【作者】 万延辉；

【导师】 佟倜；

【作者基本信息】 吉林大学 ， 外科学， 2010， 博士

【摘要】 恶性肿瘤是威胁人类健康的常见、多发疾病;目前,恶性肿瘤的临床治疗采用以多种方式进行联合治疗。如手术治疗、放射治疗、化学治疗等。因恶性肿瘤具有浸润性生长及远处转移的特征,临床上肿瘤的一些微小转移灶难以被检测到,所以近年来学者提出,对于手术的肿瘤病人选择术前、术后化学药物治疗;这样能够提高病人的生存率。同时临床实践中较大一部分恶性肿瘤,于发现时已经失去手术机会。只能进行非手术治疗,而放化疗对机体的正常、肿瘤细胞进行无选择性的杀伤,尤其化学治疗对机体的毒副作用更大。故医学工作者在积极的探索更适合肿瘤治疗方式;肿瘤靶向治疗是近来备受关注的热点问题。聚合物纳米胶束作为靶向药物载体具有稳定性好、载药量高的优点,通过修饰能靶向于特定的器官、组织和细胞,是靶向研究的焦点,同时在临床上已取得了一定的进展,给恶性肿瘤的治疗带来了机遇。靶向治疗大体上分为:主动靶向(包括物理化学靶向、生物靶向)、被动靶向两类;靶向用药是指药物选择性地到达特定器官、组织或细胞,并在该靶部位发挥药物治疗作用。经选择性用药可以增加药物在病变部位的浓度,提高药物的生物利用度,并减少或消除在正常组织细胞的毒副作用。传统被动靶向治疗多是利用网状内皮系统的监视、吞噬作用来实现。此治疗对于单核-巨噬细胞系统相关的疾病有益,但很多疾病并不存在于该系统。为避免此系统对药物的吞噬,科学工作者利用肿瘤细胞的物理、化学、生物等特点对药物进行修饰,形成回避网状内皮系统监视的靶向作用,这样可以增强非单核-巨噬细胞系统的肿瘤的治疗效果。如利用肿瘤组织EPR效应的被动靶向治疗及肿瘤细胞表面受体、抗体的主动靶向治疗等。叶酸受体是一种糖蛋白受体,目前已知它在一系列上皮肿瘤细胞表面过度表达,如卵巢、肺、肾、乳腺、骨髓、间皮等,特别是在肿瘤晚期或者已经产生耐药性的肿瘤细胞,而在正常组织中很少或几乎不表达。因此,用一般方法难以治疗的肿瘤有可能极易被叶酸导向药物所克服。紫杉醇是多种肿瘤化疗的首选药物,但其来源有限且成本较昂贵。同时紫杉醇具有亲脂性的化学结构。临床用注射液是以聚氧乙烯蓖麻油和无水乙醇的混合液溶解药物,对机体有较强的毒副作用。为减少其毒副作用,增加生物利用度,人们积极的探索其前药的开发。根据肿瘤治疗的靶向理论,本研究选用两亲性生物降解高分子聚乙二醇与带侧羧基的聚(乳酸-碳酸酯)嵌段共聚物MPEG-b-P(LA-co-MCC)同紫杉醇(paclitaxel,PTX)键合,制成高分子的前体药物,并将其与携带叶酸配体的聚合物MPEG-b-P(LA-co-DHP/FA)组装成纳米胶束。紫杉醇是一种脂溶性药物,形成胶束后呈现壳-核的表面亲水结构,紫杉醇被包裹在核内增加其在水中的溶解度,延长药物在体内的循环时间,提高生物利用度,降低毒副作用,并对过表达叶酸受体的肿瘤细胞具有主动靶向性,对少或无叶酸受体的肿瘤细胞具有被动靶向作用;为探讨其对肿瘤的抑制作用,采用Lewis肺癌细胞及荷瘤小鼠进行药效实验,并进行相关检测以期为临床应用提供理论依据。更多还原

【Abstract】 Targeted chemotherapy of cancer is a hotspot in recent years. Targeted drug delivery means that the specific drugs can reach organs, tissues or cells selectively for treatment in the target area. The concentration and the therapeutic index of specific drugs in target site would increase through selective administration. And the toxicity of non-target site would decrease.Targeted therapy of drugs consist of active targeting and passive targeting.In recent years.The targeted drug delivery system of polymer micelles prepared by the amphiphilic block copolymers received extensive attention,and become the most important direction in the field of paclitaxel delivery system.The amphiphilic block copolymers can self-assemble into a unique "core - shell" structure of micelles in aqueous solutiondue to the different solubility of its hydrophilic and hydrophobic chains. The hydrophobic segments aggregate into nuclear to increase the solubility of liposoluble drugs; and the ydrophilic segments assemble together into a shell for the stability and protection of the micelles.Compared with other delivery systems, polymer micelle drug delivery system has many advantages:such as small size and hydrophilic shell of polymer micelles makes the drug-loaded micelles can avoid the recognition of human reticuloendothelial system (RES) for prolonged metabolic period in human blood circulation. The polymer micelles can accumulated in tumor tissue because of the high permeability and high retention in its vascular endothelial (EPR effect), which is beneficial to enhance the effect of specific drugs. Micelles as targeted drug carrier materials is a new drug delivery systems which can be transported to the diseased region selectively to improve the bioavailability of drugs and to reduce the side effects.Although the EPR effect of the nanoparticles with PEG, they can not deliver drugs to any specific target cells specifically.In order to further improve the drug concentration in targeted tumor region, People started to pay attention to ligand, protein and other special materials conjugated with the nanoparticles to achieve the purpose of active targeting in recent years.Folate receptor expression is highly conserved in normal tissues, which is only expressed on certain epithelial cells but highly expressed in most of the tumor cells from epithelial tissue.Compared with other receptor-mediated drug delivery system, the unique advantages of folate receptor system are as follows:①Without synthesis and purification of ligand protein, so the micelles are non-immunogenic compared with other protein ligand;②Relatively simple structure and easy synthesis;③Smaller, better pharmacokinetic characteristics, repeatable administration of drugs;④Only highly expressed in tumor cells, so it has a good tumor-specific effect;⑤The high expression of folate receptor in human tumors. Based on the above characteristics, the Folate-mediated active targeting therapy has become a research focus.But there are more active targeting drugs using liposomes as carriers so far. Polymer nanoparticles mediated as active targeting carriers is not very common, especially the polymer micelles. The biggest problem of liposomes is fragile to oxidation, as well as poor stability, medication leakage and low drug loading capacity.Although both the polymer nanoparticles and micelles are belong to colloidal systems,the former has good stability and high drug loading capacity and the surface can be easily modified for specific requirementscomparison with liposomes.Paclitaxel (PTX) as a natural anti-cancer active substance is isolated from the Taxus species and then made into liquid preparations by the application of advanced technology Which has a good therapeutic effect for a variety of malignant tumors. Because of its low water solubility, the paclitaxel is a mixture substances dissolved by polyoxyethylene castor oil and ethanol for clinical injection, which have a strong adverse reaction to the body, so its clinical application is greatly limited.Lung cancer is one of the most common malignant tumor, as a serious threat to human health and life. In chemotherapy program of lung cancer, the Taxol is a drug of first choice.To reduce the adverse reaction, and increase drug concentration in target site, people actively use related biological materials for its modification.In this study amphiphilic biodegradable polymer MPEG-b-P (LA-co-MCC) congugated with paclitaxel (PTX) to produce polymer prodrugs, then assemble into micelles with a polymer carrier MPEG-b-P (LA-co-DHP/FA) with folic acid ligand. Then the micelles as spherical particles is produced composed of hydrophilic shell and lipophilic core. Taxol as a liposoluble drug is encapsulated in the nucleus, and its solubility in water will increase as micelles with prolonged circulation in vivo as well as increased bioavailability and reduced toxicity and a targeted effect to tumor cells with overexpression of folate ligands.To investigate the tumor inhibition effect, Lewis lung cancer mice models were established in this experiment followed by associated detection to provide the theoretical basis for clinical application.The main results of this study are as follows:1. Conjugated the amphiphilic biodegradable polymer MPEG-b-P (LA-co-MCC) with paclitaxel (PTX) to produce prodrug of polymer micelle paclitaxel (PTX Micelle) MPEG-b-P (LA-co-MCC/PTX). Another amphiphilic biodegradable polymer MPEG-b-P (LA-co-DHP) conjugated with folic acid to produce the polymer micelleswith folic acid (FA-micelles) MPEG-b-P (LA-co-DHP/FA); so that the two mixed-assemble into micelles. Thus the folic acid-paclitaxel micelles (FA-PTX micelles) is prepared.(1) The characterization of Polymer-paclitaxel Conjugate Micelles (PTX-micelles and FA-PTX micelles collectively called) are as follows: These two micelles are spherical with distribution in uniform size and good dispersibility observed by Electron microscopy, compared with non-load particlesobtained with the same preparation conditions, Drugs has little effec on size and morphology of drug-loaded nanoparticles. Observation of the characteristics of size distribution in solution by dynamic light scattering of polymer conjugated paclitaxel nanoparticles show that "PTX micelle" and FA-PTX micelles are on a basis of a single symmetric distribution. The average size of the particles were 43.6nm, 58.3nm. which can avoid the monitor of reticuloendothelial system to achive the passive and active targeting effect; (2) The drug release behavior of the micelles in PBS detected by the HPLC show that PTX micelle release fast within 24 hours and the cumulative release rate reached to 40% followed by a continuous slow release. The cumulative release in 6 days reached to 80%.Paclitaxel has a low CMC with more stable molecular chains for a longer release time.2. In vitro (1) Detection of proliferation effect of chemical drugs on Lewis lung cancer cells by MTT method,most of the drugs got statistically significant inhibition on Lewis lung cancer cell lines and the inhibition is concentration and time-dependent. (2) Observation of the effect of chemical drugs on Lewis lung cancer cells under the microscope showed the adherent lost, the pseudopodia disappeared with rounded cells and slow growth with split-phase reduction or disappearance and the accumulation of rough particles within the cytoplasm; Integrity destruction, disintegration and rupture, and the cells crack into pieces. The same concentration (including PTX) with the same time, PTX, PTX micelles, FA-PTX micelles observed abnormal cell morphology became more and more clear with gradually increased degree of multi-cellular debris.(3) Flow cytometry detection of the chemical drugs on Lewis lung cancer cells show that the effect of PTX, PTX micelles, FA-PTX Micellar with 1μg/ml concentration of PTX on Lewis lung cancer cells after 72 hours, the apoptosis rate increased gradually with significantly difference compared with the negative control group (P﹤0.05), as well as the PTX micelles, FA-PTX with significant differences between the micelle (P﹤0.05). Show that the conjugated polymer-paclitaxel micelles can induce apoptosis and to achieve tumor inhibition.3. In vivo C57BL / 6 mice were inoculated with Lewis lung cancer cells for preparation of tumor-bearing mice models, with FA-PTX micelles, PTX micelles, PTX treatment respectively, with normal saline as the control group to detecte the efficacy; 7 and 14 days after drug administration, the relevant indicators detected as follows; (1) The general state of mice after drug administration: when injection in the mice tail vein, they showed irritability and screaming in PTX group; followed by side effects such as malaise, dark coat and significantly reduced diet 2 days after administration. All the other groups showed no significant adverse reactions. (2) 7 days after the dministration, three drugs of PTX, PTX micelles and FA-PTX micelles significantly inhibited tumor, compared with the control group (P<0.01) by mice weighing. There were statistically significant difference between FA-PTX micelle group and the other two drugs groups (PTX、PTX micelles) (P<0.05). 14 days after the administration, There were statistically significant difference between FA-PTX micelle group and the other two drugs groups (PTX、PTX micelles) and the control group (P<0.05). (3) TUNEL method to detect apoptosis. The results showed that 7 days, 14 days after the administration, the comparison of gray value of each drug group and control group had statistically differences (P<0.01). The grey values followed the order of Control > Taxol > PTX micelles > FA-PTX micelles. (4) The apoptosis rates of the tumor cells were evaluated by flow cytometry using Annexin V?FITC and PI double staining. All the Annexin V positive cells were considered as apoptotic ones. As shown in Figure 3 and Table 3, the apoptosis rates of the control group were significantly lower than those of the drug groups on the 7th and 14th day after drug administration (P<0.05), and there were also significant differences in apoptosis rate between the FA-PTX micelles group and the other two drug groups (P<0.05). Moreover, on the 7th day after drug administration, the percentage of apoptosis were in the order of Control < Taxol < PTX micelles < FA-PTX micelles, while the order was Control < Taxol<PTX micelles < FA-PTX micelles on the 14th day. (5) Immunohistochemistry to detect Caspase-3 and Bcl-2 protein expression:Caspase-3 protein in the treatment group is highly expressed and in the control group with only a slight expression. There are increased expression of Caspase-3 in tumor cells in treatment groups.And between PTX, PTX micelle micelles and FA-PTX groups showed an increased protein expression in gradient; Gray value of each group were statistically significant different (P<001). Bc1-2 protein expression: Bc1-2 protein expression of the control group is the highest, which is decreased in the treatment group.And with a decreased trend of PTX, PTX micelle micelles and FA-PTX in gradient; and the gray value of each group with statistically significant difference (P<001). (6) Survival analysis curve showed that the survival rate of treated mice by PTX, PTX micelles and FA-PTX micelle group was significantly higher than the control group.And the survival rate of PTX group, PTX micelle group, FA-PTX micelle group was also increasing.And FA-PTX micelle group increased significantly.The conclusions of this study:1. Our group prepared amphiphilic block copolymers with good encapsulation efficiency, suitable preparation and purification methods, and the carrier materials is biodegradable without toxic side effects. They can avoid the monitoring of reticuloendothelial system with the appropriate particle size and grain shape; with slow-release effect after a rapid release at the beginning; as well as good water-solubility. At the same time it can improve drug targeting effect through the surface modification of the carriers.2. Drug testing in vitro validate that Polymeric micelles with PTX and paclitaxel both induce apoptosis of tumor cells and played a role of tumor inhibition.At the same time, the apoptosis and tumor inhibition effect of polymer-paclitaxel conjugate is higher than pure PTX, and the FA-PTX micelle as the highest followed by PTX micelle; and the results confirm the role of drug targeting. The maximum inhibition rate on the tumor cells of FA-micelle at every concentration was 1.83%.The cells incubated with medium containing FA-micelles at various concentrations, has no difference in morphology compare with cells cultured without FA-micelles,as a further indication that the carriers are without toxic side effects.3. The detection of tumor mass weight, tumor inhibition rate in vitro showed that the polymer- paclitaxel micellar inhibited the solid tumor significantly; and tumor suppressor effect of FA-PTX micelle is higher than PTX and PTX micelle groups. The tumor pathology examination showed the formation of apoptotic bodies in drug treatment groups; detecting cells apoptosis by Flow cytometry and TUNEL method proved the drugs can induce the apoptosis of tumor cells; meanwhile the apoptotic effect of FA-PTX micelle is higher than other treatment groups.The molecular genetic detection of oncogene and tumor suppressor genes showed that micellar paclitaxel can up-regulate the expression of tumor suppressor genes and down-regulate the expression of oncogenes.These results indicate that one of the mechanisms of drug-induced apoptosis of conjugated micelles is to regulate the tumor suppressor gene and oncogene expression levels.The innovation of this study1. The Polymer-paclitaxel Micelles prepared by conjugation change the dosage forms of paclitaxel, and the micelles is water-soluble with controlled and sustained release, and good biological compatibility and targeting effect.2. Both the in vitro and in vivo experiments confirmed the apoptosis effect of Polymer-paclitaxel Micelles and pure drug. One of the mechanisms of its antitumor effect is through inducing cells’apoptosis. At the same time it indicated that another mechanism of drug-induced apoptosis of conjugated micelles is to regulate the tumor suppressor gene and oncogene expression levels.3. The paclitaxel conjugate nano-micelles is produced by paclitaxel-polymer prodrug (MPEG-b-P (LA-co-MCC/PTX)) mixed with polymer with folic acid (MPEG-b-P (LA-co-DHP/FA)) at a certain proportion to achieve active and passive targeting effect.Therefore, the detection of receptors on tumor cells is unnecessary before using the paclitaxel conjugate micelles.更多还原