本文選題：紫杉醇 + PLGA　； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：背景:中藥治療是重要的癌癥治療手段之一,在臨床上應(yīng)用廣泛。中藥治療癌癥的優(yōu)勢不僅在于副作用少,避免腫瘤復(fù)發(fā),改善臨床癥狀,還可以提高病患的機(jī)體免疫力,延長患者的生存期。紫杉醇是臨床上常見的廣譜抗癌藥物,是紅豆杉發(fā)揮抗腫瘤作用的主要活性成分,但由于紫杉醇在水中以及常見藥用溶劑中的溶解性比較差,目前市售紫杉醇注射劑常以聚氧乙烯蓖麻油為溶劑,而該溶劑副作用大,容易引起嚴(yán)重的過敏反應(yīng)。同時紫杉醇作為一種細(xì)胞周期特異性的藥物,體內(nèi)低劑量持續(xù)給藥較一次性沖擊給藥的抑瘤效果更為理想。因此,為了解決上述問題,許多研究者將紫杉醇制成不同類型的緩釋劑型,如脂質(zhì)體、微乳、微球、納米粒等。這些新劑型一方面可以有效避免聚氧乙烯蓖麻油的使用,降低紫杉醇制劑的副作用;另一方面還可延長藥物在體內(nèi)的作用時間,增強(qiáng)療效。與普通微球相比較,多孔微球具有較大的比表面積和孔體積,藥物可吸附在多孔微球的表面或進(jìn)入孔道內(nèi)部,從而可根據(jù)機(jī)體需要制成不同時效的緩釋制劑發(fā)揮藥效,因此在藥物新劑型的開發(fā)與研究領(lǐng)域中受到人們越來越多的重視。多孔微球通過其常見骨架材料聚乳酸——輕基乙酸共聚物(poly(lactic-co-glycolic acid),PLGA)的降解作用實(shí)現(xiàn)緩釋藥物的目的,且PLGA聚合物可最終降解為水和C02,對人體沒有毒性,也沒有副作用,是FDA批準(zhǔn)的可安全使用的藥用高分子材料。因此若將紫杉醇制備成長效緩釋多孔微球制劑,一方面可以解決紫杉醇臨床應(yīng)用中注射劑水溶性差、過敏反應(yīng)嚴(yán)重等實(shí)際問題;另一方面通過對微球粒徑及孔徑的調(diào)控,可實(shí)現(xiàn)紫杉醇的精準(zhǔn)給藥。此外,在眾多的紫杉醇微球研究工作中,對于紫杉醇多孔微球的口服給藥系統(tǒng)研究未見報道。因此本論文擬詳細(xì)研究載紫杉醇長效緩釋多孔微球的可控制備,并對體內(nèi)外腫瘤治療效果進(jìn)行初步評價。目的:以紫杉醇(PTX)為模型中藥,采用牛血清白蛋白(BSA)為致孔劑,聯(lián)合復(fù)乳溶劑揮發(fā)法(water-oil-water multiple-emulsion-solvent evaporation method),制備載藥 PLGA 多孔微球。通過控制致孔劑BSA的含量,成球材料PLGA的LA/GA的比例、分子量,溶劑的種類等條件,實(shí)現(xiàn)PLGA多孔微球的可控制備,達(dá)到優(yōu)化多孔微球載藥性能的目的。此外還研究了 PLGA在12種不同有機(jī)溶劑中形成多孔薄膜的能力,為發(fā)展PLGA微球的成孔技術(shù)提供一種新的思路和實(shí)驗(yàn)方法。最后,通過體外細(xì)胞實(shí)驗(yàn)及體內(nèi)荷瘤小鼠模型驗(yàn)證載藥PLGA多孔微球的緩釋性能及抑瘤作用。方法:第一章空白PLGA多孔微球的制備及單因素考察采用致孔劑聯(lián)合復(fù)乳溶劑揮發(fā)法制備空白PLGA多孔微球,詳細(xì)研究BSA與PLGA的質(zhì)量比,PLGA的分子量以及LA/GA的比值對多孔微球粒徑和孔徑大小的影響。實(shí)驗(yàn)采用掃描電子顯微鏡(SEM)觀察多孔微球的形態(tài)特征,比較微球粒徑、孔徑大小及分散性,總結(jié)規(guī)律,優(yōu)化制備條件。第二章不同溶劑體系中PLGA多孔薄膜的制備研究12種不同有機(jī)溶劑中PLGA薄膜的成孔能力,采用光學(xué)顯微鏡對其表面形態(tài)特征進(jìn)行研究,為不使用致孔劑條件下的微球成孔提供實(shí)驗(yàn)基礎(chǔ)。第三章載紫杉醇PLGA多孔微球的制備及其體外抗腫瘤作用研究制備載紫杉醇PLGA多孔微球(PTX@PLGA),測定多孔微球的載藥量和包封率,考察生理?xiàng)l件下的釋放時效,評價不同孔徑和載藥量的PLGA多孔微球的緩慢釋藥特性。采用差示熱量掃描法、傅里葉紅外光譜法、X-射線粉末衍射法分析多孔微球中藥物的存在形式,綜合評價微球質(zhì)量,并運(yùn)用MTT法初步研究載紫杉醇PLGA多孔微球的體外抗腫瘤活性。第四章載紫杉醇PLGA多孔微球的動物體內(nèi)實(shí)驗(yàn)研究構(gòu)建荷瘤小鼠,采取灌胃隔天給藥方式,初步考察載紫杉醇PLGA多孔微球的體內(nèi)抗腫瘤作用。結(jié)果:第一章:致孔劑聯(lián)合復(fù)乳溶劑揮發(fā)法制備的空白PLGA多孔微球表面光滑圓整,SEM測得微球粒徑范圍為6-12 μm,出現(xiàn)頻率最高的粒徑為8-10 μm,微球孔徑分布于0.1-0.4μm。隨著BSA的濃度、PLGA的分子量和LA/GA的比值增大,空白PLGA多孔微球的粒徑和孔徑也與之呈正相關(guān)。第二章:在12種有機(jī)溶劑體系中考察PLGA薄膜的成孔性,結(jié)果表明:在乙腈溶液中,當(dāng)PLGA質(zhì)量濃度為10%,25℃條件下干燥,PLGA薄膜的成孔效果最佳。同樣采用復(fù)乳溶劑揮發(fā)法,在不使用致孔劑的條件下,成功制備出PLGA多孔微球。第三章:不同投藥比(10:1和10:3)及不同BSA的濃度(0.2、0.4、0.6、0.8)條件下制備的載紫杉醇PLGA多孔微球(PTX@PLGA),其釋放周期約9-10天。XRD、DSC及FTIR結(jié)果顯示PTX以無定形或分子形式成功包覆于PLGA多孔微球中。當(dāng)載藥多孔微球投藥比為10:1時,其載藥量為6.40±0.26%,包封率67.50±0.18%;當(dāng)投藥比為10:3時,其載藥量為13.74±0.21%,包封率為51.10±0.15%。MTT結(jié)果顯示不同濃度的載紫杉醇PLGA多孔微球分散液作用于人肺腺癌細(xì)胞(A549)和小鼠結(jié)腸癌細(xì)胞(CT26)時,在終濃度為4-8 mg/ml的范圍內(nèi)對A549和CT26細(xì)胞有明顯的抑制作用。第四章:CT26荷瘤小鼠模型表明給藥組PTX@PLGA 15 mg/kg能夠較明顯抑制小鼠的腫瘤生長。在灌胃給予PTX@PLGA 21天后,與模型組小鼠的瘤體積相比,治療組PTX@PLGA 15 mg/kg和25 mg/kg以及對照組PTX 15 mg/kg小鼠的瘤體積均減小,但治療組瘤體積小于單獨(dú)使用PTX組。除此而外,給藥組PTX@PLGA 15 mg/kg瘤體積小于25 mg/kg給藥組,說明PTX@PLGA 15 mg/kg是抑制CT26荷瘤小鼠腫瘤生長的更佳濃度。與模型組對比,給藥組對荷瘤小鼠的體重?zé)o顯著影響,這表明PTX@PLGA無顯著毒副作用。
[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.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R943;R96
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本文編號：1958590