本文選題：肝癌靶向　切入點：pH敏感　出處：《濱州醫(yī)學院》2015年碩士論文

【摘要】：本文旨在設計、合成功能高分子材料,構建多功能主動靶向抗腫瘤藥物納米遞藥系統(tǒng)。(1)利用肝癌細胞高表達特異性去唾液酸糖蛋白的特點,合成了含有其配體吡喃半乳糖的兩親性嵌段聚合物MPEG-b-PMaIPG,利用其自組裝行為構建了一種肝癌靶向、pH敏感的多功能納米載藥體系。(2)利用惡性腫瘤細胞較正常細胞過度表達葡萄糖轉(zhuǎn)運蛋白1(GLUT 1)的特點,將其配體N-乙酰-D-氨基葡萄糖(NAG)共價鍵合到苯乙烯-馬來酸酐嵌段共聚物上,制備了主動靶向性聚合物NAG-P(St-alt-MA)58-b-PSt130納米藥物載體。第一部分利用原子轉(zhuǎn)移自由基聚合(ATRP)技術,制備了一種分子量分布窄(Mw/Mn=1.21)的含糖嵌段共聚物。用核磁共振氫譜(1H NMR)及凝膠滲透色譜(GPC)對其結構進行了表征,確定嵌段共聚物分子式為MPEG42-b-PMaIPG20。利用熒光光譜、納米粒徑Zeta電位分析儀(DLS)、投射電子顯微鏡(TEM)等對其自組裝行為進行了表征,實驗測得該共聚物的臨界膠束濃度為0.005 mg/mL;納米粒徑約為100±4.43 nm, Zeta電位為-32.8±0.23 mV。并以DOX為模型藥物,構建了納米載藥體系,載藥量為24.77±2.68%,包封率為66.12±9.44%。透射電子顯微鏡觀察載藥前后粒徑分別約50 nm、60 nm球形納米粒子。利用透析法考察了體外藥物釋放,72 h后不同pH環(huán)境下藥物釋放率分別為：pH 5.0時DOX釋放率達85%,而在pH 6.5、pH 7.4環(huán)境下DOX的釋放分別為65%、37%,表明該納米載藥納米體系具有明顯的pH敏感特性。紅細胞溶血實驗表明,濃度1.0 mg/mL時,MPEG42-b-PMaIPG2o納米粒子的溶血率均小于5%,符合生物醫(yī)用材料對溶血的要求。MTT法檢測MPEG42-b-PMaIPG20納米粒子對小鼠成纖維細胞(L929)及肝癌細胞(HepG2)在檢測范圍內(nèi)細胞存活率均大于95%,均未表現(xiàn)出細胞毒性。利用激光共聚焦(CLSM)和流式細胞術(FCM)對載藥納米粒子DOX-NPs的攝取做了定性、定量測定,結果表明相同實驗條件下,HepG2細胞對DOX-NPs的攝取率均高于A549細胞對DOX-NPs的攝取。以HepG2和A549細胞為細胞模型,考察了DOX-NPs和游離阿霉素(DOX)對細胞的生長抑制效果,結果顯示兩種測試樣品對細胞的生長抑制均隨藥物濃度的增大而提高,DOX-NPs在實驗濃度范圍內(nèi)對HepG2細胞產(chǎn)生較強的抑制效果,而對A549細胞的抑制作用則相對較小。第二部分制備一種新型的主動靶向聚合物納米載藥體系。通過FTIR和1HNMR對NAG接枝嵌段聚合物NAG-P(St-alt-MA)58-b-PSt130進行了結構表征。芘熒光探針熒光光度法測得該兩親性嵌段共聚物的臨界膠束濃度為0.028 mg/mL。動態(tài)光散射測得粒徑為56.27±0.43 nm, PDI為0.099,pH 7.4時納米粒子表面Zeta電位為-41.46±0.99 mV。負載脂溶性藥物DOX后,載DOX納米粒子粒徑為64.21 ±0.96 nm, PDI為0.10,載藥量為25.83±1.09%,包封率為69.69±3.98%。透射電子顯微鏡觀察載藥前后納米粒子均為球形,粒徑大小分布均勻,分別為50 nm,60 nm。體外藥物釋放證明載藥體系具有較好的緩釋功能。體外細胞毒性試驗證明該納米載體在檢測濃度范圍內(nèi)對ATⅡ細胞的生存率均80%,表現(xiàn)為低毒。以MCF-7細胞和HT29細胞作為模型細胞,利用激光共聚焦(CLSM)和流式細胞術(FCM)對載藥粒子NAG-NPs-DOX和NPs-DOX的攝取進行了定性、定量分析,對比實驗表明,相同實驗條件下兩種細胞對NAG-NPs-DOX的攝取均多于對NPs-DOX的攝取。同時考察了NAG-NPs-DOX、NPs-DOX對兩種模型細胞的生長抑制效果,結果顯示相同條件下NAG-NPs-DOX載藥體系對腫瘤細胞的抑制率均強于NPs-DOX對細胞的抑制率。研究結果初步證實,含葡萄糖轉(zhuǎn)運蛋白1(GLUT1)配體N-酰-D-氨基葡萄糖分子的NAG-P(St-alt-MA)58-b-PSt130納米載體有利于腫瘤細胞吞噬,具有腫瘤細胞靶向性。本論文研究了兩種基于受體-配體介導的腫瘤細胞靶向技術,通過體外實驗評價了這兩種載體材料的生物安全性和靶向性。實驗結果表明,本文所研究的自組裝聚合物納米粒子有望成為集緩控釋、藥物靶向于一體的抗腫瘤藥物載藥系統(tǒng)。
[Abstract]:This paper aims to design and synthesis of functional polymer materials, construction of multifunctional active targeting drug delivery system to anticancer drugs. The use of nano (1) liver cancer cells with high expression of specific asialoglycoprotein, synthesized the ligand containing galactopyranose two amphiphilic block polymer MPEG-b-PMaIPG, the self-assembly behavior was constructed a target for liver cancer, multifunctional nano pH sensitive drug carrier system. (2) compared with the normal expression of glucose transporter 1 by malignant cells (GLUT 1) characteristics of the ligand N- acetyl -D- glucosamine (NAG) covalently bonded to the styrene maleic anhydride copolymer, active targeted polymer to prepare NAG-P (St-alt-MA) 58-b-PSt130 nano drug carrier. The first part using atom transfer radical polymerization (ATRP) technology, the preparation of a narrow molecular weight distribution (Mw/Mn= 1.21) containing sugar block copolymer. Using nuclear magnetic resonance spectroscopy (1H NMR) and gel permeation chromatography (GPC) were used to characterize its structure, determine the copolymer molecular formula MPEG42-b-PMaIPG20. by fluorescence spectroscopy and nano particle size Zeta potential analyzer (DLS), transmission electron microscopy (TEM) and the self assembling behaviors were characterized and measured the critical micelle concentration of the copolymer was 0.005 mg/mL; the particle size is about 100 + 4.43 nm, Zeta potential is -32.8 + 0.23 mV. and DOX as the model drug and construct nanoparticle drug delivery system, drug loading was 24.77 + 2.68%, the encapsulation rate of 66.12 + 9.44%. transmission electron microscope before and after drug particle diameters about 50 nm, 60 nm spherical nanoparticles were investigated. The in vitro drug release by dialysis, after 72 h of different pH under the environment of drug release rates were: pH 5 DOX release rate reached 85%, while in the pH 6.5 release of DOX under the environment of pH 7.4 were 65%, 37%, show PH has the obvious characteristics of the sensitive drug loaded nano nano system. That red blood cell hemolysis test, the concentration of 1 mg/mL, the hemolysis of MPEG42-b-PMaIPG2o nanoparticles were less than 5%, requirements for the hemolysis of biomedical materials.MTT detection of MPEG42-b-PMaIPG20 nanoparticles on small rat fibroblasts with (L929) and hepatocellular carcinoma (HepG2) in the detection range within the cell survival rate was more than 95%, showed no cytotoxicity. By confocal laser scanning microscope (CLSM) and flow cytometry (FCM) on the uptake of DOX-NPs nanoparticles to do a qualitative, quantitative analysis results show that under the same experimental conditions, the DOX-NPs uptake rate of HepG2 cells was higher than that of DOX-NPs uptake with HepG2 and A549 cells. A549 cells, the effects of DOX-NPs and free adriamycin (DOX) inhibitory effect on cell growth, the results showed that two kinds of test samples on cell growth inhibition Was increased with the increasing of drug concentration, DOX-NPs has strong inhibitory effect on HepG2 cells in the experimental concentration range, and the inhibitory effect on A549 cells is relatively small. The second part for the preparation of a novel active targeting polymer nano drug carrier system. Through the FTIR and 1HNMR of NAG NAG-P (graft copolymer St-alt-MA) 58-b-PSt130 were characterized. The pyrene fluorescence probe fluorescence spectrophotometry measured the two amphiphilic block copolymers of critical micelle concentration of 0.028 mg/mL. measured by dynamic light scattering particle size of 56.27 + 0.43 nm, PDI 0.099, pH 7.4 Zeta nano particle surface potential of -41.46 + 0.99 mV. fat soluble load drug DOX, DOX nanoparticle size was 64.21 + 0.96 nm, PDI was 0.10, the drug loading was 25.83 + 1.09%, the encapsulation rate of 69.69 + 3.98%. transmission electron microscope before and after drug loaded nanoparticles were spherical, the particle size of points Uniform, were 50 nm, 60 nm. demonstrated the in vitro drug release drug carrier system with slow-release function better. In vitro cytotoxicity test showed that the nanoparticles in the detection range of concentration of AT II cell survival rate were 80%, showed low toxicity. MCF-7 cells and HT29 cells as a model cell, using laser scanning focus (CLSM) and flow cytometry (FCM) quantitative analysis of drug loaded particles NAG-NPs-DOX and NPs-DOX uptake were qualitative, comparative experiments show that the two species under the same experimental conditions the cellular uptake of NAG-NPs-DOX were more than the uptake of NPs-DOX. At the same time, the effects of NAG-NPs-DOX, the inhibitory effect of NPs-DOX on the two cell model the growth results show that under the same conditions NAG-NPs-DOX drug carrier system inhibition rate of tumor cells was stronger than NPs-DOX inhibitory rate of cells. The results confirmed that the content of glucose transporter 1 (GLUT1) ligand N- acyl -D Glucosamine molecules NAG-P (St-alt-MA) 58-b-PSt130 nanoparticles to tumor cell phagocytosis, with tumor cell targeting. This paper studied two kinds of tumor cells based on the target receptor ligand mediated to technology, in vitro evaluation of these two kinds of carrier materials of biological safety and targeting experiments. The results show that the self-assembly of polymer nanoparticles is expected to become the set of controlled-release drugs, targeted anticancer drug delivery system in one.

【學位授予單位】：濱州醫(yī)學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R943;R96

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本文編號：1664899