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研究生:邱承智
研究生(外文):Chen-Chih Chiu
論文名稱:研製包覆靛氰綠及利福平之聚乳酸-聚甘醇酸奈米粒子用於破壞生物膜之抗菌治療
論文名稱(外文):Fabrication, Characterization, and Validation of Indocyanine Green-Rifampicin-Loaded PLGA Nanoparticles for Photo-Chemical Anti-Biofilm Bactericidal Application
指導教授:李宇翔李宇翔引用關係
指導教授(外文):Yu-Hsiang Lee
學位類別:碩士
校院名稱:國立中央大學
系所名稱:生醫科學與工程學系
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2018
畢業學年度:107
語文別:中文
論文頁數:90
中文關鍵詞:聚乳酸-聚甘醇酸奈米粒子靛氰綠利福平人工關節細菌感染耐甲氧西林金黃色葡萄球菌
外文關鍵詞:Poly(lactic-co-glycolic acid) nanoparticleIndocyanine greenRifampicinArtificial joint infectionMultiple drug-resistant Staphylococcus aureus
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當醫療器材置入人體時常碰到細菌感染,而其會面臨到問題為細菌容易生成生物膜,形成生物膜細菌會牢固粘附在人體中生醫器材上難以清除,而此必須手術清創置換新的生醫材料,其會需要花費大量時間以及造成病患極大痛苦。而細菌對抗生素具有抗藥性則又是另一難解的問題。近年來發現多重耐藥性細菌種類中以金黃色葡萄球菌為常見於人工關節細菌感染,而其中金黃色葡萄球菌種類中的耐甲氧西林金黃色葡萄球菌(Methicillin resistant Staphylococcus aureus,MRSA)為具有耐藥性以及毒性強並且當產生生物膜時會更難以治療。目前利福平(Rifampicin,RIF)是最常用的MRSA抗生素之一,但是由於MRSA生物膜生成而導致嚴重的耐藥性。所以本研究研發一種包覆光敏劑靛氰綠(Indocyanine Green,ICG)和抗生素利福平(RIF)之聚乳酸-甘醇酸共聚物(Poly(lactide-co-glycolide),PLGA)多功能奈米粒子(ICG-RIF-PLGA Nanoparticles; IRPNPs),應用RIF抗生素及ICG的光治療給予雙重治療效果,來達到提高臨床上的抗菌效果。在本研究結果中,IRPNPs的粒徑大小以及表面電位分別為249.02 ± 21.5 nm和-31.9 ± 3.2 mV。藥物RIF和ICG的包覆率分別為42.13 ± 0.19%和89.36± 0.08%。我們發現在體外模擬人體溫度37℃下經過48小時後,IRPNPs中ICG在吸收波長780 nm中測試吸收值與純ICG水溶液組可延緩ICG 1.89倍(p<0.05),其可表示載體具有效減緩ICG降解效果,而RIF釋放率分別為31.94%和43.19%,其可以表現出IRPNPs的穩定性。經由近紅外光照射IRPNPs中產生單態氧以及熱療效應中,從IRPNPs光治療生成單態氧機轉探討中,IRPNPs檢測到的螢光值與相同濃度下ICG螢光值有顯著差異,其在IRPNPs含20 µM可相差6.12倍( p<0.05),而此可表示ICG被包覆於IRPNPs確實能夠增強的單態氧生成量。IRPNPs光治療升溫之效能數據表明IRPNPs在近紅外光照射下會產生光熱效應造成IRPNPs中ICG含量20 µM在近紅外光照射300秒中從25.5℃達到53.42℃,而相同濃度純ICG水溶液照光組中照射近紅外光300秒中從25.1℃達到81.22℃,IRPNPs光熱治療具有升溫效果。根據IRPNPs對破壞生物膜測試中,我們的研究結果顯示最高濃度IRPNPs (ICG: 20 µM,RIF: 3.5 µM)照雷射組中與生物膜反應培養1小時與24小時候吸收值與純RIF水溶液組別作為對照可差到5.81以及5.33倍(p < 0.05),其可表示IRPNPs具有破壞生物膜能力。為了瞭解IRPNPs是否具殺菌功能,根據IRPNPs對含有生物膜的MRSA殺菌測試中,將細菌MRSA經過808 nm近紅外光照射5分鐘後IRPNPs高於濃度(10 µM ICG and 1.75 µM RIF)下反應24小時後,經由點在瓊脂盤培養10小時,並無偵測細菌,其可知IRPNPs高於(10 µM ICG and 1.75 µM RIF)濃度可以有效殺光細菌,其代表IRPNPs光治療下具有毒殺已生成生物膜的細菌能力。最後為了驗證本研究IRPNPs是否具有生物毒性,根據IRPNPs對骨細胞(MG-63)細胞毒性測試中,IRPNPs與骨細胞培養24小時,經由Hemocytometer與MTT檢測,經由計算得知細胞存活率為高於90%,其代表IRPNPs為較低生物毒性。而我們期盼開發出IRPNPs具有很高潛力應用在人工關節細菌感染治療中,而更多的研究需要我們在未來進一步證實其IRPNPs具有很好潛力。
The expansion of bacterial antibiotic resistance is a growing problem today. When medical devices are inserted into the body, it often encounters bacterial infections. For example, bacterial artificial joints infection will face problems. The bacteria will easily form biofilms, and artificial joints infection becomes especially difficult for the body to clear robustly adherent antibiotic-resistant biofilm infections. In addition, concerns about the spread of bacterial genetic tolerance to antibiotics, such as that found in multiple drug-resistant Staphylococcus aureus (MRSA), have significantly increased of late. At present, Rifampicin (RIF) is one of most commonly used MRSA antibiotics. However, serious drug resistance resulted from biofilm formation. To improve the antibacterial efficacy in the clinic, a type of multi-functional poly(lactic-co-glycolic acid) (PLGA) nanoparticles encapsulated with photosensitive substrate indocyanine green (ICG) and antibiotic of Rifampicin (RIF) (ICG-RIF-loaded PLGA Nano-Particles; IRPNPs) was developed in this study.
In this study, the mean size and surface charge of the IRPNPs were 249 ± 21.5 nm and -31.9 ± 3.2 mV, respectively, and the encapsulation efficiencies for ICG and RIF were 89.36± 0.08 % and 42.13± 0.19%, respectively. After analysis of the absorbance at λ = 780 nm for each set, we found that after 48 hours of in vitro simulated human body temperature at 37 °C, the ICG absorption of IRPNPs and the pure ICG aqueous solution group delayed the ICG by 1.89-fold (p < 0.05), which indicates that the carrier has slowed down. The ICG degradation effect, while the RIF release rates were 31.94% and 43.19%, respectively, which can show the stability of IRPNPs. The efficacy of singlet oxygen generated by light treatment of IRPNPs by near-infrared light irradiation shows that the fluorescence value detected by IRPNPs is higher than the maximum ICG fluorescence value of 6.12 at the same concentration (p < 0.05). This can indicate that the ICG is coated with the amount of singlet oxygen that the IRPNPs can indeed enhance. The efficacy data of IRPNPs in phototherapy showed that IRPNPs produced photothermal effects under near-infrared light, resulting in ICG content of 20 μM in IRPNPs reaching 53.42 °C from 25.5 °C in 300 seconds of near-infrared light irradiation, while irradiation in the same concentration of pure ICG aqueous solution group Near-infrared light reaches 81.22 °C from 25.1 °C in 300 seconds, and IRPNPs photothermal therapy has a heating effect. According to the IRPNPs for the biofilm destruction test, our results showed that the highest concentration of IRPNPs (ICG: 20 μM, RIF: 3.5 μM) in the laser group in the laser group at 1 hour and 24 hours of absorption and pure RIF aqueous solution group As a control, it can be as poor as 5.81 and 5.33 times (p < 0.05), which can indicate that IRPNPs have the ability to destroy biofilm. In order to understand whether IRPNPs have bactericidal function, according to IRPNPs for biofilm-containing MRSA bactericidal test, bacterial MRSA was irradiated by 808 nm near-infrared light for 5 minutes, and IRPNPs were reacted for 24 hours at a higher concentration (10 μM ICG and 1.75 μM RIF). After incubation for 10 hours on the agar plate, no bacteria were detected. It is known that IRPNPs are higher than (10 μM ICG and 1.75 μM RIF) and can effectively kill the bacteria, which represents the biofilm of the IRPNPs under the light treatment. Finally, in order to verify whether the IRPNPs in this study are biotoxic, according to the IRPNPs cytotoxicity test of bone cells (MG-63), IRPNPs and bone cells were cultured for 24 hours, and the cell survival rate was calculated by Trypan blue and MTT assay. At 90%, it represents lower biological toxicity of IRPNPs. We anticipate that the developed IRPNPs may exhibit a high potential for use in antimicrobial treatment.
摘要I
AbstractVIII
目錄X
圖目錄XIII
表目錄XV
第 一 章 緒論1
1.1 研究背景與動機1
1.2 研究目的3
第 二 章 原理與文獻探討4
2.1 人工關節細菌感染4
2.1.1 人工關節細菌感染介紹分期與分類4
2.1.2 人工關節細菌感染治療7
2.2 光治療11
2.2.1 光動力治療(Photodynamic therapy,PDT)11
2.2.2 光熱力治療(Photothermal therapy , PTT)14
2.3 熱療法(Thermal therapy)15
2.4 金黃色葡萄球菌(Staphylococcus aureus)16
2.4.1 金黃色葡萄球菌的特性16
2.4.2 金黃色葡萄球菌的致病性18
2.4.3 耐甲氧西林金黃色葡萄球菌(MRSA)18
2.4.4 MRSA的臨床檢驗方法19
2.5 生物膜(biofilm)21
2.6 靛青綠(Indocyanine Green,ICG)23
2.7 利福平(Rifampicin,Rifampin,RIF)25
2.8 聚乳酸-甘醇酸共聚物(Poly(lactide-co-glycolide),PLGA)介紹26
2.9 藥物載體28
2.9.1 奈米載體種類28
2.9.2 奈米載體顆粒製備原理32
第 三 章 研究材料與方法34
3.1 實驗藥品及儀器34
3.1.1 藥品34
3.1.2 儀器35
3.2 實驗流程設計架構36
3.3 製備包覆ICG、RIF之PLGA奈米載體37
3.4 IRPNPs物理特性分析38
3.4.1 IRPNPs 粒徑以及表面電位分析38
3.4.2 掃描式電子顯微鏡(SEM)分析38
3.4.3 包覆率以及包藥率分析38
3.4.4 熱穩定性分析40
3.5 IRPNPs光治療功能分析41
3.5.1 IRPNPs 光治療升溫之效能41
3.5.2 IRPNPs 光治療生成單態氧之效能41
3.6 細菌體外實驗42
3.6.1 細菌培養42
3.6.2 生物膜培養42
3.6.3 破壞生物膜分析42
3.6.4 IRPNPs殺菌分析43
3.7 IRPNPs細胞毒性分析45
3.7.1 Trypan blue細胞存活檢測45
3.7.2 MTT細胞存活率檢測45
3.8 統計分析46
第 四 章 結果與討論47
4.1 IRPNPs的物理化性分新47
4.1.1 IRPNPs粒徑以及表面電位分析47
4.1.2 IRPNPs之表面形態分析48
4.1.3 IRPNPs內RIF與ICG的包覆、包藥率分析48
4.1.4 IRPNPs熱穩定性分析49
4.2 IRPNPs光治療功能分析51
4.2.1 IRPNPs 光治療升溫之效能51
4.2.2 IRPNPs 光治療生成單態氧分析53
4.3 IRPNPs對破壞生物膜測試56
4.4 IRPNPs對MRSA殺菌測試59
4.5 IRPNPs對骨細胞毒性測試63
4.6 結論64
第 五 章 未來展望66
第 六 章 參考文獻67
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