基于Pluronic修饰的药物递送体系的构建及评价研究

发布时间：2018-05-23 07:40

本文选题：F68-VES + F127-SS-TOC　；参考：《中国中医科学院》2017年博士论文

【摘要】：目的癌症是威胁人类生命健康的严重疾病,据统计,我国癌症的发病率已经接近世界水平,但死亡率却高于世界水平。其中,乳腺癌又是我国乃至全球女性的“头号杀手”,目前,临床用于癌症治疗的手段主要有化疗、放疗、手术治疗等,其中,化疗手段较为普遍,它是一种全身性的治疗手段,给药后药物会经血液循环至全身大部分器官和组织以发挥治疗其抗肿瘤效果。化疗药物在癌症治疗过程中也面临着自身局限性的挑战,如:水溶性差、毒副作用严重、生物利用度低等这些问题往往限制了其临床应用,因此,如何提高抗肿瘤有效成分的治疗效果显得尤为重要,而纳米技术的发展恰好为开发新型抗肿瘤药物制剂提供了新的希望。聚合物胶束是由两亲性聚合物在水溶液中自组装形成的内核疏水、外壳亲水的纳米制剂,也是难溶性药物递送体系的常见形式,它能够增溶难溶性的抗肿瘤药物,并且实现药物的靶向递送。Pluronic(普朗尼克)又称泊洛沙姆,它是由聚氧乙烯(PEO)-聚氧丙烯(PPO)-聚氧乙烯(PEO)构成的三嵌段两亲共聚物,且被美国、英国药典收录为药用辅料,广泛应用于临床。本研究以Pluronic中Pluronic F68(PEO80-PPO27-PEO80)和 Pluronic F127(PEO101-PPO56-PEO101)这两种型号的聚合物作为研究对象,对其化学结构进行修饰改造,得到新合成的Pluronic F68-维生素E琥珀酸酯聚合物(F68-VES)和还原敏感的智能响应型聚合物(F127-SS-TOC),拟达到降低Pluronic的CMC值,改善其抗稀释能力,降低药物在体循环过程中泄露的风险等目的,此外,该胶束还可利用EPR效应(高通透性和滞留效应)促使药物被动靶向至肿瘤部位,提高药效,并降低对正常组织的毒副作用。方法本研究主要围绕两大药物递送体系展开,即F68-VES药物载体递送米托蒽醌(MIT)用于抗肿瘤活性的研究及还原敏感载药胶束F127-SS-TOC包载白藜芦醇(RES)后的抗乳腺癌作用研究,具体研究工作包括聚合物的合成、胶束的制备、制剂学评价、药效学评价。1.F68-VES/MIT药物递送体系的研究。(1)酯化反应合成聚合物F68-VES,并用1H NMR和FTIR验证其结构。(2)采用溶剂挥发法制备F68-VES/MIT载药胶束,DLS考察胶束粒径、Zeta电位、稳定性;TEM观察胶束形态;HPLC测定载药胶束的包封率和载药量;荧光探针法测定胶束CMC;透析法考察载药胶束的体外释放情况。(3)MTT法比较原药MIT与F68-VES/MIT载药胶束对乳腺癌细胞MCF-7及MDA-MB-231的细胞毒性。(4)采用流式细胞仪比较原药MIT与F68-VES/MIT载药胶束的促细胞凋亡作用,并用IN Cell Analyzer 2000观察细胞核形态。(5)采用流式细胞仪比较MDA-MB-231细胞对原药MIT与F68-VES/MIT载药胶束的摄取能力,IN Cell Analyzer 2000观察细胞摄取药物情况。2.F127-SS-TOC/白藜芦醇(RES)还原敏感药物递送体系的研究。(1)两步合成聚合物F127-SS-TOC后用1HNMR和FTIR对其表征。(2)溶剂挥发法制备出F127-SS-TOC/RES还原敏感载药胶束后对载药胶束的包封率和载药量、粒径、Zeta电位、胶束形态、稳定性、还原敏感性、CMC值、体外释药性能、生物相容性等性质进行考察。(3)MTT法考察原药RES、还原敏感胶束F127-SS-TOC/RES及非还原敏感胶束F127-TOC/RES对乳腺癌细胞MCF-7及MDA-MB-231的体外抗细胞增殖作用。(4)药物作用机制研究:考察还原敏感胶束F127-SS-TOC/RES对乳腺癌细胞MDA-MB-231内ROS表达、细胞凋亡的影响。结果1.F68-VES/MIT药物递送体系的研究。(1)1H NMR,FTIR及13C NMR验证F68-VES聚合物合成成功。(2)采用溶剂挥发法制备米托蒽醌载药胶束F68-VES/MIT,采用DLS测得F68-VES/MIT 载药胶束粒径为 184.33±6.53nm(PDI:0.06±0.03),Zeta 电位为-32.67±0.87 mV,且胶束在不同浓度的FBS中稳定性良好。HPLC测定载药胶束包封率和载药率分别为91.88±2.20%和5.85±0.89%。荧光探针法测定胶束临界胶束浓度约为3.311 mg/L。药物体外释放实验证明载药胶束较原药有缓释作用。(3)MTT实验表明F68-VES/MIT载药胶束能显著抑制乳腺癌细胞MCF-7及MDA-MB-231 增殖。(4)细胞凋亡实验证明原药MIT与F68-VES/MIT载药胶束组对MDA-MB-231细胞均表现出明显的促细胞凋亡作用,其中F68-VES/MIT载药胶束组的细胞凋亡率(Q2+Q4)为60.8%显著高于原药MIT的凋亡率38.9%,这也预示F68-VES/MIT 载药胶束比原药MIT更能显著诱导MDA-MB-231细胞凋亡,更好地发挥抗肿瘤效果。(5)细胞摄取实验证明MDA-MB-231细胞对原药MIT及F68-VES/MIT载药胶束均表现出时间依赖性,且随着时间增加,这两组的摄取量均有增加,且在6 h达到最大摄取量。其中,F68-VES/MIT载药胶束的细胞摄取能力又显著强于原药MIT,这可能与胶束能够促进细胞对其摄取,提高生物利用度有关。2.F127-SS-TOC/RES还原敏感药物递送体系的研究。(1)采用1H NMR及FTIR验证F127-SS-TOC聚合物的结构,结果证明该材料合成成功。(2)用新合成的载体材料包载中药抗肿瘤有效成分白藜芦醇(RES),溶剂挥发法制备出还原敏感胶束F127-SS-TOC/RES,该胶束的的包封率和载药率分别为 99.09±1.11%和13.25±2.29%,DLS测得胶束的平均粒径为 39.50±1.39 nm,PDI 为 0.16±0.01,Zeta 电位为-4.55±0.39mV。结构修饰过的 F127-SS-TOC 胶束的CMC值为7.58 mg/L,远小于F127的CMC值。该胶束7天内稳定性良好,即使在含有FBS的介质中放置一定时间,胶束粒径也无明显变化。此外,该胶束还表现出明显的还原敏感性,加入DTT 5 h后,胶束粒径发生明显变化,粒径图中的分布峰由单峰变为双峰,说明胶束开始破裂或聚集从而引发粒径改变,分布不均匀。体外释放实验表明,与无还原剂存在的条件下相比,F127-SS-TOC/RES胶束较在10 mM DTT浓度下更能有利于药物从胶束中释放出来。载体材料的安全性也是人们关注的重点,本研究还用溶血实验证明了F127-SS-TOC聚合物材料的生物相容性良好。(3)MTT实验表明还原敏感F127-SS-TOC/RES胶束较非还原敏感F127-TOC/RES胶束和原药RES对乳腺癌细胞有更强的抗增殖作用。(4)药物浓度作用 48 后,原药 RES、F127-SS-TOC/RES 及 F127-TOC/RES载药胶束对MDA-MB-231细胞均表现出不同程度的促细胞凋亡作用,其中F127-SS-TOC/RES载药胶束组的细胞凋亡率显著高于原药RES与F127-TOC/RES载药胶束。这说明F127-TOC/RES胶束能显著诱导MDA-MB-231细胞凋亡,更好地发挥抗肿瘤效果。(5)选择乳腺癌细胞MDA-MB-231研究原药RES、还原敏感胶束F127-SS-TOC/RES、非还原敏感胶束F127-TOC/RES对细胞内ROS水平影响。结果表明MDA-MB-231细胞内ROS水平高低:F127-SS-TOC/RES胶束组F127-TOC/RES胶束组原药RES对照组。结论本研究通过合成F68-VES及还原敏感的F127-SS-TOC聚合物材料,制备出用于递送难溶性抗肿瘤有效成分的给药体系,通过一系列的载体材料评价、纳米制剂性质评价、体外药效学评价揭示,基于Pluronic的聚合物载体材料能有效递送抗肿瘤药物,提高肿瘤部位的药物浓度,增强药效,降低毒副作用,为难溶性抗肿瘤有效成分的临床应用提供新的研究基础和研究方向。
[Abstract]:Objective cancer is a serious disease that threatens the life and health of human beings. According to statistics, the incidence of cancer in our country is close to the world level, but the mortality rate is higher than the world level. Among them, breast cancer is the "number one killer" of women in our country and in the world. At present, the main means of cancer treatment are chemotherapy, radiotherapy, and surgical treatment. Chemotherapy is more common. It is a systemic treatment. Drugs can be circulated through blood to most organs and tissues of the whole body after administration. The chemotherapeutic drugs also face their own limitations in the process of cancer treatment, such as poor water solubility, serious side effects and low bioavailability. Some problems often restrict its clinical application. Therefore, how to improve the therapeutic effect of anti tumor effective components is particularly important, and the development of nanotechnology provides a new hope for the development of new antitumor agents. Polymer micelles are the hydrophobic and hydrophilic shell of the two amphiphilic polymer in aqueous solution. Nanometers, a common form of insoluble drug delivery systems, can solubilized insoluble antitumor drugs, and the targeting delivery.Pluronic (Prang Nick), also known as mooring Losham, is a three block two parent copolymer composed of PEO - polyoxypropylene (PPO) - polyoxyethylene (PEO), and is the United States, UK The pharmacopoeia is used as medicinal materials and is widely used in clinic. This study uses two types of polymer, Pluronic F68 (PEO80-PPO27-PEO80) and Pluronic F127 (PEO101-PPO56-PEO101) in Pluronic as the research object, to modify the chemical structure and obtain the newly synthesized Pluronic F68- vitamin E succinate polymer (F68-VES) and The reduction of sensitive intelligent response polymer (F127-SS-TOC) is intended to reduce the CMC value of Pluronic, improve its anti dilution ability and reduce the risk of drug leakage during the body circulation. In addition, the micelles can also use the EPR effect (high permeability and retention effect) to induce the drug to target to the tumor site passively, improve the efficacy and reduce the effect. Methods the toxic and side effects on normal tissues. Methods this study was focused on two major drug delivery systems, namely, the study of the antitumor activity of the F68-VES drug carrier MIT for the antitumor activity and the anti breast cancer effect after the F127-SS-TOC encapsulated resveratrol (RES) containing the sensitive drug loaded micelles. The specific research work included the synthesis of polymers. Preparation of micelle, preparation evaluation, pharmacodynamic evaluation and pharmacodynamic evaluation of.1.F68-VES/MIT drug delivery system. (1) esterification synthesis of polymer F68-VES, and 1H NMR and FTIR to verify its structure. (2) F68-VES/MIT carrier micelles were prepared by solvent evaporation method, DLS was used to investigate micellar particle size, Zeta potential, stability; TEM observed micelle morphology; HPLC determination of drug loading The encapsulation efficiency and drug loading of micelles; fluorescence probe method to determine micelle CMC; the release of drug loaded micelles in vitro. (3) MTT method was used to compare the cytotoxicity of MIT and F68-VES/MIT drug loaded micelles to MCF-7 and MDA-MB-231 in breast cancer cells. (4) the cell apoptosis was compared by flow cytometry in the primary drug MIT and F68-VES/MIT carrier micelles. Use, observe nuclear morphology with IN Cell Analyzer 2000. (5) compare the uptake of MDA-MB-231 cells to MIT and F68-VES/MIT drug loaded micelles by flow cytometry. IN Cell Analyzer 2000 observe the uptake of drugs in cell.2.F127-SS-TOC/,.2.F127-SS-TOC/, resveratrol (RES) reduction sensitive drug delivery system. (1) two steps of synthetic polymerization After F127-SS-TOC, 1HNMR and FTIR were used to characterize it. (2) the encapsulation efficiency and drug loading of drug loaded micelles, particle size, Zeta potential, micelle morphology, stability, reduction sensitivity, CMC value, in vitro drug release performance, biocompatibility and other properties were investigated by solvent volatilization method. (3) MTT method was used to investigate the properties of RES, (3) MTT method. The anti cell proliferation effect of reduced sensitive micelle F127-SS-TOC/RES and non reduction sensitive micelle F127-TOC/RES on MCF-7 and MDA-MB-231 in breast cancer cells. (4) study on the mechanism of drug action: the effect of reduction sensitive micelle F127-SS-TOC/RES on ROS expression and apoptosis in MDA-MB-231 cells of breast cancer cells. Results 1.F68-VES/MIT drug delivery Study of system. (1) 1H NMR, FTIR and 13C NMR verify the success of F68-VES polymer synthesis. (2) using solvent evaporation method to prepare miceltrone drug micelle F68-VES/MIT, the particle size of the F68-VES/MIT carrier micelle is 184.33 + 6.53nm (PDI:0.06 + 0.03), the Zeta potential is 0.87, and the micelles are stable in different concentrations. The encapsulation efficiency and the drug loading rate of.HPLC were 91.88 + 2.20% and 5.85 + 0.89%. respectively. The determination of the critical micelle concentration of the micelles was about 3.311 mg/L. in vitro. The release experiment in vitro showed that the drug loaded micelles had a sustained release effect compared with the original drug. (3) the MTT experiment showed that the F68-VES/MIT loaded micelles could significantly inhibit the MCF-7 and MDA-MB-231 of breast cancer cells. (4) the apoptosis experiment showed that both the original drug MIT and the F68-VES/MIT carrier micelles showed obvious cell apoptosis effect on MDA-MB-231 cells, and the apoptosis rate (Q2+Q4) of the F68-VES/MIT drug loaded micelles group was significantly higher than that of the original drug MIT, and the apoptosis rate was 38.9%, which also indicated that the F68-VES/MIT drug micelles were more significant than the original drug MIT. Induction of apoptosis of MDA-MB-231 cells and better antitumor effect. (5) cell uptake experiments showed that MDA-MB-231 cells were time dependent on the drug MIT and F68-VES/MIT drug micelles of the original drug, and the uptake of these two groups increased with time, and the maximum uptake at 6 h. Among them, the cell uptake of F68-VES/MIT drug micelles was taken. The extraction capacity is significantly stronger than the original drug MIT, which may be related to the study of the micelles that can promote cell uptake by cells and improve the bioavailability of.2.F127-SS-TOC/RES reduction sensitive drug delivery systems. (1) 1H NMR and FTIR are used to verify the structure of F127-SS-TOC polymers. The results show that the material is successfully synthesized. (2) a newly synthesized carrier material is used in the loading process. The antitumor effective component of resveratrol (RES) was prepared by the solvent evaporation method. The encapsulation efficiency and drug loading rate of the micelles were 99.09 + 1.11% and 13.25 + 2.29% respectively. The average particle size of the micelles was 39.50 + 1.39 nm, PDI was 0.16 + 0.01 and Zeta potential was -4.55 + 0.39mV. structure modified F127-SS-TO. The CMC value of the C micelle is 7.58 mg/L, far less than the CMC value of F127. The micelle has good stability in 7 days, and the micelle size has no obvious change even in the medium containing FBS. In addition, the micelle also shows obvious reduction sensitivity. After adding DTT 5 h, the particle size of the micelles changes obviously, and the distribution peak in the particle size map is single peak. It is changed into Shuangfeng, which indicates that the micelles begin to break or gather so that the particle size changes and the distribution is uneven. In vitro release experiments show that, compared with the presence of no reducing agent, F127-SS-TOC/RES micelle is more beneficial to the release of drugs from the micelles at the concentration of 10 mM DTT. The safety of the carrier material is also the focus of people's attention. The study also showed that the biocompatibility of F127-SS-TOC polymer materials was good. (3) MTT experiment showed that the reduction sensitive F127-SS-TOC/RES micelles had stronger anti proliferative effect on breast cancer cells than non reduction sensitive F127-TOC/RES micelles and RES drug RES. (4) after the drug concentration was 48, RES, F127-SS-TOC/RES and F127-TOC/R of the drug were used. ES drug loaded micelles showed different degrees of apoptosis inducing effect on MDA-MB-231 cells, and the apoptosis rate of F127-SS-TOC/RES drug micelles group was significantly higher than that of RES and F127-TOC/RES drug loaded micelles. This indicated that F127-TOC/RES micelles could induce apoptosis of MDA-MB-231 cells and better play anti-tumor effect. (5) select breast cancer. Cell MDA-MB-231 studied the original drug RES, the reduction of sensitive micelle F127-SS-TOC/RES, and the effect of non reductive sensitive micelle F127-TOC/RES on the intracellular ROS level. The results showed that the level of ROS in MDA-MB-231 cells: F127-SS-TOC/RES micelle group F127-TOC/RES micelle group RES control group. Conclusion this study was conducted through the synthesis of F68-VES and reduction sensitive F127-SS-T. OC polymer material is used to prepare a drug delivery system for delivery of insoluble antitumor active ingredients. Through a series of carrier materials, evaluation of the properties of nanoscale preparations and in vitro pharmacodynamic evaluation revealed that Pluronic based polymer carrier materials can effectively deliver antitumor drugs, increase drug concentration in the tumor site, enhance drug efficiency, and reduce the effect. Toxic and side effects provide new research foundation and research direction for the clinical application of insoluble antitumor active ingredients.
【学位授予单位】：中国中医科学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943

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