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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 林?輝(Feng-Huei Lin) | |
dc.contributor.author | Yan-Jye Shyong | en |
dc.contributor.author | 熊彥傑 | zh_TW |
dc.date.accessioned | 2021-06-08T03:10:36Z | - |
dc.date.copyright | 2017-06-12 | |
dc.date.issued | 2017 | |
dc.date.submitted | 2017-05-15 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/20924 | - |
dc.description.abstract | 本研究利用多孔性的氫氧基磷灰石作為憂鬱症藥物的載體,並利用巨噬細胞的細胞特性來達成藥物釋放。經由一針劑的肌肉注射,本載體可以達到兩週以上的穩定藥物釋放。本研究利用共沉澱法製造出有適當大小的多孔性氫氧基磷灰石(mesoHAP);再經由一系列的疏水性表面改質,使的抗憂鬱症藥物奥氮平(OLZ)透過物力吸附進入材料的多孔洞表面。由於具有疏水特性,奥氮平在親水的生物體內並不會從載體內釋放出來。但當材料被巨噬細胞吞噬後,會在溶酶體(lysosome)內被分解並釋放大量的鈣磷離子,使滲透壓上升,並最終撐破溶酶體,同時使奥氮平釋放到細胞質中。此時從材料釋放出來的鈣離子也會釋放到細胞之中,提升細胞內的鈣離子濃度,並啟動細胞內的外吞作用(exocytosis),把奥氮平釋放到細胞外間質中,最終擴散到血液循環內。
在動物實驗中,我們從三個不同的角度來評估多孔性的氫氧基磷灰石-奥氮平的療效;動物行為,生理激素,跟記憶力和學習能力。憂鬱症老鼠在經由孔性的氫氧基磷灰石-奥氮平的治療後,可以長時間的提升曠場試驗和強迫游泳試驗的行為數值。血清素(serotonin)是大腦內管理情緒的激素,經由孔性的氫氧基磷灰石-奥氮平的治療後,在憂鬱症老鼠大腦內也有明顯的提升。同時經由功能性磁振造影的觀察,憂鬱症老鼠腦內的海馬迴(hippocampus)的活性也有明顯的提升。多孔性氫氧基磷灰石-奥氮平也可以提升憂鬱症老鼠在莫里斯水迷宮實驗中的記憶和學習能力。本研究提供了一個可靠的藥物釋放機制,可以經由一個一針劑的肌肉注射達到長時間的憂鬱症治療效果,希望可以解決在憂鬱症治療上,病人因經常性的忘記服藥導致療程中斷的問題。 在第二部分的研究中,我們更進一步利用含鈣離子的材料來刺激新一代的藥物載體外吐小體(exosome)的釋放。外吐小體是一種具有潛力的藥物載體,因為近期研究發現他是生物體內細胞與細胞間溝通的工具。但是在臨床的使用上卻碰到了一個問題,就是外吐小體的細胞產量太低無法達到使用上的要求。本研究發現利用磷酸鈣(calcium phosphate)材料可以刺激小鼠巨噬細胞(RAW264.7)跟人類的單核細胞(THP-1)的外吐小體產量。磷酸鈣粒子被細胞吞噬後會在溶酶體內被分解並釋放大量的鈣磷離子,使滲透壓上升,撐破溶酶體,並提高細胞質內的鈣離子濃度。同時我們發現,這些吞噬磷酸鈣粒子的細胞所釋放出的外吐小體內的鈣離子濃度並沒有顯著的提升,證明外吐小體並不會遭到磷酸鈣粒子汙染。本研究希望可以提供一個利用磷酸鈣粒子來有效提升細胞外吐小體產量的方法 | zh_TW |
dc.description.abstract | An antidepressant carrier was designed to maintain over two weeks of constant medication release. The carrier was injected into muscle where cellular activity was involved to achieve the goal of constant release. Meso-porous hydroxyapatite (mesoHAP) was synthesized into an adequate size by co-precipitation method; and then gone through a series of hydrophobic surface modification for olanzapine (OLZ) loading by physical absorption to produce mesoHAP-OLZ. Owing to its hydrophobic nature, OLZ is not effectively released from mesoHAP-OLZ in aqueous environment. However, once engulfed by macrophage, lysosome/endosome hybrid was ruptured due to osmotic pressure alteration, resulting in the release of OLZ into cytoplasma. OLZ was then exocytosed to the extracellular space due to high calcium ion (Ca2+) concentration, and finally circulated in the blood.
In the in vivo study, the efficiency of the treatment was evaluated from 3 perspectives; locomotor activities, biomarkers, and learning and memory ability. MesoHAP-OLZ can increase the locomotor activity in rats with induced depression determined by open field test (OFT) and forced swim test (FST). Serotonin (5-HT), one of the most important biomarker in depression can also be increased by mesoHAP-OLZ, leading to increased hippocampus activity as measured by functional magnetic resonance imaging (fMRI). MesoHAP-OLZ can also improve learning and memory ability in rats with induced depression using Morris water maze (MWM) test. Our findings provide a useful treatment strategy to achieve long-term drug release with a single intramuscular (IM) injection and solve the non-compliant medication intake that often occurred in antidepressant therapy. In the second part of study, we further used Ca2+-containing material to stimulate the release of exosome, a possible next generation drug carrier, exosome. Exosomes are attractive as potential carriers for drug delivery because of their natural function of transferring biomolecules among cells without eliciting immune responses. However, an obstacle to the application of exosomes for drug delivery is the difficulty in collecting sufficient numbers of these vesicles. We found that treatment with calcium phosphate (CaP) particles increased the number of exosomes secreted from macrophage-like RAW264.7 cells and monocyte-like THP-1 cells. CaP particles were easily internalized into cells and dissolved in acidic late-endosomes or lysosomes, resulting in the rupture of their membranes followed by the release of Ca2+ into cytosol. However, the Ca2+ concentrations in exosomes secreted from CaP particle-treated cells were similar to that in exosomes from untreated control cells, implying that exosomes secreted from cells treated with CaP particles are not contaminated by the Ca2+ released from CaP particles. This study highlights the potential of a new technique for the efficient production of exosomes using CaP particles. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:10:36Z (GMT). No. of bitstreams: 1 ntu-106-D00548015-1.pdf: 5271935 bytes, checksum: 884d0b374f003c68df68b2fc4c630aa3 (MD5) Previous issue date: 2017 | en |
dc.description.tableofcontents | Table of Contents
口試委員會審定書 i 致謝 ii 中文摘要 iv Abstract vi Table of Contents ix List of Charts xiii List of Tables xvii List of Abbreviations xviii Chapter 1 Introduction 1 1.1 Introduction of mesoHAP-OLZ as antidepressant drug carrier 1 1.1.1 Depression 1 1.1.2 Antidepressants Olanzapine 3 1.1.3 Non-adherence in antidepressant treatment 5 1.1.4 The purpose of study 6 1.2 Introduction of using CaP to stimulate exosome secretion from phagocytes 8 1.2.1 The next generation drug carrier exosome 8 1.2.2 The low production rate of exosome during clinical trial 9 1.2.3 The purpose of study 10 Chapter 2 Theoretical Basis 12 2.1 Hydroxyapatite as drug carrier 12 2.2 The principle of intracellular delivery of mesoHAP-OLZ 13 2.3 The animal model of depression 15 2.3.1 Behavioral despair-based 16 2.3.2 Reward-based 19 2.3.3 Genetically engineered mice 19 2.3.4 Chemically induce based 20 2.4 MesoHAP-OLZ medication and evaluation in rats 21 2.5 Exosome as the next generation drug carrier 25 2.6 Increase intracellular calcium concentration to stimulate exosome release 29 Chapter 3 Materials and methods 31 3.1 Reagents, drugs, cells, and animals 32 3.2 Materials synthesized and characterization of mesoHAP-OLZ 33 3.2.1 Synthesis of mesoporous HAP (mesoHAP) 33 3.2.2 Surface modification of mesoHAP to hydrophobicity and OLZ loading (mesoHAP-OLZ) 34 3.2.3 Materials characterization and analysis 34 3.2.4 Efficiency of OLZ loading 36 3.3 In vitro study of mesoHAP-OLZ 36 3.3.1 The evaluation of cell viability and cytotoxicity 36 3.3.2 In vitro OLZ release profile 38 3.3.3 Phagocytosis of particles uptaken by RAW-264.7 cells 39 3.4 In vivo study of mesoHAP-OLZ 39 3.4.1 In vivo OLZ release study 39 3.4.2 Animal model of rats with induced depression 40 3.4.3 Locomotor activity of rats with induced depression with and without mesoHAP-OLZ administration. 41 3.4.4 Forced swim test of rats with induced depression 42 3.4.5 5-HT determination in the rat brain 43 3.4.6 Functional imaging of rats with induced depression 44 3.4.7 Determination of learning and memory ability of rats with induced depression by MWM 44 3.4.8 Sub-chronic and chronic toxicity determination 46 3.4.9 Statistical analysis 47 3.5 Stimulation of exosome 47 3.5.1 Synthesis of CaP particles 47 3.5.2 Characterization of CaP particles: 48 3.5.3 Cytotoxicity assay 49 3.5.4 Cellular uptake and localization of CaP particles 49 3.5.5 Analysis of exosomes 50 3.5.6Assay of calcium concentration 52 Chapter 4 Results 53 4.1 Characterization of the materials 53 4.1.1 OLZ loading 59 4.2 In vitro and in vivo studies of OLZ drug release 61 4.2.1 Cell viability and cytotoxicity 61 4.2.2 Cellular uptake of mesoHAP-OLZ and OLZ releasing profile 62 4.2.3 OLZ release study in vivo 66 4.3 In vivo study of antideprssion treatment in depression induced rats 67 4.3.1 Locomotor activity of rats with induced depression 67 4.3.2 Forced swim test of rats with induced depression 70 4.3.3 Determination of 5-HT in the brain 71 4.3.4 Determination of 5-HT level in the rat hippocampus by anti-serotonin antibody staining. 72 4.3.5 Determination of hippocampal activity by fMRI 73 4.3.6 Learning and memory ability by Morris water maze 75 4.3.7 Determination of sub-chronic and chronic toxicity 78 4.4 The stimulation of exosome using CaP 82 4.4.1 Characterization of CaP particles 82 4.4.2 Uptake and intracellular behavior of CaP particles 83 4.4.3 CaP particles are not cytotoxic 85 4.4.4 CaP particles stimulate exosome secretion 86 4.4.5 Exosomes secreted by cells treated with CaP particles do not contain additional calcium 89 Chapter 5 Discussion 91 5.1 The preparation and characterization of mesoHAP-OLZ 91 5.1.1 Factors affect mesoHAP particle synthesis 91 5.1.2 Factors affect mesoHAP particle size and morphology 93 5.1.3 Hydrophobic modification of mesoHAP particles and OLZ loading 94 5.2 In vitro and in vivo OLZ drug release study 95 5.2.1 Factors affect OLZ release from mesoHAP-OLZ particles 95 5.2.2 MesoHAP-OLZ in vivo delivery and OLZ release 97 5.3 MesoHAP-OLZ antidepression treatment in depression induced rats 100 5.3.1 Recovery of activity in rats with induced depression 100 5.3.2 Biomarker measurement in the rat brain 102 5.3.3 Learning and memory ability of rats 106 5.3.4 Sub-chronic and chronic toxicity during long-term antidepressant treatment 107 5.4 CaP increase the release of exosome from cells 109 Chapter 6 Conclusion 113 References 115 Curriculum Vitae 127 Selected Publications and Conference 130 | |
dc.language.iso | en | |
dc.title | 多孔性的氫氧基磷灰石作為憂鬱症長效治療藥物載體和刺激外吐小體釋放之研究 | zh_TW |
dc.title | Mesoporous Hydroxyapatite as Drug Carrier for Long-acting Antidepression Treatment and Stimulation of Exosome | en |
dc.type | Thesis | |
dc.date.schoolyear | 105-2 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 劉華昌(Hwa-Chang Liu),王盈錦(Ying-Jiin Wang),姚俊旭(Chun-Hsu Yao),張至宏(Chih-Hung Chang),張淑真(Shwu-Jen Chang) | |
dc.subject.keyword | 氫氧基磷灰石,?氮平,藥物載體,血清素,巨噬細胞,行為表現,記憶與學習能力,外吐小體,磷酸鈣粒子, | zh_TW |
dc.subject.keyword | Hydroxyapatite,Olanzapine,Drug carrier,Serotonin,Macrophage,Locomotor activities,Learning and memory abilities,Exosome,Calcium phosphate particles, | en |
dc.relation.page | 131 | |
dc.identifier.doi | 10.6342/NTU201700808 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2017-05-16 | |
dc.contributor.author-college | 工學院 | zh_TW |
dc.contributor.author-dept | 醫學工程學研究所 | zh_TW |
顯示於系所單位: | 醫學工程學研究所 |
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