高分子/奈米金複合材料合成、鑑定及核酸傳遞應用

Synthesis and Characterizations of Polymer/Gold Nanocomplex and Its Applications on Nucleic Acid Delivery

莊鈞喬(Chun-Chiao Chuang)

指導教授 ： 張建文

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2013年)

https://doi.org/10.6843/NTHU.2013.00601

摘要

非病毒性腫瘤基因治療面臨的首要問題是如何有效地將基因送入細胞，並且維持一定時間的基因表現，並為了克服傳統上含血清環境轉染效率差及基因載體毒性限制，我們將開發具高安全性及高效率之基因傳遞載體。
第一部分研究基於開發新穎非病毒性及高生物相容性之金奈米粒子基因載體。將合成帶有雙硫鍵且具有生物可降解性之轉染高分子(SPEI)，透過1H-NMR鑑定、細胞轉染效率實驗及毒性測試。金奈米粒子基因載體部分，則是合成金奈米粒子(AuNP)後，將11-Mercaptoundecanoic acid(MUA)利用自組裝方式修飾於金奈米粒子表面(MUA-AuNP)，再將可降解高分子(SPEI)修飾於金奈米粒子表面(SPEI/MUA-AuNP)，使金奈米粒子具攜帶核酸能力，接著利用靜電作用力製備SPEI/MUA-AuNPs/nucleic acid/γglutamic acid(γPGA)複合體。並對金奈米粒子基因載體進行電泳、粒徑大小、表面電位和紫外光可見光吸收光譜之物化性鑑定。經由物化性鑑定基因片段裝載至金奈米載體後，並將SPEI/MUA-AuNPs/nucleic acids/γPGA複合材料攜帶螢光與冷光基因片段並傳送到哺乳動物細胞。從以上實驗結果顯示，金奈米粒子具有攜帶核酸能力，且相較於市售PEI25K於含血清環境下有較高轉染效率。
第二部分研究是開發具有高生物相容性極低毒性之金奈米棒基因載體。首先，利用開環反應合成帶有硫醇基之高分子(PEI800-SH)，將PEI800-SH藉由金硫共價鍵(Au-S)自組裝於金奈米棒表面，使得金奈米棒具有攜帶核酸能力，並且對金奈米棒進行洋菜膠電泳、傅立葉轉換光譜儀、粒徑大小、表面電位和紫外光可見光吸收光譜之物化性分析。最後，由細胞分布、轉染效率及存活率實驗證實，金奈米棒表面上修飾之PEI800-SH幫助金奈米棒分散性、降低細胞毒性、具有攜帶核酸能力及細胞轉染效率，綜合實驗結果顯示，PEI800-SH修飾後之金奈米棒具有較低細胞毒性及高轉染效率。

關鍵字

可降解PEI ； 金奈米粒子 ； 金奈米棒 ； 基因傳遞 ； 逐層自組裝

並列摘要

Nonviral gene delivery carriers are designed to efficiently deliver genes (nucleic acids) into the targeted cells/tissues in a safe manner. The goal of this thesis is to develop new biodegradable polymer/gold nanocomplex systems to overcome low transfection dfficiency under serum-containing environment ans carrier cytotoxicity. The thesis contains two main parts as describes below:
In the first part of the thesis, a biodegradable polymer/gold nanosphere system was prepared using layer-by-layer deposition method and assessed for its gene delivery capability. A biodegradable polymer (SPEI) was synthesized following by the structure characterizations using 1H-NMR. To prepare polymer/gold nanosphere complex, we synthesized gold nanosphere (AuNP) with uniform size distribution. 11-Mercaptoundecanoic acid (MUA) was then used to coated covalently onto the surface of AuNP. SPEI was subsequently deposited on the surface of MUA-AuNP via electrostatic interaction to form SPEI/MUA-AuNP. In order to understand the interactions between the complex and nucleic acids as well as the changes on the material's properties during layer-by-layer process, a series of physicochemical properties were studied. To assess its gene transfection capability on mammalian cells, both fluorescence gene and luciferase gene were utilized as the transgene expression reporting system. The results show that SPEI/MUA-AuNP is capable of interacting and delivering the designated genes into the cells. It is worth mentioning that the transfection efficiency of SPEI/MUA-AuNP was significantly higher than the commercial PEI25K under serum-containing environment. Importantly, SPEI/MUA-AuNP resulted in significantly lower cytotoxicity comparing to PEI25K. Taken these results together, it is anticipated that this biodegradable polymer/gold nanocomplex system has good potential on nonviral gene therapy.
In the second part of the thesis, gold nanorods (AuNR) with surface-bound low molecular weight PEI800 were synthesized and tested for the gene delivery performance. First, thiol group-bearing PEI800 (PEI800-SH) was synthesized by a ring-opening reaction. PEI800-SH was then used to assemble onto the surface of AuNR via gold-thiol covalent bonding to afford AuNR-S-PEI800 with nucleic acid-binding ability. AuNR-S-PEI800 was subsequently characterized by a series of physicochemical properties. Finally, the cellular uptake/distribution, gene transfection efficiency and cytocompatibility were investigated and the results suggest that AuNR-S-PEI800 posses well dispersity, low cytotoxicity and efficient gene transfection on mammalian cells.

並列關鍵字

biodegradable polymer ； gold nanoparticles ； gold nanorods ； layer-by-layer assembly ； gene delivery

參考文獻

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