本文關(guān)鍵詞： 透明質(zhì)酸 紫杉醇 二硫鍵 CD44受體 腫瘤治療 選擇性釋放　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：近年來(lái)藥物傳遞系統(tǒng)(Drug Delivery System, DDS)受到廣泛關(guān)注,因?yàn)榕c傳統(tǒng)制劑相比,DDS可以減少藥物毒副作用,提高藥物治療效果,提升藥物穩(wěn)定性,提高藥物的生物利用度(bioavailability)。目前已發(fā)展建立了多種類型的新型藥物遞送系統(tǒng),包擴(kuò)脂質(zhì)體(liposome),膠束(micelle)和納米粒(nanoparticle)等。但同時(shí)DDS也面臨缺少主動(dòng)靶能力和在病灶部位有效釋放活性藥物等一系列的問題。腫瘤與正常組織之間存在天然的氧化還原生理差異,即腫瘤細(xì)胞內(nèi)微環(huán)境中的還原型谷胱甘肽(glutathione, GSH)濃度遠(yuǎn)高于正常組織(100-1000倍)。利用此種特性,本研究構(gòu)建一種能主動(dòng)靶向腫瘤細(xì)胞CD44受體和在腫瘤高GSH微環(huán)境中觸發(fā)釋放的透明質(zhì)酸維生素E琥珀酸酯衍生物,以期實(shí)現(xiàn)DDS主動(dòng)靶向病灶部位和選擇性釋放藥物的目的。以親水性的透明質(zhì)酸(Hyaluronic acid, HA)為骨架,以維生素E琥珀酸酯(D-α-Tocopherol acid Succinate, α-TOS)為疏水鏈段,以具有二硫鍵的胱胺(氧化還原敏感型)和已二胺(非氧化還原敏感型)為橋鏈,通過酰胺化反應(yīng),合成了氧化還原敏感型和非氧化還原敏感型透明質(zhì)酸維生素E琥珀酸酯衍生物HA-SS-TOS (HSST)和HA-CC-TOS (HCCT),二者臨界膠束濃度分別為48.0 μg/mL和64.9μg/mL,具有良好的膠束形成能力。HSST和HCCT衍生物膠束的粒徑為114±11.6 nm和108±16.2 nm,電位為-20.9±4.3 mV和-18.2±4.2 mV。HSST和HCCT都能對(duì)紫杉醇(PTX)進(jìn)行有效包載,最高包封率和載藥量分別為88.3%,27.2%和89.7%,28.5%(w/w)。HSST在高GSH環(huán)境下快速釋放藥物,顯示出典型的氧化還原敏感性。以人卵巢癌細(xì)胞SKOV3為模型細(xì)胞,HSST和HCCT在SKOV3(細(xì)胞內(nèi)高GSH濃度)和丁硫氛酸—亞諷亞胺(Buthionine sulfoximine, BSO)預(yù)處理的SKOV3(細(xì)胞內(nèi)低GSH濃度)細(xì)胞中的攝取情況無(wú)明顯差異。HSST能選擇性在胞內(nèi)高GSH濃度的腫瘤細(xì)胞中快速釋放藥物,而在低GSH腫瘤細(xì)胞中釋放緩慢。聯(lián)合使用PTX與TOS可以增強(qiáng)PTX的抗腫瘤效果,起到協(xié)同抗腫瘤的作用。由于比HCCT釋藥更快,HSST體現(xiàn)出更強(qiáng)的腫瘤細(xì)胞增殖抑制率和選擇性殺傷腫瘤細(xì)胞的能力。采用免疫印跡技術(shù)(Western blot)和免疫熒光染色(Immunofluorescence technique)考察了CD44受體在SKOV3, A549, S180和CT26細(xì)胞組織上的表達(dá),結(jié)果顯示SKOV3, S180和CT26細(xì)胞組織為CD44受體高表達(dá),而A549為低CD44受體表達(dá),在此基礎(chǔ)上構(gòu)建了SKOV3/A549雙側(cè)皮下異位腫瘤小鼠模型。同時(shí)成功構(gòu)建了S180單側(cè)皮下異位腫瘤小鼠模型,S180雙側(cè)皮下異位腫瘤小鼠模型,CT26單側(cè)皮下異位腫瘤小鼠模型和CT26-Luc原位結(jié)腸癌腫瘤小鼠模型。系統(tǒng)地考察HSST膠束在上述腫瘤模型中CD44受體靶向和分布情況,以及膠束的體內(nèi)釋放情況。結(jié)果表明透明質(zhì)酸膠束在腫瘤中的累積是通過CD44受體介導(dǎo),具有特異性靶向腫瘤的能力；而其在高GSH腫瘤中快速釋放,說明透明質(zhì)酸膠束具有選擇性釋放藥物的能力。以S180單側(cè)皮下異位腫瘤小鼠和CT26-Luc原位結(jié)腸癌小鼠為動(dòng)物模型,紫杉醇載藥透明質(zhì)酸膠束對(duì)腫瘤的抑制效果呈現(xiàn)劑量依賴,載藥膠束具有明顯的抑制腫瘤生長(zhǎng)和轉(zhuǎn)移擴(kuò)散的作用。
[Abstract]:In recent years, drug delivery system (Drug Delivery System, DDS) received extensive attention, because compared with the traditional formulation, DDS can reduce the side effects of drugs, improve the therapeutic effect of drugs, enhance the stability of the drug and improve the bioavailability of drugs (bioavailability). Currently has developed many types of new drug delivery system, package expansion of liposomes (liposome), (micelle) and micellar nanoparticles (nanoparticle). But at the same time, DDS is also facing a lack of initiative and ability in target lesion effectively release the active drug and a series of problems. There is a natural redox physiological difference between tumor and normal tissue, the tumor microenvironment in cell type glutathione (glutathione, GSH) was much higher than that in normal tissue (100-1000 times). The use of this property, this study constructs a targeting tumor cells and CD44 receptor in tumor micro GSH Hyaluronic acid vitamin E succinate derivatives in triggering the release of the release of the drug to the lesion site and selectivity in order to achieve the DDS target. The hyaluronic acid hydrophilic (Hyaluronic acid HA) as a skeleton, with vitamin E succinate (D- -Tocopherol acid Succinate alpha, alpha -TOS) as hydrophobic chain in section, cysteamine had two disulfide (redox sensitive) and has two amine (non redox sensitive) as chain bridge, through amidation reaction, redox sensitive and non sensitive oxidation reduction synthesis of hyaluronic acid vitamin E succinate derivatives of HA-SS-TOS (HSST) and HA-CC-TOS (HCCT two), the critical micelle concentration were 48 g/mL and 64.9 g/mL, with good micelle forming ability of.HSST and derivatives of HCCT micelle size were 114 + 11.6 and 108 + 16.2 nm nm + 4.3 mV potential of -20.9 and -18.2 + 4.2 mV.HSST and HCCT The paclitaxel (PTX) effectively loaded, the highest encapsulation efficiency and drug loading were 88.3%, 27.2% and 89.7%, 28.5% (w/w).HSST quick release drugs in high GSH environment, showing typical redox sensitivity. In human ovarian cancer cells SKOV3 as model cell, HSST and HCCT in SKOV3 (high intracellular GSH concentration) and butyl sulfur atmosphere acid arylmerghylene imine (Buthionine sulfoximine, BSO a) pretreated SKOV3 (low intracellular GSH concentration) uptake in cells had no significant difference in.HSST can selectively quickly release the drug in cells with high GSH concentration in tumor cells, and tumor cells in low GSH in the slow release. The combined use of PTX and TOS can enhance the antitumor effect of PTX plays a synergistic antitumor effect. The drug release ratio HCCT faster, HSST shows the inhibition rate and selectivity of tumor cell proliferation and stronger killing tumor cells by Western blot technology ability. (Western blot) and immunofluorescence staining (Immunofluorescence technique) on CD44 receptor in SKOV3, A549, and CT26 expression of S180 cells, the results showed that SKOV3, S180 and CT26 cells for CD44 receptor expression and A549 low expression of CD44 receptor, on the basis of the construction of SKOV3/A549 bilateral subcutaneous ectopic tumor a mouse model of S180 was constructed successfully. At the same time, unilateral subcutaneous ectopic tumor model in mice, S180 bilateral subcutaneous ectopic tumor model in mice, CT26 unilateral subcutaneous ectopic tumor mice and CT26-Luc in colon cancer mouse model system to study HSST micelles CD44 in the tumor model receptor targeting and distribution, as well as the release of micelles in vivo. The results showed that the accumulation of hyaluronic acid micelles in tumors is mediated by CD44 receptor, with specific tumor targeting ability; and the high GSH in tumor Quick release that hyaluronic acid micelle has the ability to selectively release drugs. S180 unilateral subcutaneous ectopic tumor in mice and CT26-Luc mice as the animal model of colon carcinoma in situ, tumor inhibitory effect of paclitaxel hyaluronan micelles showed dose-dependent, micelle has obvious inhibition of tumor growth and metastasis diffusion effect.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R96

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1 張漢波;腫瘤還原微環(huán)境響應(yīng)的透明質(zhì)酸膠束抗腫瘤研究[D];浙江大學(xué);2017年

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本文編號(hào)：1545925