【摘要】：在惡性腫瘤的化療過(guò)程中,化療藥物缺乏對(duì)腫瘤的選擇性,并且存在嚴(yán)重的副作用,降低了治療效果。然而納米藥物載體能夠改變藥物的體內(nèi)分布,對(duì)腫瘤組織具有靶向性,可延長(zhǎng)藥物的作用時(shí)間,降低毒副作用,在腫瘤的治療領(lǐng)域具有良好的應(yīng)用前景。 本文將亞油酸(Linoleic acid)通過(guò)酯化反應(yīng)偶聯(lián)到普魯蘭多糖(Pullulan)長(zhǎng)鏈上,獲得兩親性聚合物PULA1、PULA2和PULA3。通過(guò)傅里葉變換紅外光譜(FTIR)和核磁共振氫譜(1H NMR)對(duì)聚合物的化學(xué)結(jié)構(gòu)進(jìn)行表征,采用透析法制備自組裝納米膠束PULA,芘熒光探針?lè)y(cè)得納米膠束PULA1、PULA2和PULA3的臨界膠束濃度(CMC)分別為70,62和50μ.g/mL,動(dòng)態(tài)光散射(DLS)及透射電鏡(TEM)結(jié)果顯示納米膠束PULA呈球形且均勻分布,粒徑在80.1～156.8nm之間,體外溶血實(shí)驗(yàn)表明該納米膠束具有低溶血率,表現(xiàn)出良好的生物相容性。 以抗癌藥物阿霉素(DOX)為模型藥物,通過(guò)透析法制備了載藥納米膠束PULA/DOX,納米膠束PULA3/DOX的載藥量和包封率分別達(dá)8.05%和88.6%。載藥納米膠束P ULA/DOX在pH=7.4的磷酸緩沖液(PBS)中持續(xù)釋放DOX達(dá)96h以上,DOX的累積釋放量在43%～55%之間,并且DOX的釋放速率隨釋放介質(zhì)pH的降低逐漸加快,該納米膠束的pH敏感性有利于提高DOX在腫瘤組織的累積釋放量。 通過(guò)熒光顯微鏡和流式細(xì)胞儀檢測(cè)MCF-7和SMMC-7721細(xì)胞對(duì)納米膠束PULA的攝取情況,結(jié)果表明納米膠束PULA能夠攜帶大量的DOX進(jìn)入細(xì)胞；抑制劑處理后的細(xì)胞攝取實(shí)驗(yàn)表明PULA納米膠束主要通過(guò)耗能的、肌動(dòng)蛋白聚合介導(dǎo)的內(nèi)吞作用進(jìn)入細(xì)胞；納米膠束PULA2/DOX對(duì)細(xì)胞周期影響的結(jié)果顯示該載藥納米膠束主要將MCF-7和SMMC-7721細(xì)胞阻滯在S期,并且致使部分細(xì)胞出現(xiàn)凋亡；納米膠束PULA進(jìn)入細(xì)胞后的傳遞途徑及分布結(jié)果表明納米膠束PULA攜帶DOX經(jīng)過(guò)內(nèi)吞作用進(jìn)入細(xì)胞后,傳遞到溶酶體中,在溶酶體內(nèi)釋放出來(lái)的DOX逐步遷移到細(xì)胞核發(fā)揮抗癌效果；細(xì)胞毒性試驗(yàn)表明空白納米膠束對(duì)Raw264.7和HEK293正常細(xì)胞均沒(méi)有毒性,載藥納米膠束PULA/DOX對(duì)MCF-7和SMMC-7721細(xì)胞的毒性呈現(xiàn)時(shí)間和濃度依賴性；PULA2/DOX納米膠束的體內(nèi)藥物代謝結(jié)果表明PULA納米膠束能夠延長(zhǎng)DOX在血液中的保留時(shí)間,維持血液中DOX的含量在較高濃度。 綜上所述,PULA納米膠束是一種新型的具有良好生物相容性的載體材料,有望在癌癥治療領(lǐng)域作為疏水抗癌藥物DOX的傳遞載體。
[Abstract]:In the course of chemotherapy of malignant tumor, the chemotherapeutic drugs are lack of selectivity to the tumor, and have serious side effects, which reduces the therapeutic effect. However, nanopharmaceutical carriers can change the distribution of drugs in vivo, target tumor tissues, prolong the time of action of drugs, reduce toxic side effects, and have a good application prospect in the field of tumor treatment. In this paper, linoleic acid (Linoleic acid) was coupled to the long chain of (Pullulan) by esterification, and amphiphilic polymers PULA1,PULA2 and PULA3. were obtained. The chemical structure of the polymer was characterized by Fourier transform infrared spectroscopy (FTIR) and 1H NMR. The critical micelle concentration (CMC) of PULA1,PULA2 and PULA3 were 70 ~ 62 渭 g / mL and 50 渭 路g 路mL ~ (-1), respectively. The results of dynamic light scattering (DLS) and transmission electron microscopy (TEM) showed that the PULA of nano-micelles was spherical and uniform. In vitro hemolysis test showed that the micelle had low hemolysis rate and showed good biocompatibility. Using adriamycin (DOX) as a model drug, the drug loading and encapsulation efficiency of nano-micelle PULA3/DOX were 8.05% and 88.6%, respectively. The drug loaded nano-micelle P ULA/DOX continuously released DOX in pH=7.4 phosphate buffer (PBS) for more than 96 h, and the cumulative release of DOX was between 43% and 55%, and the release rate of DOX increased with the decrease of the release medium pH. The pH sensitivity of the nano-micelle is beneficial to increase the cumulative release of DOX in tumor tissue. The uptake of nano-micelle PULA by MCF-7 and SMMC-7721 cells was detected by fluorescence microscope and flow cytometry. The results showed that nano-micelle PULA could carry a large number of DOX into the cells. The results of cell uptake test showed that PULA nanoparticles entered the cells mainly through energy-consuming, actin polymerization mediated endocytosis. The effect of PULA2/DOX on cell cycle showed that the drug loaded nano-micelles mainly blocked MCF-7 and SMMC-7721 cells in S phase and caused apoptosis in some cells. The transmission pathway and distribution of nano-micelle PULA into cells showed that nano-micelle PULA carried DOX into the cells through endocytosis, then transferred to lysosome, and gradually migrated the DOX released in lysosome to the nucleus to play an anticancer effect. The cytotoxicity test showed that the blank micelles were not toxic to Raw264.7 and HEK293 normal cells. The toxicity of drug loaded nano-micelle PULA/DOX to MCF-7 and SMMC-7721 cells was time-and concentration-dependent. The results of drug metabolism of PULA2/DOX nanoparticles in vivo showed that PULA nanoparticles could prolong the retention time of DOX in blood and maintain the content of DOX in blood at a high concentration. In conclusion, PULA nanoparticles are a new biocompatible carrier material, which is expected to be used as the delivery carrier of hydrophobic anticancer drug DOX in the field of cancer treatment.
【學(xué)位授予單位】：大連理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943;TQ460.1

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