發(fā)布時(shí)間：2018-04-29 17:00

  本文選題：二硫鍵 + 氧化還原敏感��； 參考：《山東大學(xué)》2017年碩士論文

【摘要】：癌癥已經(jīng)成為危害人類(lèi)身體健康的主要因素之一,在世界范圍內(nèi)引起了廣泛關(guān)注。目前,在臨床上用于治療癌癥的主要方法包括化學(xué)治療、放射治療、手術(shù)治療等。多西他賽(DTX)是一種在臨床上已得到廣泛應(yīng)用的化療藥物,它是紫杉醇的半合成衍生物,是一種廣譜抗癌藥,對(duì)乳腺癌、卵巢癌、前列腺癌、肺癌、頭頸癌等癌癥均具有治療作用。但是,由于DTX的水溶性差,在臨床應(yīng)用中,通常在其注射液中加入一定量的吐溫-80等增溶劑以增加其溶解性,易引起嚴(yán)重的副反應(yīng),如過(guò)敏反應(yīng)等。另外,由于DTX的組織選擇性差,容易對(duì)正常細(xì)胞造成毒副作用。為了增加DTX的溶解性,增加其對(duì)腫瘤組織的靶向性,人們近些年研究了多種藥物傳遞系統(tǒng),如囊泡、納米粒、聚合物膠束、聚合物-藥物結(jié)合物膠束等。其中,聚合物膠束是由兩親性嵌段共聚物在水中自組裝形成的納米級(jí)別的核殼結(jié)構(gòu),親水性嵌段形成聚合物膠束的外殼,而疏水性嵌段組成其疏水性的內(nèi)核,該疏水內(nèi)核可以裝載疏水性的藥物。如果將藥物與親水性或者兩親性嵌段共聚物通過(guò)化學(xué)鍵結(jié)合,則可形成聚合物-藥物結(jié)合物膠束。聚合物-藥物結(jié)合物膠束具有載藥量高、穩(wěn)定性好、藥物釋放緩慢等優(yōu)點(diǎn),具有較好的抗腫瘤效果。另外,可通過(guò)環(huán)境敏感的化學(xué)鍵將聚合物與藥物結(jié)合,使聚合物-藥物結(jié)合物膠束具有氧化還原敏感性、pH敏感性、酶敏感性等等。為了增加DTX的溶解性,降低其毒副作用,本課題將DTX通過(guò)一個(gè)含有二硫鍵的連接臂與兩親性嵌段共聚物mPEG-PBLG相連,合成了氧化還原敏感的mPEG-PBLG-SS-DTX嵌段。該嵌段可以在水中自組裝形成膠束,并通過(guò)對(duì)此膠束進(jìn)行進(jìn)一步修飾,形成多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合膠束,該混合膠束同時(shí)具有氧化還原敏感性和主動(dòng)靶向性,表現(xiàn)出更好的抗腫瘤效果。本課題的主要研究?jī)?nèi)容包括以下三點(diǎn):(1)兩種具有不同分子量的 mPEG-PBLG-SS-DTX(mPEG2000-PBLG1750-SS-DTX 和 mPEG5000-PBLG1750-SS-DTX)的合成和表征。以具有不同分子量的mPEG-NH2為大分子引發(fā)劑,與γ-Bzl-L-Glu-NCA單體發(fā)生開(kāi)環(huán)聚合反應(yīng),生成具有不同分子量的兩親性嵌段共聚物mPEG-PBLG(mPEG2000-PBLG1750和mPEG5000-PBLG1750)。然后,通過(guò)一個(gè)含有二硫鍵的連接臂將DTX與mPEG-PBLG結(jié)合,得到氧化還原敏感的聚合物-藥物結(jié)合物mPEG-PBLG-SS-DTX。并分別利用1H-NMR和FT-IR確定中間產(chǎn)物和mPEG-PBLG-SS-DTX 的合成成功。(2)氧化還原敏感的mPEG-PBLG-SS-DTX膠束的制備、表征和體外抗腫瘤評(píng)價(jià)。由于聚合物-藥物結(jié)合物mPEG-PBLG-SS-DTX具有兩親性,在水溶液中可以自組裝形成具有核殼結(jié)構(gòu)的膠束,所以在本實(shí)驗(yàn)中我們通過(guò)透析法將其制備成膠束。該膠束在TEM下呈現(xiàn)出粒徑均一、圓整的球形,利用DLS測(cè)得的粒徑分別為101.3 ± 1.4和148.9 ±1.4 nm,且粒徑布均勻,這兩種膠束的Zeta電勢(shì)分別為-20.1 ± 0.1和-14.5 ± 0.1 mV,表明這兩種mPEG-PBLG-SS-DTX膠束具有較好的穩(wěn)定性。紫外分光光度計(jì)法測(cè)得的這兩種膠束的載藥量分別為(13.9 ±0.8)%和(9.2 ± 0.4)%。利用芘探針?lè)y(cè)得的CMC均較低,分別為3.98和6.94 μg/mL,這表明這兩種膠束即使在血液循環(huán)系統(tǒng)中被稀釋,仍可以保持結(jié)構(gòu)穩(wěn)定。該膠束具有氧化還原敏感性,藥物的體外釋放實(shí)驗(yàn)結(jié)果表明,在不存在還原性DTT的環(huán)境中,120h后,約有10%的DTT從膠束中釋放出來(lái),而在DTT存在的條件下,DTX的釋放速率明顯加快,120 h后,DTX的累積釋放量達(dá)到40%左右,這說(shuō)明DTX的釋放具有氧化還原敏感性。溶血性實(shí)驗(yàn)結(jié)果表明,這兩種膠束的溶血率均較小(5%),具有較好的血液相容性。在膠束的體外抗腫瘤評(píng)價(jià)中,以A549和MCF-7/ADR細(xì)胞為模型細(xì)胞。與DTX相比,mPEG-PBLG-SS-DTX膠束對(duì)這兩種細(xì)胞表現(xiàn)出更好的細(xì)胞抑制率,mPEG-PBLG-SS-DTX膠束對(duì)MCF-7/ADR細(xì)胞24 h的IC50值約為DTX的十五分之一。另外,體外攝取實(shí)驗(yàn)結(jié)果表明,A549和MCF-7/ADR細(xì)胞對(duì)mPEG-PBLG-SS-DTX/C-6膠束的攝取效率要強(qiáng)于對(duì)C-6的攝取效率,這與細(xì)胞毒性實(shí)驗(yàn)的結(jié)果一致。(3)多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合膠束的制備、表征和體外抗腫瘤評(píng)價(jià)。本部分選用具有較大載藥量的mPEG2000-PBLG1750-SS-DTX與含有主動(dòng)靶向分子的mPEG-FA混合,通過(guò)透析法制備了多功能的mPEG-PBLG-SS-DTX/mPEG-FA混合膠束,該混合膠束不僅具有氧化還原敏感性,還具有主動(dòng)靶向性。在加入mPEG-FA后,mPEG-PBLG-SS-DTX/mPEG-FA混合膠束呈現(xiàn)出粒徑均一、形態(tài)圓整的球形,且粒徑和載藥量分別為129.7±2.1 nm和(9.0 ± 1.7)%。利用芘探針?lè)y(cè)得的CMC為5.08 μg/mL,這與mPEG-PBLG-SS-DTX膠束的相似,表明該混合膠束在血液循環(huán)系統(tǒng)中可保持結(jié)構(gòu)的穩(wěn)定。溶血實(shí)驗(yàn)結(jié)果表明,不同濃度的mPEG-PBLG-SS-DTX/mPEG-FA混合膠束溶液的溶血率均小于5%,說(shuō)明該混合膠束不會(huì)引起紅細(xì)胞的溶血。藥物的體外釋放實(shí)驗(yàn)結(jié)果表明,mPEG-PBLG-SS-DTX/mPEG-FA混合膠束仍然具有氧化還原敏感性。另外,將葉酸受體(FR)低表達(dá)的A549細(xì)胞和FR高表達(dá)的MCF-7細(xì)胞作為模型細(xì)胞,對(duì)mPEG-PBLG-SS-DTX/mPEG-FA混合膠束的抗腫瘤效果進(jìn)行評(píng)價(jià)。細(xì)胞毒性實(shí)驗(yàn)結(jié)果表明,對(duì)于A549細(xì)胞,mPEG-PBLG-SS-DTX/mPEG-FA 混合膠束的抑制率與 mPEG-PBLG-SS-DTX 膠束無(wú)明顯差別,而對(duì)于MCF-7細(xì)胞,前者要明顯高于后者,這說(shuō)明mPEG-PBLG-SS-DTX/mPEG-FA混合膠束具有主動(dòng)靶向性,對(duì)FR高表達(dá)的腫瘤細(xì)胞具有更好的抗腫瘤效果。在細(xì)胞攝取實(shí)驗(yàn)中,A549細(xì)胞對(duì)mPEG-PBLG-SS-DTX/C-6 膠束、mPEG-PBLG-SS-DTX/mPEG-FA/C-6 混合膠束和mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合膠束+2 mM FA的細(xì)胞攝取效率無(wú)明顯的差別,而在MCF-7細(xì)胞中,mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合膠束進(jìn)入細(xì)胞的效率要明顯高于其他兩者,這表明mPEG-PBLG-SS-DTX/mPEG-FA/C-6混合膠束可通過(guò)FR介導(dǎo)的細(xì)胞內(nèi)吞作用進(jìn)入細(xì)胞,這使其細(xì)胞攝取效率得到了顯著的提高。細(xì)胞凋亡實(shí)驗(yàn)展現(xiàn)出了類(lèi)似的結(jié)果,mPEG-PBLG-SS-DTX膠束和mPEG-PBLG-SS-DTX/mPEG-FA混合膠束孵育12 h后,A549細(xì)胞的凋亡并無(wú)明顯差異,而在MCF-7細(xì)胞中,后者的凋亡效率要明顯高于前者。這些結(jié)果均表明,mPEG-PBLG-SS-DTX/mPEG-FA混合膠束具有主動(dòng)靶向性,對(duì)FR高表達(dá)的腫瘤細(xì)胞表現(xiàn)出更好的抑制效果。
[Abstract]:Cancer has become one of the major factors that harm the health of human beings and has aroused widespread concern around the world. The main clinical methods for the treatment of cancer include chemical therapy, radiation therapy, and surgical treatment. DTX is a clinically widely used chemotherapeutic drug, it is taxol Semisynthetic derivatives, a broad-spectrum anticancer drug, have therapeutic effects on breast, ovarian, prostate, lung and head and neck cancers. However, due to the poor solubility in water of DTX, a certain amount of Twain -80 is usually added to its injection to increase its solubility and cause serious side effects, such as excessive side effects. In addition, due to the poor tissue selectivity of DTX, it is easy to cause toxic and side effects to normal cells. In order to increase the solubility of DTX and increase the targeting of tumor tissue, a variety of drug delivery systems have been studied in recent years, such as vesicles, nanoparticles, polymer micelles, polymer micelles, polymer micelles and so on. It is a nanoscale nuclear shell structure formed by self assembly of two amphiphilic block copolymers in water. The hydrophilic block forms the shell of the polymer micelle, and the hydrophobic block consists of its hydrophobic core. The hydrophobic core can be loaded with hydrophobic drugs. If drug and hydrophilic or two amphiphilic block copolymers are bonded through chemical bonding, the hydrophobic core can be used as a hydrophobic core. Polymer drug binding micelles can form polymer drug binding micelles. Polymer drug binding micelles have the advantages of high drug loading, good stability and slow release of drugs, and have good antitumor effects. In addition, the polymer can be combined with drugs by environmental sensitive chemical bonds to make the polymer drug binding micelles have redox sensitivity. PH sensitivity, enzyme sensitivity and so on. In order to increase the solubility of DTX and reduce its toxic and side effects, DTX has synthesized a redox sensitive mPEG-PBLG-SS-DTX block by connecting a two parent block copolymer with a two sulfur bond arm, which can form micelles by self assembly in water and through this micelle. Further modification was carried out to form a multifunction mPEG-PBLG-SS-DTX/mPEG-FA mixed micelle. The hybrid micelle had both redox sensitivity and active targeting, and showed better anti-tumor effects. The main contents of this study included the following three points: (1) two kinds of mPEG-PBLG-SS-DTX (mPEG2000-PBLG1750-SS-) with different molecular weights. Synthesis and characterization of DTX and mPEG5000-PBLG1750-SS-DTX. Using mPEG-NH2 with different molecular weights as a macromolecular initiator and an open ring polymerization with gamma -Bzl-L-Glu-NCA monomer, two amphiphilic block copolymers with different molecular weights, mPEG-PBLG (mPEG2000-PBLG1750 and mPEG5000-PBLG1750), are produced. Then, a two sulfur bond is used. The joint arm combines DTX with mPEG-PBLG to obtain the redox sensitive polymer drug binding mPEG-PBLG-SS-DTX. and determine the synthesis of intermediate products and mPEG-PBLG-SS-DTX using 1H-NMR and FT-IR respectively. (2) the preparation, characterization and antitumor evaluation of the redox sensitive mPEG-PBLG-SS-DTX micelles. The compound mPEG-PBLG-SS-DTX has two affinity and can be self assembled into a micelle with nuclear shell structure in aqueous solution. So in this experiment, we prepared the micelles by dialysis. The micelles showed a uniform size and round ball under TEM. The size of the particles was 101.3 + 1.4 and 148.9 + 1.4 nm, respectively. The Zeta potential of these two micelles is -20.1 + 0.1 and -14.5 + 0.1 mV, respectively, indicating that the two mPEG-PBLG-SS-DTX micelles have good stability. The dosage of the two micelles obtained by UV spectrophotometer is (13.9 + 0.8)% and (9.2 + 0.4)% respectively. The CMC measured by pyrene probe method are lower, 3.98 and 6.94 mu, respectively. G/mL, which indicates that the two micelles can remain stable even if they are diluted in the blood circulation system. The micelles have redox sensitivity and the drug release in vitro shows that about 10% of DTT released from the micelles in the absence of reductive DTT environment, and the release of DTX under the presence of DTT. After 120 h, the cumulative release of DTX was about 40%, which indicated that the release of DTX was redox sensitivity. The hemolysis results of the two micelles showed that the hemolysis of these two kinds of micelles were smaller (5%) and had better blood compatibility. In the anti tumor evaluation of micelles, A549 and MCF-7/ADR cells were used as model cells. And DTX In contrast, mPEG-PBLG-SS-DTX micelles showed better cell inhibition rates for these two cells, and the IC50 value of mPEG-PBLG-SS-DTX micelles to MCF-7/ADR cells 24 h was about 1/15 of DTX. In addition, the uptake of A549 and MCF-7/ADR cells in vitro showed that the uptake efficiency of A549 and MCF-7/ADR cells to mPEG-PBLG-SS-DTX/C-6 micelles was stronger than that of C-6. This is in accordance with the results of cytotoxicity test. (3) preparation, characterization and in vitro anti-tumor evaluation of multifunction mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles. This part uses a mixture of mPEG2000-PBLG1750-SS-DTX with a larger dose and a mPEG-FA containing active target molecules, and the multifunction mPEG-PBLG-SS-DTX/mPEG-FA is prepared by dialysis method. The mixed micelles not only have redox sensitivity, but also have active targeting. After adding mPEG-FA, the mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles show a uniform particle size and round shape, and the particle size and drug loading are 129.7 + 2.1 nm and (9 + 1.7)% respectively. The CMC of the pyrene probe is 5.08 mu g/mL, which is with M The similarity of PEG-PBLG-SS-DTX micelles shows that the mixed micelles can maintain the stability of the structure in the blood circulation system. The hemolysis test results show that the hemolysis rate of the mixed micelle solution of different concentrations is less than 5%, indicating that the mixed micelle does not cause hemolysis of the red cells. The results of the drug release in vitro show that the mixture micelle does not cause hemolysis. MPEG-PBLG-SS-DTX/mPEG-FA mixed micelles still have redox sensitivity. In addition, the antitumor effect of mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles was evaluated by using A549 cells with low expression of folic acid receptor (FR) and MCF-7 cells with high expression of FR as model cells. The cytotoxicity test results showed that for A549 cells, mPEG-PBLG-SS-DTX. The inhibition rate of /mPEG-FA mixed micelles was not significantly different from that of mPEG-PBLG-SS-DTX micelles, but for MCF-7 cells, the former was significantly higher than that of the latter, which indicated that mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles had active targeting and had better anti-tumor effects on FR highly expressed tumor cells. In cell uptake experiments, A549 cells were to mPEG-PBLG There was no significant difference in the cell uptake efficiency of -SS-DTX/C-6 micelles, mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles and mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles, +2 mM FA, but in MCF-7 cells, the efficiency of mPEG-PBLG-SS-DTX/mPEG-FA/C-6 mixed micelles into cells was significantly higher than that of both of them, which indicated that mPEG-PBLG-SS-DTX/mPEG-FA/C-6 The mixed micelles can enter cells through the cell endocytosis mediated by FR, which significantly improves the efficiency of cell uptake. Apoptosis experiments show similar results. After incubation of 12 h micelles and mPEG-PBLG-SS-DTX/mPEG-FA micelles of mPEG-PBLG-SS-DTX micelles, there is no significant difference in the apoptosis of A549 cells, but in MCF-7 cells. The results showed that the mPEG-PBLG-SS-DTX/mPEG-FA mixed micelles had active targeting and showed a better inhibitory effect on the tumor cells with high expression of FR.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R943

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