基于载姜黄素的肝素修饰还原氧化石墨烯纳米粒的研究

发布时间：2018-05-03 16:10

  本文选题：还原氧化石墨烯 + 生物相容性　； 参考：《山东大学》2017年博士论文

【摘要】：还原氧化石墨烯(rGO)是一种平面内含氧基团减少的氧化石墨烯(GO),是近十年来迅速发展起来的高分子材料,由于载药量高、光热转换效率高、可修饰性等性能,使其在生物医药领域的多个方向如药物传递、基因转导、DNA操纵、肿瘤成像、光热治疗、骨组织工程等有广泛的研究。本文分为三部分,第一部分以溶血试验为评价手段,以溶血指数(HI)为评价指标,寻找生物相容性高的GO衍生物所具备的理化特征,为载姜黄素(Cur)肝素修饰的rGO纳米粒中GO载体的选择、修饰和制剂研究奠定基础。第二部分对载Cur的rGO纳米粒进行药学研究。该纳米粒以Cur为模型药物、以二次氧化的GO为起始的载体材料、以未分级肝素(UFH)为亲水性修饰高分子,以叶酸(FA)为靶头,经还原处理,分别制成非FA修饰纳米粒(记为Cur-rGO-UFH)和FA修饰纳米粒(记为Cur-rGO-UFH-FA)。以体外释放度试验、体外细胞试验、药代动力学试验、组织分布试验、组织切片等为评价手段,对这两种纳米粒进行体内外评价。第三部分为该纳米粒中rGO相关的分子模拟研究。从机理上直观地解释了:(1)未修饰的rGO是热力学不稳定的,需用亲水性高分子加以修饰;(2)rGO载Cur的过程。以溶菌酶(Lyso)和肌红蛋白(Myo)为模型蛋白质、以未修饰的rGO为典型的载体材料、以石墨烯(GRO)为参照,说明了纳米粒进入生物体后载体材料的生物相容性,如:与Myo含量丰富的心脏的相容性,与Lyso含量丰富的肝、脾、肺和肾的相容性。本论文主要的研究方法与结果如下:第1章通过从合成和表征了 9类25个GO的衍生物。第2章建立了新的求溶血指数(HI)的方法和溶血试验条件。求HI的方法是利用540nm、576nm和600nm三处紫外吸收的540-576-600法,它是一种典型的三点吸收差值法。为说明该法的准确性,比较了文献中常用到的溶血百分数(HP)方法,包括以540法为代表的单点吸收值法和以540-655法为代表的两点吸收差值法,同时还建立了求HI的以576-600法为代表的两点吸收值法。通过三个实例说明了新建的540-576-600法具有求GO衍生物的HI准确度高的特点。而常见的求HP的方法存在结果偏大,甚至有可能出现超过100%的溶血现象。合理的试验条件:5%葡萄糖溶液为溶血介质、家兔来源终浓度为1%新鲜健康的RBCs作为理想的红细胞(RBCs),3000rpm离心5min,37℃孵育3～6h。在红细胞浓度、离心速度等问题上,对GO与RBCs之间相互作用进行定量计算。本研究未见文献报道。第3章对第1章中25个GO衍生物进行溶血评价,找到了生物相容性高的GO衍生物理化特征,如粒径减小、亲水性高分子修饰、还原处理、电荷减少。第4章对两种肝素化rGO材料进行评价。在生物相容性高的结构特征基础上,选择以UFH为亲水性材料,以FA为靶头、GO-2为起始载体材料,制成了两种肝素化rGO材料,分别为肝素-己二酸二酰肼-rGO(记为rGO-UFH)和叶酸修饰的肝素-己二酸二酰肼-rGO(记为rGO-UFH-FA)。这两种材料具有浓度、时间依赖性的光热效应。在浓度为10μg·mL-1且808nm光照5min条件下,两种材料均能达到肿瘤光热治疗所需的下限温度50℃。在非光照条件下,两种材料对MCF-7或A549细胞基本无毒;在光照下,rGO-UFH-FA对MCF-7细胞的杀伤性较强,说明rGO-UFH-FA通过叶酸受体(FR)介导作用进入胞内,而rGO-UFH可能通过EPR效应进入到细胞。第5章对两种姜黄素rGO纳米粒进行体外研究。首先建立了计算载药量(DL)和包封率(EE)的方法,以星点设计-效应面法优化了 Cur-rGO-UFH-FA的制备工艺。体外释放研究表明:纳米粒中Cur的释放受温度的影响较大,受pH的影响较小,说明rGO-UFH-FA包载Cur的过程是以π-π堆积作用为主,氢键和静电作用较小。细胞实验表明:在光照和非光照条件下,三制剂(Cur溶液组、Cur-rGO-UFH-FA组和Cur-rGO-UFH组)对MCF-7和A549两种细胞的细胞毒性差异较大。光照下细胞毒性显著增强,说明两种纳米粒中rGO在光照作用下发挥了光热效应,且光热效应与Cur对细胞的毒性具有协同作用。在非光照下,与溶液组相比,Cur-rGO-UFH对MCF-7和A549的细胞毒性显著增强,说明Cur-rGO-UFH可能通过EPR效应进入细胞,溶液组中Cur通过被动扩散进入细胞。Cur-rGO-UFH-FA 的细胞毒性比 Cur-rGO-UFH 的高,说明 Cur-rGO-UFH-FA除通过EPR效应途径外,还通过FR介导进入细胞。细胞凋亡实验表明:三组制剂中Cur的细胞凋亡率具有浓度依赖性;低浓度时,Cur-rGO-UFH-FA诱导细胞凋亡能力最强,Cur-rGO-UFH次之,溶液最差,说明低浓度时FR介导的胞饮作用发挥明显,高浓度时三组Cur制剂在被动扩散的驱使下进入细胞内浓度均较高,并产生了大面积细胞凋亡和坏死。细胞摄取实验表明:两种Cur靶向制剂对MCF-7细胞摄取均具有浓度和时间依赖性。本研究未见文献报道。第6章是对两种Cur纳米粒进行体内研究。大鼠静注Cur溶液组后,Cur的半衰期较短(仅0.32h),而两种纳米粒中Cur的半衰期大大延长,达到了 rGO纳米粒延长Cur半衰期的预期目标。小鼠体内分布实验表明:与Cur溶液相比,两种纳米粒具有肝靶向性,Cur-rGO-UFH-FA还具有肺靶向性。2h时的组织切片观察到两种Cur纳米制剂对心、脾组织没有影响,对肝、肺和肾有一过性影响,在48h时对这些组织的影响基本消除。第7章rGO分子的自聚集和载姜黄素过程进行模拟。结果表明:低浓度且亲水性较强的rGO仍是热力学不稳定体系,说明需采用亲水性大分子对rGO加以修饰。rGO包载Cur的本质是rGO吸附Cur;由于尺寸较大的rGO结构中含有sp2区域,故也模拟了 GRO包载Cur的过程。观察到rGO与Cur在5.01ns时发生了吸附,但疏水性更强的GRO在20ns内没有与Cur发生吸附,说明吸附过程不仅与π-π堆积作用有关,还与范德华力、静电力、氢键和水分子的碰撞等多种因素有关,长时间搅拌能增加分子间的碰撞几率,是提高载药量的途径之一。本研究未见文献报道。第8章研究了 GRO或rGO与Lyso和Myo两个蛋白质的相互作用。结果表明:蛋白质中各种氨基酸对rGO或GRO的结合作用是不固定的,rGO或GRO与蛋白之间的作用是复杂的,氨基酸的作用可随环境而变。rGO的结合能低,说明以rGO为载体的纳米粒体内生物相容性好。本研究未见文献报道。
[Abstract]:Reduced graphene oxide (rGO) is a kind of graphene oxide (GO) with reduced oxygen group in the plane. It has been developed rapidly in the past ten years. Due to high drug loading, high photothermal conversion efficiency and modifiable properties, it has made it in many directions in the field of biomedicine, such as drug delivery, gene transduction, DNA manipulation, tumor imaging, and light. Thermal treatment, bone tissue engineering and other extensive studies. This article is divided into three parts. The first part is a hemolysis test as the evaluation method, the hemolysis index (HI) as the evaluation index, to find the physicochemical characteristics of the GO derivatives with high biocompatibility, the selection, modification and preparation of GO carrier in the rGO nanoparticles loaded with curcumin (Cur) heparin modification The second part carries out a pharmaceutical study on the rGO nanoparticles carrying Cur, which uses Cur as a model drug, with two oxidized GO as the carrier material, with unfractionated heparin (UFH) as the hydrophilic polymer, and with folic acid (FA) as the target, and by reduction, to make non FA modified nanoparticles (Cur-rGO-UFH) and FA modification respectively. Nanoparticles (Cur-rGO-UFH-FA). In vitro release test, in vitro cell test, pharmacokinetic test, tissue distribution test, tissue section and so on as evaluation means, the two nanoparticles are evaluated in vitro and in vivo. The third part is the molecular simulation study of the nanoparticles in the nanoparticles. The mechanism is explained from the mechanism: (1) unmodified R GO is thermodynamically unstable and needs to be modified with hydrophilic polymer; (2) the process of carrying Cur in rGO. Using the lysozyme (Lyso) and myoglobin (Myo) as the model protein, the unmodified rGO as the typical carrier material, and using graphene (GRO) as the reference to illustrate the biocompatibility of the carrier materials after the nanoparticles enter the organism, such as the abundance of Myo content. The compatibility of the rich heart, and the compatibility of Lyso rich liver, spleen, lung and kidney. The main research methods and results in this paper are as follows: the first chapter is through the synthesis and characterization of 9 classes of 25 GO derivatives. The second chapter establishes a new method for solving the hemolysis index (HI) and the hemolysis test conditions. The method of seeking HI is to use 540nm, 576nm and 600nm three. The 540-576-600 method for ultraviolet absorption is a typical three point absorption difference method. In order to illustrate the accuracy of the method, the method of hemolysis percentage (HP) commonly used in the literature is compared, including the single point absorption value method represented by the 540 method and the two point absorption difference method represented by the 540-655 method. At the same time, the 576-600 method for the HI is also established. The two-point absorption method represented by three examples shows that the new 540-576-600 method has the characteristics of high accuracy of HI for finding GO derivatives. The common method for seeking HP has a large result, even more than 100% of hemolysis. The reasonable test conditions: 5% glucose solution is hemolytic medium and the final concentration of rabbit's source is 1%. Fresh and healthy RBCs was used as ideal red cell (RBCs), 3000rpm centrifugation 5min, incubating 3 ~ 6h. at 37 degrees centigrade in red blood cell concentration and centrifuge speed, etc. the interaction between GO and RBCs was quantitatively calculated. The study was not reported in the literature. The third chapter was used to evaluate the hemolysis of 25 GO derivatives in the first chapter, and found the GO derivative with high biocompatibility. Biological physicochemical characteristics, such as particle size reduction, hydrophilic polymer modification, reduction treatment, charge reduction. Fourth chapters are used to evaluate two kinds of heparinated rGO materials. On the basis of high biocompatibility structure characteristics, UFH is used as hydrophilic material, FA as the target and GO-2 as starting carrier material, and two kinds of heparinated rGO materials are made, respectively liver. Two Acylhydrazine -rGO (rGO-UFH) and folic acid modified heparin two hydrazine hydrazine -rGO (recorded as rGO-UFH-FA). These two materials have concentration, time dependent photothermal effect. Under the concentration of 10 mu g / mL-1 and 808nm light 5min, the two materials can reach the lower temperature of 50 centigrade for the photothermal treatment of tumor. Under conditions, two kinds of materials are basically non-toxic to MCF-7 or A549 cells; under light, rGO-UFH-FA is more lethal to MCF-7 cells. It shows that rGO-UFH-FA enters the cell through the mediating effect of folic acid receptor (FR), and rGO-UFH may enter the cell through the EPR effect. The fifth chapter studies the two kinds of Zingiber rGO nanoparticles in vitro. First, the calculation is established. The preparation process of Cur-rGO-UFH-FA was optimized by the method of drug loading (DL) and encapsulation efficiency (EE). The release study in vitro showed that the release of Cur in the nanoparticles was greatly influenced by the temperature, which was less affected by pH, indicating that the rGO-UFH-FA loading Cur process was mainly pion pion accumulation, and the hydrogen bond and electrostatic action were smaller. The cytotoxicity of three preparations (Cur solution group, Cur-rGO-UFH-FA group and Cur-rGO-UFH group) on the cytotoxicity of MCF-7 and A549 two cells was significantly different under light and non light conditions. The cytotoxicity of the cells was significantly enhanced under light, indicating that the rGO in the two nanoparticles had a light heat effect under the light of light, and the photothermal effect and the toxicity of Cur to the cells. In non light, the cytotoxicity of Cur-rGO-UFH to MCF-7 and A549 increased significantly compared with the solution group, indicating that Cur-rGO-UFH may enter cells through the EPR effect, and the cytotoxicity of Cur through passive diffusion into cell.Cur-rGO-UFH-FA is higher than that of Cur-rGO-UFH in solution group, indicating that Cur-rGO-UFH-FA is not only effective in EPR. In addition to the pathway, the cell apoptosis was mediated by FR. Apoptosis experiments showed that the apoptosis rate of Cur in the three groups was concentration dependent; at low concentration, Cur-rGO-UFH-FA induced the strongest apoptosis, Cur-rGO-UFH times and the worst solution, indicating that FR mediated cytotoxicity was obvious at low concentration, and three groups of Cur preparations at high concentration were in high concentration. In the passive diffusion, the intracellular concentration was high and the cell apoptosis and necrosis were produced in large areas. Cell uptake experiments showed that two kinds of Cur targeting agents had concentration and time dependence on the uptake of MCF-7 cells. The study was not reported in the literature. The sixth chapter was in vivo study on two kinds of Cur nanofilms. Rats were injected with Cur solution. After the group, the half-life of Cur was shorter (only 0.32h), and the half-life of Cur in the two nanoparticles was greatly extended, which reached the expected target of prolonging the Cur half-life of rGO nanoparticles. In vivo distribution experiment in mice showed that two nanoparticles had liver targeting compared with Cur solution, and two kinds of tissues were observed when Cur-rGO-UFH-FA also had lung targeting.2h. The Cur nanoscale has no effect on the heart and spleen, and has an over effect on the liver, lung and kidney. The effect of the 48h on these tissues is basically eliminated. The self aggregation of the seventh rGO molecules and the process of carrying curcumin are simulated. The results show that the low concentration and hydrophilic rGO is still a thermodynamic instability system, indicating that hydrophilic macromolecules need to be used. The essence of the modification of rGO for the.RGO loading Cur is that rGO adsorbs Cur; because the larger rGO structure contains the SP2 region, it also simulates the GRO loading Cur process. It is observed that rGO and Cur are adsorbed in 5.01ns, but the stronger hydrophobicity is not adsorbed with the GRO, indicating that the adsorption process is not only associated with the pion pion accumulation. It is also related to many factors such as van Edward force, static electricity, hydrogen bond and water molecules. Long time agitation can increase the probability of collisions between molecules. It is one of the ways to increase the amount of drug loading. This study has not been reported in the literature. The eighth chapter studies the interaction between GRO and rGO and the two proteins of Lyso and Myo. The results show that various amino acids in the protein are amino acids. The binding of rGO or GRO is not fixed, the role of rGO or GRO and protein is complex, and the effect of amino acids can be reduced with the environment with the low binding energy of.RGO. It shows that the biocompatibility of the nanoparticles with rGO as the carrier is good.

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

本文编号：1839181