壳聚糖基金纳米棒的构建及其在肿瘤治疗中的应用

发布时间：2018-05-09 02:39

  本文选题：金纳米棒 + 壳聚糖　； 参考：《北京协和医学院》2012年博士论文

【摘要】：金纳米棒(gold nanorods、GNRs)具有独特的光、电性能,成为材料学家关注的热点,被越来越多地应用于医学成像、生物检测、基因及药物载体和光热治疗等生物医学研究领域,展现出广阔的应用前景。壳聚糖具有良好的生物相容性、低毒性、可降解性、对生物黏膜较强的粘附性及组织相容性,可用于生物医用材料、基因载体及新型药物传递系统的研究中。尤其是壳聚糖作为抗肿瘤药物载体的研究已经非常广泛。 本文基于GNRs的光热疗以及抗肿瘤药物的化疗,构建了一种毒性低、生物相容性好、适合生物医学领域运用的壳聚糖基金纳米棒杂化材料,实现光热疗-化疗联合治疗肿瘤的目的。主要研究工作如下： 1.合成了不同PEG取代度、不同PEI取代度的壳聚糖衍生物。首先,以聚乙二醇(PEG2000)与丁二酸酐(SA)为原料,合成了双羧基的聚乙二醇化合物(化合物1),通过1H NMR和13C NMR进行结构表征结果显示,合成产物为化合物1且转化完全。同时,由聚乙烯亚胺(PEI)合成了部分巯基化的PEI(化合物2),反应中投料比增加或反应时间延长均会使巯基化程度加深,合成了三种巯基取代度的化合物2,即PEI-SH2、PEI-SH2.4和PEI-SH2.7。以壳聚糖、化合物1和2为原料,通过NHS/EDC催化法分别合成了壳聚糖-PEG衍生物(化合物3)和壳聚糖-PEG-PEI衍生物(化合物4),并通过1HNMR和FT-IR进行结构表征,分别探讨化合物3和化合物4合成过程中投料比对各步产物取代度的影响,随着化合物1和2投料比例的增加取代度增大,但增大到一定程度时不会再出现明显增大,得到了一系列壳聚糖衍生物。 2.合成了长径比可控的金纳米棒(GNRs)并进行了表面修饰。通过晶种生长法合成了GNRs,CTAB包裹于GNRs表面,端位相对裸露巯基优先结合于端位。将化合物2与GNRs混合,通过Au-S键2结合于GNRs表面,透射电镜图可见化合物2与GNRs摩尔比较低时,GNRs以端位连接为主,摩尔比较高时出现部分并肩排列情况。化合物4与GNRs混合,混合液被透析处理所得产物(CS-GNRs)结构以并肩排列为主,且化合物4的PEI取代度高、与GNRs摩尔比高时有利于并肩排列组装形式的出现；混合液被静置处理所得CS-GNRs在摩尔比低时,呈现聚合物被包裹于内部,GNRs排列在外的组装结构,摩尔比高时也出现了并肩排列的趋势。综合产物稳定性、生物相容性等方面,静置条件下的并肩结构更适合用于下一步研究。 3.考察了CS-GNRs用于药物载体研究的性质。以阿霉素(DOX)与化合物4为原料,在NHS/DCC催化下合成了阿霉素-壳聚糖衍生物(DOX-CS,化合物5),参照DOX的浓度-吸光度标准曲线,计算化合物5中DOX取代度为18.4%。通过UV-vis光谱检测表明CS-GNRs的光学稳定性、温度敏感性和激光的稳定性良好,可用于体内外研究。DOX-CS与GNRs偶联产物DOX-CS-GNRs的UV-vis光谱中纵轴吸收峰的位置发生了红移TEM图观察到产物DOX-CS-GNRs为侧面排列的GNRs团簇。 4.体外评价了DOX-CS-GNRs的细胞毒性、细胞摄取以及光热疗-化疗联合抗肿瘤效果。通过细胞毒性实验证明,GNRs在很低浓度便对细胞表现出了很强的毒性作用。CS-GNRs和DOX-CS-GNRs则表现出了较好的生物相容性,细胞毒性很小。DOX-CS-GNRs与肿瘤细胞共同孵育2h后,通过激光共聚焦显微镜在细胞内观察到红色荧光(DOX的荧光)表明,DOX-CS-GNRs已进入了肿瘤细胞。在近红外激光照射条件下,体外评价CS-GNRs对肿瘤细胞的光热疗杀伤作用,DOX-CS-GNRs对肿瘤细胞光热疗-化疗联合治疗作用,结果显示CS-GNRs对肿瘤细胞具有明显的杀伤抑制效果,而DOX-CS-GNRs的杀伤效果更高。以上结果表明,光热疗-化疗的联合治疗作用要比单纯光热疗的治疗作用更强,具有更理想的治疗效果。 综上所述,壳聚糖衍生物与金纳米棒通过Au-S键化学键合方式构建具有特定组装结构的壳聚糖基金纳米棒,同时负载抗肿瘤药物阿霉素,其具有良好的生物相容性和肿瘤杀伤作用,通过光热疗-化疗联合治疗方式比单纯的光热疗具有更强的肿瘤杀伤效果。
[Abstract]:Giner Mi Bar (gold nanorods, GNRs), with its unique light and electrical properties, has become a hot spot of attention by materials scientists. It has been more and more applied to biomedical research fields such as medical imaging, biological detection, gene and drug carrier and photothermal therapy, and has a broad application prospect. Chitosan has good biocompatibility, low toxicity and can be reduced. The solubility, strong adhesion and histocompatibility of biological mucous membrane can be used in the research of biomedical materials, gene carriers and new drug delivery systems. Especially, chitosan has been widely used as an antitumor drug carrier.
Based on the phototherapy of GNRs and the chemotherapy of antitumor drugs, this paper constructs a chitosan fund nanorod hybrid material with low toxicity and good biocompatibility and suitable for the biomedical field. The main research work is to achieve the purpose of phototherapy and chemotherapy combined with chemotherapy.
1. the chitosan derivatives with different degree of substitution and different degree of substitution of PEG were synthesized. First, polyglycol (PEG2000) and butylene two anhydride (SA) were used as raw materials to synthesize the polycarboxylic polyethylene glycol compound (compound 1). The structural characterization results were shown by 1H NMR and 13C NMR, and the synthetic product was compound 1 and the transformation was complete. At the same time, polyethylene was made from polyethylene. Partially mercapto PEI (compound 2) was synthesized by Imide (PEI). The degree of sulfhydryl group was deepened by the increase of feed ratio or the prolongation of reaction time, and three kinds of sulfhydryl compounds 2, namely, PEI-SH2, PEI-SH2.4 and PEI-SH2.7., were prepared with chitosan, compound 1 and 2 as raw materials. The derivatization of chitosan -PEG was synthesized by NHS/EDC catalysis. Biological (compound 3) and chitosan -PEG-PEI derivatives (compound 4) were characterized by structural characterization by 1HNMR and FT-IR. The effect of feeding ratio on the degree of substitution of each step in the synthetic process of compound 3 and compound 4 was investigated. The degree of substitution increased with the increase of the proportion of compound 1 and 2, but the increase to a certain extent did not appear to be obvious. In addition, a series of chitosan derivatives were obtained.
2. the length ratio controlled gold nanorod (GNRs) was synthesized and the surface modification was carried out. GNRs was synthesized by seed growth method, CTAB was wrapped on the surface of GNRs, and the end position was combined with the nude sulfhydryl group at the end position. Compound 2 and GNRs were mixed with Au-S key 2 on the GNRs surface, and the compound 2 and GNRs mole were lower, GNRs The compound 4 and GNRs are mixed with GNRs, and the structure of the mixture is based on shoulder arrangement, and the PEI substitution of compound 4 is high. When the molar ratio is higher than the GNRs molar ratio, it is beneficial to the appearance of shoulder arrangement, and the mixed solution is treated by the static treatment of CS. When the mole ratio is low, -GNRs shows that the polymer is wrapped inside and the GNRs is arranged outside the assembly structure, and the trend of shoulder arrangement appears at the higher molar ratio. The stability of the synthetic products, biocompatibility and so on, the shoulder structure under the static condition is more suitable for the next research.
3. the properties of CS-GNRs used for drug carrier study were investigated. Adriamycin (DOX) and compound 4 were used as raw materials to synthesize adriamycin chitosan derivatives (DOX-CS, compound 5) under the catalysis of NHS/DCC. According to the concentration absorption standard curve of DOX, the DOX substitution degree in compound 5 was determined by UV-vis spectroscopy to show the optical stability of CS-GNRs. The qualitative, temperature sensitive and laser stability are good. It can be used in the study of the UV-vis spectra of.DOX-CS and GNRs coupling product DOX-CS-GNRs in vitro and in vivo. The position of the longitudinal axis absorption peak in the UV-vis spectrum of DOX-CS-GNRs occurred in the red shift TEM diagram and observed the GNRs clusters of the product DOX-CS-GNRs in side arrangement.
4. in vitro, the cytotoxicity, cell uptake, phototherapy and chemotherapy combined with anti tumor effect were evaluated in vitro. By cytotoxicity test, the cytotoxicity test showed that GNRs showed a strong toxic effect on the cells at very low concentration,.CS-GNRs and DOX-CS-GNRs showed good biocompatibility, and the cytotoxicity was very small.DOX-CS-GNRs and the tumor was fine. After the co incubation of 2h, the red fluorescence (fluorescence of DOX) was observed by laser confocal microscopy in the cells. DOX-CS-GNRs had entered the tumor cells. Under the conditions of near infrared laser irradiation, the photothermotherapy killing effect of CS-GNRs on the tumor cells was evaluated in vitro. DOX-CS-GNRs was combined with phototherapy and chemotherapy of tumor cells. The results show that CS-GNRs has an obvious killing effect on tumor cells, and the killing effect of DOX-CS-GNRs is higher. The above results show that the combined therapeutic effect of phototherapy and chemotherapy is more effective than simple phototherapy, and has more ideal therapeutic effect.
To sum up, chitosan derivatives and gold nanorods construct a chitosan fund nanorod with a specific assembly structure through the Au-S bond chemical bond, which is also loaded with antitumor drug adriamycin, which has good biocompatibility and tumor killing effect, and is better than simple phototherapy by photothermotherapy combined therapy. A strong tumor killing effect.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R318.08;R730.5
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本文编号：1864221