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Please use this identifier to cite or link to this item: http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48746

Title: 三段式適合體修飾之金奈米粒子於凝血酶活性抑制
Gold Nanoparticles Modified with Triblock Aptamers for Inhibition of Thrombin Activity
Authors: Huang, San-Shan
黃三珊
Contributors: 國立臺灣海洋大學:生命科學暨生物科技學系
Keywords: 適合體;單層自組裝;金奈米粒子;凝血酶;抗凝血藥物
aptamers;self-assembled hybrid monolayer;gold nanoparticles;thrombin;anticoagulants
Date: 2015
Issue Date: 2018-08-22T06:30:57Z
Abstract: 本研究中製備出三段式自組裝飾合體之金奈米粒子,是由自組裝之三段式適合體與金奈米粒子 (Au NPs; 粒徑大小13奈米) 所合成,對抑制凝血酶 (thrombin) 剪切纖維蛋白原 (fibrinogen) 有極佳的效果。第一段以聚單股腺嘌呤 [poly(adenine), poly-A] 與金奈米粒子表面作用;第二段則是可和另一股雜合 (hybridize, h) 的核苷酸;第三段分別是由15個與29個鹼基組成的專一辨識凝血酶之適合體(TBA15, TBA29),提供凝血酶雙價作用 (bivalent interaction)。自組裝之三段式適合體可調控: TBA15與TBA29之最佳立體空間距離、適合體在Au NPs 上之密度。這些增強材料與凝血酶的作用 (解離常數為1.5 × 10−11 M)。經過優化後修飾於 Au NPs 表面的三段式適合體為TBA15 h20A20和TBA29 h20A20 (A20為20個腺嘌呤鹼基組成的單股序列; h20為雜交序列的鹼基數)。TBA15/29h20-A20-Au NPs 在血漿中延長凝血酶凝集時間 (thrombin clotting time) 至少為市售凝血藥物肝素、阿加曲班、水蛭素、華法林的10倍。此外,TBA15/29h20-A20-Au NPs在血漿中有極高的穩定度 (半衰期大於14天) 與極好的生物相容性 (低細胞毒性與溶血效果)。最有趣的是 TBA15/29h20-A20-Au NPs 的抗凝血效果具可控性。當 TBA15/29h20-A20-Au NPs 照射綠光雷射 (波長532奈米, 1 瓦/平方公分),透過熱傳導促使TBA從Au NPs脫附,使其抗凝血效果為可逆的。我們的材料擁有簡易製備 (自組裝)、低成本 (未官能化之適合體)、光調控性、高穩定性以及良好的抑制效果等優點,這些在未來應用於治療凝血失調相當有潛力。這項實驗揭示自組裝奈米材料於調控分子結合、蛋白辨識以及酶活性的調控的可能性。
We demonstrated that thrombin-binding aptamer-conjugated gold nanoparticles (TBA–Au NPs), prepared from a self-assembled hybrid monolayer (SAHM) of triblock aptamers on Au NPs (13 nm), can effectively inhibit thrombin activity toward fibrinogen. The first block [poly(adenine): poly-A] at the end of the triblock TBA was used for the self-assembly on Au NP surface. The second block, in the middle of TBA, was composed of oligonucleotides that could hybridize with each other. The third block, containing TBA15 (15-base, binding to the exosite I of thrombin) and TBA29 (29-base, binding to the exosite II of thrombin) provided bivalent interaction with thrombin. The SAHM triblock aptamers have optimal distances between TBA15 and TBA29, aptamer density, and orientation on the Au NP surfaces. These properties strengthen the interactions with thrombin (Kd was 1.5 × 10−11 M), resulting in an extremely high anticoagulant potency. Modifications of Au NPs with triblock aptamers were optimized [TBAh20A20; A20: number of adenines (20), h20: hybrid length (20)]. The thrombin clotting time (TCT) mediated by TBA15h20A20/TBA29h20A20–Au NPs was >10 times longer than the TCT of commercially available drugs (heparin, argatroban, hirudin, or warfarin). The TBA15h20A20/TBA29h20A20–Au NPs exhibited excellent stability in the human plasma (half-life >14 days) and good biocompatibility (low cytotoxicity and hemolysis). Most interestingly, we found that the inhibition by SAHM TBA15h20A20/TBA29h20A20–Au NPs was controllable. It can be reversed using green laser irradiation (532 nm, 1 W), via heat transfer-induced TBA release from Au NPs. Therefore, these easily prepared (self-assembled), low cost (non-thiolated aptamer), photo-controllable, multivalent TBA15h20A20/TBA29h20A20–Au NPs (high density of TBA15/TBA29 on Au NPs) show good potential for the treatment of various diseases related to blood-clotting disorders. Our study opens up the possibility of regulation of molecule binding, protein recognition, and enzyme activity using SAHM aptamer-functionalized nanomaterials.
URI: http://ethesys.lib.ntou.edu.tw/cgi-bin/gs32/gsweb.cgi?o=dstdcdr&s=G0010336013.id
http://ntour.ntou.edu.tw:8080/ir/handle/987654321/48746
Appears in Collections:[生命科學暨生物科技學系] 博碩士論文

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