介孔二氧化硅纳米材料的制备及在药物递送方面的应用研究

发布时间：2018-01-11 19:35

本文关键词：介孔二氧化硅纳米材料的制备及在药物递送方面的应用研究　出处：《湖北中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：由于纳米材料的独特性质,使其在医药领域尤其是癌症的治疗中起着很重要的作用。介孔二氧化硅纳米粒(Mesoporous silica nanoparticles,MSN)是1992年由Stober发现的无机纳米材料,是最有前途的药物载体之一。介孔二氧化硅纳米粒的种类繁多,可以通过控制条件制备出不同的型号,如MCM-41、MCM-48、SBA-15等。本课题拟以两种不同种类的介孔二氧化硅为载体SBA-15和MCM-41,分别装载3种不同性质的药物,通过不同的给药途径来研究其在递药方面的应用。具体研究内容如下:(1)通过水热合成法制备SBA-15介孔二氧化硅纳米粒,建立艾塞那肽体外分析方法,以SBA-15介孔二氧化硅纳米粒为载体材料,采用浸渍吸附法制备EXT-SBA-15,并考察其体外释药特征;以EXT-Sol为对照组,研究EXT-SBA-15经皮下注射给药后在大鼠体内的药动学过程,Exendin-4 ELISA试剂盒测定不同时间点大鼠体内EXT的含量,计算药动学参数,以考察EXT-SBA-15的体内释药情况;采用糖尿病小鼠为模型动物,设置空白对照组、模型对照组、EXT-Sol组、EXT-SBA-15组以及空白SBA-15组,初步考察糖尿病小鼠皮下注射EXT-SBA-15后的降血糖效果。结果表明,成功制备了EXT-SBA-15纳米粒,制备工艺简单,载药量高,体内外研究表明EXT-SBA-15能延长药物在体内滞留时间,具有明显缓释特征和降血糖作用。本研究为SBA-15等介孔硅材料的应用和多肽类药物缓释制剂的研究开发提供了实验依据和理论参考。(2)采用共沉淀法制备氨基改性介孔二氧化硅纳米粒,静电吸附载入ATO,PAA酸碱共轭制备PAA-ATO-MSN。采用透射电镜、扫描电镜观察纳米粒的形态结构,小角X射线衍射仪考察其介孔结构,氮气吸脱附仪测定其比表面积、孔径、孔容,红外光谱仪考察氨基和PAA修饰情况,热重分析仪测定PAA接枝率,激光粒度Zeta电位仪测定粒径和Zeta电位;高速离心法结合电感耦合等离子发射光谱(Inductively coupledplasmaemissionspectrum,icp)测定其包封率及载药量;选用不同ph的磷酸盐缓冲液(phosphatebufferedsaline,pbs)(ph5.0,6.0和7.4)作为释放介质,透析袋法考察其在不同ph条件下体外释药特性;以smmc-7721细胞为体外模型,考察空白载体msn、nh2-msn和paa-msn毒性和ato溶液(atosolution,ato-sol),三氧化二砷氨基改性介孔二氧化硅纳米粒(arsenictrioxideloadedmesoporoussilicananoparticles,ato-msn)和聚丙烯酸修饰氨基改性介孔二氧化硅纳米粒(polyacrylicacidgraftedarsenictrioxideamino-functionedmesoporoussilicananoparticles,paa-ato-msn)的体外肿瘤细胞抑制率及周期阻滞情况;以ato-sol、ato-msn为对照组,大鼠尾静脉注射(intravenousinjection,iv)给药后,股动脉、颈静脉插管技术采集血样,采用电感耦合等离子质谱(inductivelycoupledplasmamassspectrometry,icp-ms)测定血药浓度计算药动学相关参数,考察其体内药动学行为。以昆明(km)小鼠异位移植肝癌(h22)为药效模型,设置空白对照组、ato-sol组、ato-msn组和paa-ato-msn组考察各制剂抗肿瘤效果。结果表明成功制备了载三氧化二砷ph响应介孔二氧化硅纳米粒(paa-ato-msn),其粒径较小、具有较高的包封率和载药量,体外释药具有明显的ph响应性及缓释特性,且paa-msn能明显改善ato在大鼠体内的药动学行为,paa-ato-msn体内外抗肿瘤效果均明显优于ato,该载体具有较好的应用前景。(3)采用改良的经典stober法制备介孔二氧化硅纳米粒(msn),通过热重分析法、氮气脱吸附法、xrd衍射等技术进行表征分析;发明反复饱和溶液吸附法制备载入ptx的msn(msn-ptx),然后以msn-ptx为核,采用自组装和薄膜水化法构建核/壳结构的angiopep-2修饰介孔二氧化硅脂质囊纳米粒(ang-msn-lp-ptx);透析袋法考察体外释药特性;mtt法考察空白载体对hbmec和c6的细胞毒性,以及载药纳米粒对c6细胞的抗肿瘤作用;建立体外bbb模型研究载体对ptx的跨膜转运能力和对c6细胞周期的影响。建立荷脑胶质瘤大鼠模型,对肿瘤组织进行病理切片观察脑胶质瘤的侵袭情况,考察载药纳米粒对荷脑胶质瘤大鼠治疗作用;采用血-脑同步微透析技术,考察载药纳米粒在血液和脑组织中的药动学特性。结果表明制备的MSN轮廓圆整光滑,粒径分布均匀,比表面积大,孔径均一,性质稳定;反复饱和溶液吸附法可有效提高药物载药量,解决纳米载体载药量低的问题,值得应用和借鉴;所构建的ANG-MSN-LP载体系统,融合了介孔材料和脂质材料的优点,同时解决二氧化硅易团聚、易突释的问题和脂质体载药低、易漏药的问题,是一种安全、可靠、有良好生物相容性的纳米粒,经Angiopep-2修饰后的二氧化硅脂质囊纳米粒可以有效的透过血脑屏障,实现脑靶向递药,其作为一种优良的递药载体并有望应用于脑胶质瘤的治疗;本论文改进的体外BBB模型,将HBMEC和C6脑胶质瘤细胞通过transwell嵌套共建了双细胞模型,体外考察递药载体透过BBB后对脑胶质瘤细胞的抑制作用,较为逼真模拟递药载体在体内的运行过程,具有较高的科学性;血-脑同步微透析技术研究脑靶向递药系统的药动学特性,能够同时和实时的测定脑内和血液内的游离型药物的浓度,准确考察药物的药动学特性,具有无可比拟。
[Abstract]:Due to the unique properties of nano materials, making it especially in the field of medicine plays a very important role in the treatment of cancer. The mesoporous silica nanoparticles (Mesoporous silica nanoparticles, MSN) is inorganic nano materials discovered by Stober in 1992, is the most promising drugs carrier. One of the types of mesoporous silica nanoparticles are by controlling the preparation conditions of different types, such as MCM-41, MCM-48, SBA-15 and so on. This paper intends to two different kinds of mesoporous silica SBA-15 and MCM-41, respectively. The loading of 3 different types of drugs, administered through different ways to study its application in drug delivery. The specific research the contents are as follows: (1) preparation of SBA-15 mesoporous silica nanoparticles by hydrothermal synthesis method, analysis method establishment of exenatide in vitro by SBA-15 mesoporous silica nanoparticles as carrier material, by dipping suction With the preparation of EXT-SBA-15, and investigate its in vitro release characteristics; using EXT-Sol as the control group, EXT-SBA-15 subcutaneous injection after administration in rats in vivo pharmacokinetic process, the content of Exendin-4 ELISA kit for determination of EXT in rats at different time points, the pharmacokinetic parameters were calculated, in order to study the in vivo release of EXT-SBA-15 drug use; diabetic mice as a model animal, set the blank control group, model control group, EXT-Sol group, EXT-SBA-15 group and control group SBA-15, preliminary study of hypoglycemic effect in diabetic mice after subcutaneous injection of EXT-SBA-15. The results show that EXT-SBA-15 nanoparticles were successfully prepared, simple preparation process, high drug loading, in vitro and in vivo research shows that EXT-SBA-15 can prolong the residence time of the drug in the body, has obvious sustained-release characteristics and hypoglycemic effect. The research for the SBA-15 mesoporous silica materials and application of polypeptide drug sustained-release preparation. The experimental and theoretical basis for the development. (2) the preparation of amino modified mesoporous silica nanoparticles coprecipitation method, electrostatic adsorption loading ATO, pH PAA conjugate preparation of PAA-ATO-MSN. by transmission electron microscope observation on the morphology of nanoparticles by scanning electron microscopy, small angle X ray diffraction on the mesoporous structure, nitrogen adsorption determination the specific surface area, pore size, Kong Rong desorption instrument, infrared spectroscopy of amino and PAA modification, determination of the grafting ratio of PAA thermogravimetric analyzer, laser particle and Zeta potential analyzer was used to measure the particle size and Zeta potential; high speed centrifugation combined with inductive coupled plasma atomic emission spectrometry (Inductively coupledplasmaemissionspectrum ICP) to determine the entrapment efficiency and loading dosage; phosphate buffer with different pH (phosphatebufferedsaline, PBS) (ph5.0,6.0 and 7.4) as the release medium, the dialysis bag was used to investigate the pH in different conditions in vitro drug release. ; SMMC-7721 cells in vitro model to study the control vector MSN, nh2-msn and paa-msn toxicity and ATO solution (atosolution, ato-sol), arsenic trioxide amine modified mesoporous silica nanoparticles (arsenictrioxideloadedmesoporoussilicananoparticles, ato-msn) and polyacrylic acid modified amine modified mesoporous silica nanoparticles (polyacrylicacidgraftedarsenictrioxideamino-functionedmesoporoussilicananoparticles, paa-ato-msn) in vitro and the inhibition rate of tumor cell cycle block; ato-sol, ato-msn for the control group, rats were given intravenous injection (intravenousinjection, IV) of femoral artery after administration, jugular vein intubation technique to collect blood samples by inductively coupled plasma mass spectrometry (inductivelycoupledplasmamassspectrometry, ICP-MS) for determination of blood concentration to calculate the pharmacokinetic parameters related, investigate the in vitro and in pharmacokinetic behavior in Kunming (km) mice. Ectopic transplantation for hepatocellular carcinoma (H22) as efficacy model, set the blank control group, ato-sol group, ato-msn group and paa-ato-msn group to investigate the antitumor effect of preparation. The results show that the prepared carrier response of arsenic trioxide pH mesoporous silica nanoparticles (paa-ato-msn), the smaller particle size, high encapsulation efficiency and drug loading, in vitro the drug release has obvious pH response and release characteristics, and paa-msn can significantly improve the ATO pharmacokinetic behavior in rats, paa-ato-msn in vitro and in vivo antitumor effects were better than ATO, the carrier has good application prospects. (3) using a modified classical Stober preparation of mesoporous silica nanoparticles (MSN), by thermogravimetric analysis, nitrogen adsorption, XRD diffraction characterization of the invention; repeated saturated solution adsorption preparation of loaded PTX MSN (msn-ptx), and then to msn-ptx as the core, by self assembling and water film The construction of core / shell structure of angiopep-2 modified mesoporous silica nanoparticles liposome method (ang-msn-lp-ptx); the dialysis bag method to study drug release in vitro; cytotoxic MTT method to study the control vector to HBMEC and C6, and the drug loaded nanoparticles on the anti-tumor effect of C6 cell; effects of transmembrane ability of carrier in vitro the BBB model of PTX and C6 on the cell cycle. To establish the model of glioma bearing rats were invasion of glioma pathological observation of tumor tissue, effects of drug loaded nanoparticles on the therapeutic effect of glioma bearing rats; using synchronous blood brain microdialysis, investigation of drug loaded nanoparticles in the blood and brain tissue pharmacokinetic characteristics. The results show that the MSN circle contour prepared smooth, uniform particle size distribution, large surface area, uniform pore size, stable property; repeated saturated solution adsorption method can effectively improve the drug loading capacity, To solve the problem of low drug loading nanoparticles, it is worthy of application and reference; ANG-MSN-LP vector system constructed by combining the advantages of mesoporous materials and lipid materials, at the same time to solve the problem of silica agglomerate, and liposome easily burst release of drug loaded low leakage of the medicine, is a safe, reliable and compatible the good biological nanoparticles modified by Angiopep-2, silica nanoparticles liposome can penetrate the blood-brain barrier effectively, realize the brain targeting drug delivery, as a kind of excellent drug delivery and is expected to be used for brain glioma treatment; in vitro BBB model this paper improved, HBMEC and C6 in brain glioma the double cell through the cell model and the nested Transwell, in vitro study drug delivery through BBB inhibition of glioma cells, a more realistic simulation of drug delivery in the operation process of the body, with the science high; The blood brain synchronous microdialysis technology can be used to study the pharmacokinetic characteristics of the brain targeting drug delivery system. It can simultaneously determine the concentration of free drugs in the brain and blood simultaneously and accurately, and accurately investigate the pharmacokinetic characteristics of the drug.

【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R943;TB383.1

【相似文献】