pH响应二氧化硅纳米药物递送系统的构建及抗肿瘤活性研究

发布时间：2018-03-04 15:13

本文选题：二氧化硅纳米颗粒　切入点：pH响应　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：临床上传统的癌症治疗手段包括手术治疗、放疗和化疗等,其中化疗被用得最为普遍,但是在化疗过程中,化疗药物的毒副作用以及多重耐药性(MDR)使得化疗的效果被受限制,同时病人也承受巨大的痛苦。为了解决这两个难题,可在正常组织环境下减少药物泄漏的刺激-响应性纳米药物递送系统已被发明和广泛利用,当纳米药物递送系统被癌细胞摄取进入到特定靶向位置时,通过环境的刺激性诱导药物的释放进而减小药物对正常组织和细胞的毒副作用。诺贝尔医学奖获得者Christian de Duve提出可以通过溶酶体引起细胞死亡从而克服癌细胞MDR。由于溶酶体膜的破损使得部分细胞自溶,当溶酶体膜通透性提高时,某些特定的组织蛋白酶将从溶酶体释放到细胞质中,这被认为可引起细胞凋亡或类似凋亡途径的细胞死亡。这一途径能有效地克服癌细胞的MDR,为癌症的治疗提供了新方向。二氧化硅纳米粒子作为药物递送系统载体的应用已成为纳米医药研究的一大热点,主要因为二氧化硅纳米粒子具有无毒性、粒径均一、比表面积大、生物相容性好等特点。特别是具有某种响应性的纳米载体,通过刺激因子的作用达到对负载药物的可控释放。基于此,本论文以两种不同结构的二氧化硅纳米粒子为载体构建多种pH响应的药物递送系统,详细探究了药物递送系统在不同pH环境下药物的释放行为及研究其对人乳腺癌细胞和人乳腺癌耐药细胞活性的抑制作用。具体包括以下三部分内容:一、基于介孔二氧化硅(MSNs)的纳米载药系统的构建及可控释放研究。以十六烷基三甲基氯化铵(CTAC)为表面活性剂,正硅酸乙酯(TEOS)为硅源,三乙醇胺(TEA)为碱源成功制得粒径均一,排列规整的MSNs。通过透射电子显微镜(TEM)、红外光谱仪(IR)、氮气吸附与解析和比表面积分析仪(BET、BJH)等手段对MSNs的理化性质进行了表征。以广谱抗癌药物阿霉素(DOX)作为药物模型分子,得到负载DOX的药物递送系统(DOX@MSNs):考察了DOX在MSNs中的载药情况及DOX@MSNs在不同pH条件下药物的释放行为。实验结果表明,其载药量和包封率分别为5.8%、27.7%,药物的释放具有很好的pH响应性,在中性pH 7.4条件下药物释放较少(7.62%)且非常缓慢,而在弱酸性环境下随时间的延长药物释放增大(达42.2%)。这种pH响应药物释放特性能够更好地发挥抑制肿瘤的作用,细胞毒性实验结果表明,MSNs本身对细胞无毒性,具有很好的生物相容性。另外,载阿霉素纳米药物递送系统DOX@MSNs对细胞的杀伤能力较游离DOX强,即纳米载药系统具有很好的抗肿瘤作用。该纳米载药系统具有潜在的应用价值,有望用于纳米给药递送系统在肿瘤治疗中的可控释放研究。二、pH响应的空心介孔二氧化硅纳米药物递送系统的构建用改进的Stober法合成空心介孔二氧化硅纳米粒子(HMSNs)。首先以十八烷基三甲氧基硅烷(C18TMS)为模板剂,正硅酸乙酯(TEOS)为硅源,氨水为碱源制得二氧化硅纳米球,然后用0.2 M碳酸钠溶液在80oC条件下腐蚀2 h,最后在600oC高温下煅烧6h去除模板剂,得到大小均一、单分散性好的HMSNs。以HMSNs为载体负载DOX,得到载DOX的药物递送系统DOX@HMSNs(DMSNs):接着用碳酸氢钠溶液(NaHCO3)和碳酸铵溶液((NH4)2CO3)处理DMSNs得到两种气泡产生型的药物递送系统,简称为BGNSs-SBC和BGNSs-AC。结果表明,碳酸氢钠和碳酸铵处理后可提高系统的载药率。对两种载药系统进行了TEM电镜等表征。体外释放实验结果显示,载药系统在正常生理环境pH 7.4条件下,药物释放较少;而在溶酶体微环境pH 5.0条件下,药物释放加快且释放量增大,两者释放量分别为51.4%和44.9%,相比之下,BGNSs-SBC较BGNSs-AC有更高的释放量。超声成像实验证实载药系统在pH 5.0的作用下产生大量CO_2气泡,且BGNSs-SBC较BGNSs-AC气泡多。当BGNSs-SBC和BGNSs-AC被细胞摄取进入溶酶体内后,由于CO_2的产生加快药物DOX的释放。三、BGNSs-SBC和BGNSs-AC抑制人乳腺癌细胞和人乳腺癌耐药细胞活性的应用用人乳腺癌细胞(MCF-7)和人乳腺癌细胞耐药株(MCF-7/ADR)为细胞模型,考察BGNSs-SBC和BGNSs-AC的抗癌活性及其克服耐药性的作用。MCF-7细胞活性实验结果表明,BGNSs-SBC和BGNSs-AC都具有较高的抗癌活性。MCF-7/ADR细胞活性测试结果显示,BGNSs-SBC和BGNSs-AC的抗耐药细胞活性比游离DOX高,且BGNSs-SBC对细胞的抑制作用最强。说明这两种药物递送系统可有效克服癌细胞的耐药性。荧光共定位实验得到游离的DOX分布在细胞核,而BGNSs-SBC和BGNSs-AC中释放的DOX主要分布在细胞质和溶酶体中,没有进入细胞核却有较高的抗肿瘤活性,表明载药系统的抗肿瘤机制与众不同。溶酶体成像实验和Caspase-3酶活性检测实验都证实BGNSs-SBC和BGNSs-AC处理后的癌细胞溶酶体膜通透性显著提高,且BGNSs-SBC提高得更明显,这一结果使得某些特定的组织蛋白酶将从溶酶体释放到细胞质中,引起细胞凋亡和类似凋亡途径的细胞死亡。相比BGNSs-AC,BGNSs-SBC能更有效克服癌细胞的耐药性。
[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.

【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：TQ460.1;TB383.1

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