基于纳米石墨烯的基因与药物载体研究

发布时间：2018-05-10 05:05

本文选题：纳米石墨烯 + 基因和药物输送　；参考：《苏州大学》2015年博士论文

【摘要】：癌症是当前影响人类最为主要的疾病之一,给整个人类健康带来了严重的威胁。目前常用的癌症治疗手段,尤其是化学药物疗法,虽然能够在一定的程度上抑制肿瘤的发生,但是其往往由于对肿瘤细胞缺少良好的选择性而会对正常组织造成较强的毒副作用并会诱导肿瘤细胞产生多耐药性,从而不能有效地抑制肿瘤生长,最终导致了治疗失败。因此,开发具有肿瘤靶向性、能够克服肿瘤细胞多耐药性的新型高效的癌症治疗手段得到了广发的关注和深入的研究。在本博士论文中,我们以纳米石墨烯为核心材料,通过不同的表面修饰方法筛选出了一种低毒、高效并具有良好血清稳定性的基因转染载体;接下来,我们利用石墨烯在近红外光区较强的光热转换能力,在细胞水平上实现了光热促进的基因转染;最后,我们以Polo样激酶I(Plk1)基因为治疗靶点,通过RNA干扰手段在细胞水平上实现了光热促进的基因治疗。在另一个工作中,我们设计了一种基于纳米石墨烯的对肿瘤组织和肿瘤细胞内的微酸环境具有双重响应性的纳米药物输送载体,并在细胞水平上通过光热治疗和化疗的联合使用有效抑制了多耐药性肿瘤细胞的生长。本博士论文研究为发展新型癌症治疗手段、克服肿瘤细胞多耐药性、提高癌症治疗效果提供了一个新的实验依据。本论文的研究内容和主要结论如下:1、通过静电吸附方法在纳米石墨烯的表面修饰上一层带正电荷的聚乙烯亚胺分子,得到了表面带正电荷的纳米石墨烯复合物GO-PEI,并在细胞水平上研究了其基因转染效果。研究发现:通过静电吸附方法可以有效地将聚乙烯亚胺包裹到纳米石墨烯表面,有效提高了石墨烯在生理溶液中的稳定性,为后续的基因转染研究提供了可能;凝胶电泳实验发现纳米石墨烯/聚乙烯亚胺复合物可以有效地包裹质粒DNA;体外细胞活力测试发现,通过与纳米石墨烯结合可以有效降低聚乙烯亚胺的细胞毒性;最后,发现纳米石墨烯/聚乙烯亚胺复合物可以有效地将质粒DNA转染到细胞内,并与单独的聚乙烯亚胺相比,其基因转染效果有了一定的提高。2、在前一个工作的基础上,我们通过共价修饰的方法首先在纳米石墨烯表面偶联上枝状聚乙二醇分子,以提高其在含血清生理溶液中的稳定性并降低其细胞毒性;然后再通过共价修饰的方法将聚乙烯亚胺偶联到石墨烯表面,得到了聚乙二醇和聚乙烯亚胺双功能化的纳米石墨烯ngo-peg-pei,并研究其在含有血清的培养基中的基因转染效果。接下来,利用石墨烯在近红外光区的光热转换能力,我们研究了低功率近红外激光照射对ngo-peg-pei针对质粒dna和小干扰rna转染能力的影响。研究发现:纳米石墨烯表面共价偶联上聚乙二醇后可以进一步提高其在多种生理溶液中的稳定性以及在含血清培养基中的基因转染效果,并在一定程度上降低了共价偶联的聚乙烯亚胺的细胞毒性;利用石墨烯在近红外光区的光热转换能力,发现低功率的近红外激光照射可以在短时间内有效提高材料的进细胞能力,极大地提高了质粒dna在细胞内的表达以及小干扰rna的干扰效果。3、利用肿瘤组织和正常组织酸度的不同,我们设计了一种基于纳米石墨烯的对肿瘤组织和肿瘤细胞内不同酸度环境具有双重响应性的药物输送载体,研究了其克服肿瘤细胞多耐药性的能力。首先通过共价偶联的方法,分别将聚乙二醇和聚烯丙基胺偶联到石墨烯表面,得到了在生理溶液中具有良好稳定性的表面带正电荷的纳米石墨烯复合物ngo-peg-pah;然后,通过酸酐水解的方法将上一步得到的ngo-peg-pah表面的氨基用2,3-二甲基马来酸酐封闭,得到了表面带负电荷的纳米氧化石墨烯复合物ngo-peg-da。研究发现该材料在正常生理ph值下可以维持其表面的负电性,丧失其原本良好的进细胞能力;一旦将其孵育在微酸环境中(ph6.8),该材料的表面电荷就会发生反转变为正电荷,从而恢复其进细胞能力。进一步研究发现,石墨烯表面的疏水区域可以被用于装载疏水的抗肿瘤药物(例如:阿霉素),并具有ph值响应性的药物释放性质和细胞杀伤能力。最后,结合石墨烯优良的光热转换能力,我们在细胞水平上通过光热治疗和化学治疗的联合使用有效克服了肿瘤细胞的多耐药性。在本博士论文中,我们系统研究了石墨烯在基因与药物的可控运输方面的应用潜能。研究发现,石墨烯自身优良的光热转换性可以有效的提高材料的基因转染效果以及克服肿瘤多耐药性的能力,并证明了石墨烯的光热效应在与基因治疗或化疗联用以用于癌症治疗的优势。本论文研究为开发新型癌症治疗方法提供了一个新策略,为后续光热响应性以及肿瘤微环境响应性的智能型基因与药物载体的设计提供了一定的参考。
[Abstract]:Cancer is one of the most important diseases that affect human being. It poses a serious threat to human health. The commonly used methods of cancer treatment, especially chemical therapy, can inhibit the occurrence of cancer to a certain extent, but it is often due to the lack of good selectivity for the tumor cells. Strong toxic and side effects can induce tumor cells to produce multi drug resistance, which can not effectively inhibit tumor growth and eventually lead to treatment failure. Therefore, the development of new and efficient cancer therapy with tumor targeting and overcoming the multidrug resistance of tumor cells has received extensive attention and in-depth study. In this paper, we use nano graphene as the core material to screen a gene transfection carrier with low toxicity, high efficiency and good serum stability by different surface modification methods. Next, we use the strong photothermal conversion ability of graphene in the near infrared light region, and realize the gene transfer of light and heat promoted at the cell level. In the end, we use the Polo like kinase I (Plk1) gene as the target to achieve a photothermal gene therapy at the cell level by RNA interference. In another work, we designed a nano drug delivery carrier based on the dual responsiveness of the nano graphene to the tumor tissue and the micro acid environment in the tumor cells. The combined use of photothermal therapy and chemotherapy at the cell level effectively inhibits the growth of multidrug-resistant tumor cells. This thesis provides a new experimental basis for the development of new cancer therapies, overcoming the multidrug resistance of cancer cells and improving the effectiveness of cancer treatment. 1, 1, by modifying a positive charged polyethylenimide molecule on the surface of graphene by electrostatic adsorption, the nano graphene complex with positive charge on the surface was obtained, and the gene transfection effect was studied at the cell level. It was found that polyethylenimide could be effectively wrapped by electrostatic adsorption. Coated with the surface of graphene, the stability of graphene in the physiological solution is improved effectively. It is possible for the subsequent gene transfection research. The gel electrophoresis experiment found that the nano graphene / polyethyleneimine complex can effectively wrap the plasmid DNA. In vitro cell viability test found that the combination of graphene with nano graphene can be effective. The cytotoxicity of polyethyleneimine was reduced. Finally, it was found that the nano graphene / polyethyleneimine complex could effectively transfect plasmid DNA into the cell. Compared with the individual polyethyleneimine, the gene transfection effect had a certain increase of.2. On the basis of the previous work, we first made the covalent modification in nanoscale. The branched polyethylene glycol is coupled to the surface of graphene to improve its stability in the serum containing serum and reduce its cytotoxicity. Then, the polyethylenimide is coupled to the surface of graphene by covalent modification, and the diethylene glycol and polyethylenimide nano graphene ngo-peg-pei has been obtained and studied. The effect of gene transfection in the medium containing serum. Next, we studied the effect of low power near infrared laser irradiation on the transfection capacity of plasmid DNA and small interfering RNA using the light heat transfer ability of graphene in near infrared light region. We found that the covalent coupling of the surface of graphene on the surface of graphene can be obtained by ngo-peg-pei. To further improve its stability in a variety of physiological solutions and the effect of gene transfection in the serum containing medium, the cytotoxicity of covalent conjugated polyethyleneimine is reduced to a certain extent, and the photothermal conversion ability of graphene in near infrared light area is used, and the low power near infrared laser irradiation can be found in a short time. The expression of plasmid DNA in the cell and the interference effect of small interfering RNA were greatly improved, and the interference effect of small interference was greatly improved. Using the difference of the acidity of the tumor tissue and normal tissue, we designed a kind of drug delivery based on the dual responsiveness of the nano graphene to the tumor tissues and the different acidity environments in the tumor cells. The ability to overcome the multidrug resistance of tumor cells was studied. First, polyethylene glycol and polyallyl amine were coupled to the surface of graphene by covalent coupling, and the positive charged nano graphene complex ngo-peg-pah with good stability in the physiological solution was obtained. The method has closed the amino group on the surface of ngo-peg-pah with 2,3- two methyl maleic anhydride, and obtained the nanocomposite ngo-peg-da. with negative charge on the surface. It was found that the material could maintain the negative electricity on the surface under normal physiological pH, and lost its original good cellular ability; once it was incubated in micro In an acid environment (ph6.8), the surface charge of the material turns into a positive charge to restore its cellular capacity. Further studies have found that the hydrophobic region of the graphene surface can be used to load hydrophobic antitumor drugs (such as doxorubicin), with the response to the pH value of drug release and cell killing. Finally, Combined with the excellent photothermal conversion ability of graphene, we have effectively overcome the multidrug resistance of tumor cells at the cellular level by combination of photothermal therapy and chemical therapy. In this doctoral thesis, we systematically studied the potential of graphene in the controllable transport of genes and drugs. The photothermal conversion can effectively improve the gene transfection effect and overcome the multidrug resistance of the tumor, and prove the advantages of the photothermal effect of graphene in combination with gene therapy or chemotherapy for cancer treatment. This paper provides a new strategy for the development of new cancer treatment methods for subsequent photothermal response. It provides a reference for the design of intelligent genes and drug carriers in response to stress and tumor microenvironment.

【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：O613.71;TQ460.1

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