本文選題：二氧化硅納米顆粒　切入點(diǎn)：pH響應(yīng)　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：臨床上傳統(tǒng)的癌癥治療手段包括手術(shù)治療、放療和化療等,其中化療被用得最為普遍,但是在化療過(guò)程中,化療藥物的毒副作用以及多重耐藥性(MDR)使得化療的效果被受限制,同時(shí)病人也承受巨大的痛苦。為了解決這兩個(gè)難題,可在正常組織環(huán)境下減少藥物泄漏的刺激-響應(yīng)性納米藥物遞送系統(tǒng)已被發(fā)明和廣泛利用,當(dāng)納米藥物遞送系統(tǒng)被癌細(xì)胞攝取進(jìn)入到特定靶向位置時(shí),通過(guò)環(huán)境的刺激性誘導(dǎo)藥物的釋放進(jìn)而減小藥物對(duì)正常組織和細(xì)胞的毒副作用。諾貝爾醫(yī)學(xué)獎(jiǎng)獲得者Christian de Duve提出可以通過(guò)溶酶體引起細(xì)胞死亡從而克服癌細(xì)胞MDR。由于溶酶體膜的破損使得部分細(xì)胞自溶,當(dāng)溶酶體膜通透性提高時(shí),某些特定的組織蛋白酶將從溶酶體釋放到細(xì)胞質(zhì)中,這被認(rèn)為可引起細(xì)胞凋亡或類似凋亡途徑的細(xì)胞死亡。這一途徑能有效地克服癌細(xì)胞的MDR,為癌癥的治療提供了新方向。二氧化硅納米粒子作為藥物遞送系統(tǒng)載體的應(yīng)用已成為納米醫(yī)藥研究的一大熱點(diǎn),主要因?yàn)槎趸杓{米粒子具有無(wú)毒性、粒徑均一、比表面積大、生物相容性好等特點(diǎn)。特別是具有某種響應(yīng)性的納米載體,通過(guò)刺激因子的作用達(dá)到對(duì)負(fù)載藥物的可控釋放�；诖�,本論文以兩種不同結(jié)構(gòu)的二氧化硅納米粒子為載體構(gòu)建多種pH響應(yīng)的藥物遞送系統(tǒng),詳細(xì)探究了藥物遞送系統(tǒng)在不同pH環(huán)境下藥物的釋放行為及研究其對(duì)人乳腺癌細(xì)胞和人乳腺癌耐藥細(xì)胞活性的抑制作用。具體包括以下三部分內(nèi)容:一、基于介孔二氧化硅(MSNs)的納米載藥系統(tǒng)的構(gòu)建及可控釋放研究。以十六烷基三甲基氯化銨(CTAC)為表面活性劑,正硅酸乙酯(TEOS)為硅源,三乙醇胺(TEA)為堿源成功制得粒徑均一,排列規(guī)整的MSNs。通過(guò)透射電子顯微鏡(TEM)、紅外光譜儀(IR)、氮?dú)馕脚c解析和比表面積分析儀(BET、BJH)等手段對(duì)MSNs的理化性質(zhì)進(jìn)行了表征。以廣譜抗癌藥物阿霉素(DOX)作為藥物模型分子,得到負(fù)載DOX的藥物遞送系統(tǒng)(DOX@MSNs):考察了DOX在MSNs中的載藥情況及DOX@MSNs在不同pH條件下藥物的釋放行為。實(shí)驗(yàn)結(jié)果表明,其載藥量和包封率分別為5.8%、27.7%,藥物的釋放具有很好的pH響應(yīng)性,在中性pH 7.4條件下藥物釋放較少(7.62%)且非常緩慢,而在弱酸性環(huán)境下隨時(shí)間的延長(zhǎng)藥物釋放增大(達(dá)42.2%)。這種pH響應(yīng)藥物釋放特性能夠更好地發(fā)揮抑制腫瘤的作用,細(xì)胞毒性實(shí)驗(yàn)結(jié)果表明,MSNs本身對(duì)細(xì)胞無(wú)毒性,具有很好的生物相容性。另外,載阿霉素納米藥物遞送系統(tǒng)DOX@MSNs對(duì)細(xì)胞的殺傷能力較游離DOX強(qiáng),即納米載藥系統(tǒng)具有很好的抗腫瘤作用。該納米載藥系統(tǒng)具有潛在的應(yīng)用價(jià)值,有望用于納米給藥遞送系統(tǒng)在腫瘤治療中的可控釋放研究。二、pH響應(yīng)的空心介孔二氧化硅納米藥物遞送系統(tǒng)的構(gòu)建用改進(jìn)的Stober法合成空心介孔二氧化硅納米粒子(HMSNs)。首先以十八烷基三甲氧基硅烷(C18TMS)為模板劑,正硅酸乙酯(TEOS)為硅源,氨水為堿源制得二氧化硅納米球,然后用0.2 M碳酸鈉溶液在80oC條件下腐蝕2 h,最后在600oC高溫下煅燒6h去除模板劑,得到大小均一、單分散性好的HMSNs。以HMSNs為載體負(fù)載DOX,得到載DOX的藥物遞送系統(tǒng)DOX@HMSNs(DMSNs):接著用碳酸氫鈉溶液(NaHCO3)和碳酸銨溶液((NH4)2CO3)處理DMSNs得到兩種氣泡產(chǎn)生型的藥物遞送系統(tǒng),簡(jiǎn)稱為BGNSs-SBC和BGNSs-AC。結(jié)果表明,碳酸氫鈉和碳酸銨處理后可提高系統(tǒng)的載藥率。對(duì)兩種載藥系統(tǒng)進(jìn)行了TEM電鏡等表征。體外釋放實(shí)驗(yàn)結(jié)果顯示,載藥系統(tǒng)在正常生理環(huán)境pH 7.4條件下,藥物釋放較少;而在溶酶體微環(huán)境pH 5.0條件下,藥物釋放加快且釋放量增大,兩者釋放量分別為51.4%和44.9%,相比之下,BGNSs-SBC較BGNSs-AC有更高的釋放量。超聲成像實(shí)驗(yàn)證實(shí)載藥系統(tǒng)在pH 5.0的作用下產(chǎn)生大量CO_2氣泡,且BGNSs-SBC較BGNSs-AC氣泡多。當(dāng)BGNSs-SBC和BGNSs-AC被細(xì)胞攝取進(jìn)入溶酶體內(nèi)后,由于CO_2的產(chǎn)生加快藥物DOX的釋放。三、BGNSs-SBC和BGNSs-AC抑制人乳腺癌細(xì)胞和人乳腺癌耐藥細(xì)胞活性的應(yīng)用用人乳腺癌細(xì)胞(MCF-7)和人乳腺癌細(xì)胞耐藥株(MCF-7/ADR)為細(xì)胞模型,考察BGNSs-SBC和BGNSs-AC的抗癌活性及其克服耐藥性的作用。MCF-7細(xì)胞活性實(shí)驗(yàn)結(jié)果表明,BGNSs-SBC和BGNSs-AC都具有較高的抗癌活性。MCF-7/ADR細(xì)胞活性測(cè)試結(jié)果顯示,BGNSs-SBC和BGNSs-AC的抗耐藥細(xì)胞活性比游離DOX高,且BGNSs-SBC對(duì)細(xì)胞的抑制作用最強(qiáng)。說(shuō)明這兩種藥物遞送系統(tǒng)可有效克服癌細(xì)胞的耐藥性。熒光共定位實(shí)驗(yàn)得到游離的DOX分布在細(xì)胞核,而B(niǎo)GNSs-SBC和BGNSs-AC中釋放的DOX主要分布在細(xì)胞質(zhì)和溶酶體中,沒(méi)有進(jìn)入細(xì)胞核卻有較高的抗腫瘤活性,表明載藥系統(tǒng)的抗腫瘤機(jī)制與眾不同。溶酶體成像實(shí)驗(yàn)和Caspase-3酶活性檢測(cè)實(shí)驗(yàn)都證實(shí)BGNSs-SBC和BGNSs-AC處理后的癌細(xì)胞溶酶體膜通透性顯著提高,且BGNSs-SBC提高得更明顯,這一結(jié)果使得某些特定的組織蛋白酶將從溶酶體釋放到細(xì)胞質(zhì)中,引起細(xì)胞凋亡和類似凋亡途徑的細(xì)胞死亡。相比BGNSs-AC,BGNSs-SBC能更有效克服癌細(xì)胞的耐藥性。
[Abstract]:The traditional clinical cancer treatment include surgery, radiotherapy and chemotherapy, chemotherapy is the most widely used, but in the course of chemotherapy, the side effects of chemotherapy drugs, multi drug resistance (MDR) makes the effect of chemotherapy is limited, and the patient also suffered great pain. In order to solve these two the problem, in normal tissue environment to reduce stimulus drug spill response of nano drug delivery systems have been developed and widely used, when the nano drug delivery system by cancer cells into specific target position, by stimulating the release of drug induced environment and reduce the toxicity to normal tissues and cells drug. Nobel prize winner Christian de Duve proposed by lysosomes causes cell death in cancer cells MDR. to overcome the dilapidation of the lysosomal membrane makes some cells from the solution, when Lysosomal membrane permeability increased, specific cathepsin will be released from lysosomes to the cytoplasm, which are considered to be caused by apoptosis or apoptosis like cell death. This approach can effectively overcome the cancer cells of MDR, for the treatment of cancer provides a new direction. The application of silica nanoparticles as carrier of drug delivery system has become a hot research topic of nano medicine, mainly because silica nanoparticles is non-toxic, uniform particle size, large surface area, good biocompatibility and other characteristics. Especially with some sort of response of nano carrier, to release of drug loaded by controllable stimulating factor. Based on this, the paper takes two different structures of silica nanoparticles for vector delivery system pH response, to detail the drug delivery system in different pH environment The drug release behavior and Study on human breast cancer cells and human breast cancer cells. The inhibitory effects on the activity of specific content includes the following three parts: first, based on mesoporous silica (MSNs) on the release of construction and controllable nano drug carrier system. By sixteen alkyl three methyl ammonium chloride (CTAC) as surfactant agent, tetraethylorthosilicate (TEOS) as silicon source and triethanolamine (TEA) prepared with uniform size as the alkali source, regular array MSNs. by transmission electron microscopy (TEM), infrared spectrometer (IR), nitrogen adsorption and desorption and surface area analyzer (BET, BJH) on the physiochemical properties of MSNs characterized. With broad-spectrum anticancer drug doxorubicin (DOX) as a model drug molecules, drug loaded DOX delivery system (DOX@MSNs): drug loading and release behavior of DOX@MSNs DOX in MSNs under different pH drugs were investigated. Experimental results show Next, the drug loading and encapsulation rate were 5.8%, 27.7%, the release of the drug has good pH response, in the neutral condition of pH 7.4 release less (7.62%) and very slow, while in the weak acid environment along with the prolongation of time of drug release increased (up to 42.2%). The pH response drug release characteristics are better able to play a role in tumor suppression, toxicity test showed no cytotoxicity to MSNs itself, with good biocompatibility. In addition, doxorubicin loaded nano drug delivery system of DOX@MSNs cell killing ability compared with free DOX, namely nano drug delivery system has a good anti-tumor effect the nano drug delivery system has potential application value, is expected to be used to nano drug delivery system in cancer treatment and controlled release study. Two, hollow mesoporous silica nano drug delivery system pH response construction with improved Stobe Synthesis of hollow mesoporous silica nanoparticles by R (HMSNs). The first eighteen alkyl trimethoxysilane (C18TMS) as template, tetraethyl orthosilicate (TEOS) as silicon source, ammonia as alkali source to prepare silica nanospheres, then with 0.2 M sodium carbonate solution under the condition of 80oC corrosion for 2 h, the last in the 600oC under high temperature calcination to remove the template 6h, have uniform size and good monodispersity of HMSNs. HMSNs as a carrier to load DOX, get a drug delivery system containing DOX DOX@HMSNs (DMSNs): (NaHCO3) followed by sodium bicarbonate and ammonium carbonate solution ((NH4) 2CO3) DMSNs drug two bubbles the type of delivery system, referred to as BGNSs-SBC and BGNSs-AC.. The results showed that sodium bicarbonate and ammonium carbonate treatment could improve the drug loading rate of the system. Two kinds of drug delivery system of TEM electron microscopy. The in vitro release experiment results indicated that the drug carrier system in the normal ring PH 7.4 environmental conditions, drug release and less; in the lysosomes micro environment under the condition of pH 5, the drug release and the release speed increases, both emissions were 51.4% and 44.9%, compared with BGNSs-SBC compared with the BGNSs-AC release amount higher. Ultrasound imaging experiments confirmed that the drug loaded system produces a large number of bubbles in the role of CO_2 pH 5, BGNSs-AC and BGNSs-SBC than the bubble much. When BGNSs-SBC and BGNSs-AC are cell uptake into the lysosomes, due to the production of CO_2 to accelerate drug release of DOX. Three, application of BGNSs-SBC and BGNSs-AC on human breast cancer cells and human breast cancer cell activity in human breast cancer cells (MCF-7) and drug resistance of breast cancer cell line (MCF-7/ADR) as the cell model, the influences of BGNSs-SBC and BGNSs-AC and anticancer activity of.MCF-7 cells to overcome the resistance activity test results showed that BGNSs-SBC and BGNSs-AC have high resistance The activity of.MCF-7/ADR cell carcinoma activity test results showed that BGNSs-SBC and BGNSs-AC resistant cell activity than free DOX high, and BGNSs-SBC on the cell was inhibited. The two drug delivery system can effectively overcome the drug resistance of cancer cells. Fluorescence co localization experiments to get the free DOX distribution in the nucleus, and the release of BGNSs-SBC and BGNSs-AC the DOX is mainly distributed in the cytoplasm and in lysosomes, did not enter the nucleus has high antitumor activity, showed that the mechanism of anti tumor drug carrier system. Imaging experiments and Caspase-3 out of the ordinary lysosomal enzyme activity detection experiments have confirmed that the cancer cell BGNSs-SBC and lysosomal membrane permeability increased significantly after BGNSs-AC treatment, and BGNSs-SBC increased more obviously this result makes certain cathepsin will be released from lysosomes to the cytoplasm, induce apoptosis and similar apoptosis Cell death in the pathway. Compared to BGNSs-AC, BGNSs-SBC can be more effective to overcome the drug resistance of cancer cells.

【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：TQ460.1;TB383.1

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本文編號(hào)：1566172