本文選題：還原氧化石墨烯 + 生物相容性�。� 參考：《山東大學(xué)》2017年博士論文

【摘要】：還原氧化石墨烯(rGO)是一種平面內(nèi)含氧基團減少的氧化石墨烯(GO),是近十年來迅速發(fā)展起來的高分子材料,由于載藥量高、光熱轉(zhuǎn)換效率高、可修飾性等性能,使其在生物醫(yī)藥領(lǐng)域的多個方向如藥物傳遞、基因轉(zhuǎn)導(dǎo)、DNA操縱、腫瘤成像、光熱治療、骨組織工程等有廣泛的研究。本文分為三部分,第一部分以溶血試驗為評價手段,以溶血指數(shù)(HI)為評價指標(biāo),尋找生物相容性高的GO衍生物所具備的理化特征,為載姜黃素(Cur)肝素修飾的rGO納米粒中GO載體的選擇、修飾和制劑研究奠定基礎(chǔ)。第二部分對載Cur的rGO納米粒進(jìn)行藥學(xué)研究。該納米粒以Cur為模型藥物、以二次氧化的GO為起始的載體材料、以未分級肝素(UFH)為親水性修飾高分子,以葉酸(FA)為靶頭,經(jīng)還原處理,分別制成非FA修飾納米粒(記為Cur-rGO-UFH)和FA修飾納米粒(記為Cur-rGO-UFH-FA)。以體外釋放度試驗、體外細(xì)胞試驗、藥代動力學(xué)試驗、組織分布試驗、組織切片等為評價手段,對這兩種納米粒進(jìn)行體內(nèi)外評價。第三部分為該納米粒中rGO相關(guān)的分子模擬研究。從機理上直觀地解釋了:(1)未修飾的rGO是熱力學(xué)不穩(wěn)定的,需用親水性高分子加以修飾;(2)rGO載Cur的過程。以溶菌酶(Lyso)和肌紅蛋白(Myo)為模型蛋白質(zhì)、以未修飾的rGO為典型的載體材料、以石墨烯(GRO)為參照,說明了納米粒進(jìn)入生物體后載體材料的生物相容性,如:與Myo含量豐富的心臟的相容性,與Lyso含量豐富的肝、脾、肺和腎的相容性。本論文主要的研究方法與結(jié)果如下:第1章通過從合成和表征了 9類25個GO的衍生物。第2章建立了新的求溶血指數(shù)(HI)的方法和溶血試驗條件。求HI的方法是利用540nm、576nm和600nm三處紫外吸收的540-576-600法,它是一種典型的三點吸收差值法。為說明該法的準(zhǔn)確性,比較了文獻(xiàn)中常用到的溶血百分?jǐn)?shù)(HP)方法,包括以540法為代表的單點吸收值法和以540-655法為代表的兩點吸收差值法,同時還建立了求HI的以576-600法為代表的兩點吸收值法。通過三個實例說明了新建的540-576-600法具有求GO衍生物的HI準(zhǔn)確度高的特點。而常見的求HP的方法存在結(jié)果偏大,甚至有可能出現(xiàn)超過100%的溶血現(xiàn)象。合理的試驗條件:5%葡萄糖溶液為溶血介質(zhì)、家兔來源終濃度為1%新鮮健康的RBCs作為理想的紅細(xì)胞(RBCs),3000rpm離心5min,37℃孵育3～6h。在紅細(xì)胞濃度、離心速度等問題上,對GO與RBCs之間相互作用進(jìn)行定量計算。本研究未見文獻(xiàn)報道。第3章對第1章中25個GO衍生物進(jìn)行溶血評價,找到了生物相容性高的GO衍生物理化特征,如粒徑減小、親水性高分子修飾、還原處理、電荷減少。第4章對兩種肝素化rGO材料進(jìn)行評價。在生物相容性高的結(jié)構(gòu)特征基礎(chǔ)上,選擇以UFH為親水性材料,以FA為靶頭、GO-2為起始載體材料,制成了兩種肝素化rGO材料,分別為肝素-己二酸二酰肼-rGO(記為rGO-UFH)和葉酸修飾的肝素-己二酸二酰肼-rGO(記為rGO-UFH-FA)。這兩種材料具有濃度、時間依賴性的光熱效應(yīng)。在濃度為10μg·mL-1且808nm光照5min條件下,兩種材料均能達(dá)到腫瘤光熱治療所需的下限溫度50℃。在非光照條件下,兩種材料對MCF-7或A549細(xì)胞基本無毒;在光照下,rGO-UFH-FA對MCF-7細(xì)胞的殺傷性較強,說明rGO-UFH-FA通過葉酸受體(FR)介導(dǎo)作用進(jìn)入胞內(nèi),而rGO-UFH可能通過EPR效應(yīng)進(jìn)入到細(xì)胞。第5章對兩種姜黃素rGO納米粒進(jìn)行體外研究。首先建立了計算載藥量(DL)和包封率(EE)的方法,以星點設(shè)計-效應(yīng)面法優(yōu)化了 Cur-rGO-UFH-FA的制備工藝。體外釋放研究表明:納米粒中Cur的釋放受溫度的影響較大,受pH的影響較小,說明rGO-UFH-FA包載Cur的過程是以π-π堆積作用為主,氫鍵和靜電作用較小。細(xì)胞實驗表明:在光照和非光照條件下,三制劑(Cur溶液組、Cur-rGO-UFH-FA組和Cur-rGO-UFH組)對MCF-7和A549兩種細(xì)胞的細(xì)胞毒性差異較大。光照下細(xì)胞毒性顯著增強,說明兩種納米粒中rGO在光照作用下發(fā)揮了光熱效應(yīng),且光熱效應(yīng)與Cur對細(xì)胞的毒性具有協(xié)同作用。在非光照下,與溶液組相比,Cur-rGO-UFH對MCF-7和A549的細(xì)胞毒性顯著增強,說明Cur-rGO-UFH可能通過EPR效應(yīng)進(jìn)入細(xì)胞,溶液組中Cur通過被動擴散進(jìn)入細(xì)胞。Cur-rGO-UFH-FA 的細(xì)胞毒性比 Cur-rGO-UFH 的高,說明 Cur-rGO-UFH-FA除通過EPR效應(yīng)途徑外,還通過FR介導(dǎo)進(jìn)入細(xì)胞。細(xì)胞凋亡實驗表明:三組制劑中Cur的細(xì)胞凋亡率具有濃度依賴性;低濃度時,Cur-rGO-UFH-FA誘導(dǎo)細(xì)胞凋亡能力最強,Cur-rGO-UFH次之,溶液最差,說明低濃度時FR介導(dǎo)的胞飲作用發(fā)揮明顯,高濃度時三組Cur制劑在被動擴散的驅(qū)使下進(jìn)入細(xì)胞內(nèi)濃度均較高,并產(chǎn)生了大面積細(xì)胞凋亡和壞死。細(xì)胞攝取實驗表明:兩種Cur靶向制劑對MCF-7細(xì)胞攝取均具有濃度和時間依賴性。本研究未見文獻(xiàn)報道。第6章是對兩種Cur納米粒進(jìn)行體內(nèi)研究。大鼠靜注Cur溶液組后,Cur的半衰期較短(僅0.32h),而兩種納米粒中Cur的半衰期大大延長,達(dá)到了 rGO納米粒延長Cur半衰期的預(yù)期目標(biāo)。小鼠體內(nèi)分布實驗表明:與Cur溶液相比,兩種納米粒具有肝靶向性,Cur-rGO-UFH-FA還具有肺靶向性。2h時的組織切片觀察到兩種Cur納米制劑對心、脾組織沒有影響,對肝、肺和腎有一過性影響,在48h時對這些組織的影響基本消除。第7章rGO分子的自聚集和載姜黃素過程進(jìn)行模擬。結(jié)果表明:低濃度且親水性較強的rGO仍是熱力學(xué)不穩(wěn)定體系,說明需采用親水性大分子對rGO加以修飾。rGO包載Cur的本質(zhì)是rGO吸附Cur;由于尺寸較大的rGO結(jié)構(gòu)中含有sp2區(qū)域,故也模擬了 GRO包載Cur的過程。觀察到rGO與Cur在5.01ns時發(fā)生了吸附,但疏水性更強的GRO在20ns內(nèi)沒有與Cur發(fā)生吸附,說明吸附過程不僅與π-π堆積作用有關(guān),還與范德華力、靜電力、氫鍵和水分子的碰撞等多種因素有關(guān),長時間攪拌能增加分子間的碰撞幾率,是提高載藥量的途徑之一。本研究未見文獻(xiàn)報道。第8章研究了 GRO或rGO與Lyso和Myo兩個蛋白質(zhì)的相互作用。結(jié)果表明:蛋白質(zhì)中各種氨基酸對rGO或GRO的結(jié)合作用是不固定的,rGO或GRO與蛋白之間的作用是復(fù)雜的,氨基酸的作用可隨環(huán)境而變。rGO的結(jié)合能低,說明以rGO為載體的納米粒體內(nèi)生物相容性好。本研究未見文獻(xiàn)報道。
[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.

【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R943
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本文編號：1839181