金黃色葡萄球菌(Staphylococcus aureus)為皮膚和軟組織感染最常見的致病菌。由於抗生素的濫用，導致細菌逐漸演化為具有抗藥性的菌株，如多重抗藥性金黃色葡萄球菌(Methicillin-resistant Staphylococcus aureus, MRSA)。因此急需發展出更有效，且不會產生抗藥性菌株的治療方式。本研究開發出一多功能之奈米抗菌材料，利用共沉澱法將氧化鐵粒子(IONP)生成於石墨烯(GE)的表面，藉由石墨烯可將近紅外光轉換成熱能的特性，以及氧化鐵可藉由Fenton reaction (Fe+2+H2O2→Fe+3+OH-+OH.)催化過氧化氫，產生毒性更高的氫氧自由基的特性，對細菌進行毒殺。本研究發現，將氧化鐵粒子分散於石墨烯表面，可減少氧化鐵粒子聚集的現象，增加氧化鐵的表面積以提高催化能力，並且使石墨烯能有效分散於水溶液中。在照射近紅外光(Near-infrared, NIR)後，GE-IONP能升溫到50℃以上，造成細菌的死亡。更重要的是，升溫可以進一步增加氧化鐵催化過氧化氫產生氫氧自由基的效果，使高溫和氫氧自由基能協同作用以達到殺菌之目的。本研究以小鼠皮下感染MRSA的模式來測試GE-IONP對於細菌感染的治療效果。因發炎部位的過氧化氫濃度較正常組織高，因此將有利於氧化鐵催化產生高毒性的氫氧自由基來進行殺菌。實驗結果顯示，GE-IONP結合NIR照射的治療，可以有效的抑制MRSA的生長，並且加速感染後皮膚的修復。
關鍵字: 石墨烯、氧化鐵奈米粒子、多重抗藥性金黃色葡萄球菌、光熱治療、氫氧自由基

Alternative approaches to treating subcutaneous abscesses—especially those associated with antibiotic-resistant pathogenic bacterial strains—that eliminate the need for antibiotics are urgently needed. This work reports an injectable system of graphene-iron oxide nanocomposite (GE-IONP), which combines the photothermal effect of graphene and the ability of iron oxide to catalyze the production of hydroxyl radical from hydrogen peroxide. The photothermal effect of GE-IONP nanocomposite can further enhance the formation of hydroxyl radical from hydrogen peroxide. The system can rapidly produce localized heat and hydroxyl radical by near-infrared (NIR) light irradiation, as a combination strategy for treating subcutaneous abscesses. In vitro and in vivo antibacterial experimental results show strong synergistic bactericidal effects when hyperthermia is combined with reactive oxygen species (ROS) toxicity, demonstrating that the system is an effective and useful modality in the treatment of subcutaneous infection.
Keywords: Graphene, Iron-Oxide Nanoparticle, Photothermal Therapy, MRSA, Subcutaneous Infection

目錄
摘要 I
Abstract II
目錄 III
圖目錄 V
第一章 緒論 1
1-1細菌感染(Bacterial infection) 1
1-2光熱治療(Photothermal therapy) 2
1-3石墨烯(Graphene) 3
1-4活性氧化物質(Reactive oxygen species, ROS) 4
1-5氧化鐵(Iron oxide) 6
1-6研究目的與動機 7
1-7實驗設計流程圖 7
第二章 製程與實驗 8
2-1 實驗藥品 8
2-2製備石墨稀-氧化鐵奈米複合物 (GE-IONP) 9
2-3物化性分析 (Characterization) 10
2-3-1穿透式電子顯微鏡 (Transmission Electron Microscopy, TEM) 10
2-3-2紫外光/可見光吸收光譜 (UV/Vis spectra) 10
2-3-3 X-射線繞射 (X-ray diffraction, XRD) 11
2-3-4傅里葉轉換紅外光譜 (FT-IR spectra) 12
2-4 照射近紅外光(NIR)產生熱 12
2-5偵測氫氧自由基的產生 12
2-6細菌培養 13
2-7體外殺菌實驗 (In vitro antibacterial activity) 14
2-8細菌之SEM 樣品製作 15
2-9動物實驗 ( Animal study ) 15
第三章 結果與討論 16
3-1以TEM 觀察表面型態 16
3-2以XRD 分析晶型 18
3-3吸收光譜 19
3-4以FT-IR spectra 分析化學鍵結 19
3-5照射近紅外光產生熱 20
3-6偵測氫氧自由基的產生 23
3-7體外殺菌實驗 (In vitro antibacterial activity) 25
3-8動物實驗 ( Animal Study ) 28
第四章 結論 32
參考資料 33

參考資料
[1] Huh AJ, Kwon YJ. "Nanoantibiotics": A new paradigm for treating infectious diseases using nanomaterials in the antibiotics resistant era. J Controlled Release. 2011;156:128-45.
[2] Kojic N, Pritchard EM, Tao H, Brenckle MA, Mondia JP, Panilaitis B, et al. Focal Infection Treatment using Laser-Mediated Heating of Injectable Silk Hydrogels with Gold Nanoparticles. Adv Funct Mater. 2012;22:3793-8.
[3] Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med. 2004;10:S122-S9.
[4] Ahmed K, Zaidi SF. Treating cancer with heat: hyperthermia as promising strategy to enhance apoptosis. J Pak Med Assoc. 2013;63:504-8.
[5] Wu MC, Deokar AR, Liao JH, Shih PY, Ling YC. Graphene-based photothermal agent for rapid and effective killing of bacteria. ACS Nano. 2013;7:1281-90.
[6] Janssen LJ. Airway Smooth Muscle as a Target in Asthma and the Beneficial Effects of Bronchial Thermoplasty. Journal of Allergy. 2012;2012:9.
[7] Welsher K, Liu Z, Sherlock SP, Robinson JT, Chen Z, Daranciang D, et al. A route to brightly fluorescent carbon nanotubes for near-infrared imaging in mice. Nat Nano. 2009;4:773-80.
[8] 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. J Am Chem Soc. 2011;133:6825-31.
[9] You J, Zhang R, Zhang G, Zhong M, Liu Y, Van Pelt CS, et al. Photothermal-chemotherapy with doxorubicin-loaded hollow gold nanospheres: A platform for near-infrared light-trigged drug release. J Controlled Release. 2012;158:319-28.
[10] Zhou F, Xing D, Ou Z, Wu B, Resasco DE, Chen WR. Cancer photothermal therapy in the near-infrared region by using single-walled carbon nanotubes. BIOMEDO. 2009;14:021009--7.
[11] Lin D, Qin T, Wang Y, Sun X, Chen L. Graphene oxide wrapped SERS tags: multifunctional platforms toward optical labeling, photothermal ablation of bacteria, and the monitoring of killing effect. ACS Appl Mater Interfaces. 2014;6:1320-9.
[12] Yang X, Li Z, Ju E, Ren J, Qu X. Reduced graphene oxide functionalized with a luminescent rare-earth complex for the tracking and photothermal killing of drug-resistant bacteria. Chemistry. 2014;20:394-8.
[13] Tanaka Y, Tsunemi Y, Kawashima M, Nishida H. The impact of near-infrared in plastic surgery. Plastic Surgery. 2013.
[14] Naebe M, Wang J, Amini A, Khayyam H, Hameed N, Li LH, et al. Mechanical property and structure of covalent functionalised graphene/epoxy nanocomposites. Sci Rep. 2014;4:4375.
[15] Luo J, Jang HD, Huang J. Effect of sheet morphology on the scalability of graphene-based ultracapacitors. ACS Nano. 2013;7:1464-71.
[16] Huang M, Pascal TA, Kim H, Goddard WA, Greer JR. Electronic--mechanical coupling in graphene from in situ nanoindentation experiments and multiscale atomistic simulations. Nano Lett. 2011;11:1241-6.
[17] Yuan J, Liew KM. High-temperature thermal stability and in-plane compressive properties of a graphene and a boron-nitride nanosheet. J Nanosci Nanotechnol. 2012;12:2617-24.
[18] An X, Butler TW, Washington M, Nayak SK, Kar S. Optical and sensing properties of 1-pyrenecarboxylic Acid-functionalized graphene films laminated on polydimethylsiloxane membranes. ACS Nano. 2011;5:1003-11.
[19] Lightcap IV, Kamat PV. Graphitic design: prospects of graphene-based nanocomposites for solar energy conversion, storage, and sensing. Acc Chem Res. 2013;46:2235-43.
[20] Behura SK, Mahala P, Nayak S, Yang Q, Mukhopadhyay I, Janil O. Fabrication of bi-layer graphene and theoretical simulation for its possible application in thin film solar cell. J Nanosci Nanotechnol. 2014;14:3022-7.
[21] Kang X, Wang J, Wu H, Liu J, Aksay IA, Lin Y. A graphene-based electrochemical sensor for sensitive detection of paracetamol. Talanta. 2010;81:754-9.
[22] Zhang W, Guo Z, Huang D, Liu Z, Guo X, Zhong H. Synergistic effect of chemo-photothermal therapy using PEGylated graphene oxide. Biomaterials. 2011;32:8555-61.
[23] Tao Y, Ju E, Ren J, Qu X. Immunostimulatory oligonucleotides-loaded cationic graphene oxide with photothermally enhanced immunogenicity for photothermal/immune cancer therapy. Biomaterials. 2014.
[24] Lin CW, Wei KC, Liao SS, Huang CY, Sun CL, Wu PJ, et al. A reusable magnetic graphene oxide-modified biosensor for vascular endothelial growth factor detection in cancer diagnosis. Biosens & bioelectron. 2014.
[25] Chiu NF, Huang TY, Lai HC, Liu KC. Graphene oxide-based SPR biosensor chip for immunoassay applications. Nanoscale Res Lett. 2014;9:445.
[26] Qin XC, Guo ZY, Liu ZM, Zhang W, Wan MM, Yang BW. Folic acid-conjugated graphene oxide for cancer targeted chemo-photothermal therapy. J Photochem Photobiol B. 2013;120:156-62.
[27] 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.
[28] Gurunathan S, Han JW, Dayem AA, Eppakayala V, Kim JH. Oxidative stress-mediated antibacterial activity of graphene oxide and reduced graphene oxide in Pseudomonas aeruginosa. Int J Nanomedicine. 2012;7:5901-14.
[29] Liu Z, Robinson JT, Sun X, Dai H. PEGylated nanographene oxide for delivery of water-insoluble cancer drugs. J Am Chem Soc. 2008;130:10876-7.
[30] Krishnamoorthy K, Veerapandian M, Zhang LH, Yun K, Kim SJ. Antibacterial Efficiency of Graphene Nanosheets against Pathogenic Bacteria via Lipid Peroxidation. J Phys Chem C. 2012;116:17280-7.
[31] Li D, Muller MB, Gilje S, Kaner RB, Wallace GG. Processable aqueous dispersions of graphene nanosheets. Nat Nanotechnol. 2008;3:101-5.
[32] Mocsai A. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. J Exp Med. 2013;210:1283-99.
[33] Legodi MA, de Waal D. The preparation of magnetite, goethite, hematite and maghemite of pigment quality from mill scale iron waste (vol 74, pg 161, 2007). Dyes and Pigments. 2008;78:177-.
[34] Berry CC, Wells S, Charles S, Aitchison G, Curtis AS. Cell response to dextran-derivatised iron oxide nanoparticles post internalisation. Biomaterials. 2004;25:5405-13.
[35] Wu H, Yin JJ, Wamer WG, Zeng M, Lo YM. Reactive oxygen species-related activities of nano-iron metal and nano-iron oxides. J Food Drug Anal. 2014;22:86-94.
[36] Lindsey ME, Tarr MA. Quantitation of hydroxyl radical during fenton oxidation following a single addition of iron and peroxide. Chemosphere. 2000;41:409-17.
[37] Voinov MA, Sosa Pagan JO, Morrison E, Smirnova TI, Smirnov AI. Surface-mediated production of hydroxyl radicals as a mechanism of iron oxide nanoparticle biotoxicity. J Am Chem Soc. 2011;133:35-41.
[38] Cabiscol E, Tamarit J, Ros J. Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol. 2000;3:3-8.
[39] Hyslop PA, Hinshaw DB, Scraufstatter IU, Cochrane CG, Kunz S, Vosbeck K. Hydrogen-Peroxide as a Potent Bacteriostatic Antibiotic - Implications for Host-Defense. Free Radical Bio Med. 1995;19:31-7.
[40] Auffan M, Achouak W, Rose J, Roncato MA, Chaneac C, Waite DT, et al. Relation between the redox state of iron-based nanoparticles and their cytotoxicity toward Escherichia coli. Environ Sci Technol. 2008;42:6730-5.
[41] Yamaguchi S, Kishikawa N, Ohyama K, Ohba Y, Kohno M, Masuda T, et al. Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species. Analytica chimica acta. 2010;665:74-8.
[42] Cheng J, Zhang M, Wu G, Wang X, Zhou JH, Cen KF. Photoelectrocatalytic Reduction of CO2 into Chemicals Using Pt-Modified Reduced Graphene Oxide Combined with Pt-Modified TiO2 Nanotubes. Environ Sci Technol. 2014;48:7076-84.