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研究生:黃齡儀
研究生(外文):Ling-Yi Huang
論文名稱:以聚乳酸高分子結合光感藥物曁化療藥物之奈米載體克服癌細胞抗藥性之研究
論文名稱(外文):Reversal of doxorubicin resistance by branched star porphyrin-polylactide composed nanoparticle in cancer cells
指導教授:謝銘鈞謝銘鈞引用關係
指導教授(外文):Ming-Jium Shieh
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:醫學工程學研究所
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:英文
論文頁數:55
中文關鍵詞:光動力療法奈米粒子抗藥性P-醣蛋白
外文關鍵詞:PolylactidePorphyrinDoxorubicinNanoparticlesPhotodynamic therapyChemotherapyP-glycoproteinDrug resistance
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光感藥物的研發一直是光動力療法中的研究主軸,發展更具有潛力的光感藥物,是許多研究的目標。而隨著最近幾年奈米藥物載體的蓬勃發展,如:奈米粒子、微胞、微脂體等,皆為研究的重點。經由奈米載體所包覆的藥物,可以胞噬作用進入細胞內。根據許多研究指出,奈米載體可降低許多癌症化療藥物的毒性及副作用,在癌症組織中,亦具有EPR效果(Enhanced Permeation and Retention effect)。因此,在此篇研究中,將光感藥物接上高分子聚乳酸,使同時為一藥物載體並具有光感藥物的功能。首先確定此光感藥物紫質接上聚乳酸後,並不影響其光學特性。接著測試此光感奈米粒子的光毒性,確定其在特定波長光照後可大幅減少癌症細胞存活率。再進一步包覆化療藥物doxorubicin (俗稱小紅莓),使此光感奈米粒子同時具有化療效果,成為一雙功能性的奈米粒子。而又鑒於許多癌細胞對於化療藥物具有抗藥性,使得化療大幅降低療效,根據先前研究指出,聚乙二醇琥珀酸酯(TPGS)不但具有抑制抗藥性機制P-醣蛋白的活性之功能,且為一有效的介面活性劑,可幫助奈米粒子的大小更加集中。最後希望藉由光化學內化(photochemical internalization),成功扭轉抗藥性細胞的抗藥機制。
One potential way to overcome the side effects of chemotherapy is to develop therapeutic drug delivery systems that enhance tumor cytotoxicity but reduce the adverse effects to the normal cells. Recently the studies of nanoparticle formulation of anticancer devices via drugs associated with synthetic polymers have been used as delivery systems for this purpose. Because of enhanced permeability and retention effects, polymer-drug conjugates with nano-sizes can more easily permeate tumor tissues and accumulate in the tumor microenvironment over time. In this study, we attempt to synthesize a novel star-shaped biodegradable polylactide (PLA) with photodynamic and chemo- therapeutics for cancer treatments. The branched star porphyrin-PLA conjugate was synthesized successfully by ring-opening polymerization of lactides from porphyrins with benzyl alcohol under a novel [(DAIP)2Ca]2 catalyst as the red polymer powder. The doxorubicin-loaded nanoparticles were fabricated by a modified oil-in-water single-emulsion solvent evaporation/extraction technique. The size of branched star porphyrin-PLA composed nanoparticle (BSPPLA-NP) without or with dug loading was 67.26nm or 79.7nm, respectively.
This novel BSPPLA-NP was characterized for intracellular distribution, cell viability and phototoxicity in vitro. The results show that BSPPLA-NP, mainly localized in endosome/lysosome compartments, was non-toxic below 10μM, but significantly induced cell death after suitable irradiation (1.4J/cm2) in HeLa cells and MCF-7 cells. Furthermore, the photodynamic treatment obviously improved the cytotoxicity of doxorubicin-loaded BSPPLA-NP through synergistic effects by median effect analysis. Therefore, the BSPPLA-NP with an efficient chemotherapeutic agents loading, showed considerable potential as a bimodal biomaterial for chemo-photodynamic drug delivery system for cancer therapy.
To solve the drug resistant problem used TPGS, a water-soluble vitamin E derivative that can inhibit P-glycoprotein and also a good surfactant. PCI (photochemical internalization) that combined with BSPPLA-NP and doxorubicin is the other way to reverse drug resistant.
目 錄
中文摘要……………………………………………1
英文摘要……………………………………………2
1. Intruductoin………………………… 4
2. Materials and Methods………….……7
2.1 Chemicals and Materials…………….8
Materials for Cell Culture……….9
2.2 Synthesis of branched star porphyrin-PLA……10
2.3 Nanoparticle formation……………………………10
BSPPLA-NP, DOX-BSPPLA-NP
2.4 Nanoparticle Characterization……..…………11
Size, Size Distribution, and Morphology of the Particles,
Drug-encapsulation efficiency,
In vitro release study
2.5 Cell culture and incubation conditions……………………………………...12
2.6 In vitro cytotoxicity using MTT Assay………13
BSPPLA-NP and DOX-BSPPLA-NP Cytotoxicity and phototoxicity measurement,
Free Dox combined with TPGS Cytotoxicity,
PCI measurement(light before, light after)
2.7 Cellular nanoparticle uptake: (CLSM)…………15
Intracellular localization of Dox-loaded nanoparticles
Intracellular localization of doxorubicin combined with TPGS
Intracellular localization of PCI effect
2.8 Flow Cytometry Studies for cellular uptake…17
2.9 Multidrug resistant assay Uptake of calcein-AM in MDR cells.........18
Measure by microplate reader
Measure by flow cytometry
3. Results………………………………………20
Part I. Developing a new PDT nanocarrier that have dual function :
BSPPLA NPs based-PDT combined with Dox
3.1 Synthesis and characterize BSPPLA…………20
3.2 Nanoparticle Characterization………………………………………20
Size, Size Distribution, and Drug- encapsulation efficiency of the Particles
Stability of nanoparticles
In vitro release study
3.3 Cytotoxicity and phototoxicity measurement..21
BSPPLA-NP and DOX-BSPPLA-NP Cytotoxicity and phototoxicity measurement
3.4 Cellular nanoparticle uptake(DOX-BSPPLA-NP: ADR and MCF7)………23
Part II. Reversal the drug resistant of MCF-7/ADR
1. TPGS combined with Dox
3.5 Intracellular localization of doxorubicin combined with TPGS…………25
3.6 Flow Cytometry Studies for cellular uptake……25
3.7 Cytotoxicity measurement……………………………26
3.8 Multidrug resistant assay Uptake of calcein-AM in MDR cells……………..26
2. PCI : combined BSPPLA-NP with Dox ( light before & light after)
3.9 Cytotoxic effects of photochemical internalization27
3.10 Intracellular localization of PCI effect..………...27
4. Discussions
Part I. Developing a new PDT nanocarrier that have dual function .........28
Part II. Reversal the drug resistant of MCF-7/ADR
1. TPGS combined with Dox...………………………………29
2. PCI : combined BSPPLA-NP with Dox ( light before & light after)……………31
5. Conclusion………………………………………33
參考文獻………………………………………………………34
附錄…………………………………………………… ..38-55
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