指導教授：牟中原臺灣大學：化學研究所林郁萱Lin, Yu-HsuanYu-HsuanLin牟中原指導2014-11-252018-07-102014-11-252018-07-102014http://ntur.lib.ntu.edu.tw//handle/246246/261244中孔洞氧化矽奈米粒子由於具有高表面積、單一孔洞大小以及容易進行官能基修飾等特性，使其常作為多功能型的奈米材料。在生物應用方面，因為中孔洞氧化矽奈米粒子具有極好的生物相容性及可分解性，因此被廣泛地運用在奈米生醫方面的研究，例如控制藥物進行定點釋放、蛋白質及基因的載體、醫學造影以及生物偵測等。 活性氧化物質 (ROS) 在生物體內扮演重要的角色，除了抵禦外來有機體及幫助細胞間訊號傳遞外，過去也有許多研究顯示ROS和許多疾病 (癌症、高血壓、糖尿病等) 的形成習習相關。在人體內，有許多抗氧化物質能夠精準地控制ROS含量，其中，過氧化物歧化酶 (SOD) 以及谷胱甘肽過氧化物酶 (GPx) 是主要的兩種蛋白質。過氧化物歧化酶將超氧陰離子 (O2‧-) 還原成過氧化氫；谷胱甘肽過氧化物酶再將過氧化氫分解成水。 這篇論文中，我們設計出具有催化功能的中孔洞氧化矽奈米粒子，其可有效地減少ROS並進一步的降低細胞內的氧化壓力。此奈米粒子可分為三個部分，分別是中孔洞二氧化矽奈米粒子、連接鏈以及抗氧化蛋白。首先，先利用共縮合及後修飾的方式將螢光分子FITC和NTA-Ni 修飾在中孔洞氧二化矽奈米粒子上。接著，利用基因工程的方式使TAT分別和SOD及GPx形成融合蛋白 (fusion protein)。最後，利用鎳及His-tag所形成的配位共價鍵來連接奈米粒子及抗氧化蛋白。我們利用人類免疫缺陷病毒中的反活化轉錄因子蛋白 (TAT) 能高效率穿越細胞膜的特性，希望能將奈米粒子更有效的送進細胞內。 在這研究的最後，我們將兩種帶有不同蛋白的奈米粒子 (FMSN-TAT-SOD、FMSN-TAT-GPx) 共同送進細胞內，最後我們找到了降低細胞內ROS效率最好的蛋白比例。在這個比例之下，我們可以有效的提升受到氧化壓力刺激的細胞存活率。我們預期這結果能使中孔洞二氧化矽奈米粒子在蛋白質治療上有更進一步的運用。Mesoporous silica nanoparticle (MSN) was used as a multifunctional material because of its characteristics, such as high surface area, uniform pore size and easy functionalization. Besides, MSN is very suitable for biological applications such as controlled drug release, cell labeling and enzyme delivery due to the properties of biocompatibility and degradability. As well known, ROS played a crucial role in many diseases and was defensed by superoxide dismutase (SOD) and glutathione peroxidase (GPx) which are two major antioxidant enzymes in physiological condition. The SOD scavenges superoxide radicals into hydrogen peroxide and GPx can convert hydrogen peroxide into water. In this study, we designed a catalytic MSN as a nanoreactor for scavenging ROS to reduce oxidative damages. The nanoreactor consists of three parts; namely, MSN, tether and enzymes. MSN was first synthesized with FITC and then modified by conjugating silane tether (NTA-Ni) to form FMSN-NTA-Ni. The fusion proteins of His-tagged TAT-hSOD and His-tagged TAT-hGPx were constructed and expressed by using genetic engineering method. Finally, the two fusion proteins are conjugated on the MSN surface respectively through the nickel and His-tagged interaction to compose sequential ROS scavengers. HIV transactivator protein (TAT) containing MSN was considered as an effective method in order to increasing the transmembrane permeability of nanoparticles. We finally demonstrated a two different functionalized-MSNs co-delivery into cells which can decline ROS efficiently. This study becomes a novel and promising tool for future therapeutic approach.口試委員會審定書 # 謝誌 i 中文摘要 iii ABSTRACT iv CONTENTS v LIST OF FIGURES viii LIST OF TABLES xii Chapter 1 Introduction 1 1.1 Ordered Mesoporous Silica 1 1.2 Synthesis of Mesoporous Silica Nanoparticles 3 1.2.1 Silica Sol-Gel Chemistry 3 1.2.2 Modified Stober Methods 5 1.2.3 Mechanism 7 1.2.4 Functionalization of MSNs 8 1.3 Biological Application of MSNs 11 1.3.1 MSNs-Based Drug Delivery 15 1.3.2 Protein Delivery 16 1.4 Reactive Oxygen Species (ROS) and Diseases 17 1.5 Motivation 20 Chapter 2 Materials and Methods 21 2.1 Synthesis of Fluorescent Mesoporous Silica Nanoparticles 21 2.2 Synthetic Scheme for NTA-Silane Linker 21 2.3 Conjugation of NTA-silane and Ni (II) with FMSN 22 2.4 Immobilization of His-TAT-Protein with FMSN-NTA-Ni 22 2.5 Characterization 23 2.6 Cell Liens and Reagents 23 2.7 Plasmid Construction 24 2.7.1 Human Cu, Zn-SOD 24 2.7.2 Human glutathione peroxidase 1 Sec49C mutant 25 2.8 Expression and Purification of Recombinant Proteins 26 2.9 Cell Culture 26 2.10 Western Blotting Analysis 27 2.11 Determination of Superoxide Dismutase and Glutathione Peroxidase Activity 27 2.12 Cell Viability Assay 28 2.13 Flow Cytometry Analysis 29 2.14 Immunocytochemical Staining for Proteins Expression 29 Chapter 3 Results and Discussion 30 3.1 Characterization of Fluorescence Mesoporous Silica Nanoparticles 31 3.2 Delivery of FMSN-TAT-Proteins into HeLa cell 35 3.2.1 Cytotoxicity of Nanoparticles 35 3.2.2 Delivery efficiency and distribution pattern of Nanoparticles 36 3.2.3 Refolded Enzymes Activity Assay 41 3.2.4 FMSN-TAT-Proteins Decrease Superoxide Anion Stress 44 3.2.5 Sequential Reactions in Co-delivery of FMSN-TAT-SOD and FMSN-TAT-GPx 46 Chapter 4 Conclusions 49 Chapter 5 References 502525627 bytesapplication/pdf論文公開時間：2019/08/08論文使用權限：同意有償授權(權利金給回饋本人)中孔洞矽奈米粒子抗氧化蛋白活性氧化物質[SDGs]SDG3thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/261244/1/ntu-103-R01223207-1.pdf