究了叶酸靶向P(HB-HO)载阿霉素纳米粒的制备技术及基本特性。以纳米粒粒径作为主要指标,比较了纳米粒的不同制备方法,最终确定W1/O/W2超声乳化法为最佳制备方法。以L9(34)正交设计法优化了W1/O/W2超声乳化法制备纳米粒的工艺条件,得出最佳的工艺条件为：阿霉素、Ⅰ’A-PEG-P(HB-HO)、PVA、Tween80和Span80的浓度分别为10%、6.5%、3%、5%和1%(W/V),油相与内水相体积比为3:1,超声功率为200W,超声时间180s。对载药纳米粒的基本特性进行了检测,结果表明：叶酸靶向P(HB-HO)载阿霉素纳米粒的平均粒径为241.6±9.3nm,载药量、包封率分别为29.6±2.9%、83.5±5.7%,体外累积释药率11d可达80%,其释放规律符合Higuchi方程。该载药纳米粒在4℃下放置6个月后,其粒径、载药量及包封率均无显著变化,稳定性较好。通过体外细胞试验考察了纳米粒对模型细胞株HeLa的细胞相容性、细胞靶向性、细胞毒性及凋亡率。HeLa细胞与纳米粒进行共培养后,经MTT法检测,结果表明：空白P(HB-HO)纳米粒和空白叶酸靶向P(HB-HO)纳米粒均未呈现出细胞毒性,证明P(HB-HO)和FA-PEG-P(HB-HO)具有良好的细胞相容性。当载药纳米粒的药物浓度范围在0.005-50μM时,分别对游离阿霉素、P(HB-HO)载阿霉素纳米粒、叶酸靶向P(HB-HO)载阿霉素纳米粒和添加1mM游离叶酸的叶酸靶向P(HB-HO)载阿霉素纳米粒进行了细胞毒性试验,结果表明：叶酸靶向P(HB-HO)载阿霉素纳米粒对HeLa细胞生长的抑制率最大,IC50最小,为0.87μM。说明叶酸对HeLa细胞具有靶向作用,由叶酸介导的载药纳米粒能更加有效的抑制HeLa细胞的增殖。纳米粒的药物浓度、培育时间、游离叶酸影响试验和倒置荧光显微镜观察结果进一步证明了叶酸靶向P(HB-HO)载阿霉素纳米粒对HeLa细胞具有靶向作用。经显微镜观察发现,叶酸靶向P(HB-HO)载阿霉素纳米粒组的细胞凋亡现象最为严重。当药物浓度为50μM时,流式细胞仪测得叶酸靶向P(HB-HO)载阿霉素纳米粒组的细胞凋亡率最高,达84.25±2.43%。与其它各给药组相比,有着显著性差异(p<0.05)。采用体内动物试验研究了叶酸靶向P(HB-HO)载阿霉素纳米粒在荷瘤裸鼠体内组织的分布、靶向作用及抗肿瘤效果。构建HeLa荷瘤裸鼠模型后随机分组,初步研究了纳米粒在体内组织的分布及靶向作用。结果表明：将阿霉素制备成纳米粒后,可以延长其在体内的半衰期,提高机体对阿霉素的生物利用度。同时能大大降低阿霉素对心脏的毒副作用。叶酸靶向P(HB-HO)载药纳米粒组在肿瘤组织中的阿霉素浓度明显高于其它给药组,在48h时该组肿瘤中阿霉素浓度分别为游离阿霉素组、P(HB-HO)载阿霉素纳米粒组的10.81、3.33倍,说明该纳米粒具有良好的肿瘤靶向性。抑瘤试验结果表明：叶酸靶向P(HB-HO)载阿霉素纳米粒组的瘤重与生理盐水组相比具有极显著性差异(p<0.01),其抑瘤率可达76.91%,且明显高于其它给药组。该组肿瘤组织在试验期间体积增长速率最慢,实验动物体重变化较为平稳。试验结束后该组瘤块体积最小,为178.91±17.43 mm3。瘤块组织学观察发现,叶酸靶向P(HB-HO)载阿霉素纳米粒组肿瘤细胞数目明显减少,分布较为稀疏,可以看到肿瘤细胞严重坏死。由此可见,叶酸靶向P(HB-HO)载阿霉素纳米粒具有良好的抑瘤效果。通过本文研究,我们首次建立了二步法合成FA-PEG-P(HB-HO)的技术,并获得了FA-PEG-P(HB-HO)产品。建立了叶酸靶向P(HB-HO)载阿霉素纳米粒制备工艺,并确定了主要技术参数,研发出了一种治疗宫颈癌的新型靶向给药纳米制剂。体内外试验结果表明,该纳米粒靶向性好,对宫颈癌疗效显著,对机体的毒副作用小,有着良好的应用前景。更多还原

【Abstract】 It is well known that cancer has become one of the most serious threats to human at present, and more than 20 million people die of cervical cancer worldwide each year. In the currently clinical treatments to cervical cancer, chemotherapy drugs play a very important role in killing local tumor tissues and removing systemic cancer cells. However, these drugs, owing to its drastic side effects, often bring cancer patients with great pain. Thus, it is necessary to design and develop a novel anticancer drug.Targeting drug delivery system (TDDS) with effective therapy and lower side effect has been one of the hottest topics in current pharmacology. TDDS is mainly composed of targeting ligand, carrier, anticancer drug and corresponding technique. The targeting effect and binding sites of TDDS is determined by the targeting ligand, and the drug loading and delivery is depended on carrier with biocompatibility, biodegradability and processing feasibility. Anticancer drug gives TDDS specific anticancer effects, and the technology is the core element of TDDS.After reading a large number of literatures, we have designed the research program of this paper. Because of over-expression of folate receptor (FR) on the surface of cancer cells, we selected its corresponding ligand folic acid (FA) as targeting ligand. In order to delivery drug to targeting site and extend its duration of action, we chose poly (3-hydroxybutyrate-co-3-hydroxyoctanoate) [P(HB-HO)] with good physical-chemical properties and biological characteristics as drug carrier. The DOX-loaded, folate-mediated P(HB-HO) nanoparticles (DOX/FA-PEG-P(HB-HO) NPs) were prepared by W1/O/W2 solvent extraction/evaporation method, adopting doxorubicin (DOX) as the model anticancer drug. Their physicochemical properties, targeting effect and pharmacodynamics were also investigated.The DOX/FA-PEG-P(HB-HO) NPs have been prepared in two stages. First, an original FA-PEG-P(HB-HO) conjugate with 86.39% yield was synthesized by amide bonds, and its chemical structure was confirmed by FTIR and’H NMR spectroscopy. The DOX/FA-PEG-P(HB-HO) NPs were then prepared by W1/O/W2 ultrasonic emulsification method. The correlative parameters of the method were optimized by L9(34) orthogonal design, and the optimal conditions were as follows:the concentration of DOX, FA-PEG-P(HB-HO), PVA, Tween80 and Span80 were 10%,6.5%,3%,5% and 1%(W/V), separately. The volume ratio of oil phase/internal water phase was 3:1, and the ultrasonic output and time was 200W and 180s, respectively. The average size, drug loading capacity and encapsulation efficiency of such NPs were found to be 241.6±9.3nm,29.6±2.9% and 83.5±5.7%. The in vitro release profile displayed that nearly 80% DOX was released in the first 11 days and its release formulation fitted to Higuchi equation. There were no great changes in the properties of the NPs when they stored for 6 months at 4℃. Therefore, the DOX/FA-PEG-P(HB-HO) NPs have excellent stability.The cellular compatibility, cellular targeting, cytotoxicity and apoptosis of these NPs to HeLa cells were investigated by in vitro cell line experiments. The results of MTT showed that no toxicity was observed after incubation with blank P(HB-HO) NPs and blank FA-PEG-P(HB-HO) NPs, and the two polymers displayed satisfactory cellular compatibility. The DOX/FA-PEG-P(HB-HO) NPs (IC50=0.87μM) displayed greater cytotoxicity to HeLa cells than other treated groups. The intracellular uptake tests of the NPs in vitro and fluorescence observation showed that the DOX/FA-PEG-P(HB-HO) NPs were efficiently taken up by HeLa cells. In addition, the results of microscope observation and flow cytometry exhibited that the apotosis of HeLa cells treated with 50μM DOX/FA-PEG-P(HB-HO) NPs was 84.25±2.43%, with significant difference (p<0.05). These data in vitro demonstrated that DOX/FA-PEG-P(HB-HO) NPs could target the HeLa cells efficiently and lead to a strong cytotoxicity due to high affinity of FA and FR.The tissue distribution, targeting effect and anti-tumor activity of DOX/FA-PEG-P(HB-HO) NPs were evaluated by in vivo experiments. After building HeLa xenograft tumor models, the BALB/c nude tumor-bearing mice were randomly divided into several groups. The results of tissue distribution and targeting tests showed that the half-life of the DOX was prolonged, its bioavailability was enhanced and its toxicity to heart was greatly reduced when DOX was prepared as NPs, the DOX concentration of DOX/FA-PEG-P(HB-HO) NPs group was much higher than other treated groups, and the DOX concentration in the tumors of this group was 10.81 and 3.33 times higher than free DOX group and DOX/P(HB-HO) NPs group, respectively. Thus, the DOX/FA-PEG-P(HB-HO) NPs have good targeting to HeLa tumors. In vivo anti-tumor activity demonstrated that the tumor weight of DOX/FA-PEG-P(HB-HO) NPs group was much smaller than control group (p<0.01), and the inhibit tumor rate was 76.91%. The final mean tumor load of the NPs was 178.91±17.43 mm3, remarkably smaller than other treated groups. Furthermore, the body weight change of DOX/FA-PEG-P(HB-HO) NPs group was very stable. Histopathology observations of tumors displayed that necrosis areas after DOX/FA-PEG-P(HB-HO) NPs treatment were severe. All these results have illustrated that DOX/FA-PEG-P(HB-HO) NPs are effective in targeting treatment of tumors.In conclusion, the technique of synthesizing FA-PEG-P(HB-HO) was built, and the product was achieved for the first time. Moreover, the technique and technical parameters of preparing DOX/FA-PEG-P(HB-HO) NPs were established, and a novel targeting nano-agent for treatment of cervical cancer was developed. The results of in vitro and in vivo experiments have illustrated that the NPs with little side effects are effective in the ability of active targeting, and are remarkable in treating cervical cancer. The new TDDS may represent a promising prospect in future.更多还原