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研究生： 亞瑪菈
Arham, Marannu
論文名稱： 聚賴胺酸之接枝研究：合成、自組裝及其為載體之應用
Studies of Poly(L-lysine)-Based Graft Copolypeptides: Synthesis, Self Assembly and Their Applications as Carriers
指導教授： 詹正雄
Jan, Jeng-Shiung
學位類別： 碩士
Master
系所名稱： 工學院 - 化學工程學系
Department of Chemical Engineering
論文出版年： 2011
畢業學年度： 99
語文別： 英文
論文頁數： 91
中文關鍵詞： 聚賴胺酸 、自組裝 、雙醣 、肌紅素 、小紅莓
外文關鍵詞： poly(L-lysine), self-assembly, disaccharide, myoglobin, doxorubicin
相關次數： 點閱：136  下載：2 
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    • 摘要
    在此探討兩性共聚合高分子聚賴胺酸-接枝六碳鏈及雙醣(Poly(L-lysine)-g-hexanoyl and Poly(L-lysine)-g-(hexanoyl, lactobionolactone)) 於水相之自組裝行為及藉由包覆親水性蛋白質-肌紅素(myoglobin)及抗癌親水性藥物- Doxorubicin以評估其為載體或包覆體的應用。於水相中自組裝後之兩性共聚合高分子其形成粒子大小會因疏水比例、pH值及離子濃度而不同，故此更經由動態光散射儀(DLS)及穿透式電子顯微鏡(TEM)證實於不同條件下所製備的奈子粒子大小約為100至400奈米大小。更甚，將此生物相容之共聚合高分子於薄膜水合法中加入交聯劑 (genipin) 以穩定其結構以提高肌紅素包覆率，並經DLS測試所形成的囊泡(vesicle) 大小在180~280奈米之間，並定出包覆肌紅素的效率可達50% 且證實為一良好攜氧載體，並於水相中穩定存在達數月。 雙醣為一良好標的受器於癌細胞，故在相同程序下，改變包覆物為水溶性抗癌藥物DOX，測其包覆效果，其效率可達30%。實驗發現，在藥物釋放實驗中，所製備的包覆藥物之奈米液胞於酸性及中性環境下都不足以阻擋藥物的快速釋放，推測原因為六碳鏈所形成的疏水作用力不足而顯鬆散而導致DOX小分子易穿透疏水膜。對此欲嘗試藉由Genipin交聯以穩定結構來延緩藥物釋放時間。利用雙醣修飾不只提高雙性共聚胺基酸高分子之生物相容性，這些兩性共聚胺酸並能應用於生醫領域中的標靶特藥物載體、仿生包覆體及功能性生物奈米反應器。

    ABSTRACT
    The synthesis and self-assembly of poly(L-lysine)-based graft copolypeptide, poly(L-lysine)-graft-hexanoyl (PLH) and poly(L-lysine)-graft-(hexanoyl,lactobionolactone) (PLL-g-(Hex,Lac)), and their evaluation as carriers/encapsulants for myoglobin (Mb) and doxorubicin (DOX) encapsulation have been investigated. The interplay between the hydrophobic interactions and the chain conformational changes upon hexanoyl and disaccharide substitution determined the self-assembly behavior of PLH and PLL-g-(Hex,Lac), which further influenced the change of the assembled nanostructures. The DLS and TEM data revealed that these amphiphilic copolypeptides can form vesicles with mean sizes between 100 and 400 nm in acidic and neutral aqueous solution. The hexanoyl substitution can regulate the hydrophobic interaction, chain conformation of PLH and PLL-g-(Hex,Lac), and subsequently the size of the assembled vesicles at different pH values and ionic strengths. Taking advantage of the self-assembly capability, the PLH was employed for protein encapsulation, i.e. myoglobin (Mb). The encapsulation efficiency was higher than 50% and the encapsulated protein was still capable of carrying oxygen, which demonstrated that the PLH vesicles are promising carriers or encapsulants. The Mb-loaded PLH particles were further crosslinked by genipin to form stable nanostructures with mean sizes between 180 and 280 nm. The as-prepared particles were found to be well suspended in solution for months. Lactobionolactone was also used here as a target ligand for tumor cells. An anticancer drug, doxorubicin (DOX), has been encapsulated in these vesicles with efficiency around 27-30%. The release curves show faster release, either in acidic or neutral condition and no difference with non-encapsulated drug, which is possibly because the hydrophobic membrane is not strong enough to retain the drug inside the core. In the future, by modification of these vesicles, such as crosslinking the bilayer or deposition of inorganic compound onto the vesicles, it is expected to increase the time release of DOX. With grafting lactobionolactone, one can expect not only the biocompatibility of poly(L-lysine) can be improved but also these amphiphilic copolypeptides can be useful as targeted drug carriers, biomimetic encapsulants, and functional nanobioreactors in the biomedical fields.

    ABSTRACT i 摘要 ii ACKNOWLEDGEMENTS iii TABLE OF CONTENTS v LIST OF TABLES ix LIST OF SCHEMES x LIST OF FIGURES xi CHAPTER ONE INTRODUCTION 1 1.1 Overview 1 1.2 Polypeptide 2 1.3 Self Assembly of Peptide-Based Copolymer 2 1.4 Applications of Polypeptide in Biomedical 4 1.5 Research Motivation 5 CHAPTER TWO LITERATURE REVIEW 8 2.1 Graft Copolymer 8 2.1.1 Nature of The Backbone 9 2.1.2 Effect of Monomer 10 2.1.3 Effects of Solvent 10 2.1.4 Effect of Initiator 11 2.1.5 Role of Additives on Grafting 11 2.1.6 Effects of Temperature 11 2.2 Polymeric Vesicles (Polymersomes) 12 2.3 Polymeric Vesicles as Carriers 14 2.3.1 Oxygen Carriers 14 2.3.1.1 Myoglobin 16 2.3.1.2 Hemoglobin 17 2.3.1.3 Oxygen Delivery and Storage 18 2.3.2 Drug Carriers 19 2.4 Conformational Changes of Polypeptide 21 2.5 Genipin as Crosslinking Agent 23 CHAPTER THREE EXPERIMENTAL SECTION 28 3.1 Materials 28 3.2 Sample Preparation 29 3.2.1 Synthesis of Poly(L-lysine)-graft-Hexanoyl (PLH) 29 3.2.2 Synthesis of Poly(L-lysine)-graft-(Hexanoyl, Lactobionolactone) (PLL-g-(Hex,Lac)) 31 3.3 Characterization and Measurements 33 3.3.1 Gel Permeation Chromatography (GPC) 33 3.3.2 1H Nuclear Magnetic Resonance (1H-NMR) 34 3.3.3 Fourier Transform Infrared (FT-IR) Spectroscopy 34 3.3.4 Particle Size and Zeta Potential 35 3.3.5 Transmission Electron Microscopy (TEM) 36 3.3.6 Critical Aggregation Concentration (CAC) 36 3.3.7 Ultraviolet-Visible (UV-vis) Spectroscopy 37 3.3.8 Circular Dichroism (CD) 37 3.3.9 Titration Analysis 38 3.4 Application as Oxygen Carrier 38 3.4.1 Encapsulation of Mb in The PLH Vesicles 38 3.4.2 Evaluation of Mb Activity 40 3.4.3 Crosslinking of PLH Vesicles 40 3.4.4 Crosslinking of Mb-Loaded PLH Particles 41 3.5 Application as Drug Carrier 41 3.5.1 Evaluation of Dox Encapsulation 41 3.5.2 Evaluation of Dox Release in Vitro 43 CHAPTER FOUR RESULTS AND DISCUSSION 44 4.1 Synthesis and Characterization of Graft Copolypeptide 44 4.1.1 Synthesis and Characterization of Poly(L-lysine)-graft-Hexanoyl (PLH) 44 4.1.2 Synthesis and Characterization of Poly(L-lysine)-graft-(Hexanoyl, Lactobionolactone) (PLL-g-(Hex,Lac)) 48 4.2 Self-Assembly of Graft Copolypeptides 51 4.2.1 Self-Assembly of Poly(L-lysine)-graft-Hexanoyl (PLH) 51 4.2.2 Self-Assembly of Poly(L-lysine)-graft-(Hexanoyl, Lactobionolactone) (PLL-g-(Hex,Lac)) 66 4.3 Applications of Graft Copolypeptide 72 4.3.1 Application of Poly(L-lysine)-graft-Hexanoyl (PLH) as Oxygen Carrier 72 4.3.1.1 Encapsulation of Myoglobin in the PLH Vesicles 72 4.3.1.2 Crosslinking of Mb-Loaded PLH Particles 77 4.3.2 Application of PLH and PLL-g-(Hex,Lac) as Drug Carrier 81 4.3.2.1 Drug Loading and Release in Vitro 81 CHAPTER FIVE CONCLUSION AND SUGGESTIONS 83 REFERENCES 85 APPENDIX 89 CURRICULUM VITAE 91

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