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研究生: Andri Hardiansyah
Andri - Hardiansyah
論文名稱: 具磁性及pH敏感性之脂體質的製備及其標靶性的控釋機制
Development of Liposomes for Controlled Released via pH and Magnetic Stimuli-Triggering Mechanism
指導教授: 楊銘乾
Ming-Chien Yang
口試委員: 施劭儒
Shao-Ju Shih
高震宇
Chen-Yu Kao
劉定宇
Ting-Yu Liu
楊智勇
Chih-Yung Yang
學位類別: 博士
Doctor
系所名稱: 工程學院 - 材料科學與工程系
Department of Materials Science and Engineering
論文出版年: 2015
畢業學年度: 103
語文別: 英文
論文頁數: 177
中文關鍵詞: 脂質體pH感測行為磁感測行為藥物控釋細胞相容性
外文關鍵詞: Liposomes, pH-sensitive behavior, magnetic-sensitive behavior, drug controlled release, cytocompatibility
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具有標靶性及緩控釋性多種功能的藥物傳輸體系,有助於實現腫瘤的標靶治療,並將毒副作用降低到較低的水平。當脂質體載體運送藥物至目的後,需將藥物由載體中釋出才得以發揮藥效。因此近年研究設計脂質體載體的組成,希望藉由內在的刺激或外在的刺激,促使到達目的的脂質體載體加速將藥物釋放。載體的藥物釋放可由內在的刺激(包括pH值,酶的濃度或氧化還原梯度)和外在的刺激(包括溫度變化,磁場,超聲強度,光或電脈衝)或二者的刺激使經特別設計的載體釋出藥物送到人體特定的器官。
此研究的第一部分是製備pH敏感的脂質體,透過雙性幾丁聚醣(Chitosonic® Acid,CA)的表面修飾。表面修飾後的脂質體研究分析是透過透射電子顯微鏡,X射線光電子光譜,傅利葉變換紅外光譜,動態光散射, 界達電位,接觸角和螢光顯微鏡。TEM及介達電位分析結果表明CA-修飾脂質體為奈米尺度及表面為正電。熒光和共聚焦顯微鏡分析結果表明CA-修飾脂質體增強滲透性和滯留效應和細胞內化。此外,在較低的pH環境,低於生力pH值 (7.4) CA-修飾脂質體釋放更多的小紅莓 (doxorubicin)。體外細胞毒性試驗表明,CA-修飾脂質體對於哺乳動物細胞沒有細胞毒性。這些結果表明,CA-修飾脂質體可以作為內在pH敏感性釋放系統,並可用於癌症治療(較低的pH環境中可釋放抗癌藥物)
此研究的第二部分是製備有磁性的脂質體。在這項研究中,將親水性的磁性奈米顆粒和小紅莓抗癌藥包覆在脂質體裡,以提供磁特性和化學療法的效果。利用高頻磁場(high-frequency magnetic field)的外在刺激做為外部標靶性導引和藥物釋放的機制。藥物載體的細胞毒性實驗結果顯示, 磁性脂質體對哺乳動物細胞無毒性作用。此外,組合包覆小紅莓的磁性特質與HFMF的治療降低大腸癌(CT-26)的細胞存活率。這些結果表明,結合化療和熱療透過HFMF可是一個有效的策略來殺死癌細胞。


Selective, targeted and controlled drug release have been received greatest attention in the drug delivery system to get the desirable therapeutic activity of the disease treatment. In this respect, development of drug carriers system that could release the contents of encapsulated drug or moiety as the response to the stimuli is urgently needed to achieve the optimum disease treatment. Intrinsic stimuli-triggered release mechanism (including pH, enzyme concentration or redox gradients) and extrinsic stimuli-triggered release mechanism (including variations in temperature, magnetic field, ultrasound intensity, light or electric pulses) or the combination of both stimuli-triggered mechanisms could be used to achieve the optimum controlled specific drug release at the specific targeted pathological sites.
The first part of this research is focused on the development of pH-sensitive liposomes through surface modification of liposomes using carboxymethyl-hexanoyl chitosan (Chitosonic® Acid, CA). The resultant of CA-modified liposomes was investigated using transmission electron microscope, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, dynamic light scattering, zeta (ζ) potential, contact angle and fluorescence microscope. TEM observation and zeta (ζ) potential measurements revealed that CA-modified liposomes were developed in nanometer scale and positive surface charges, respectively. These properties provide an enhanced permeability and retention effects and arising cellular internalization as revealed by fluorescence and confocal microscope observation. Furthermore, CA-modified liposomes exhibited an enhancement of doxorubicin release in the lower pH environment instead of in the physiological pH. In vitro cytotoxicity assay revealed that the CA-modified liposomes exhibited no cytotoxicity to the normal mammalian cells. These result suggested that CA-modified liposomes could offer the intrinsically pH-sensitivity release-system which is promising for cancer therapy (lower pH environment).
The second part is focused on the development of magnetic-sensitive liposomes. In this study, the aqueous stable magnetic nanoparticles and doxorubicin, an anticancer drug, have been encapsulated in the liposomes compartment to provide the magnetic properties and chemotherapy effect, respectively. High-frequency magnetic field (HFMF) exposure was used as a modality to provide the externally-targeting guided and drug release-triggered mechanism, simultaneously. In vitro cytotoxicity assay of drug carrier against normal mammalian cell revealed that this drug carrier platform exhibited no cytotoxicity towards mammalian cell. Furthermore, the combination of doxorubicin-loaded magnetic liposomes with HFMF treatment decreasing the colorectal cancer (CT-26) cell viability. These result suggested that this combination of chemotherapy and thermotherapy through HFMF could be a promising strategies to killing the cancer cells.

摘要 I Abstract III Acknowledgements V Contents VIII Figures XII Table XVII List of Abbreviation XVIII Chapter 1 Introduction 1 1.1 Background 1 1.2 Research Objectives and Aims of the study 3 Chapter 2 Literature Review 6 2.1 Cancer and conventional chemotherapy 6 2.2 Nanotechnology and Liposomes 7 2.3 Smart Liposomes 13 2.3.1 Development of pH-sensitive liposomes 13 2.3.1.1 Amide bonding 16 2.3.1.2 Chitosan and carboxymethyl-hexanoyl chitosan (Chitosonic® Acid) 17 2.3.2 Development of magnetic-sensitive liposomes 21 2.3.2.1 Magnetic field as a drug carriers transport guidance 22 2.3.2.2 Magnetic liposomes 23 2.3.2.3 Hyperthermia, inductive heating and high-frequency magnetic field (HFMF) 25 2.4 Introduction to cancer cells: Colorectal cancer (CT-26) and human breast cancer Michigan Cancer Foundation-7 (MCF-7) cells 30 Chapter 3 Experimental Method 32 3.1 Flow chart of experiment 32 3.1.1 Part I: Novel pH-Sensitive Drug Carriers of Carboxymethyl-Hexanoyl Chitosan (Chitosonic® Acid) Modified Liposomes 32 3.1.2 Part II: Magnetic liposomes for colorectal cancer cell therapy by high-frequency magnetic field treatment 33 3.2 Experimental materials 34 3.3 Experimental apparatus and instruments 36 3.4 Physicochemical Characterizations 38 3.4.1 Transmission electron microscopy (TEM) 38 3.4.2 Dynamic light scattering (DLS) and zeta (ζ) potential 39 3.4.3 XPS (Tarasova, Vision Science, Tarasova & Vision Science, 2007) 40 3.4.4 Fourier transform infrared (FTIR) spectroscopy 41 3.4.5 X-ray diffraction (XRD) 42 3.4.6 Fluorescence microscope 42 3.4.7 Contact angle (Yuan & Lee, 2013b) 42 3.4.8 Degree of coupling 43 3.5 Cellular studies 44 3.5.1 Cell culture 44 3.5.2 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) Assay 45 3.5.3 Labeling nuclear DNA 47 Chapter 4 Novel pH-Sensitive Drug Carriers of Carboxymethyl-Hexanoyl Chitosan (Chitosonic® Acid) Modified Liposomes 48 4.1 Introduction 49 4.2 Materials and Methods 52 4.2.1 Materials 52 4.2.2 Preparation of liposomes 52 4.2.3 Preparation of Chitosonic® Acid (CA)-modified liposomes 53 4.2.4 Physicochemical characterizations 54 4.2.5 In vitro drug release studies 56 4.2.6 Cell culture 57 4.2.7 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay 57 4.2.8 Fluorescence microscope 58 4.2.9 Confocal Microscopy 58 4.2.10 In vitro cytotoxicity assay toward human breast cancer (MCF-7) cell 59 4.2.11 Cellular internalization toward human breast cancer (MCF-7) cell 60 4.2.12 Statistical analysis 60 4.3 Results and Discussion 60 4.3.1 Characterizations of CA-modified liposomes 60 4.3.2 pH-sensitive behaviors and in vitro drug release studies 74 4.3.3 Cytotoxicity study 79 4.3.4 Cellular uptake 80 4.3.5 In vitro cytotoxicity assay toward human breast cancer (MCF-7) cell 84 4.3.6 Cellular uptake and internalization of CA-modified liposomes toward human breast cancer (MCF-7) cells 87 4.4 Conclusion 90 Acknowledgements 91 Chapter 5 Magnetic liposomes for colorectal cancer cells therapy by high-frequency magnetic field treatment 92 5.1 Introduction 93 5.2 Materials and Methods 97 5.2.1 Materials 97 5.2.2 Synthesis of aqueous stable citric acid-coated magnetic nanoparticles (CAMNP) 97 5.2.3 Preparation of magnetic liposomes 99 5.2.4 Characterization 100 5.2.5 Hyperthermia experiment 101 5.2.6 In vitro drug release studies 101 5.2.7 Cell culture 102 5.2.8 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay 102 5.2.9 In vitro evaluation of chemo-hyperthermia studies 103 5.2.10 Statistical analysis 104 5.3 Results and discussion 104 5.3.1 Characterizations of magnetic nanoparticles and magnetic liposomes 104 5.3.2 Magnetic-sensitive behaviors and in vitro drug release studies 112 5.3.3 Cytotoxicity study 115 5.3.4 In vitro chemo-hyperthermia 117 5.4 Conclusions 121 Acknowledgments 122 Chapter 6 Overall conclusions and future works 123 6.1 Conclusions 123 6.2 Future works 123 References 127 Author Introduction 151 Publications 152

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