载紫杉醇PLGA多孔微球的制备及其抗肿瘤活性研究

发布时间：2018-05-31 06:08

  本文选题：紫杉醇 + PLGA　； 参考：《扬州大学》2017年硕士论文

【摘要】：背景:中药治疗是重要的癌症治疗手段之一,在临床上应用广泛。中药治疗癌症的优势不仅在于副作用少,避免肿瘤复发,改善临床症状,还可以提高病患的机体免疫力,延长患者的生存期。紫杉醇是临床上常见的广谱抗癌药物,是红豆杉发挥抗肿瘤作用的主要活性成分,但由于紫杉醇在水中以及常见药用溶剂中的溶解性比较差,目前市售紫杉醇注射剂常以聚氧乙烯蓖麻油为溶剂,而该溶剂副作用大,容易引起严重的过敏反应。同时紫杉醇作为一种细胞周期特异性的药物,体内低剂量持续给药较一次性冲击给药的抑瘤效果更为理想。因此,为了解决上述问题,许多研究者将紫杉醇制成不同类型的缓释剂型,如脂质体、微乳、微球、纳米粒等。这些新剂型一方面可以有效避免聚氧乙烯蓖麻油的使用,降低紫杉醇制剂的副作用;另一方面还可延长药物在体内的作用时间,增强疗效。与普通微球相比较,多孔微球具有较大的比表面积和孔体积,药物可吸附在多孔微球的表面或进入孔道内部,从而可根据机体需要制成不同时效的缓释制剂发挥药效,因此在药物新剂型的开发与研究领域中受到人们越来越多的重视。多孔微球通过其常见骨架材料聚乳酸——轻基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)的降解作用实现缓释药物的目的,且PLGA聚合物可最终降解为水和C02,对人体没有毒性,也没有副作用,是FDA批准的可安全使用的药用高分子材料。因此若将紫杉醇制备成长效缓释多孔微球制剂,一方面可以解决紫杉醇临床应用中注射剂水溶性差、过敏反应严重等实际问题;另一方面通过对微球粒径及孔径的调控,可实现紫杉醇的精准给药。此外,在众多的紫杉醇微球研究工作中,对于紫杉醇多孔微球的口服给药系统研究未见报道。因此本论文拟详细研究载紫杉醇长效缓释多孔微球的可控制备,并对体内外肿瘤治疗效果进行初步评价。目的:以紫杉醇(PTX)为模型中药,采用牛血清白蛋白(BSA)为致孔剂,联合复乳溶剂挥发法(water-oil-water multiple-emulsion-solvent evaporation method),制备载药 PLGA 多孔微球。通过控制致孔剂BSA的含量,成球材料PLGA的LA/GA的比例、分子量,溶剂的种类等条件,实现PLGA多孔微球的可控制备,达到优化多孔微球载药性能的目的。此外还研究了 PLGA在12种不同有机溶剂中形成多孔薄膜的能力,为发展PLGA微球的成孔技术提供一种新的思路和实验方法。最后,通过体外细胞实验及体内荷瘤小鼠模型验证载药PLGA多孔微球的缓释性能及抑瘤作用。方法:第一章空白PLGA多孔微球的制备及单因素考察采用致孔剂联合复乳溶剂挥发法制备空白PLGA多孔微球,详细研究BSA与PLGA的质量比,PLGA的分子量以及LA/GA的比值对多孔微球粒径和孔径大小的影响。实验采用扫描电子显微镜(SEM)观察多孔微球的形态特征,比较微球粒径、孔径大小及分散性,总结规律,优化制备条件。第二章不同溶剂体系中PLGA多孔薄膜的制备研究12种不同有机溶剂中PLGA薄膜的成孔能力,采用光学显微镜对其表面形态特征进行研究,为不使用致孔剂条件下的微球成孔提供实验基础。第三章载紫杉醇PLGA多孔微球的制备及其体外抗肿瘤作用研究制备载紫杉醇PLGA多孔微球(PTX@PLGA),测定多孔微球的载药量和包封率,考察生理条件下的释放时效,评价不同孔径和载药量的PLGA多孔微球的缓慢释药特性。采用差示热量扫描法、傅里叶红外光谱法、X-射线粉末衍射法分析多孔微球中药物的存在形式,综合评价微球质量,并运用MTT法初步研究载紫杉醇PLGA多孔微球的体外抗肿瘤活性。第四章载紫杉醇PLGA多孔微球的动物体内实验研究构建荷瘤小鼠,采取灌胃隔天给药方式,初步考察载紫杉醇PLGA多孔微球的体内抗肿瘤作用。结果:第一章:致孔剂联合复乳溶剂挥发法制备的空白PLGA多孔微球表面光滑圆整,SEM测得微球粒径范围为6-12 μm,出现频率最高的粒径为8-10 μm,微球孔径分布于0.1-0.4μm。随着BSA的浓度、PLGA的分子量和LA/GA的比值增大,空白PLGA多孔微球的粒径和孔径也与之呈正相关。第二章:在12种有机溶剂体系中考察PLGA薄膜的成孔性,结果表明:在乙腈溶液中,当PLGA质量浓度为10%,25℃条件下干燥,PLGA薄膜的成孔效果最佳。同样采用复乳溶剂挥发法,在不使用致孔剂的条件下,成功制备出PLGA多孔微球。第三章:不同投药比(10:1和10:3)及不同BSA的浓度(0.2、0.4、0.6、0.8)条件下制备的载紫杉醇PLGA多孔微球(PTX@PLGA),其释放周期约9-10天。XRD、DSC及FTIR结果显示PTX以无定形或分子形式成功包覆于PLGA多孔微球中。当载药多孔微球投药比为10:1时,其载药量为6.40±0.26%,包封率67.50±0.18%;当投药比为10:3时,其载药量为13.74±0.21%,包封率为51.10±0.15%。MTT结果显示不同浓度的载紫杉醇PLGA多孔微球分散液作用于人肺腺癌细胞(A549)和小鼠结肠癌细胞(CT26)时,在终浓度为4-8 mg/ml的范围内对A549和CT26细胞有明显的抑制作用。第四章:CT26荷瘤小鼠模型表明给药组PTX@PLGA 15 mg/kg能够较明显抑制小鼠的肿瘤生长。在灌胃给予PTX@PLGA 21天后,与模型组小鼠的瘤体积相比,治疗组PTX@PLGA 15 mg/kg和25 mg/kg以及对照组PTX 15 mg/kg小鼠的瘤体积均减小,但治疗组瘤体积小于单独使用PTX组。除此而外,给药组PTX@PLGA 15 mg/kg瘤体积小于25 mg/kg给药组,说明PTX@PLGA 15 mg/kg是抑制CT26荷瘤小鼠肿瘤生长的更佳浓度。与模型组对比,给药组对荷瘤小鼠的体重无显著影响,这表明PTX@PLGA无显著毒副作用。
[Abstract]:Background: Traditional Chinese medicine is one of the important means of cancer treatment, which is widely used in clinical practice. The advantages of traditional Chinese medicine in the treatment of cancer are not only less side effects, avoid tumor recurrence, improve clinical symptoms, but also improve the immunity of the patients and prolong the life period of the patients. Taxol is a common broad spectrum anticancer drug in clinic, and it is Taxus chinensis. The main active component of antitumor activity is played, but the solubility of paclitaxel in water and common medicinal solvents is poor. At present, Paclitaxel injection is often used as a solvent of polyoxyethylene castor oil, and the side effect of the solvent is large and causes severe allergic reactions. Meanwhile, taxol is a cell cycle specific drug. In order to solve the above problems, many researchers have made paclitaxel into different types of sustained-release formulations, such as liposomes, microemulsion, microspheres, nanoparticles and so on. These new formulations can effectively avoid the use of polyoxyethylene castor oil and reduce the violet. The side effects of taxol preparation, on the other hand, can also prolong the action time of the drug in the body and enhance the effect. Compared with the ordinary microspheres, the porous microspheres have a larger specific surface area and pore volume. The drug can be adsorbed on the surface of the porous microspheres or into the inside of the channel, so that according to the needs of the body, the drug can be made into different aging sustained-release agents. As a result, more and more attention has been paid to the development and research of new drug dosage forms. Porous microspheres can be used to achieve the order of sustained-release drugs through the degradation of poly (lactic acid) copolymer (poly (lactic-co-glycolic acid), PLGA), which are common skeleton materials, and PLGA polymers can be degraded into water and C02 to the human body. There is no toxicity and no side effects. It is a safe and safe medicinal material approved by FDA. Therefore, if paclitaxel is prepared for the preparation of growth effect and sustained release porous microspheres, on the one hand, it can solve the practical problems such as poor water solubility and severe allergic reaction in the clinical application of taxol, and on the other hand, the particle size and pore size of the microspheres are adjusted. In addition, in the research work of paclitaxel microspheres, the oral administration of paclitaxel porous microspheres has not been reported. Therefore, this paper intends to study the controllable preparation of paclitaxel long effect sustained release porous microspheres and to evaluate the effect of tumor treatment in vitro and in vivo. Paclitaxel (PTX) is a model Chinese medicine, using bovine serum albumin (BSA) as a pore agent, combined with water-oil-water multiple-emulsion-solvent evaporation method to prepare porous PLGA porous microspheres. By controlling the content of BSA of the pore agent, the proportion of LA/GA for the PLGA of the ball material, the molecular weight and the type of solvent, etc. The controllable preparation of PLGA porous microspheres can achieve the purpose of optimizing the properties of porous microspheres. In addition, the ability of PLGA to form porous films in 12 different organic solvents is also studied. It provides a new idea and experimental method for the development of the pore forming technology of PLGA microspheres. Finally, it is verified by in vitro cell experiments and in vivo tumor bearing mice model. The sustained release properties and tumor suppressor effects of the porous PLGA microspheres. Methods: the preparation of PLGA porous microspheres in the first chapter and the single factor investigation were made by using the pore agent combined with the solvent evaporation method to prepare the blank PLGA porous microspheres. The mass ratio of BSA to PLGA, the molecular weight of PLGA and the ratio of LA/ GA to the particle size and pore size of the porous microspheres were studied in detail. The morphological characteristics of porous microspheres were observed by scanning electron microscope (SEM). The size, size and dispersion of microspheres were compared. The rules were summarized and the conditions for preparation were optimized. In the second chapter, the preparation of porous PLGA films in different solvent systems studied the pore forming ability of the PLGA films in 12 different organic solvents, and the optical microscope was used to determine the pore formation of the porous microspheres. The study of surface morphological characteristics provides an experimental basis for microspheres without pore agent. Third the preparation of paclitaxel PLGA porous microspheres and its anti-tumor effect in vitro, study the preparation of paclitaxel PLGA porous microspheres (PTX@PLGA), determine the drug loading and encapsulation efficiency of porous microspheres, evaluate the release aging under physiological conditions, and evaluate the release effect under physiological conditions. The slow release characteristics of PLGA porous microspheres with different pore size and drug loading were analyzed by differential thermal scanning, Fu Liye infrared spectroscopy and X- ray powder diffraction, the quality of microspheres was evaluated synthetically, and the antitumor activity of the poro microspheres loaded with PLGA was preliminarily studied by MTT method. Fourth The experimental study of paclitaxel PLGA porous microspheres in animals was carried out to construct the tumor bearing mice. The antitumor effect of paclitaxel PLGA porous microspheres was preliminarily investigated. Results: the first chapter: the surface of the porous microspheres prepared by the pore agent combined with the solvent evaporation method was smooth and round, and the particle size of the microspheres was measured by SEM. The range is 6-12 mu m, the highest particle size is 8-10 m, the microsphere pore size distribution is 0.1-0.4 mu m. with the concentration of BSA, the ratio of the molecular weight of PLGA and the ratio of LA/GA is increased. The diameter and pore size of the blank PLGA porous microspheres are also positively correlated. The second chapter: the pore formation of the PLGA thin film is investigated in the 12 organic solvent systems. The results show that: in B In nitrile solution, when the PLGA mass concentration is 10% and 25 C is dry, the pore forming effect of PLGA film is the best. The PLGA porous microspheres are successfully prepared by the reemulsion solvent evaporation method without the pore agent. The third chapters: the paclitaxel P prepared under the different dosage ratio (10:1 and 10:3) and the concentration of different BSA (0.2,0.4,0.6,0.8). The release period of LGA microspheres (PTX@PLGA) is about 9-10 days.XRD, and DSC and FTIR results show that PTX is successfully coated with PLGA porous microspheres in amorphous or molecular form. When the dosage of drug loading porous microspheres is 10:1, the drug loading is 6.40 + 0.26% and the encapsulation efficiency is 67.50 + 0.18%. When the dosage is 10:3, the drug loading is 13.74 + 0.21% and the encapsulation efficiency is 51.. The results of 10 + 0.15%.MTT showed that the different concentration of paclitaxel PLGA porous microspheres dispersed in human lung adenocarcinoma cells (A549) and mouse colon cancer cells (CT26), and had obvious inhibitory effects on A549 and CT26 cells in the range of final concentration of 4-8 mg/ml. The fourth chapter: CT26 bearing mice model showed that the drug group PTX@PLGA 15 mg/kg could be compared. The tumor growth of mice was significantly inhibited. Compared with the tumor volume in the model group, the volume of PTX@PLGA 15 mg/kg and 25 mg/kg in the treatment group and the PTX 15 mg/kg mice in the control group decreased after 21 days of gavage, but the volume of the tumor in the treatment group was less than that of the PTX group alone. In addition, the volume of PTX@PLGA 15 mg/kg tumor of the administration group was less than 25. The mg/kg administration group showed that PTX@PLGA 15 mg/kg was the better concentration of tumor growth in CT26 bearing mice. Compared with the model group, the group had no significant effect on the weight of the tumor bearing mice, which showed that PTX@PLGA had no significant toxic and side effects.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943;R96
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本文编号：1958590