非共价功能化修饰的氧化石墨烯作为抗癌药物载体的研究

发布时间：2018-01-03 02:44

本文关键词：非共价功能化修饰的氧化石墨烯作为抗癌药物载体的研究　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：随着空气质量的恶化和人类生活方式的改变,癌症的发病率和致死率正逐年上升,寻求有效的癌症治疗手段刻不容缓。化疗是有效而经典的治疗手段,但是大多数化疗药物如多柔比星具有选择性低,毒副作用尤其是心脏毒性严重,半衰期短,易产生多药耐药等缺点。因此为了提高药物的治疗效果,最大化的减轻毒副作用,开发新型有效的药物载体至关重要。2004年有学者首次报道了石墨烯,凭借其独特的结构特征,优良的机械、热力学及光学特征,在各个领域都引起了极大地研究热忱。氧化石墨烯(GO)是石墨烯的氧化衍生物,具有超大的比表面积和优良的光热效果,已经成为纳米医学领域的研究热点。GO在水中可以分散,但在生理溶液中易聚集产生沉淀,极大地限制了其在药物载体方面的应用,因此必须对其进行合适的表面功能化修饰提高其在生理溶液中的稳定性和分散性。一般可以利用GO大的共轭结构及其表面和边缘存在的丰富的含氧官能团对其进行非共价或共价功能化修饰,共价键修饰在引入化学反应和有机溶剂的同时,GO的共轭结构和物理性质也会发生变化,而非共价键修饰可以避免繁琐的化学反应,最大化的保留GO的性质,因此非共价键修饰得到了越来越多的关注。理想的药物传递系统应该是使运载的药物在血液循环和正常组织中不释放,通过主动或被动靶向作用浓集于肿瘤部位,然后进入细胞,受细胞内环境影响快速释放药物,才会使药物具有最好的抗肿瘤效果和最小的副作用。因此环境刺激响应型药物传递系统得到了广泛的关注,我们可以利用肿瘤细胞内部的环境特征(低pH,高GSH浓度等)设计药物载体,使载药系统在肿瘤细胞内由于环境变化而释放药物发挥治疗效果。本课题以充分发挥GO自身的功能和性质为目的,采用两种不同的生物相容性的大分子通过非共价键方式修饰GO,分别获得的pH敏感的主动靶向药物传递系统和氧化还原敏感的药物传递系统,并分别对其进行了体内外评价。本课题的研究内容如下:(1)利用某些肿瘤细胞表面过度表达的叶酸受体,制备了叶酸受体介导的主动靶向的药物传递系统。将主动靶向分子FA通过酰胺反应与生物相容性的BSA结合,获得的FA-BSA通过非共价方式修饰GO制备主动靶向药物载体FA-BSA/GO,其中FA-BSA既可作为稳定剂,分散剂,又具有靶向功能。通过TEM,AFM,DLS观察其形貌,片层尺寸及厚度,Zeta电势监测其稳定性,溶血实验初步判定其良好的生物相容性。(2)将抗肿瘤药物DOX通过π-π和氢键作用吸附到FA-BSA/GO表面获得pH敏感的主动靶向药物传递系统。当DOX与FA-BSA/GO的质量比为3:1时,载药量达到最大为30.43%,即1 mg FA-BSA/GO可以吸附437.43μgDOX,远高于传统制剂。体外释放实验表明,与游离DOX相比,该药物传递系统具有明显的pH敏感性和缓释行为。体外细胞毒性及细胞摄取结果表明,FA可以进一步提高肿瘤细胞对FA-BSA/GO/DOX的摄取,从而使肿瘤细胞内DOX浓度增高,提高DOX治疗效果,降低其对正常组织的毒副作用。(3)为了进一步解决GO在生理溶液中的稳定性及基于GO药物传递系统的不完全的,缓慢的药物释放行为,本课题利用肿瘤细胞内外GSH浓度的差异,采用氧化还原敏感的前药分子mPEG2K-PCL2K-SS-DOX作为稳定剂、分散剂、通过非共价键修饰GO,获得了氧化还原敏感的药物传递系统GOPN,并对其体内外抗肿瘤活性进行了研究。(4)前药分子的存在使得制备的GOPN在生理溶液中能够稳定存在,同时由于PEG的长循环作用,可延长GOPN在体内循环时间,从而避免重复用药。同时通过TEM观察其形貌,DLS测定其粒径,并通过粒径观察其在血浆中的稳定性,结果表明GOPN具有良好的稳定性。(5)体外释放结果表明GOPN具有良好的氧化还原敏感性,并且与NGO/DOX相比,释药更完全。体外细胞毒性实验结果表明GOPN的抑制率高于游离DOX和NGO/DOX,这是由于氧化还原敏感的GOPN通过内吞作用进入细胞,在细胞内高的GSH浓度下,二硫键断裂,释放药物,快速杀死癌细胞,因此我们制备的GOPN具有更好的治疗效果。体外细胞摄取实验结果表明A549细胞对GOPN的摄取量高于游离的DOX,高的摄取量可以保证药物浓集于肿瘤部位,是提高肿瘤杀伤力的重要原因。(6)最后通过荷瘤小鼠对GOPN的体内抗肿瘤活性进行了评价,通过相对肿瘤体积变化可以看出,GOPN对肿瘤增长有一定的抑制作用。通过考察荷瘤小鼠相对体重变化和组织切片对GOPN的用药安全性进行了评价,结果表明GOPN可以明显降低DOX导致的心脏毒性,因此我们制备的GOPN具有有效的体内抑瘤效果和良好的生物安全性。综上所述,本课题首次成功将两种生物相容性的大分子(FA-BSA和mPEG2K-PCL2K-SS-DOX)通过非共价方式修饰GO,分别构建了 pH敏感的主动靶向药物载体和氧化还原敏感的药物传递系统,不仅成功地解决了 GO在生理溶液中不稳定的问题及基于GO的药物传递系统释放缓慢和不完全的问题,而且可以提高DOX的治疗效果,降低毒副作用,提高病人的耐受性,同时还拓宽了GO在生物医学领域中的应用,因此本课题具有重要研究意义。
[Abstract]:With the deterioration of air quality and the changes of human life style, the incidence rate of cancer and the death rate is rising year by year, to seek the effective means of treating cancer chemotherapy is effective and urgent. The classic means of treatment, but most chemotherapy drugs such as doxorubicin has low selectivity, side effects especially serious cardiac toxicity, short half-life that is easy to produce multidrug resistance. Therefore in order to improve the therapeutic effect of drugs, the maximum reduce side effects, the development of new and effective drug carrier.2004 is reported for the first time years scholar Shi Moxi, by virtue of its unique structure, excellent mechanical, thermodynamic and optical characteristics in various fields have attracted great study enthusiasm. Graphene oxide (GO) is a derivative of graphene oxide, with large surface area and excellent thermal effect, has become the nano medical field Study on the hot.GO can be dispersed in water, but in a physiological solution, prone to aggregation, resulting in precipitation, which greatly limits its application in drug delivery, so it must be appropriate to the function of surface modification to improve its stability in physiological solution and dispersion. Generally non covalent or covalent functionalization of the use of GO to the conjugate structure and surface and the edge of the rich oxygen containing functional groups, covalent modification in the chemical reaction and organic solvent, conjugate structure and physical properties of GO will change, and non covalent modification can avoid the cumbersome nature of chemical reaction, the maximum retention of GO therefore, the non covalent modification is gaining more and more attention. An ideal drug delivery system should be to carry the drug was not released in the circulation of the blood and normal tissues, through active or passive target To the effect of concentration on the tumor, and then enter the cell, affected by environmental cell rapid release of the drug will make the drug have antitumor effect best and minimal side effects. Therefore, environmental stimuli responsive drug delivery system has received wide attention, we can use the internal environmental characteristics of tumor cells (low pH, high profit GSH concentration) design of drug carrier, the drug loading system in tumor cells due to changes in the environment and release drugs play a therapeutic effect. This issue in order to give full play to the function of GO and the nature of their own for the purpose of using two kinds of molecular differences in biocompatibility through non covalent modification of GO, respectively pH active target sensitive delivery system and drug delivery system to oxidation reduction sensitive drug evaluation, and has separately carried on the in vitro and in vivo. The content of this research are as follows: (1) the use of certain tumor cells The surface over expression of folate receptor, preparation of pharmaceutical active targeting of folate receptor mediated delivery system. To combine the active targeting molecule FA through amide reaction and biological compatibility of BSA, access to FA-BSA via non covalent modification of GO preparation of active targeting drug carrier FA-BSA/GO FA-BSA, which can be used as a stabilizer also, dispersant, targeted function. Through the TEM, AFM, DLS to observe the morphology, size and thickness of layers, Zeta potential of monitoring the stability, hemolysis test preliminary determination of its good biocompatibility. (2) the antitumor drug DOX by tt-tt and hydrogen bonding adsorption onto the surface of FA-BSA/GO pH active target sensitive delivery system to drugs. When DOX and FA-BSA/GO mass ratio was 3:1, the maximum loading capacity is 30.43%, which is 1 mg FA-BSA/GO was 437.43 gDOX, much higher than that of traditional formulations. The in vitro release experiment showed that with free DOX Compared to the drug delivery system has pH sensitivity and sustained release behavior significantly. In vitro cytotoxicity and cellular uptake results show that FA can further improve the uptake of FA-BSA/GO/DOX in tumor cells, so that the concentration of DOX in tumor cells increased, DOX improve the therapeutic effect, reduce the side effects on normal tissue (3) in order to further. To solve the stability of GO in physiological solution and GO based drug delivery system is not completely, the slow drug release behavior, the differences of tumor cells inside and outside of the GSH concentration, the redox sensitive prodrug molecule mPEG2K-PCL2K-SS-DOX as stabilizer, dispersant, through non covalent modification of GO, obtained the redox sensitive drug delivery system of GOPN, and the in vivo antitumor activity were studied. (4) existence of prodrug molecules allows the preparation of GOPN can stably exist in physiological solution In the meanwhile, due to the long cycle of PEG, GOPN in vivo can prolong the circulation time, thus avoiding repeated use. At the same time to observe the morphology of TEM by DLS, determine the particle size, and particle size were observed by in the plasma stability. The results show that GOPN has good stability. (5) the results showed that GOPN in vitro release has good redox sensitivity, and compared with NGO/DOX, the drug release more completely. In vitro cytotoxicity test results show that the inhibition rate of GOPN was higher than that of the free DOX and NGO/DOX, which is due to the redox sensitive GOPN by endocytosis into the cell, the concentration of GSH in cells under high, two sulfur bond breaking, release drug fast to kill cancer cells, so we prepared GOPN has a better therapeutic effect. The experimental results show that the cell uptake amount of GOPN uptake in A549 cells was higher than that of the free DOX, intake of high concentration can be ensured At the tumor site, is an important reason to improve tumor lethality. (6) the antitumor activity of GOPN in tumor bearing mice was evaluated by the relative tumor volume changes can be seen, GOPN has inhibitory effect on tumor growth in tumor bearing mice. Through the investigation of changes of body weight and tissue sections of GOPN the safety was evaluated. The results show that GOPN can significantly reduce the cardiac toxicity caused by DOX, so we prepared GOPN with effective anti-tumor effect and good biological safety. To sum up, this paper first made two biocompatible macromolecules (FA-BSA and mPEG2K-PCL2K-SS-DOX) by non covalent method modified GO, we constructed pH active targeting drug carrier and sensitive to redox sensitive drug delivery system, not only successfully solve the GO instability problem in physiological solution As well as the slow and incomplete release of drug delivery system based on GO, it can improve the therapeutic effect of DOX, reduce the side effects and increase the tolerance of patients. It also broadens the application of GO in biomedicine. Therefore, this topic has important research significance.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R943

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