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研究生: 楊景勛
Yang, Ching-Hsun
論文名稱: 利用小角度散射研究添加聚乙二醇脂質之磷脂質圓盤微胞及高分子微胞與DNA之吸附作用
Small Angle Scattering Studies on PEGylated Lipid Bicelles and Polymer Micelles/DNA Complexes
指導教授: 林滄浪
Lin, Tsang-Lang
口試委員: 鄭有舜
Jeng, U-Ser
李明道
Lee, Ming-Tao
蕭百沂
Hsiao, Pai-Yi
王本誠
Wang, Pen-Cheng
學位類別: 博士
Doctor
系所名稱: 原子科學院 - 工程與系統科學系
Department of Engineering and System Science
論文出版年: 2019
畢業學年度: 107
語文別: 英文
論文頁數: 147
中文關鍵詞: 小角度散射磷脂質圓盤微胞高分子微胞DNA吸附
外文關鍵詞: Small angle scattering, Lipid bicelle, Polymer micelle, DNA adsorption
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  • 研究主要目的是更深入了解自組裝生物材料的結構與作用機制,進而對於其應用如藥物輸送及基因治療有所幫助,主要利用小角度散射技術研究添加聚乙二醇脂質之磷脂質圓盤微胞及高分子微胞。添加聚乙二醇脂質之磷脂質在圓盤微胞,可以提昇圓盤微胞抗聚集的能力,聚乙二醇脂質的分佈會影響到微胞與其他分子的作用,也會影響到微胞的尺寸,因此在載體設計上是非常重要的參數;研究發現聚乙二醇脂質不只分佈在圓盤微胞的邊緣,也會有一定數量分佈在圓盤微胞的核心平面區域,然而其分佈在圓盤微胞邊緣之數量密度約為核心平面區域的1.7倍,因此圓盤微胞的尺寸會隨著加入越多聚乙二醇脂質之磷脂質而越來越小。另外我們也研究由聚(苯乙烯)-聚(碘化N-甲基4-乙烯基吡啶)嵌段共聚物所形成之高分子微胞吸附去氧核醣核酸的作用,由於該嵌段共聚物在水溶液中形成具有帶正電高分子刷外層微胞,因此其高分子刷殼層可以經由電荷作用吸附多量之去氧核醣核酸;研究發現的確如此,但當去氧核醣核酸濃度較高時,高分子刷殼層大致已吸滿去氧核醣核酸,吸附在表層的去氧核醣核酸會開始使高分子微胞產生團塊聚集,對於應用較為不利,須保持去氧核醣核酸濃度低於聚集臨界濃度。除此之外,在研究圓盤微胞與去氧核醣核酸或多價離子的作用時,我們發現會有沉澱再溶解的現象,利用小角度散射觀察到帶電荷圓盤微胞在加入聚電解質時會形成的有序層狀堆疊結構,但所產生的散射峰在聚電解質濃度超過某特定臨界濃度時,散射峰開始減弱至消失,顯示無法再維持有序緊密的聚集結構。由穿透式電子顯微鏡的觀察可以發現高聚電解質濃度不會使圓盤微胞的片層聚集結構完全再溶解分散,只是變成排列變得較為無序的聚集結構。


    Our studies mainly focus on the biological self-assembly materials which can be used in biomedical applications, such as drug delivery and gene therapy. The studies include the lipid bicelles incorporated with PEGylated lipids and the cationic core-shell polymer micelles. Small angle scattering (SAS) is used to investigate these complex structures. The distributed PEGylated lipids affects the interaction of the bicelles as well as the size of the bicelle. The added C16-PEG2000 ceramide lipids are found to distribute both in rim and also the planar region of the bicelle. However, the lipid number density of the added C16-PEG2000-Ceramide in the rim is found to be about 1.7 times of the C16-PEG2000-Ceramide lipid density in the planar region. The bicelle size becomes smaller due to the incorporation of the added C16-PEG2000-Ceramide lipid in the rim. Other than studying the bicelles, we also investigated the interaction of PS-b-P4VPQ micelles with DNA. The thick cationic brush shell of the PS-b-P4VPQ micelles is expected to be able to adsorb appreciable amounts of DNA within the brush layer. Indeed, it is found that appreciable amount of DNA is adsorbed in the cationic brush layer and the brush layer is swelled due to the adsorption of DNA. At high DNA concentrations, some adsorbed DNA start to build up at the edge or surface of the brush layer which could induce aggregation of the polymer micelle/DNA complexes. This means that it is possible to prepare mostly dispersed polymer/DNA complexes by keeping the DNA concentration below the aggregation concentration. Moreover, the interaction of charged bicelles with DNA and multivalent ions is found to have the reentrant condensation phenomena. From SAXS measurement, it is found that the lamellar diffraction peak of bicelle stacks gradually diminish when the spermidine concentration reaches a threshold. However, the ordered lamellar structure only becomes disordered instead of complete re-dissolution as evidenced by the observation of TEM.

    ABSTRACT i TABLE OF CONTENTS iii LIST OF TABLES vi LIST OF FIGURES vii Chapter 1: Introduction 1 1.1 Gene therapy and Drug delivery 1 1.2 Non-viral vector: Bicelles 6 1.3 Non-viral vector: Polymer complexes 8 1.4 Research Objectives 10 Chapter 2: Review of Lipid and Polymer Complexes 13 2.1 PEGylated bicelles 13 2.2 Phase diagram of lipid complexes 16 2.3 Application of lipid complexes 21 2.4 Application of polymer complexes 22 2.5 Reentrant condensation 27 Chapter 3: Experimental Section 31 3.1 Materials 31 3.1.1 Lipid 31 3.1.2 Block-copolymer 34 3.1.3 DNA 34 3.1.4 Polycation 34 3.2 Sample preparations 35 3.2.1 SAXS sample 35 3.2.2 TEM sample 36 3.3 Methods 36 3.3.1 Small angle X-ray scattering (SAXS) 36 3.3.2 Small angle neutron scattering (SANS) 42 3.3.3 Transmission electron microscopy (TEM) 44 3.4 Igor fitting analysis 45 3.4.1 Form factor: PolyCoreBicelle model 45 3.4.2 Form factor: PolyCoreShellCylinder model 47 3.4.3 Structure factor: HardSphere model 48 3.4.3 Structure factor: StickyHardSphere model 49 3.4.5 Structure factor: HayterPenfoldMSA model 49 Chapter 4: PEG-Cermide Bicelles 51 4.1 PEG-Cer. bicelles 51 4.2 PEG-Cer. bicelles v.s. Temperature 67 4.3 PEG-Cer./DC-Chol. bicelles with DNA in solution 70 Chapter 5: PS-b-P4VPQ Block-copolymer Micelles 73 5.1 BCP micelles with low DNA concentrations 82 5.2 BCP micelles with high DNA concentrations 85 Chapter 6: Bicelle Reentrant Condensation Systems 89 6.1 Cationic ions condensation with bicelle/DNA complexes 89 6.2 Sp3+ condensation with anionic bicelles 94 Chapter 7: Conclusions 103 References 105 Appendix-A: SANS profiles of PEG2000 Cer. Bicelles 115 Appendix-B: PEG2000 Bicelles 120 B1. SAXS profiles 120 B2. SANS profiles 125 Appendix-C: Neutralized PEG2000 Bicelles 132 C1. PEG2000/DC-Chol. bicelles 132 C2. PEG2000/DOTAP bicelles 133 Appendix-D: PEG5000 Bicelles 136 D1. PEG5000 bicelles 136 D2. PEG5000 bicelles v.s. Temperature 138 Appendix-E: DPPC/PEG2000 complexes 141 E1. DPPC/PEG2000 complexes 141 E2. DPPC/PEG2000 complexes v.s. Temperature 143 Appendix-F: DPPC/PEG5000 complexes 145

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