臺灣博碩士論文加值系統

氧化石墨烯（graphene oxide）是石墨粉末經過化學氧化及脫層後的產物，以強氧化劑與石墨粉末反應，氧化後在邊緣形成羧酸基，且在其上形成酚羥基與環氧基團，由於其可均勻的分散於水中，現今應用在許多生醫材料之用途上。在本研究中，我們發現以金奈米粒子還原於氧化石墨烯片上後，在特定的激發光下可有效用於抗菌治療中，產生充分的氧化壓力對細菌造成損害，且在金奈米粒子所提供的偕效效果下，可將效果發揮得更為顯著；同時，氧化石墨烯也是項良好的抗菌材料，其不規則狀的邊緣已在諸多的文獻中顯示能對細胞膜造成無法修復的傷害。因此，本研究旨在開發一非抗生素之藥物，避免細菌產生抗藥性的同時，更對細菌產生毒殺的效果，降低細菌的滋生，而非消耗性的結構更帶來新概念的抗菌方式，可以在低損耗的情況下產生多次的治療，期望能對骨髓炎等容易復發性之疾病產生有效的應用。

Graphene oxide, produced through chemical oxidation and delamination after adding a strong oxidizing agent with graphite powder product, which forms carboxylic acid group at the edges and the epoxy phenolic hydroxyl groups thereon. Due to its hydrophilic property, it can be favorably dispersed in water; therefore, being used in many biomedical applications. In the present study, we found that the reduction of gold nanoparticles on the graphene oxide sheets will be stimulated under specific radiation, which makes the material significantly useful in antimicrobial therapy. In this case, gold nanoparticles play an important role in enhancing the amount of oxidative stress. Moreover, the graphene oxide is also a good antibacterial material items, its irregular shape of the edge has been shown that are capable of causing irreparable damage to the bacteria cell membrane. Hence, this study aimed at developing a non-antibiotic drugs in order to prevent the multi-drug resistant of bacteria, simultaneously, reducing the growth of bacteria and dealing serious damages. Furthermore, this non-consumed material can bring a new concept of generating multiple treatment. We expect this research can produce effective applications for osteomyelitis or any recurrent disease.

致謝 I
摘要 II
Abstract III
目錄 IV
圖目錄 VII
第一章 緒論 1
1. 前言 1
第二章 文獻回顧 3
2.1 慢性骨髓炎的症狀與治療 3
2.2 抗菌系統（Antibacterial system） 5
2.3 石墨烯類材料 10
2.3.1 氧化石墨烯 12
2.3.2 石墨烯類材料抗菌機制 14
2.3.3 石墨烯類材料產生氧化壓力機制 17
2.4 輻射 19
2.4.1 X 射線（X-ray） 20
2.4.2 金奈米粒子與X-ray之交互作用 22
第三章 材料與方法 24
3-1 實驗設計 24
3-2 實驗藥品 26
3-3 實驗儀器 27
3-4 材料製備方法 28
3-4.1 氧化⽯墨烯的製備 28
3-4.2 穿透式電⼦顯微鏡 29
3-4.3 動態散射粒徑分析儀 29
3-4.4 氧化石墨烯攜載奈米金粒子 29
3-4.5 細菌菌株與培養條件 30
3-4.6 細菌存活率測試 30
3-4.7 細胞培養與⽣物相容性測試 31
3-4.8 單一菌落培養實驗 31
3-4.9 掃描式電子顯微鏡觀測 32
3-4.10 原⼦⼒學顯微鏡 33
3-4.11 GME 於 Ti6Al4V 上之抗菌實驗 33
3-4.12 重複性抗菌實驗 34
3-4.13 輻射模擬實驗 34
第四章 結果與討論 35
4.1 氧化石墨烯及GME之鑑定 35
4.2 氧化石墨烯及GME對細胞毒性之探討 40
4.3 氧化石墨烯及GME產生活性氧之機制探討 43
4.4 氧化石墨烯及GME之抗菌機制探討 46
4.5 氧化石墨烯及GME不同狀態之抗菌性質探討 51
4.6 氧化石墨烯及GME之無消耗性抗菌探討 53
4.7 輻射劑量模擬實驗 55
第五章 結論 57
參考文獻 58

參考文獻
[1] Waldvogel FA, Medoff G, Swartz MN. Osteomyelitis: a review of clinical features, therapeutic considerations and unusual aspects. New England Journal of Medicine. 1970;282:198-206.
[2] Haas DW, McAndrew MP. Bacterial osteomyelitis in adults: evolving considerations in diagnosis and treatment. The American journal of medicine. 1996;101:550-61.
[3] Ciampolini J, Harding K. Pathophysiology of chronic bacterial osteomyelitis. Why do antibiotics fail so often? Postgraduate medical journal. 2000;76:479-83.
[4] Lew DP, Waldvogel FA. Osteomyelitis. The Lancet. 2004;364:369-79.
[5] Sun X, Liu Z, Welsher K, Robinson JT, Goodwin A, Zaric S, et al. Nano-graphene oxide for cellular imaging and drug delivery. Nano research. 2008;1:203-12.
[6] Yang K, Zhang S, Zhang G, Sun X, Lee S-T, Liu Z. Graphene in mice: ultrahigh in vivo tumor uptake and efficient photothermal therapy. Nano letters. 2010;10:3318-23.
[7] Tian B, Wang C, Zhang S, Feng L, Liu Z. Photothermally enhanced photodynamic therapy delivered by nano-graphene oxide. Acs Nano. 2011;5:7000-9.
[8] Hu Z, Huang Y, Sun S, Guan W, Yao Y, Tang P, et al. Visible light driven photodynamic anticancer activity of graphene oxide/TiO 2 hybrid. Carbon. 2012;50:994-1004.
[9] Hu Z, Li J, Li C, Zhao S, Li N, Wang Y, et al. Folic acid-conjugated graphene–ZnO nanohybrid for targeting photodynamic therapy under visible light irradiation. Journal of Materials Chemistry B. 2013;1:5003-13.
[10] Ristic BZ, Milenkovic MM, Dakic IR, Todorovic-Markovic BM, Milosavljevic MS, Budimir MD, et al. Photodynamic antibacterial effect of graphene quantum dots. Biomaterials. 2014;35:4428-35.
[11] Ma L, Zou X, Chen W. A new X-ray activated nanoparticle photosensitizer for cancer treatment. Journal of biomedical nanotechnology. 2014;10:1501-8.
[12] Zou X, Yao M, Ma L, Hossu M, Han X, Juzenas P, et al. X-ray-induced nanoparticle-based photodynamic therapy of cancer. Nanomedicine. 2014;9:2339-51.
[13] Chen H, Wang GD, Chuang Y-J, Zhen Z, Chen X, Biddinger P, et al. Nanoscintillator-mediated X-ray inducible photodynamic therapy for in vivo cancer treatment. Nano letters. 2015;15:2249-56.
[14] Falconer SB, Brown ED. New screens and targets in antibacterial drug discovery. Current opinion in microbiology. 2009;12:497-504.
[15] Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nature medicine. 2004;10:S122-S9.
[16] Organization WH. Antimicrobial resistance: global report on surveillance: World Health Organization; 2014.
[17] Sigler K, Chaloupka J, Brozmanova J, Stadler N, Höfer M. Oxidative stress in microorganisms—I. Folia microbiologica. 1999;44:587-624.
[18] Cabiscol E, Tamarit J, Ros J. Oxidative stress in bacteria and protein damage by reactive oxygen species. International Microbiology. 2010;3:3-8.
[19] Basu AK, Loechler EL, Leadon SA, Essigmann JM. Genetic effects of thymine glycol: site-specific mutagenesis and molecular modeling studies. Proceedings of the National Academy of Sciences. 1989;86:7677-81.
[20] West SC. Enzymes and molecular mechanisms of genetic recombination. Annual review of biochemistry. 1992;61:603-40.
[21] Bitounis D, Ali‐Boucetta H, Hong BH, Min DH, Kostarelos K. Prospects and challenges of graphene in biomedical applications. Advanced Materials. 2013;25:2258-68.
[22] Liu J, Cui L, Losic D. Graphene and graphene oxide as new nanocarriers for drug delivery applications. Acta biomaterialia. 2013;9:9243-57.
[23] Robinson JT, Burgess JS, Junkermeier CE, Badescu SC, Reinecke TL, Perkins FK, et al. Properties of fluorinated graphene films. Nano letters. 2010;10:3001-5.
[24] Xu Y, Sheng K, Li C, Shi G. Self-assembled graphene hydrogel via a one-step hydrothermal process. ACS nano. 2010;4:4324-30.
[25] Zhu Y, Murali S, Cai W, Li X, Suk JW, Potts JR, et al. Graphene and graphene oxide: synthesis, properties, and applications. Advanced materials. 2010;22:3906-24.
[26] Ma J, Zhang J, Xiong Z, Yong Y, Zhao X. Preparation, characterization and antibacterial properties of silver-modified graphene oxide. Journal of Materials Chemistry. 2011;21:3350-2.
[27] Robinson JT, Tabakman SM, Liang Y, Wang H, Sanchez Casalongue H, Vinh D, et al. Ultrasmall reduced graphene oxide with high near-infrared absorbance for photothermal therapy. Journal of the American Chemical Society. 2011;133:6825-31.
[28] Şahin H, Topsakal M, Ciraci S. Structures of fluorinated graphene and their signatures. Physical Review B. 2011;83:115432.
[29] Georgakilas V, Otyepka M, Bourlinos AB, Chandra V, Kim N, Kemp KC, et al. Functionalization of graphene: covalent and non-covalent approaches, derivatives and applications. Chemical reviews. 2012;112:6156-214.
[30] Yue K, Gao W, Huang R, Liechti KM. Analytical methods for the mechanics of graphene bubbles. Journal of Applied Physics. 2012;112:083512.
[31] He Y, Wu F, Sun X, Li R, Guo Y, Li C, et al. Factors that affect Pickering emulsions stabilized by graphene oxide. ACS applied materials &; interfaces. 2013;5:4843-55.
[32] Lim D-K, Barhoumi A, Wylie RG, Reznor G, Langer RS, Kohane DS. Enhanced Photothermal Effect of Plasmonic Nanoparticles Coated with Reduced Graphene Oxide. Nano letters. 2013;13:4075-9.
[33] Radich JG, Kamat PV. Making graphene holey. Gold-nanoparticle-mediated hydroxyl radical attack on reduced graphene oxide. ACS nano. 2013;7:5546-57.
[34] Wang Y, Wang H, Liu D, Song S, Wang X, Zhang H. Graphene oxide covalently grafted upconversion nanoparticles for combined NIR mediated imaging and photothermal/photodynamic cancer therapy. Biomaterials. 2013;34:7715-24.
[35] Jung HS, Kong WH, Sung DK, Lee M-Y, Beack SE, Keum DH, et al. Nanographene oxide–hyaluronic acid conjugate for photothermal ablation therapy of skin cancer. ACS nano. 2014;8:260-8.
[36] Hu Y, Li F, Han D, Niu L. Biocompatible Graphene for Bioanalytical Applications: Springer; 2015.
[37] Chung C, Kim Y-K, Shin D, Ryoo S-R, Hong BH, Min D-H. Biomedical applications of graphene and graphene oxide. Accounts of chemical research. 2013;46:2211-24.
[38] Wang A, Pu K, Dong B, Liu Y, Zhang L, Zhang Z, et al. Role of surface charge and oxidative stress in cytotoxicity and genotoxicity of graphene oxide towards human lung fibroblast cells. Journal of Applied Toxicology. 2013;33:1156-64.
[39] Chen J, Liu H, Zhao C, Qin G, Xi G, Li T, et al. One-step reduction and PEGylation of graphene oxide for photothermally controlled drug delivery. Biomaterials. 2014;35:4986-95.
[40] Fiorillo M, Verre AF, Iliut M, Peiris-Pagés M, Ozsvari B, Gandara R, et al. Graphene oxide selectively targets cancer stem cells, across multiple tumor types: Implications for non-toxic cancer treatment, via “differentiation-based nano-therapy”. Oncotarget. 2015;6:3553.
[41] Hu W, Peng C, Luo W, Lv M, Li X, Li D, et al. Graphene-based antibacterial paper. Acs Nano. 2010;4:4317-23.
[42] Liu L, Liu J, Wang Y, Yan X, Sun DD. Facile synthesis of monodispersed silver nanoparticles on graphene oxide sheets with enhanced antibacterial activity. New Journal of Chemistry. 2011;35:1418-23.
[43] Liu S, Zeng TH, Hofmann M, Burcombe E, Wei J, Jiang R, et al. Antibacterial activity of graphite, graphite oxide, graphene oxide, and reduced graphene oxide: membrane and oxidative stress. Acs Nano. 2011;5:6971-80.
[44] Xu W-P, Zhang L-C, Li J-P, Lu Y, Li H-H, Ma Y-N, et al. Facile synthesis of silver@ graphene oxide nanocomposites and their enhanced antibacterial properties. J Mater Chem. 2011;21:4593-7.
[45] Gurunathan S, Han JW, Dayem AA, Eppakayala V, Kim J-H. Oxidative stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa. International journal of nanomedicine. 2012;7:5901.
[46] Liu S, Hu M, Zeng TH, Wu R, Jiang R, Wei J, et al. Lateral dimension-dependent antibacterial activity of graphene oxide sheets. Langmuir. 2012;28:12364-72.
[47] Sivudu K, Mahajan Y. Mass production of high quality graphene: An analysis of worldwide patents. NanoWerk. 2012.
[48] Wang X, Zhou N, Yuan J, Wang W, Tang Y, Lu C, et al. Antibacterial and anticoagulation properties of carboxylated graphene oxide–lanthanum complexes. Journal of Materials Chemistry. 2012;22:1673-8.
[49] Notley SM, Crawford RJ, Ivanova EP. Bacterial interaction with graphene particles and surfaces. Advances in graphene science and technology. 2013.
[50] Tang J, Chen Q, Xu L, Zhang S, Feng L, Cheng L, et al. Graphene oxide–silver nanocomposite as a highly effective antibacterial agent with species-specific mechanisms. ACS applied materials &; interfaces. 2013;5:3867-74.
[51] Li J, Wang G, Zhu H, Zhang M, Zheng X, Di Z, et al. Antibacterial activity of large-area monolayer graphene film manipulated by charge transfer. Scientific reports. 2014;4.
[52] Nanda SS, An SSA, Yi DK. Oxidative stress and antibacterial properties of a graphene oxide-cystamine nanohybrid. International Journal of Nanomedicine. 2015;10:549.
[53] Ruiz ON, Brown NA, Fernando KS, Harruff-Miller BA, Gunasekera TS, Bunker CE. Graphene oxide-based nanofilters efficiently remove bacteria from fuel. International Biodeterioration &; Biodegradation. 2015;97:168-78.
[54] áde Leon ACC. On the antibacterial mechanism of graphene oxide (GO) Langmuir–Blodgett films. Chemical Communications. 2015.
[55] Tu Y, Lv M, Xiu P, Huynh T, Zhang M, Castelli M, et al. Destructive extraction of phospholipids from Escherichia coli membranes by graphene nanosheets. Nature nanotechnology. 2013;8:594-601.
[56] Krishnamoorthy K, Veerapandian M, Zhang L-H, Yun K, Kim SJ. Antibacterial efficiency of graphene nanosheets against pathogenic bacteria via lipid peroxidation. The Journal of Physical Chemistry C. 2012;116:17280-7.
[57] He J, Zhu X, Qi Z, Wang C, Mao X, Zhu C, et al. Killing Dental Pathogens Using Antibacterial Graphene Oxide. ACS applied materials &; interfaces. 2015;7:5605-11.
[58] Chen J, Peng H, Wang X, Shao F, Yuan Z, Han H. Graphene oxide exhibits broad-spectrum antimicrobial activity against bacterial phytopathogens and fungal conidia by intertwining and membrane perturbation. Nanoscale. 2014;6:1879-89.
[59] Liu X, Sen S, Liu J, Kulaots I, Geohegan D, Kane A, et al. Antioxidant deactivation on graphenic nanocarbon surfaces. Small. 2011;7:2775-85.
[60] Seibert JA, Boone JM. X-ray imaging physics for nuclear medicine technologists. Part 2: X-ray interactions and image formation. Journal of nuclear medicine technology. 2005;33:3-18.
[61] Hainfeld JF, Dilmanian FA, Slatkin DN, Smilowitz HM. Radiotherapy enhancement with gold nanoparticles. Journal of Pharmacy and Pharmacology. 2008;60:977-86.
[62] Misawa M, Takahashi J. Generation of reactive oxygen species induced by gold nanoparticles under x-ray and UV irradiations. Nanomedicine: Nanotechnology, Biology and Medicine. 2011;7:604-14.
[63] Hummers Jr WS, Offeman RE. Preparation of graphitic oxide. Journal of the American Chemical Society. 1958;80:1339-.
[64] Mao W. X-ray-induced Dissociation of H2O and Formation of an O2-H2 Compound at High Pressure.
[65] Ershov B, Gordeev A. A model for radiolysis of water and aqueous solutions of H 2, H 2 O 2 and O 2. Radiation Physics and Chemistry. 2008;77:928-35.
[66] Kavitha T, Gopalan AI, Lee K-P, Park S-Y. Glucose sensing, photocatalytic and antibacterial properties of graphene–ZnO nanoparticle hybrids. Carbon. 2012;50:2994-3000.