本文選題：金納米棒 + 殼聚糖��； 參考：《北京協(xié)和醫(yī)學(xué)院》2012年博士論文

【摘要】：金納米棒(gold nanorods、GNRs)具有獨(dú)特的光、電性能,成為材料學(xué)家關(guān)注的熱點(diǎn),被越來越多地應(yīng)用于醫(yī)學(xué)成像、生物檢測、基因及藥物載體和光熱治療等生物醫(yī)學(xué)研究領(lǐng)域,展現(xiàn)出廣闊的應(yīng)用前景。殼聚糖具有良好的生物相容性、低毒性、可降解性、對生物黏膜較強(qiáng)的粘附性及組織相容性,可用于生物醫(yī)用材料、基因載體及新型藥物傳遞系統(tǒng)的研究中。尤其是殼聚糖作為抗腫瘤藥物載體的研究已經(jīng)非常廣泛。 本文基于GNRs的光熱療以及抗腫瘤藥物的化療,構(gòu)建了一種毒性低、生物相容性好、適合生物醫(yī)學(xué)領(lǐng)域運(yùn)用的殼聚糖基金納米棒雜化材料,實(shí)現(xiàn)光熱療-化療聯(lián)合治療腫瘤的目的。主要研究工作如下： 1.合成了不同PEG取代度、不同PEI取代度的殼聚糖衍生物。首先,以聚乙二醇(PEG2000)與丁二酸酐(SA)為原料,合成了雙羧基的聚乙二醇化合物(化合物1),通過1H NMR和13C NMR進(jìn)行結(jié)構(gòu)表征結(jié)果顯示,合成產(chǎn)物為化合物1且轉(zhuǎn)化完全。同時(shí),由聚乙烯亞胺(PEI)合成了部分巰基化的PEI(化合物2),反應(yīng)中投料比增加或反應(yīng)時(shí)間延長均會(huì)使巰基化程度加深,合成了三種巰基取代度的化合物2,即PEI-SH2、PEI-SH2.4和PEI-SH2.7。以殼聚糖、化合物1和2為原料,通過NHS/EDC催化法分別合成了殼聚糖-PEG衍生物(化合物3)和殼聚糖-PEG-PEI衍生物(化合物4),并通過1HNMR和FT-IR進(jìn)行結(jié)構(gòu)表征,分別探討化合物3和化合物4合成過程中投料比對各步產(chǎn)物取代度的影響,隨著化合物1和2投料比例的增加取代度增大,但增大到一定程度時(shí)不會(huì)再出現(xiàn)明顯增大,得到了一系列殼聚糖衍生物。 2.合成了長徑比可控的金納米棒(GNRs)并進(jìn)行了表面修飾。通過晶種生長法合成了GNRs,CTAB包裹于GNRs表面,端位相對裸露巰基優(yōu)先結(jié)合于端位。將化合物2與GNRs混合,通過Au-S鍵2結(jié)合于GNRs表面,透射電鏡圖可見化合物2與GNRs摩爾比較低時(shí),GNRs以端位連接為主,摩爾比較高時(shí)出現(xiàn)部分并肩排列情況�；衔�4與GNRs混合,混合液被透析處理所得產(chǎn)物(CS-GNRs)結(jié)構(gòu)以并肩排列為主,且化合物4的PEI取代度高、與GNRs摩爾比高時(shí)有利于并肩排列組裝形式的出現(xiàn)；混合液被靜置處理所得CS-GNRs在摩爾比低時(shí),呈現(xiàn)聚合物被包裹于內(nèi)部,GNRs排列在外的組裝結(jié)構(gòu),摩爾比高時(shí)也出現(xiàn)了并肩排列的趨勢。綜合產(chǎn)物穩(wěn)定性、生物相容性等方面,靜置條件下的并肩結(jié)構(gòu)更適合用于下一步研究。 3.考察了CS-GNRs用于藥物載體研究的性質(zhì)。以阿霉素(DOX)與化合物4為原料,在NHS/DCC催化下合成了阿霉素-殼聚糖衍生物(DOX-CS,化合物5),參照DOX的濃度-吸光度標(biāo)準(zhǔn)曲線,計(jì)算化合物5中DOX取代度為18.4%。通過UV-vis光譜檢測表明CS-GNRs的光學(xué)穩(wěn)定性、溫度敏感性和激光的穩(wěn)定性良好,可用于體內(nèi)外研究。DOX-CS與GNRs偶聯(lián)產(chǎn)物DOX-CS-GNRs的UV-vis光譜中縱軸吸收峰的位置發(fā)生了紅移TEM圖觀察到產(chǎn)物DOX-CS-GNRs為側(cè)面排列的GNRs團(tuán)簇。 4.體外評價(jià)了DOX-CS-GNRs的細(xì)胞毒性、細(xì)胞攝取以及光熱療-化療聯(lián)合抗腫瘤效果。通過細(xì)胞毒性實(shí)驗(yàn)證明,GNRs在很低濃度便對細(xì)胞表現(xiàn)出了很強(qiáng)的毒性作用。CS-GNRs和DOX-CS-GNRs則表現(xiàn)出了較好的生物相容性,細(xì)胞毒性很小。DOX-CS-GNRs與腫瘤細(xì)胞共同孵育2h后,通過激光共聚焦顯微鏡在細(xì)胞內(nèi)觀察到紅色熒光(DOX的熒光)表明,DOX-CS-GNRs已進(jìn)入了腫瘤細(xì)胞。在近紅外激光照射條件下,體外評價(jià)CS-GNRs對腫瘤細(xì)胞的光熱療殺傷作用,DOX-CS-GNRs對腫瘤細(xì)胞光熱療-化療聯(lián)合治療作用,結(jié)果顯示CS-GNRs對腫瘤細(xì)胞具有明顯的殺傷抑制效果,而DOX-CS-GNRs的殺傷效果更高。以上結(jié)果表明,光熱療-化療的聯(lián)合治療作用要比單純光熱療的治療作用更強(qiáng),具有更理想的治療效果。 綜上所述,殼聚糖衍生物與金納米棒通過Au-S鍵化學(xué)鍵合方式構(gòu)建具有特定組裝結(jié)構(gòu)的殼聚糖基金納米棒,同時(shí)負(fù)載抗腫瘤藥物阿霉素,其具有良好的生物相容性和腫瘤殺傷作用,通過光熱療-化療聯(lián)合治療方式比單純的光熱療具有更強(qiáng)的腫瘤殺傷效果。
[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.

【學(xué)位授予單位】：北京協(xié)和醫(yī)學(xué)院
【學(xué)位級別】：博士
【學(xué)位授予年份】：2012
【分類號】：R318.08;R730.5
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本文編號：1864221