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研究生: 郭文碩
Kuo, Wen-Shuo
論文名稱: 具治療性之奈米材料於細菌、癌細胞與幹細胞上之應用
The Therapeutic Nanomaterials Applied in Bacteria, Cancer Cells and Stem Cells
指導教授: 葉晨聖
Yeh, Chen-Sheng
學位類別: 博士
Doctor
系所名稱: 理學院 - 化學系
Department of Chemistry
論文出版年: 2009
畢業學年度: 97
語文別: 中文
論文頁數: 144
中文關鍵詞: 間質幹細胞光熱治療劑光動力抗菌化學治療
外文關鍵詞: mesenchymal stem cells, photodynamic, photothermal therapy
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  • 本論文主要著重在具有治療性之奈米材料在生物上,且分四部份,分別為細菌表面包覆一層金奈米殼當作癌細胞之光熱治療劑、金奈米棒表面連接光敏劑經由光動力治療與光熱治療法殺死抗藥性金紅色葡萄球菌、包覆紫杉醇之聚乳酸/聚甘醇酸共聚物奈米粒子於癌細胞抗藥性之癌細胞的抗藥反轉性探討與細胞影像之偵測、與聚乳酸/聚甘醇酸共聚物奈米粒子當做一載體應用於人類幹細胞上。
    第一部份,經由進行氧化還原反應後,將氯金酸之四氯化金離子還原成金奈米粒子沉積在細菌的表面,形成一細菌包覆金奈米殼層之奈米材料(bacteria@Au composites);且發現隨著觀察時間增加,包覆金奈米殼層之細菌依舊具有生命力,經由細胞毒性測試可知此奈米材料有不錯之生物相容性。再加上此奈米材料其表面電漿共振在近紅外光區有吸收之特性,可將此奈米材料接上專一性之抗體後,變成一有效率之光熱治療劑並應用於殺死癌症細胞上。這些結果提供了我們在操作與運用細菌時一個有趣的方法,使得我們在奈米科學上有了新的概念,可將此奈米材料應用於在生醫領域上與提供了我們另一個癌症治療的方法。
    第二部份,我們已於金奈米棒表面依序連接上聚丙烯酸與TBO光敏劑。此奈米材料可同時當作光動力抗菌化學治療與光熱治療雙功能治療劑;當同時發揮兩功能來殺MRSA時,其殺菌效果比單一功能之殺菌效果還來得有效果,換句話說,可明顯提升殺死多重抗藥性細菌株之效率。
    第三部份,我們合成出於聚乳酸/聚甘醇酸共聚物內部包覆紫杉醇且於其表面共價連接上量子點之奈米粒子(paclitaxel-loaded PLGA-QD655 nanoparticles),且此奈米材料內包覆之紫杉醇抗癌藥物被細胞攝入之機率、停留於細胞內之時間與降低細胞增生之能力等皆較單純之紫杉醇還來的好上許多。結果也顯示,此paclitaxel-loaded PLGA-QD655奈米粒子可增加對紫杉醇具有抗藥性之KB paclitaxel-50細胞株其毒性,並同時以PLGA奈米粒子證明了KB paclitaxel-50為具有MDR機制之細胞株。由於連接了量子點後,此奈米材料也可當作一生物影像探針。因此,paclitaxel-loaded PLGA-QD655奈米粒子可當做一個新的癌症治療工具,且連接上量子點後可有效率的在生醫方面發揮廣泛之應用。
    第四部份,我們已成功的以聚乳酸/聚甘醇酸共聚物於其表面共價連接上量子點之奈米粒子(PLGA-QD nanoparticles)標識人類間質幹細胞。經由MTT毒性測試與活性氧分析,結果顯示奈米粒子有很好的生物相容性;且經由流式細胞儀定量與半定量之方式和誘導耦合電漿結果可知奈米粒子於3小時,幾乎都已被細胞經由胞飲作用被攝入細胞內,且可停留於細胞內長達4週之久。除此之外,這些已標識奈米粒子之細胞其增生與分化能力並不會被影響或抑制。從藥物釋放之結果可知,我們證實了PLGA奈米粒子確實可當一奈米攜帶者於細胞內來釋放所包覆的藥物或其他物質。在未來,PLGA奈米粒子不只可當做一可包覆誘導幹細胞分化成特定細胞、表現特定蛋白之藥物的奈米膠囊也可當做一能夠控制所包覆之藥物其釋放之生物材料。可使得PLGA在幹細胞治療上有很大的應用。

    This thesis is principally emphasized that therapeutic nanomaterials applied in bacteria, cancer cells and stem cell. We discuss it for four parts and they are bacteria coated Au nanoshells as photothermal agent in cancer cells, antimicrobial gold nanorods with dual-modality photodynamic inactivation and hyperthermia, paclitaxel loaded poly(D,L-lactide-co-glycolide) nanoparticles for reversion of drug resistance and cancer cellular imaging, poly(lactide-co-glycolide) as a potential carrier applied in human mesenchymal stem cells, respectively.
    Part 1, when the E. coli bacteria were incubated with HAuCl4 precursors without the addition of a reducing agent, gold nanoshells were generated and covered the bacteria surface. Interestingly, the bacteria coating with gold nanomaterials remained alive and showed no toxicity to the tested mammalian cells. Gold nanoshell formation rendered E. coli inactive. The bacteria@Au composites exhibited near infrared (NIR) absorption and can be applied to photothermal therapy. The bacteria@Au composites can be conveniently conjugated with anti-EGFR antibodies for targeted NIR photothermal destruction of cancer cells. These results might provide an intriguing method to use bacteria and bacteria@Au composites in biomedical applications.
    Part 2, polyacrylic acid (PAA) polymer and a hydrophilic photosensitizer, TBO, were conjugated on the surface of Au nanorods. The resulting Au-PAA-TBO nanorods served as photodynamic and hyperthermia agents. Combined PACT and hyperthermia more efficiently killed bacteria compared with PACT or hyperthermia treatment alone, and it improved antimicrobial efficiency.
    Part 3, we have demonstrated to synthesize paclitaxel-loaded PLGA-QD655 nanoparticles then showed that loading PLGA nanoparticles with paclitaxel resulted in greater cellular drug uptake, sustained intracellular drug retention, and decreased the proliferation of paclitaxel-treated cancer cells. The data with paclitaxel-loaded nanoparticles in a drug resistant cell line suggest not only that drug resistance can be overcome by sustaining intracellular drug retention, but also that the paclitaxel-resistant cell line we used is associated with MDR mechanisms. We showed that conjugating quantum dots with PLGA nanoparticles turns the PLGA nanoparticles into molecular imaging probes. Paclitaxel-loaded PLGA-QD655 nanoparticles seem to be a new therapeutic tool for curing cancers, and PLGA nanoparticles coated with quantum dots appear to be useful for additional biomedical applications.
    Part 4, we have successfully labeled hMSCs with PLGA-QD NPs. The MTT cytotoxic investigation and ROS analysis both showed that these NPs exhibited a good cytocompatibility. Qualitative and semi-quantitative flow cytometry and ICP results indicated that the maximum internalization of NPs in cells was achieved within 3 hours of incubation and can be retained for long periods of time. Moreover, NPs labeling did not hinder the proliferation and differentiation capability of hMSCs. From the results of the substrate-encapsulated delivery system, we demonstrate that PLGA NPs can indeed be used as nano-carriers to release encapsulated materials in stem cells. In the future, it is promising that PLGA NPs will not only be used as nano-capsules to load drugs that induce stem cell differentiate into specific cells or genes that express desired proteins but they will also be used for controlled release of biomaterials. It is concluded that these experiments may lead to further applications of PLGA in stem cell therapy.

    目錄 中文摘要……………………………………………………………………………..Ⅰ 英文摘要……………………………………………………………………………..Ⅱ 誌謝………………………………………………………………………………….Ⅳ 目錄…………………………………………………………………………………..Ⅴ 表目錄……………………………………………………………………………..ⅩⅡ 圖目錄…………………………………………………………………………….ⅩⅢ 縮寫檢索表……………………………………………………………………..ⅩⅤⅢ 第一章 緒論..................................................................................................................1 1-1. 奈米科學............................................................................................................... 1 1-1-1. 奈米材料之發展簡介…………….………..……………………………….1 1-1-2. 奈米材料之基本性質………………………………………………………..1 a. 表面效應………………………………………………………………………...2 b. 尺寸效應………………………………………………………………………...2 c. 量子效應………………………………………………………………………...2 1-1-3. 奈米材料之表面電漿共振…………………………………………………..3 1-1-4. 奈米材料之穩定性…………………………………………………………..3 a. 吸引力…………………………………………………………………………...4 b. 排斥力…………………………………………………………………………...4 1-1-5. 奈米材料之表面修飾………………………………………………………..5 1-1-6. 奈米材料之形狀與特性……………………………………………………..5 1-1-7. 奈米材料製備方式…………………………………………………………..6 1-2. 生醫奈米材料……………………………………………………………………6 1-2-1. 生醫奈米材料之簡介………………………………………………………..6 1-2-2. 金奈米粒子與生物系統之應用……………………………………………..7 1-2-3. 磁性奈米粒子與其生醫應用………………………………………………..8 1-2-4. 量子點於生醫上之應用……………………………………………………11 1-2-5. 奈米碳管於生醫上之應用…………………………………………………12 1-2-6. 奈米微脂體於生醫上之應用………………………………………………13 第二章 具生物相容性之Bacteria@Au奈米材料利用光熱治療破壞癌細胞之應用 ……………………………………………………………………………………..…19 2-1. 癌症治療法………………………………………………………………..……19 2-1-1. 外科手術切除………………………………………………………………19 2-1-2. 局部放射線照射治療…...………………………………………………….19 2-1-3. 化學藥物治療…………...………………………………………………….20 2-1-4. 溫熱治療 (hyperthermia therapy)………...………………………………..20 2-1-5. 標靶治療(targeting therapy)…………….……………………………….....21 2-2. 奈米材料應用於癌症之光熱治療(photothermal therapy)……...21 2-2-1. 癌細胞對熱敏感之原因……………………………………………………22 2-2-2. 應用於光熱治療之奈米材料………………………………………………23 2-2-2-1. 以金為主之奈米材料(Au-based material)…………………………..….23 a. 金奈米棒……………………………………………………………………...23 b. 二氧化矽-金核殼材料(SiO2/gold nanoshells)…………………………….23 c. 金奈米籠(gold nanocages)………………………………………………....24 2-2-2-2. 石墨碳管………………………………………………………………...24 2-2-2-3. FeCo/graphic-shell奈米粒子………………………………………….....25 2-2-3. 生物性模版與金奈米粒子作用之簡介........................................................25 2-2-3-1. 病毒模板………………………………………………………………...25 2-2-3-2. 真菌模板…………………………………………………………….......26 2-2-3-3. 細菌模板...................................................................................................26 a. 巨大芽孢桿菌(Bacillus megatherium)…………………………………….…26 b. 綠膿桿菌(Pseudomonas aeruginosa)………………………………………...27 2-2-4. 研究動機……………………………………………………………………27 2-2-5. 藥品…………………………………………………………………………28 2-2-6. 材料與方法…………………………………………………………………28 1. 穿透式電子顯微鏡(TEM)…………………………………………………28 2. 藉由負染色法(negative staining)染細菌並利用TEM觀察細菌型態……….28 3. 經由切片之細菌利用TEM觀察細菌型態………………...…………………29 4. 利用能量分散光譜(Energy Dispersive X-ray Spectrometer,EDX或EDS …………………………………………………………………………………29 5. X光粉末繞射儀(X-ray Diffractometer)…………………………………..29 6. 細菌包覆金奈米殼層之奈米材料(DH5α@Au composites)之三倍頻(third harmonic generation,THG)影像……………………………………………..29 7. 細胞培養……………………………………………………………………….29 8. 細胞毒性(WST-1)測試………………………………………………………...30 9. 具專一性之anti-EGFR抗體與DH5α@Au composites奈米材料之連接 (DH5α@Au composites with anti-EGFR)…………………………………….30 10. 光熱治療法(photothermal therapy)…………………………………………....30 11. DH5@Au composites奈米材料之升溫效率測試…………………………30 2-2-7. 結果與討論……………………...………………………………………….31 2-2-7-1. 反應機制與鑑定………………………………….……………………..31 2-2-7-2. Bacteria@DH5α composites之三倍頻影像……………………………..31 2-2-7-3. 表面電漿共振…………………………………………………………...32 2-2-7-4. 細胞毒性(WST-1)…………………………………………………….…33 ##毒素(Toxin)…………………………………………………………………33 2-2-7-5. 光熱治療………………………………………………………………...36 2-2-8. 結論…………………………………………………………………………37 第三章 具光動力殺菌與光熱治療雙功能之金奈米棒……………………………47 3-1. 抗藥性微生物(drug-resistant microorganisms)………………………………..47 3-2. 抗藥性細菌的作用機制………………………………………………………47 a. TMP……………………………………………………………………………...48 b. TAR……………………………………………………………………………...49 c. DIE........................................................................................................................49 3-3. 抗藥性之金黃色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)………………………………………………………………………………50 3-4. 光動力抗菌化學治療(photodynamic antimicrobial chemotherapy,PACT)………………………………………………………………………………...50 3-4-1. 發展史………………………………………………………………………50 3-4-2. 原理…………………………………………………………………………51 3-4-3. 光動力治療抑制腫瘤相關研究……………………………………………52 3-4-4. 光動力治療抑制微生物相關研究…………………………………………53 3-4-5. 光敏劑………………………………………………………………………53 3-4-5-1. 光敏劑的種類...........................................................................................54 a. 第一代光敏劑...................................................................................................54 b. 第二代光敏劑………………………………………………………………...54 3-4-6. 光敏劑與一般化療藥物的區別……………………………………………55 3-5. 奈米粒子於光動力治療之應用…………………………………….………….56 a. 金奈米粒子……………………………………………………………………...56 b. silica 奈米粒子………………………………………………………………...56 c. 量子點…………………………………………………………………………..56 3-6. 研究動機………………………………………………………………………..57 3-7. 藥品……………………………………………………………………………..58 3-8. 材料與方法……………………………………………………………………..58 1. Au-PAA-TBO金奈米棒之製備…………………………………………...…….58 2. 估計每條金奈米棒上連接之TBO光敏劑平均數量………………………….59 3. 鑑定……………………………………………………………………………...59 4. MRSA之培養……………………………………………………………………59 5. 估計經由雷射照射後MRSA細菌之存活率…………………………………..59 6. 單態氧(singlet oxygen)之偵測………………………………………………….60 7. Au-PAA-TBO金奈米棒之升溫實驗…………………………………………....60 3-9. 結果與討論…………………………………………………………………….61 3-9-1. 金奈米棒的製備…………………………………………………………....61 3-9-2. 金奈米棒連接上光敏劑之表面電漿共振…………………………………62 3-9-3. 金奈米棒連接上光敏劑之PACT…………………………………………..62 3-9-4. 金奈米棒連接上光敏劑之單態氧(singlet oxygen)測定…………………..63 3-9-5. 具光動力抗菌化學治療與光熱治療雙功能之金奈米棒殺菌效率之測定… ……………………………………………………………………………………....63 3-10. 結論…………………………………………………………………………....64 第四章 紫杉醇包覆於無穩定劑之聚乳酸/聚甘醇酸共聚物奈米粒子內並於其表面連接量子點,應用於對抗癌藥物有抗藥性之癌細胞的抗藥反轉性探討與細胞影像之偵測…………………………………………………………………..…………73 4-1. 太平洋紫杉醇 (學名: Taxus brevifolia Nutt)…………………………….....73 4-1-1. 發展史…………………………………………………………………..73 4-1-2. 紫杉醇之結構與作用機制…………………………………………….73 4-1-3. 紫杉醇溶液之配製…………………………………………………………74 4-1-4. 紫杉醇之臨床研究…………………………………………………………74 4-2. 癌細胞的MDR…………………………………………...………………….75 4-2-1. P-glycoprotein mediated MDR………………………………………………75 4-2-2. 非P-glycoprotein mediated MDR………………………………………......76 4-2-3. MDR逆轉劑(reversal reagent)………………………………………………77 4-3. 藥物控制釋放傳遞……………………………………………………………..78 4-4. 生物可降解性之高分子………………………………………………………..78 4-4-1. PLA………………………………………….……………………………….80 4-4-2. PGA…………………………………………………………………...……..80 4-4-3. PLGA………………………………………………………………………...81 4-5. 奈米粒子應用於藥物傳輸系統……………….……………………………….81 4-5-1. 具可降解性奈米粒子之製備………………………………………………82 a. PLA與PLGA奈米粒子……………………………………………………......82 b. 聚己內酯(poly(ε-carprolactone),PCL)奈米粒子……………………….……82 c. 幾丁聚糖(chitosan)奈米粒子………………………………………………….83 4-5-2. 奈米粒子之藥物控制釋放…………………………………………………83 4-5-3. 奈米粒子搭載DNA之應用………………………………………………..84 4-5-4. 奈米粒子於靜脈注射之應用………………………………………………84 4-5-5. 生物可降解性奈米粒子於癌細胞之多重抗藥性之研究…………………85 4-5-5-1. Polyisohexylcyanoacrylate (PIHCA)與polyisobutylcyanoacrylate (PI- BCA)奈米粒子………………………………………………………………........85 4-5-5-2. PEO-modified PCL奈米粒子……………………………………….…..85 4-5-5-3. PLGA奈米粒子…………………………………………………………85 4-6. 研究動機………………………………………………………………………..86 4-7. 藥品……………………………………………………………………………..89 4-8. 材料與方法……………………………………………………………………..89 1. PLGA奈米粒子之製備………………………………………………………….89 2. PLGA奈米粒子包覆paclitaxel (paclitaxel-loaded PLGA NPs)之製備............................................................................................................................89 3. 於已包覆paclitaxel之PLGA奈米粒子表面連接上PEG-NH2-QD655 (PLGA- QD655 NPs)之製備…………………………….………………………………....90 4. PLGA奈米粒子之鑑定………………………………….…...………………….90 5. Paclitaxel於體外(in vitro)之藥物釋放研究………………………………….....90 6. 細胞培養………………………………………………………………………...90 7. MTT細胞毒性測試(培養時間24小時)………………………………………...90 8. MTT細胞毒性測試(培養時間120小時)……………………………………….91 9. 以共軛焦顯微鏡觀測已分別與paclitaxel和paclitaxel-PLGA-QD655 奈米粒 子作用之癌細胞影像……………………………………………………………..91 4-9. 結果與討論……………………………………………………………………..92 4-9-1. 表面無穩定劑之PLGA奈米粒子製備………………………………….....92 4-9-2. 包覆於PLGA奈米粒子內paclitaxel之藥物釋放…………………………92 4-9-3. 人類子宮頸癌細胞對paclitaxel之抗藥性測試……………………………92 4-9-4. Verapamil對MDR機制之測試……………………………………………..93 4-9-5. Paclitaxel-loaded PLGA-QD655奈米粒子之共軛焦顯微影像……………...94 4-10. 結論……………………………………………………………………………94 第五章 無穩定劑之聚乳酸/聚甘醇酸共聚物奈米粒子之表面連接量子點當作一具潛力之載體並應用於人類間質幹細胞…………………………………………105 5-1. 組織工程……………………………………………………………………....105 5-2. 幹細胞簡介……………………………………………………………………105 a. 全能性幹細胞(totipotent stem cells)………………………………………..107 b. 豐富潛能性幹細胞(pluripotent stem cells)…………………………………107 c. 特定潛能性幹細胞(multipotent stem cells)………………………………...107 d. 單能性幹細胞或稱祖源細胞 (unipotent stem cells , progenitor cells)………107 5-3. 現階段幹細胞在疾病治療之應用…………………………………………....107 5-4. 奈米材料於幹細胞上之應用............................................................................108 5-4-1. 金奈米粒子..................................................................................................108 5-4-2. 鑭系元素......................................................................................................108 5-4-3. 二氧化鈦奈米粒子與鈦合金奈米材料…………………………………..108 5-4-4. 量子點與磁性奈米粒子…………………………………………………..108 5-4-5. silica奈米粒子……………………………………………………………..109 5-4-6. PLGA粒子....................................................................................................109 5-5. 研究動機............................................................................................................109 5-6. 藥品……………………………………………………………………………112 5-7. 材料與方法........................................................................................................112 1. PLGA奈米粒子之製備………………………………………………………...112 2. PLGA奈米粒子連接上PEG-NH2-QD655 (PLGA-QD655 NPs)之製備………..113 3. PLGA奈米粒子之鑑定………………………………………………………...113 4. 人類間質幹細胞之培養……………………………………………………….113 5. MTT細胞毒性測試(培養時間24小時………………………………………..113 6. MTT細胞毒性測試(培養時間28天)………………………………………….113 7. ROS之分析…………………………............................................................…..113 8. 流式細胞儀(flow cytometry)…………………………………………………..114 9. 奈米粒子被人類間質幹細胞攝入之效率(uptake efficiency)………………...114 10. 以共軛焦顯微鏡觀測已標識PLGA-QD655 奈米粒子之人類間質幹細胞影像 …………………………..…………………………………………………………114 11. 脂肪細胞分化之分析(Adipogenic differentiation analysis)…………………114 12. 骨細胞分化之分析(Osteogenic differentiation analysis)….…………………114 13. 軟骨細胞分化之分析(Chondrogenic differentiation analysis)………………115 14. Oil Red O染色………………………………………………………………..115 15. Alizarin Red S染色…………………………………………………………...115 16. Safranin Red 染色………………………………………………………….....115 17. 以共軛焦顯微鏡觀測已標識包覆FITC之PLGA-QD565 (FITC-loaded PLG- A-QD565)奈米粒子的人類間質幹細胞影像………………………………..115 5-8. 結果與討論……………………………………………………………………116 5-8-1. 表面無穩定劑之PLGA奈米粒子製備…………………………………..116 5-8-2. 連接量子點之PLGA(PLGA-QD)奈米粒子其MTT毒性測試……….....116 5-8-3. PLGA-QD奈米粒子其ROS測試………………………………………...116 ## 流式細胞儀(flow cytometry)……………………………………………...116 5-8-4. PLGA-QD奈米粒子停留於間質幹細胞內部之分析…………………….117 5-8-5. 含有PLGA-QD奈米粒子之間質幹細胞其分化能力之分析…………...118 5-8-6. PLGA奈米粒子可作為一載體之分析……………………………………118 5-9. 結論……………………………………………………………………………119 第六章 參考文獻…………………………………………….…………………….129 自述………………………………………………………………………………....144 表目錄 第一章 緒論………………………………………………………………………….1 表 1-1. 球體奈米粒子之比表面積與粒徑變化對照表。…………………………15 表1-2. 奈米粒子中所含的原子數及表面原子所佔的比率與粒徑的係。……….15 表1-3.奈米材料之製作方法。…………………………………………….………..16 第二章 具生物相容性之Bacteria@Au奈米材料利用光熱治療破壞癌細胞之應用 ………………………………………………………………………………………..19 表2-1. 此研究中,三種不同品系之大腸桿菌的基因組。……………………….40 表2-2. 此三種大腸桿菌其平均之長與寬。………………………………………..41 圖目錄 第一章 緒論………………………………………………………….……………….1 圖1-1. 奈米維度與能隙圖。………………………………………….…………...15 圖1-2. A-I之TEM照片分別為球形、三角型、線狀、方型、中空、螺旋槳、 花型、珊瑚與管狀。………………………………………………….………….......15 圖1-3. 不同尺寸(3 nm、13 nm和35 nm)的金奈米粒子之穿透式電子顯微鏡圖和 吸收光譜圖。球型、棒型、中空型奈米金材料之穿透式電子顯微鏡圖和吸收光譜 圖。……………………………………………………………………………………15 第二章 具生物相容性之Bacteria@Au奈米材料利用光熱治療破壞癌細胞之應用 ………………………………………………………………………………………..19 圖2-1. 金奈米棒(a)各種不同長寬比的吸收光譜。(b)長寬比為3.9金奈米棒之穿透式電子顯微鏡圖。(c)細胞體外光熱治療實驗。………………….……………....38圖2-2. 核殼比值(core diameter/shell thickness)越大時,最大吸收波長會隨之產生紅位移最大吸收波。………………………………………………………………38 圖2-3. 二氧化矽-氧化鐵-金的核殼材料。(a)二氧化矽-氧化鐵-金的核殼材料之製作流程。(b)TEM下的二氧化矽-氧化鐵-金的核殼材料。(c)二氧化矽-氧化鐵-金的核殼材料於紫外光可見光下的吸收光譜圖。…………………….……………......39 圖2-4. 金、銀標準氧化還原電位以及還原反應。………………….……………..39 圖2.5. (a)銀奈米方體之TEM與SEM。(b)金奈米籠之TEM與SEM。(c)調控四氯氫化金加入濃度,控制金奈米籠吸收光譜位置。(d-1,d-2)為實驗組,(d-3,d-4)為控制組。螢光染劑calcein AM使活細胞呈綠色,螢光染劑ethidium homodimer-1 (EthD-1)使死細胞呈紅色。……………………….…………………………………39 圖2-6. (a)奈米碳管修飾上基因(DNA)蛋白質示意圖,(b)不同材料濃度在808 nm的吸收值之關係圖,(c)以1.4 W cm-2的雷射功率照射濃度為25 mg L-1碳管水溶液升溫曲線關係圖。…………………………………………….…………………...40 圖2-7. (a) FeCo/graphitic - shell金屬奈米粒子修飾PL - PEG示意圖,(b) FeCo/graphitic-shell金屬奈米粒子之TEM圖,(c)材料在不同濃度下的UV-Vis吸收圖,(d)材料溫度變化關係圖。……………………….………………………….40 圖2-8. 經由方法一與方法二製備金奈米殼層之示意圖。……….………………..41 圖2-9. 單根大腸桿菌與包覆金奈米殼層之大腸桿菌其TEM圖。………….…....41 圖2-10. 多根大腸桿菌(DH5α)與包覆金奈米殼層之大腸桿菌其TEM圖。.….....42 圖2-11. 大腸桿菌利用切片技術(ultramicrotoming technique)鑑定其奈米殼層之厚度。……………………………………………………………………………………42 圖 2-12. 經(a)方法一與(b)方法二所合成的奈米材料之XRD圖譜。………...….43 圖2-13. 經由TEM之EDX設備對(a)方法一與(b)方法二所合成的奈米材料進行元素鑑定。……………………………………………………………………...…….43 圖2-14. 經由(a)方法一與(b)方法二所合成之包覆完金殼層的大腸桿菌此奈米材料(bacteria@DH5α composites)之三倍頻影像。…………………………..………44 圖2-15. 觀察經由(a)方法一與(b)方法二所合成的bacteria@DH5α composites隨著與金成長液培養時間增加(1-32小時),其表面電漿共振變化狀況。…….…......44 圖2-16. 經由方法一隨著與金成長液培養時間增加至(a)1小時與(b)32小時所合成的bacteria@DH5α composites。(c)經方法二培養32小時所得之奈米材料。………………………………………………………………………….………...44 圖2-17. 利用人類角質素細胞HaCaT與A549這兩細胞株來測由兩方法合成之bacteria@DH5α composites其細胞毒性。……….…….…………………………..45 圖2-18. 利用HaCaT與A549這兩細胞株來測由兩方法合成之bacteria@DH5α composites其細胞毒性。…………………………………………………….………45 圖2-19. 把接上anti-EGFR抗體的兩種bacteria@DH5α composites (DH5α@Au-AbEGFR composites)分別與A549、HaCaT細胞混合施打近紅外光雷射6分鐘(功率範圍為24 W cm-2-30 W cm-2)。………………….……………………45 圖2-20. 經由紅外光雷射照射純水與100 μg mL-1 DH5α@Au composites(方法一與方法二)8分鐘,溫度上升的情形(功率為26 W cm-2)。…….………………….46 圖2-21. 將兩種DH5α@Au-AbEGFR composites (100 μg mL-1) 與A549細胞混合並施打近紅外光雷射4-6分鐘(功率範圍為26 W cm-2)。活細胞經由酯化鈣黃綠素(calcein AM)染色後,發出綠色螢光來辨別。…………….……………………….46 第三章 具光動力殺菌與光熱治療雙功能之金奈米棒……………………………47 圖3-1. 利用phthalocyanine光敏劑當作金奈米粒子表面之保護劑。…..……….65 圖3-2. 於金奈米粒子表面共價連接上toluidine blue O (TBO)光敏劑。……..…..65 圖3-3. 將水溶性之2-devinyl-2-(1-hexyloxyethyl)pyropheophorbide (HPPH)光敏劑摻入silica奈米粒子中。……………………………………………..………………66 圖3-4. 金奈米棒的成長機制。……………………………………………………...66 圖3-5. 雙層狀結構的CTAB吸附於金奈米棒表面。…………………..………….66 圖3-6. Au-PAA-TBO金奈米棒之合成圖。……………………………..…………..67 圖3-7. (a)金奈米棒、(b)Au-PAA金奈米棒與(c)Au-PAA-TBO金奈米棒之TEM圖。……………………………………………………………………………………67 圖3-8. Mass (electron impact) spectroscopy質譜儀分析。………..……………….67 圖3-9. 金奈米棒、Au-PAA金奈米棒與Au-PAA-TBO金奈米棒之UV-Vis光譜圖。………………………………………………………………….………………...68 圖3-10. (a)PAA、(b)TBO與(c)Au-PAA-TBO金奈米棒之FTIR光譜。……..…..68 圖3-11. MRSA與Au-PAA-TBO nanorods 於暗室混合培養24小時(37 ℃),再以(a) Live/Dead kit (Invitrogen)估算細菌之存活率。(b) 塗盤法(colony forming unit counting method)計數細菌之菌落數。……………………………………………..69 圖3-12. 藉由PACT法來進行殺死MRSA。…………...…………………………..69 圖3-13. 分別測量經Au-PAA-TBO金奈米棒與TBO混合之MRSA其單態氧產生量。…………………………………………………..……………………………….70 圖3-14. Au-PAA-TBO金奈米棒(1.8  109)經由808 nm NIR雷射(最大輸出功率3.1 W cm2, beam spot: 16 mm2) 照射5-40分鐘之升溫速率圖。………...………70 圖3-15. 以hyperthermia殺死MRSA。經Au-PAA-TBO金奈米棒作用之細菌(1.8  109 金奈米棒) 照射NIR 808 nm (最大輸出功率3.1 W cm2, beam spot: 16 mm2) (a) Live/Dead kit (Invitrogen)估算細菌之存活率。(b) 塗盤法計數細菌之菌落數。……………………………………………………………..…………………….70 圖3-16. 與Au-PAA-TBO金奈米棒混合之MRSA依序施予HeNe 633 nm雷射(0.06 W cm-2, beam spot: 40.8 mm2) 1分鐘與NIR 808 nm雷射(3.1 W cm-2, beam spot: 16 mm2)25分鐘,Live/Dead kit (Invitrogen)估算細菌之存活率。………….71 圖3-17. 與Au-PAA-TBO金奈米棒混合之MRSA依序施予HeNe 633 nm雷射(0.06 W cm-2, beam spot: 40.8 mm2) 1分鐘與NIR 808 nm雷射(3.1 W cm-2, beam spot: 16 mm2)25分鐘,以塗盤法計數細菌之菌落數。……………………….…….72 第四章 紫杉醇包覆於無穩定劑之聚乳酸/聚甘醇酸共聚物奈米粒子內並於其表面連接量子點,應用於對抗癌藥物有抗藥性之癌細胞的抗藥反轉性探討與細胞影像之偵測………………………………………………………………………..……73 圖4-1. 包覆paclitaxel之PLGA奈米粒子共價連接上量子點(paclitaxel-PLGA- QD655 NPs)之製備,應用於癌細胞之抗藥性研究。…………...………………….95 圖4-2. (a)無穩定劑之PLGA奈米粒子、(b)已包覆paclitaxel之PLGA奈米粒子與(c) 已包覆paclitaxel之PLGA奈米粒子共價連接上量子點(paclitaxel-loaded PLGA-QD655 NPs)之TEM圖(inset圖顯示單一顆paclitaxel-loaded PLGA-QD655奈米粒子)。(d)共軛焦顯微鏡所拍攝之paclitaxel-loaded PLGA-QD655奈米粒子影像。………………………………………………………………….……..………….95 圖4-3. Paclitaxel-loaded PLGA與paclitaxel-loaded PLGA-QD655奈米粒子於體外(in vitro)PBS溶液中(pH7.4,37 ℃)之藥物釋放研究。…………………..….………95 圖4-4.人類子宮頸癌KB與KB paclitaxel-50細胞分別加入(a) PLGA奈米粒子與(b) PLGA-QD655奈米粒子培養24小時後,藉由MTT法測其細胞存活率。………………………………………………………………………..…………..96 圖4-5.人類子宮頸癌KB細胞(paclitaxel-sensitive KB cells)分別加入(a) paclitaxel與(b) paclitaxel-loaded PLGA-QD655 奈米粒子培養3小時至5天後,藉由MTT法測其細胞存活率。……………………………………………………………........97 圖4-6.人類子宮頸癌KB paclitaxel-50細胞(paclitaxel-resistant KB cells)分別加入(a) paclitaxel與(b) paclitaxel-loaded PLGA-QD655 奈米粒子培養3小時至5天後,藉由MTT法測其細胞存活率。…………………………….………………….............98 圖4-7.人類子宮頸癌KB細胞加入(±)-verapamil培養5天後,藉由MTT法測其細胞存活率。…………………………………………………..……………………..99 圖4-8.人類子宮頸癌KB paclitaxel-50細胞加入(±)-verapamil培養5天後,藉由MTT法測其細胞存活率。………………………………...………………………...99 圖4-9.人類子宮頸癌KB paclitaxel-50細胞分別加入paclitaxel、paclitaxel-loaded PLGA-QD655 奈米粒子、paclitaxel + verapamil(5.0mM)與paclitaxel-loaded PLGA-QD655 奈米粒子 + verapamil(5.0mM)培養3小時至5天後,藉由MTT法測其細胞存活率。……………………………………….……………………….100 圖4-10.人類子宮頸癌KB paclitaxel-50細胞分別加入paclitaxel、paclitaxel-loaded PLGA-QD655 奈米粒子、paclitaxel + verapamil(2.5mM)與paclitaxel-loaded PLGA-QD655 奈米粒子 + verapamil(2.5mM)培養3小時至5天後,藉由MTT法測其細胞存活率。………………………………………………………………..101 圖4-11.人類子宮頸癌KB細胞分別加入paclitaxel、paclitaxel-loaded PLGA-QD655 奈米粒子、paclitaxel + verapamil(5.0 mM)與paclitaxel-loaded PLGA-QD655 奈米粒子 + verapamil(5.0 mM)培養3小時至5天後,藉由MTT法測其細胞存活率。…………………………………………………………………………………..102 圖.4-12 KB細胞加入paclitaxel與paclitaxel-loaded PLGA-QD655奈米粒子(paclitaxel劑量4.45 mM)1、3、5天之共軛焦顯微影像。………………………...103 圖.4-13 KB paclitaxel-50細胞加入paclitaxel與paclitaxel-loaded PLGA-QD655奈米粒子(paclitaxel劑量4.45 mM)1、3、5天之共軛焦顯微影像。……………………..103 第五章 無穩定劑之聚乳酸/聚甘醇酸共聚物奈米粒子之表面連接量子點當作一具潛力之載體並應用於人類間質幹細胞…………………………………………105 圖5-1. 表面帶負電金奈米粒子與帶正電之Jet-PEI試劑利用靜電吸附力連接,再與有表現GFP的pEGFP-N1載體(vector)混合。......................................................120 圖5-2. 合成的鑭系元素NaYF4:Yb/Er奈米粒子表面利用靜電吸附力包覆了一層polyethyleneimine (PEI)高分子聚合物於粒子表面之TEM圖。.............................120 圖5-3. 玻璃基材上先塗覆一層poly(dimethyldiallyl ammonium chloride)(PDDA )/PSS sodium,之後再連接二氧化鈦奈米粒子,依序重複層與層(layer by layer)包覆,之後再將間質幹細胞培養於基材上,活細胞以綠色染劑染色呈現綠色螢光。.............................................................................................................................120 圖5-4. hydroxyapatite-coated Ti13Nb13Zr合金奈米材料(a)蝕刻前與(b)後之SE -M圖。………………………...………………………………………………….…121 圖5-5. 孔洞性silica奈米粒子於表面連接上fluorescein isothiocyanate(FITC)有機螢光染劑混合之TEM圖。……..…………………………………………………121 圖5-6. silica奈米粒子包覆上SPIO與於表面連接上已包覆了孔洞性silica奈米粒子之FITC混合。…………….……………………………………………………..121 圖5-7. 孔洞性silica奈米粒子表面連接上gadolinium(Gd)-FITC之TEM圖。..122 圖5-8. PLGA之結構。…..…………………………………………………………122 圖5-9. PLGA奈米粒子共價連接上量子點(PLGA-QD NP)之製備,與對人類間質幹細胞之分化研究。……………..…………………………………………………122 圖5-10. (a)無穩定劑之PLGA奈米粒子與(b) PLGA奈米粒子共價連接上量子點(PLGA-QD NPs)之TEM圖。(c)共軛焦顯微鏡所拍攝之PLGA-QD NPs影像……. …………..……………………..……………………………………………………123 圖5-11. 人類間質幹細胞分別加入量子點與PLGA-QD奈米粒子培養24小時後 ,藉由(a)MTT法測其細胞存活率,與(b)藉由流式細胞儀(flow cytometry)測其H2O2之產生量。(c) 利用MTT法,量測人類間質幹細胞分別加入量子點(QDs-labeled hMSCs)與PLGA-QD奈米粒子(PLGA-QD NPs-labeled hMSCs)培養4週後,其細胞存活率(奈米粒子劑量200 μg mL-1)。………………..…….................................123 圖5-12. 細胞經雷射光激發產生的散射光與螢光。………..…………………….124 圖5-13. 人類間質幹細胞分別加入量子點(QDs-labeled hMSCs)與PLGA-QD奈米粒子(PLGA-QD NPs-labeled hMSCs)培養4週(奈米粒子劑量200 μg mL-1)使細胞增生之共軛焦顯微影像圖。………………………………………………………..124 圖5-14. 流式細胞儀分析人類間質幹細胞分別加入(a)量子點(QDs-labeled hMSCs)與(b)PLGA-QD奈米粒子後培養時間長達4週的訊號測定(奈米粒子劑量200 μg mL-1)。(c)流式細胞儀量化之訊號強度。(d) 藉由誘導偶極電漿(inductively coupled plasma,ICP)分析量子點中鎘離子含量。……………..………………...................125 圖5-15. 於體外(in vitro),經奈米粒子標識後之已分化的人類間質幹細胞其螢光顯微影像。(a-c)未被誘導分化之幹細胞;(d-f)經誘導分化14天後所分化之脂肪細胞(adipocyte),以Oil Red O染劑染色;(j-l) 經誘導分化14天後所分化之骨細胞(osteocyte),以Alizarin Red S染劑染色;(p-r) 經誘導分化28天後所分化之軟骨細胞(chondrocyte),以Safranin O染劑染色。藉由奈米粒子所放射之紅光(Em: 655 nm)同時觀察不同細胞內奈米粒子放光之情形(g-i,m-o與s-u)。...……….................126 圖5-16. (a-b)人類間質幹細胞分化能力之量化分析。當奈米粒子與幹細胞混合培 養並經誘導分化後,得到分化之脂肪細胞與骨細胞,分別以Oil Red O與Alizarin Red S染色。再利用DMSO分別將已染脂肪細胞內之Oil Red O染劑與染骨細胞 組織染劑Alizarin Red S洗下,測其含量。……………………………..………….127 圖5-17. (a)為FITC包覆於PLGA-QD奈米粒子內之圖示法,其中QD為放射黃光(Em: 565 nm)之量子點。圖5-16(b-c)分別表示FITC包覆於PLGA奈米粒子與包覆於PLGA-QD奈米粒子之TEM圖。圖5-16(d)表示將FITC加入到人類間質幹細胞後培養12小時之共軛焦顯微影像。圖5-16(e)為PLGA-QD經過12小時之培養之顯微影像。圖5-16(f)證實FITC確實可經由PLGA奈米粒子之生物降解而被釋放出來。………………………………………………………………………..127 圖5-18. 體外(in vitro)的FITC-loaded PLGA-QD565 NPs釋放實驗。………..…127

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