臺灣博碩士論文加值系統

Biocompatible nanoparticles for cancer therapy are of an interesting area across a number of science, engineering and biomedical disciplines. Particles with different sizes, shapes, and physical properties are considered to influence the organ accumulation and cell uptake. Herein, gold nanoparticle will be used as the inner core of a drug carrier for immobilizing anti-cancer drugs and PEG polymers. The outer shell of PEG polymers will act to avoid mononuclear phagocyte system recognition while to allow much tumor uptake in association with passive or active targeting. The density of PEG polymers will be regulated to prepare a suitable long circulation nanoparticles. Additionally, anti-cancer drug derivatives with hydrazone linkers will be designed to be sensitive to intracellular pH changes and degradable to release drugs from gold nanoparticles.
In this study, we have successfully prepared different mPEG-SH densities and different sizes of gold nanoparticles. The in vitro BSA stability and flow cytometer study showed that the minimized mPEG-SH density had the best BSA stability and the best size for cell endocytosis was about 50 nm. Furthermore, doxorubicin (DOX) was conjugated to gold nanoparticles via acidic pH-sensitive hydrazone linkers. DOX conjugated gold nanopartilces showed higher drug release at endosome pH 4.5~6.5 as compared to physiological pH 7.4. By cell proliferation assays, DOX conjugated 50 nm-PEG min gold nanopartilces showed more cytotoxicity compared to DOX conjugated 20 nm-PEG min gold nanopartilces, which was similar to free DOX, against HCT116 and HeLa cells.
In conclusion, this study demonstrated the optimal size and mPEG-SH density of gold nanopartilces in combination with pH sensitive doxorubicin derivative as a long circulation drug carrier for cancer therapy.

在化學、工程以及生物醫學等領域，生物可相容性的奈米粒子一直被廣泛的應用在癌症治療上。不同材料大小、形狀與物理性質的奈米粒子皆會影響細胞的吞噬能力以及體內器官的累積。在本實驗中，我們設計一藥物載體以金奈米粒子做為內核，並在表面上接枝聚乙烯醇高分子以及抗癌藥物。在奈米粒子表面接枝聚乙烯醇高分子，可避免體內單核吞噬細胞系統的辨識以增加在血液循環的時間，並透過主動或被動標的大量累積在腫瘤的位置。藉由調控表面聚乙烯醇高分子的密度製備出一具有長時間血液循環的金奈米粒子，並將抗癌藥物透過hydrazone的鍵結修飾成具酸鹼敏感性的衍生物。當藥物載體被細胞吞噬時，進入細胞endosome，因酸鹼值下降使得hydrazone的鍵結水解斷裂，進而將藥物從載體上釋放出來。
本研究成功製備出不同大小的金奈米粒子以及不同表面聚乙烯醇高分子的密度，透過胎牛血清穩定度與流式細胞儀的實驗，證明了表面接枝防止金奈米粒子聚集的最小值PEG高分子數量即具有良好的血清穩定度以及50奈米的大小是細胞吞噬較佳的粒徑。成功透過hydrazone鍵結以修飾DOX抗癌藥物使其能夠接枝上金奈米粒子的表面。在體外藥物釋放的實驗中，在pH值7.4模擬血液的環境之下，DOX抗癌藥物能夠穩定的存在金奈米粒子的表面上，而當pH值降低到4.5~6.5時，hydrazone的鍵結會快速水解斷裂使DOX抗癌藥物能夠釋放出來。在化學材料對於細胞毒性的實驗上，聚乙烯醇高分子接枝上金奈米粒子對於細胞是幾乎沒有毒性，而在接有DOX抗癌藥物的金奈米粒子，對於細胞的毒性則是50奈米大於20奈米並與單純的DOX抗癌藥物不相上下。本研究利用聚乙烯醇高分子以及抗癌藥物的衍生物修飾金奈米粒子並探討高分子密度與粒子大小對於細胞吞噬的影響，未來希望能成為一種安全的藥物傳遞的載體及應用於癌症治療。

List of Contents
Abstract
Chapter 1 Motivation and purpose 1
Chapter 2 Introduction 3
2.1 Nanoparticles: an excellent scaffold for biological applications. 4
2.2 Nanoparticles as drug delivery systems. 5
2.2.1 Positive targeting of drug delivey 8
2.2.2 Long-circulating nanoparticles 10
2.3 Material property 12
2.3.1 The property of polyethylene glycol. 13
2.3.2 Pegylated modification. 14
2.4 Important properties of gold nanoparticles. 16
2.4.1 Surface plasmon absorption property of gold nanoparticles. 18
2.4.2 Cellular uptake of gold nanoparticles. 22
2.4.3 Current Applications of Gold Nanoparticles. 24
Chapter 3 Materials and methods 28
3.1 Reagents 28
3.2 Equipment and Instruments 30
3.3 Cell lines and buffers 31
3.4 Methods 32
3.4.1 Synthesis of methoxy poly(ethylene glycol)-undecyl mercaptane 32
3.4.2 Determination of free thiol group content. 33
3.4.3 General gold nanoparticles synthesis technique. 34
3.4.4 To quantitatively determine the maximum of mPEG-SH on gold nanoparticles surface. 35
3.4.5 Preparation of pegylated gold nanoparticles. 36
3.4.6 The copper grid of pegylated gold nanoparticles for TEM. 36
3.4.7 Stability of pegylated gold nanoparticles in phosphate buffer and bovine serum albumin (BSA) solution. 37
3.4.8 Cell uptake experiments of different PEG density by Flow cytometer. 37
3.4.9 Synthesis of doxorubicin derivatives for pH inductive. 38
3.4.10 Prepare Dox conjugated 20 nm-PEG min and 50 nm-PEG min gold nanoparticles. 39
3.4.11 The drug loading efficiency and loading content of the nanoparticles. 40
3.4.12 Drug releasing of Dox conjugated gold nanoparticles in phosphate buffer. 40
3.4.13 Cytotoxicity of pegylated gold nanoparticles and Dox conjugated gold nanoparticles. 41
Chapter 4 Results and discussion 42
4.1 Characterization of mPEG derivative. 42
4.1.1 Methoxy poly(ethylene glycol)-undecene. 42
4.1.2 Methoxy poly(ethylene glycol)-undecane thioacetate ester. 44
4.1.3 Methoxy poly(ethylene glycol)-undecyl mercaptane. 45
4.2 Determination of free thiol group content using Ellman’s assay. 48
4.3 Colloidal gold nanoparticles synthesis. 49
4.4 To quantitatively determine the maximum of mPEG-SH on gold nanoparticles surface. 52
4.5 Characterization of PEGylated gold nanoparticles. 53
4.6 Stability of pegylated gold nanoparticles in phosphate buffer and bovine serum albumin (BSA) solution. 55
4.7 Cell uptake experiments of different PEG density by Flow cytometry. 58
4.8 Characterization of doxorubicin derivatives. 61
4.8.1 3-(2-Pyridyldithio) propionyl hydrazide (PDPH). 61
4.8.2 Doxorubicin 13-{3-(2-pyridyldithio) propionyl} hydrazone hydrochloride. 62
4.8.3 Doxorubicin hydrazone thiol. 63
4.9 The drug loading efficiency and loading content of the Dox conjugated gold nanoparticles. 65
4.10 Drug releasing of DOX conjugated gold nanoparticles in phosphate buffer. 67
4.11 Cytotoxicity of pegylated GNPs. 69
4.12 Cytotoxicity of DOX conjugated gold nanopartilces. 70
Chapter 5 Conclusion 74
Reference 76

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