本文選題：F68-VES + F127-SS-TOC��； 參考：《中國中醫(yī)科學(xué)院》2017年博士論文

【摘要】：目的癌癥是威脅人類生命健康的嚴(yán)重疾病,據(jù)統(tǒng)計,我國癌癥的發(fā)病率已經(jīng)接近世界水平,但死亡率卻高于世界水平。其中,乳腺癌又是我國乃至全球女性的“頭號殺手”,目前,臨床用于癌癥治療的手段主要有化療、放療、手術(shù)治療等,其中,化療手段較為普遍,它是一種全身性的治療手段,給藥后藥物會經(jīng)血液循環(huán)至全身大部分器官和組織以發(fā)揮治療其抗腫瘤效果�；熕幬镌诎┌Y治療過程中也面臨著自身局限性的挑戰(zhàn),如:水溶性差、毒副作用嚴(yán)重、生物利用度低等這些問題往往限制了其臨床應(yīng)用,因此,如何提高抗腫瘤有效成分的治療效果顯得尤為重要,而納米技術(shù)的發(fā)展恰好為開發(fā)新型抗腫瘤藥物制劑提供了新的希望。聚合物膠束是由兩親性聚合物在水溶液中自組裝形成的內(nèi)核疏水、外殼親水的納米制劑,也是難溶性藥物遞送體系的常見形式,它能夠增溶難溶性的抗腫瘤藥物,并且實現(xiàn)藥物的靶向遞送。Pluronic(普朗尼克)又稱泊洛沙姆,它是由聚氧乙烯(PEO)-聚氧丙烯(PPO)-聚氧乙烯(PEO)構(gòu)成的三嵌段兩親共聚物,且被美國、英國藥典收錄為藥用輔料,廣泛應(yīng)用于臨床。本研究以Pluronic中Pluronic F68(PEO80-PPO27-PEO80)和 Pluronic F127(PEO101-PPO56-PEO101)這兩種型號的聚合物作為研究對象,對其化學(xué)結(jié)構(gòu)進(jìn)行修飾改造,得到新合成的Pluronic F68-維生素E琥珀酸酯聚合物(F68-VES)和還原敏感的智能響應(yīng)型聚合物(F127-SS-TOC),擬達(dá)到降低Pluronic的CMC值,改善其抗稀釋能力,降低藥物在體循環(huán)過程中泄露的風(fēng)險等目的,此外,該膠束還可利用EPR效應(yīng)(高通透性和滯留效應(yīng))促使藥物被動靶向至腫瘤部位,提高藥效,并降低對正常組織的毒副作用。方法本研究主要圍繞兩大藥物遞送體系展開,即F68-VES藥物載體遞送米托蒽醌(MIT)用于抗腫瘤活性的研究及還原敏感載藥膠束F127-SS-TOC包載白藜蘆醇(RES)后的抗乳腺癌作用研究,具體研究工作包括聚合物的合成、膠束的制備、制劑學(xué)評價、藥效學(xué)評價。1.F68-VES/MIT藥物遞送體系的研究。(1)酯化反應(yīng)合成聚合物F68-VES,并用1H NMR和FTIR驗證其結(jié)構(gòu)。(2)采用溶劑揮發(fā)法制備F68-VES/MIT載藥膠束,DLS考察膠束粒徑、Zeta電位、穩(wěn)定性;TEM觀察膠束形態(tài);HPLC測定載藥膠束的包封率和載藥量;熒光探針法測定膠束CMC;透析法考察載藥膠束的體外釋放情況。(3)MTT法比較原藥MIT與F68-VES/MIT載藥膠束對乳腺癌細(xì)胞MCF-7及MDA-MB-231的細(xì)胞毒性。(4)采用流式細(xì)胞儀比較原藥MIT與F68-VES/MIT載藥膠束的促細(xì)胞凋亡作用,并用IN Cell Analyzer 2000觀察細(xì)胞核形態(tài)。(5)采用流式細(xì)胞儀比較MDA-MB-231細(xì)胞對原藥MIT與F68-VES/MIT載藥膠束的攝取能力,IN Cell Analyzer 2000觀察細(xì)胞攝取藥物情況。2.F127-SS-TOC/白藜蘆醇(RES)還原敏感藥物遞送體系的研究。(1)兩步合成聚合物F127-SS-TOC后用1HNMR和FTIR對其表征。(2)溶劑揮發(fā)法制備出F127-SS-TOC/RES還原敏感載藥膠束后對載藥膠束的包封率和載藥量、粒徑、Zeta電位、膠束形態(tài)、穩(wěn)定性、還原敏感性、CMC值、體外釋藥性能、生物相容性等性質(zhì)進(jìn)行考察。(3)MTT法考察原藥RES、還原敏感膠束F127-SS-TOC/RES及非還原敏感膠束F127-TOC/RES對乳腺癌細(xì)胞MCF-7及MDA-MB-231的體外抗細(xì)胞增殖作用。(4)藥物作用機(jī)制研究:考察還原敏感膠束F127-SS-TOC/RES對乳腺癌細(xì)胞MDA-MB-231內(nèi)ROS表達(dá)、細(xì)胞凋亡的影響。結(jié)果1.F68-VES/MIT藥物遞送體系的研究。(1)1H NMR,FTIR及13C NMR驗證F68-VES聚合物合成成功。(2)采用溶劑揮發(fā)法制備米托蒽醌載藥膠束F68-VES/MIT,采用DLS測得F68-VES/MIT 載藥膠束粒徑為 184.33±6.53nm(PDI:0.06±0.03),Zeta 電位為-32.67±0.87 mV,且膠束在不同濃度的FBS中穩(wěn)定性良好。HPLC測定載藥膠束包封率和載藥率分別為91.88±2.20%和5.85±0.89%。熒光探針法測定膠束臨界膠束濃度約為3.311 mg/L。藥物體外釋放實驗證明載藥膠束較原藥有緩釋作用。(3)MTT實驗表明F68-VES/MIT載藥膠束能顯著抑制乳腺癌細(xì)胞MCF-7及MDA-MB-231 增殖。(4)細(xì)胞凋亡實驗證明原藥MIT與F68-VES/MIT載藥膠束組對MDA-MB-231細(xì)胞均表現(xiàn)出明顯的促細(xì)胞凋亡作用,其中F68-VES/MIT載藥膠束組的細(xì)胞凋亡率(Q2+Q4)為60.8%顯著高于原藥MIT的凋亡率38.9%,這也預(yù)示F68-VES/MIT 載藥膠束比原藥MIT更能顯著誘導(dǎo)MDA-MB-231細(xì)胞凋亡,更好地發(fā)揮抗腫瘤效果。(5)細(xì)胞攝取實驗證明MDA-MB-231細(xì)胞對原藥MIT及F68-VES/MIT載藥膠束均表現(xiàn)出時間依賴性,且隨著時間增加,這兩組的攝取量均有增加,且在6 h達(dá)到最大攝取量。其中,F68-VES/MIT載藥膠束的細(xì)胞攝取能力又顯著強(qiáng)于原藥MIT,這可能與膠束能夠促進(jìn)細(xì)胞對其攝取,提高生物利用度有關(guān)。2.F127-SS-TOC/RES還原敏感藥物遞送體系的研究。(1)采用1H NMR及FTIR驗證F127-SS-TOC聚合物的結(jié)構(gòu),結(jié)果證明該材料合成成功。(2)用新合成的載體材料包載中藥抗腫瘤有效成分白藜蘆醇(RES),溶劑揮發(fā)法制備出還原敏感膠束F127-SS-TOC/RES,該膠束的的包封率和載藥率分別為 99.09±1.11%和13.25±2.29%,DLS測得膠束的平均粒徑為 39.50±1.39 nm,PDI 為 0.16±0.01,Zeta 電位為-4.55±0.39mV。結(jié)構(gòu)修飾過的 F127-SS-TOC 膠束的CMC值為7.58 mg/L,遠(yuǎn)小于F127的CMC值。該膠束7天內(nèi)穩(wěn)定性良好,即使在含有FBS的介質(zhì)中放置一定時間,膠束粒徑也無明顯變化。此外,該膠束還表現(xiàn)出明顯的還原敏感性,加入DTT 5 h后,膠束粒徑發(fā)生明顯變化,粒徑圖中的分布峰由單峰變?yōu)殡p峰,說明膠束開始破裂或聚集從而引發(fā)粒徑改變,分布不均勻。體外釋放實驗表明,與無還原劑存在的條件下相比,F127-SS-TOC/RES膠束較在10 mM DTT濃度下更能有利于藥物從膠束中釋放出來。載體材料的安全性也是人們關(guān)注的重點,本研究還用溶血實驗證明了F127-SS-TOC聚合物材料的生物相容性良好。(3)MTT實驗表明還原敏感F127-SS-TOC/RES膠束較非還原敏感F127-TOC/RES膠束和原藥RES對乳腺癌細(xì)胞有更強(qiáng)的抗增殖作用。(4)藥物濃度作用 48 后,原藥 RES、F127-SS-TOC/RES 及 F127-TOC/RES載藥膠束對MDA-MB-231細(xì)胞均表現(xiàn)出不同程度的促細(xì)胞凋亡作用,其中F127-SS-TOC/RES載藥膠束組的細(xì)胞凋亡率顯著高于原藥RES與F127-TOC/RES載藥膠束。這說明F127-TOC/RES膠束能顯著誘導(dǎo)MDA-MB-231細(xì)胞凋亡,更好地發(fā)揮抗腫瘤效果。(5)選擇乳腺癌細(xì)胞MDA-MB-231研究原藥RES、還原敏感膠束F127-SS-TOC/RES、非還原敏感膠束F127-TOC/RES對細(xì)胞內(nèi)ROS水平影響。結(jié)果表明MDA-MB-231細(xì)胞內(nèi)ROS水平高低:F127-SS-TOC/RES膠束組F127-TOC/RES膠束組原藥RES對照組。結(jié)論本研究通過合成F68-VES及還原敏感的F127-SS-TOC聚合物材料,制備出用于遞送難溶性抗腫瘤有效成分的給藥體系,通過一系列的載體材料評價、納米制劑性質(zhì)評價、體外藥效學(xué)評價揭示,基于Pluronic的聚合物載體材料能有效遞送抗腫瘤藥物,提高腫瘤部位的藥物濃度,增強(qiáng)藥效,降低毒副作用,為難溶性抗腫瘤有效成分的臨床應(yīng)用提供新的研究基礎(chǔ)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.
【學(xué)位授予單位】：中國中醫(yī)科學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R943

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