【摘要】：基因治療作為一種全新的、革命性的治療手段，在過去幾十年取得了巨大發(fā)展，成為治療疾病的新技術，越來越受到關注。而基因治療的關鍵在于如何構建生物安全性好、基因遞送效率高的輸運載體。金納米籠（Au NCs）具有特殊的局域表面等離子體共振(LSPR)現(xiàn)象，可吸收近紅外波段(700～1200nm)的光波，將光能轉換為熱能，可實現(xiàn)對疾病的熱療效應。聚乙烯亞胺(PEI)分子上含有大量氨基基團而顯正電性，可負載負電性基因?qū)崿F(xiàn)目標基因的遞送，從而達到基因治療的效果。 基于此，本研究通過制備銀納米立方體，經(jīng)氯金酸刻蝕得到金納米籠（AuNCs）；將PEI進行巰基化修飾，通過Au-S鍵將PEI錨定到金納米籠表面，得到一種新型的基因載體(AuNCs-PEI)，并初步研究了該載體對質(zhì)粒DNA的運載效果。結果如下：1、銀納米立方體及金納米籠的制備及表征 通過控制硫化鈉的濃度、滴加量，及通入體系的氬氣速率，得到邊長為45nm的單分散銀納米立方體顆粒，其形貌單一且粒徑均勻；同時控制氯金酸的滴加體積和濃度，可制得LSPR峰在810nm處的金納米籠。2、AuNCs-PEI的構建及其運載基因效果的研究 將巰基化修飾的1.8KDa PEI和10KDa PEI，與AuNCs表面的PVP進行配體交換，得到相應的基因載體，分別記為AuNCs-PEI(1.8)和AuNCs-PEI(10)。采用波長為800nm的飛秒激光器照射AuNCs-PEI分散液，可檢測到分散液的溫度明顯上升，說明該載體具有明顯的熱效應。凝膠阻滯實驗結果顯示，AuNCs-PEI與EGFP(綠色熒光蛋白質(zhì)粒)質(zhì)量比(Au/DNA)高于1：1時，可將DNA全部結合到載體上；MTT毒性檢測結果顯示，AuNCs-PEI (10)在Au/DNA質(zhì)量比為15時，293T細胞仍保持較高的活力(85%)，同時AuNCs-PEI (1.8)處理的細胞活力與對照組相當，說明二者均具有較高的生物相容性；倒置熒光顯微鏡及流式細胞儀檢測結果發(fā)現(xiàn)，AuNCs-PEI (10)在與DNA質(zhì)量比為15時，，細胞轉染效率可達15%以上，說明AuNCs-PEI (10)載體具有一定基因遞送能力。因此，AuNCs-PEI(10)在基因治療相關疾病方面，具有一定的應用前景。 此外，為闡明特殊形貌(如方形、棒狀、線型等)的納米銀顆粒在印刷電子領域可能存在的應用前景，我們比較了銀納米立方體與同粒徑球形銀納米顆粒的導電性，結果顯示采用球形銀納米顆粒印制的導電圖樣，其電導率高于銀納米立方體的電導率。另外，由于球形銀納米顆粒合成方法更為簡單，且可大批量合成；而銀納米立方體合成條件苛刻，批次產(chǎn)量小。說明基于銀納米立方體制備的導電銀漿實現(xiàn)實際應用較為困難。
[Abstract]:Gene therapy, as a new and revolutionary therapy, has made great progress in the past few decades, and has become a new technology in the treatment of diseases, which has attracted more and more attention. The key of gene therapy is how to construct transport vector with good biological safety and high gene delivery efficiency. Gold nano-cage (Au NCs) has a special local surface plasmon resonance (LSPR) phenomenon. It can absorb the near infrared (700~1200nm) light wave and convert the light energy into heat energy, which can realize the effect of hyperthermia on disease. Polyethyleneimide (PEI) molecule contains a large number of amino groups and is positively charged, which can load negative electricity gene to realize the delivery of the target gene, thus achieving the effect of gene therapy. Based on this, the silver nanocrystals were prepared, and the gold nanocages (AuNCs); was prepared by chlorauric acid etching. The PEI was modified by sulfhydrylation, and PEI was anchored to the surface of the gold nanowires by Au-S bond. A novel gene vector (AuNCs-PEI) was obtained, and the effect of the vector on carrying plasmid DNA was preliminarily studied. The results are as follows: 1. The preparation and characterization of silver nanocrystals and gold nanometers were studied. By controlling the concentration of sodium sulphide, the amount of drops and the rate of argon gas flowing into the system, the monodisperse silver nanocrystalline particles with side length of 45nm were obtained. Its morphology is single and its particle size is uniform. At the same time, the drop volume and concentration of chlorgold acid are controlled. The construction of AuNCs-PEI with LSPR peak at 810nm and its effect on gene delivery were studied. The ligand exchange of mercapto-modified 1.8KDa PEI and 10KDa PEI, with PVP on the surface of AuNCs was carried out, and the corresponding gene vectors were recorded as AuNCs-PEI (1.8) and AuNCs-PEI (10), respectively. Using femtosecond laser with wavelength of 800nm to irradiate AuNCs-PEI dispersions, the temperature of the dispersions can be detected to rise obviously, which indicates that the carrier has obvious thermal effect. The results of gel block test showed that when the mass ratio of AuNCs-PEI and EGFP (green fluorescent protein particle) (Au/DNA) was higher than 1:1, all DNA could be bound to the carrier. The results of MTT toxicity test showed that 293T cells maintained high activity (85%) when the Au/DNA mass ratio was 15:00, and the cell viability of AuNCs-PEI (1.8) was similar to that of the control group, indicating that both of them had high biocompatibility. The results of reverse fluorescence microscopy and flow cytometry showed that the transfection efficiency of AuNCs-PEI _ (10) was more than 15% when the mass ratio of AuNCs-PEI _ (10) to DNA was 15:00, indicating that the AuNCs-PEI _ (10) vector had the ability of gene delivery. Therefore, AuNCs-PEI (10) has a certain application prospect in gene therapy related diseases. In addition, in order to elucidate the potential applications of silver nanoparticles with special morphologies (such as square, rod, line, etc.) in the field of printed electronics, we compared the electrical conductivity of silver nanometers with spherical silver nanoparticles of the same size. The results show that the electrical conductivity of spherical silver nanoparticles is higher than that of silver nanocrystals. In addition, the synthesis method of spherical silver nanoparticles is simpler and can be synthesized in large quantities, while the synthesis conditions of silver nanocrystals are harsh and the batch yields are small. It is difficult to realize the practical application of conductive silver paste based on silver nano cube.
【學位授予單位】：北京印刷學院
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：TB383.1

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