本研究利用高溫摻雜氮原子製備氮摻雜氧化石墨稀量子點(NGODs)。含氮官能基可修補其結構上的缺陷、調整電子結構，另外調控尺寸大小可增強量子侷限效應，進而提升光催化之活性。在犧牲試劑抗壞血酸的存在下，NGODs在照光下能催化產生大量的活性氧物質(reactive oxygen species)，且具有良好的生物相容性，使其在光動力治療上有極大潛力。
然而光的穿透性會降低光動力治療的效率，進一步影響細胞的毒殺效果，因此我們利用結合療法來提升治療效率，以光動力治療及一氧化碳氣體療法共同殺死腫瘤細胞。本研究以NGODs作為光敏化劑，並以抗壞血酸(ascorbic acid, AA)做為犧牲試劑(sacrificial reagent)，在照光下能產生H2O2，達成光動力治療殺癌細胞，同時促使一氧化碳釋放分子(CORM-401)釋放一氧化碳達成氣體療法。我們以中孔二氧化矽奈米粒子(mesoporous silicate nanoparticle, MSN)做為藥物載體負載CORM-401，可以避免CORM-401在運送過程中受到周圍環境影響而失活。
由研究結果可知，NGODs與抗壞血酸在照光下能催化氧氣並產生大量的過氧化氫，同時釋放CORM-401上的CO，且其釋放率於一小時內高達92%。經過腫瘤細胞之毒殺測試，PDT結合MSN (ext-MSN-TA)包覆之CORM-401，其細胞毒殺效果優於單一療法，細胞存活率可進一步降至35%，此結果證明此PDT結合CO氣體療法有相當優異的毒殺效果，因此使得NGODs應用在協同治療上有相當大的潛力。

Carbon monoxide (CO) can cause the dysfunction of mitochondria and then induces the apoptosis of cancer cells. The CO-based gas therapy recently attracted scholar attention on cardiovascular diseases and cancer treatment. To date, a variety of CO-releasing molecules (CORMs) which can be stimulated by light or H2O2 have been developed. However, CO is well known as silent killer, so the in situ-triggered and controllable release of CO is vitally important and greatly challenging. Photodynamic therapy (PDT) is an emerging therapy which is less invasive and has minor side-effects compared to other local treatment such as surgery or radiotherapy. Photodynamic therapy is composed of photosensitizers, oxygen and light. Unlike traditional or organic photosensitizers which have poor water dispersibility and high cytotoxicity related to heavy metal, graphene-based nanomaterials are environmental-friendly and are widely applied to biomedical field. Graphene oxide dots (GODs) which have tunable optical and electronic properties and excellent biocompatibility are applicable to biomedical field. The alien atom doping on graphene oxide dots can repair the vacancy defect, reduce charge recombination efficiency, and largely enhance ROS generation. Herein, we employed nitrogen-doped graphene oxide quantum dots (NGODs) as the photosensitizer in PDT and H2O2 sensitive CO-releasing molecules (CORMs) for gas therapy to develop a PDT-driven CO controllable release system. Under light illumination, the NGODs effectively catalyze considerable H2O2 or hydroxyl radical (OH•) generation and simultaneously trigger CO release from CORMs to synergistically diminish all cancer cells. This strategy to combine PDT and CO gas therapy shows high toxicity to cancer cells and provides a synergistic anticancer effect and therapeutic safety in vitro.

[1] Kessel, D., Photodynamic therapy: A brief history. J. Clin. Med. 2019, 8, 1581.
[2] Moan, J.; Peng, Q., An outline of the history of PDT. Photodyn. Ther. 2003, 2, 1-18.
[3] Dolmans, D. E.; Fukumura, D.; Jain, R. K., Photodynamic therapy for cancer. Nat. Rev. Cancer 2003, 3, 380-387.
[4] Baskaran, R.; Lee, J.; Yang, S.-G., Clinical development of photodynamic agents and therapeutic applications. Biomater. Res. 2018, 22, 1-8.
[5] Leem, J. W.; Kim, S.-R.; Choi, K.-H.; Kim, Y. L., Plasmonic photocatalyst-like fluorescent proteins for generating reactive oxygen species. Nano Convergence 2018, 5, 1-14.
[6] Abrahamse, H.; Hamblin, M. R., New photosensitizers for photodynamic therapy. Biochem. J. 2016, 473, 347-364.
[7] Maeda, K., Photocatalytic water splitting using semiconductor particles: history and recent developments. J. Photochem. Photobiol., C 2011, 12, 237-268.
[8] Kudo, A., Photocatalyst materials for water splitting. Catal. Surv. Asia 2003, 7, 31-38.
[9] Manoharan, D.; Li, W.-P.; Yeh, C.-S., Advances in controlled gas-releasing nanomaterials for therapeutic applications. Nanoscale Horiz. 2019, 4, 557-578.
[10] Chen, L.; Zhou, S.-F.; Su, L.; Song, J., Gas-mediated cancer bioimaging and therapy. ACS Nano 2019, 13, 10887-10917.
[11] Gholipour, M. R.; Dinh, C.-T.; Béland, F.; Do, T.-O., Nanocomposite heterojunctions as sunlight-driven photocatalysts for hydrogen production from water splitting. Nanoscale 2015, 7, 8187-8208.
[12] Shen, S.; Shi, J.; Guo, P.; Guo, L., Visible-light-driven photocatalytic water splitting on nanostructured semiconducting materials. Int. J. Nanotechnol. 2011, 8, 523-591.
[13] Detty, M. R.; Gibson, S. L.; Wagner, S. J., Current clinical and preclinical photosensitizers for use in photodynamic therapy. J. Med. Chem. 2004, 47, 3897-3915.
[14] Lucky, S. S.; Soo, K. C.; Zhang, Y., Nanoparticles in photodynamic therapy. Chem. Rev. 2015, 115, 1990-2042.
[15] Kwiatkowski, S.; Knap, B.; Przystupski, D.; Saczko, J.; Kędzierska, E.; Knap-Czop, K.; Kotlińska, J.; Michel, O.; Kotowski, K.; Kulbacka, J., Photodynamic therapy–mechanisms, photosensitizers and combinations. Biomed. Pharmacother. 2018, 106, 1098-1107.
[16] Wang, L.; Xu, D.; Gao, J.; Chen, X.; Duo, Y.; Zhang, H., Semiconducting quantum dots: Modification and applications in biomedical science. Sci. China Mater. 2020, 63, 1631-1650.
[17] Yaghini, E.; Seifalian, A. M.; MacRobert, A. J., Quantum dots and their potential biomedical applications in photosensitization for photodynamic therapy. 2009.
[18] Samia, A. C.; Chen, X.; Burda, C., Semiconductor quantum dots for photodynamic therapy. J. Am. Chem. Soc. 2003, 125, 15736-15737.
[19] Bakalova, R.; Ohba, H.; Zhelev, Z.; Ishikawa, M.; Baba, Y., Quantum dots as photosensitizers? Nat. Biotechnol. 2004, 22, 1360-1361.
[20] Shen, Y.; Shuhendler, A. J.; Ye, D.; Xu, J.-J.; Chen, H.-Y., Two-photon excitation nanoparticles for photodynamic therapy. Chem. Soc. Rev. 2016, 45, 6725-6741.
[21] Samia, A. C.; Dayal, S.; Burda, C., Quantum dot‐based energy transfer: perspectives and potential for applications in photodynamic therapy. Photochem. Photobiol. 2006, 82, 617-625.
[22] Juzenas, P.; Chen, W.; Sun, Y.-P.; Coelho, M. A. N.; Generalov, R.; Generalova, N.; Christensen, I. L., Quantum dots and nanoparticles for photodynamic and radiation therapies of cancer. Adv. Drug Delivery Rev. 2008, 60, 1600-1614.
[23] Derfus, A. M.; Chan, W. C.; Bhatia, S. N., Probing the cytotoxicity of semiconductor quantum dots. Nano Lett. 2004, 4, 11-18.
[24] Falcao, E. H.; Wudl, F., Carbon allotropes: beyond graphite and diamond. J. Chem. Technol. Biotechnol. 2007, 82, 524-531.
[25] Ströbel, R.; Garche, J.; Moseley, P.; Jörissen, L.; Wolf, G., Hydrogen storage by carbon materials. J. Power Sources 2006, 159, 781-801.
[26] Valencia, F.; Romero, A. H.; Ancilotto, F.; Silvestrelli, P. L., Lithium adsorption on graphite from density functional theory calculations. The Journal of Physical Chemistry B 2006, 110, 14832-14841.
[27] Hannay, N.; Geballe, T.; Matthias, B.; Andres, K.; Schmidt, P.; MacNair, D., Superconductivity in graphitic compounds. Phys. Rev. Lett. 1965, 14, 225.
[28] Ando, T., The electronic properties of graphene and carbon nanotubes. NPG Asia Mater. 2009, 1, 17-21.
[29] Xiang, Q.; Yu, J.; Jaroniec, M., Graphene-based semiconductor photocatalysts. Chem. Soc. Rev. 2012, 41, 782-796.
[30] Chung, C.; Kim, Y.-K.; Shin, D.; Ryoo, S.-R.; Hong, B. H.; Min, D.-H., Biomedical applications of graphene and graphene oxide. Acc. Chem. Res. 2013, 46, 2211-2224.
[31] Singh, D. P.; Herrera, C. E.; Singh, B.; Singh, S.; Singh, R. K.; Kumar, R., Graphene oxide: An efficient material and recent approach for biotechnological and biomedical applications. Materials Science and Engineering: C 2018, 86, 173-197.
[32] Yeh, T.-F.; Cihlář, J.; Chang, C.-Y.; Cheng, C.; Teng, H., Roles of graphene oxide in photocatalytic water splitting. Mater. Today 2013, 16, 78-84.
[33] Ahmad, H.; Fan, M.; Hui, D., Graphene oxide incorporated functional materials: A review. Composites, Part B 2018, 145, 270-280.
[34] Chen, L.-C.; Xiao, Y.-K.; Ke, N.-J.; Shih, C.-Y.; Yeh, T.-F.; Lee, Y.-L.; Teng, H., Synergy between quantum confinement and chemical functionality of graphene dots promotes photocatalytic H 2 evolution. J. Mater. Chem. A 2018, 6, 18216-18224.
[35] Guo, B.; Fang, L.; Zhang, B.; Gong, J. R., Graphene doping: a review. Insciences J. 2011, 1, 80-89.
[36] Li, M.; Liu, C.; Zhao, H.; An, H.; Cao, H.; Zhang, Y.; Fan, Z., Tuning sulfur doping in graphene for highly sensitive dopamine biosensors. Carbon 2015, 86, 197-206.
[37] Nguyen, B.-S.; Xiao, Y.-K.; Shih, C.-Y.; Nguyen, V.-C.; Chou, W.-Y.; Teng, H., Electronic structure manipulation of graphene dots for effective hydrogen evolution from photocatalytic water decomposition. Nanoscale 2018, 10, 10721-10730.
[38] Yuan, X., Control of semiconductor quantum dots by tailored static and dynamic strain fields. 2019.
[39] Berends, A. C.; de Mello Donega, C., Ultrathin one-and two-dimensional colloidal semiconductor nanocrystals: Pushing quantum confinement to the limit. The journal of physical chemistry letters 2017, 8, 4077-4090.
[40] Li, S.; Dong, G.; Hailili, R.; Yang, L.; Li, Y.; Wang, F.; Zeng, Y.; Wang, C., Effective photocatalytic H2O2 production under visible light irradiation at g-C3N4 modulated by carbon vacancies. Appl. Catal., B 2016, 190, 26-35.
[41] Lu, N.; Liu, N.; Hui, Y.; Shang, K.; Jiang, N.; Li, J.; Wu, Y., Characterization of highly effective plasma-treated g-C3N4 and application to the photocatalytic H2O2 production. Chemosphere 2020, 241, 124927.
[42] Shi, L.; Yang, L.; Zhou, W.; Liu, Y.; Yin, L.; Hai, X.; Song, H.; Ye, J., Photoassisted construction of holey defective g‐C3N4 photocatalysts for efficient visible‐light‐driven H2O2 production. Small 2018, 14, 1703142.
[43] Xiong, Z.; Wang, Z.; Muthu, M.; Zhang, Y., Construction of an in-situ Fenton-like system based on a g-C3N4 composite photocatalyst. J. Hazard. Mater. 2019, 373, 565-571.
[44] Shiraishi, Y.; Kanazawa, S.; Sugano, Y.; Tsukamoto, D.; Sakamoto, H.; Ichikawa, S.; Hirai, T., Highly selective production of hydrogen peroxide on graphitic carbon nitride (g-C3N4) photocatalyst activated by visible light. ACS Catal. 2014, 4, 774-780.
[45] Zhu, Z.; Pan, H.; Murugananthan, M.; Gong, J.; Zhang, Y., Visible light-driven photocatalytically active g-C3N4 material for enhanced generation of H2O2. Appl. Catal., B 2018, 232, 19-25.
[46] Kofuji, Y.; Ohkita, S.; Shiraishi, Y.; Sakamoto, H.; Tanaka, S.; Ichikawa, S.; Hirai, T., Graphitic carbon nitride doped with biphenyl diimide: efficient photocatalyst for hydrogen peroxide production from water and molecular oxygen by sunlight. ACS Catal. 2016, 6, 7021-7029.
[47] Hou, W.-C.; Wang, Y.-S., Photocatalytic generation of H2O2 by graphene oxide in organic electron donor-free condition under sunlight. ACS Sustainable Chem. Eng. 2017, 5, 2994-3001.
[48] He, Q., Precision gas therapy using intelligent nanomedicine. Biomater. Sci. 2017, 5, 2226-2230.
[49] Wang, Y.; Yang, T.; He, Q., Strategies for engineering advanced nanomedicines for gas therapy of cancer. Natl. Sci. Rev. 2020, 7, 1485-1512.
[50] Jin, Q.; Deng, Y.; Jia, F.; Tang, Z.; Ji, J., Gas therapy: An emerging “green” strategy for anticancer therapeutics. Adv. Ther. 2018, 1, 1800084.
[51] Lee, H. J.; Park, D. J.; Choi, G. H.; Yang, D.-N.; Heo, J. S.; Lee, S. C., pH-Responsive mineralized nanoparticles as stable nanocarriers for intracellular nitric oxide delivery. Colloids Surf., B 2016, 146, 1-8.
[52] Heilman, B. J.; St. John, J.; Oliver, S. R.; Mascharak, P. K., Light-triggered eradication of acinetobacter baumannii by means of NO delivery from a porous material with an entrapped metal nitrosyl. J. Am. Chem. Soc. 2012, 134, 11573-11582.
[53] Wang, X.; An, L.; Tian, Q.; Cui, K., Recent progress in H 2 S activated diagnosis and treatment agents. RSC Adv. 2019, 9, 33578-33588.
[54] Powell, C. R.; Dillon, K. M.; Matson, J. B., A review of hydrogen sulfide (H2S) donors: Chemistry and potential therapeutic applications. Biochem. Pharmacol. 2018, 149, 110-123.
[55] Foster, J. C.; Radzinski, S. C.; Zou, X.; Finkielstein, C. V.; Matson, J. B., H2S-releasing polymer micelles for studying selective cell toxicity. Mol. Pharmaceutics 2017, 14, 1300-1306.
[56] Yan, H.; Du, J.; Zhu, S.; Nie, G.; Zhang, H.; Gu, Z.; Zhao, Y., Emerging delivery strategies of carbon monoxide for therapeutic applications: from CO gas to CO releasing nanomaterials. Small 2019, 15, 1904382.
[57] Jin, Z.; Wen, Y.; Xiong, L.; Yang, T.; Zhao, P.; Tan, L.; Wang, T.; Qian, Z.; Su, B.-L.; He, Q., Intratumoral H 2 O 2-triggered release of CO from a metal carbonyl-based nanomedicine for efficient CO therapy. Chem. Commun. 2017, 53, 5557-5560.
[58] Wu, L.; Cai, X.; Zhu, H.; Li, J.; Shi, D.; Su, D.; Yue, D.; Gu, Z., PDT‐Driven Highly Efficient Intracellular Delivery and Controlled Release of CO in Combination with Sufficient Singlet Oxygen Production for Synergistic Anticancer Therapy. Adv. Funct. Mater. 2018, 28, 1804324.
[59] Zhao, B.; Zhao, P.; Jin, Z.; Fan, M.; Meng, J.; He, Q., Programmed ROS/CO-releasing nanomedicine for synergetic chemodynamic-gas therapy of cancer. J. Nanobiotechnol. 2019, 17, 1-12.
[60] Zhou, Y.; Yu, W.; Cao, J.; Gao, H., Harnessing carbon monoxide-releasing platforms for cancer therapy. Biomaterials 2020, 120193.
[61] Li, W.-P.; Su, C.-H.; Tsao, L.-C.; Chang, C.-T.; Hsu, Y.-P.; Yeh, C.-S., Controllable CO release following near-infrared light-induced cleavage of iron carbonyl derivatized Prussian blue nanoparticles for CO-assisted synergistic treatment. ACS Nano 2016, 10, 11027-11036.
[62] Chakraborty, I.; Carrington, S. J.; Hauser, J.; Oliver, S. R.; Mascharak, P. K., Rapid eradication of human breast cancer cells through trackable light-triggered CO delivery by mesoporous silica nanoparticles packed with a designed photoCORM. Chem. Mater. 2015, 27, 8387-8397.
[63] Yin, H.; Fang, J.; Liao, L.; Nakamura, H.; Maeda, H., Styrene-maleic acid copolymer-encapsulated CORM2, a water-soluble carbon monoxide (CO) donor with a constant CO-releasing property, exhibits therapeutic potential for inflammatory bowel disease. J. Controlled Release 2014, 187, 14-21.
[64] Guan, Q.; Zhou, L.-L.; Li, Y.-A.; Dong, Y.-B., A nanoscale metal–organic framework for combined photodynamic and starvation therapy in treating breast tumors. Chem. Commun. 2019, 55, 14898-14901.
[65] Hummers Jr, W. S.; Offeman, R. E., Preparation of graphitic oxide. J. Am. Chem. Soc. 1958, 80, 1339-1339.
[66] Martínez-Carmona, M.; Ho, Q. P.; Morand, J.; García, A.; Ortega, E.; Erthal, L. C.; Ruiz-Hernandez, E.; Santana, M. D.; Ruiz, J.; Vallet-Regí, M., Amino-Functionalized Mesoporous Silica Nanoparticle-Encapsulated Octahedral Organoruthenium Complex as an Efficient Platform for Combatting Cancer. Inorg. Chem. 2020, 59, 10275-10284.
[67] Pedraza, D.; Díez, J.; Colilla, M.; Vallet-Regí, M., Amine-functionalized mesoporous silica nanoparticles: A new nanoantibiotic for bone infection treatment. Biomedical Glasses 2018, 4, 1-12.
[68] Hosseinpour, S.; Walsh, L. J.; Xu, C., Biomedical application of mesoporous silica nanoparticles as delivery systems: A biological safety perspective. J. Mater. Chem. B 2020, 8, 9863-9876.
[69] Karakuzu, O.; Cruz, M. R.; Liu, Y.; Garsin, D. A., Amplex Red Assay for Measuring Hydrogen Peroxide Production from Caenorhabditis elegans. Bio-Protoc. 2019, 9.
[70] Van Der Vlies, A. J.; Inubushi, R.; Uyama, H.; Hasegawa, U., Polymeric framboidal nanoparticles loaded with a carbon monoxide donor via phenylboronic acid-catechol complexation. Bioconjugate Chem. 2016, 27, 1500-1508.
[71] Li, Y.; Zhao, Y.; Cheng, H.; Hu, Y.; Shi, G.; Dai, L.; Qu, L., Nitrogen-doped graphene quantum dots with oxygen-rich functional groups. J. Am. Chem. Soc. 2012, 134, 15-18.
[72] Stankovich, S.; Dikin, D. A.; Dommett, G. H.; Kohlhaas, K. M.; Zimney, E. J.; Stach, E. A.; Piner, R. D.; Nguyen, S. T.; Ruoff, R. S., Graphene-based composite materials. nature 2006, 442, 282-286.
[73] Wang, H.; Maiyalagan, T.; Wang, X., Review on recent progress in nitrogen-doped graphene: synthesis, characterization, and its potential applications. ACS Catal. 2012, 2, 781-794.
[74] Yeh, T.-F.; Chen, S.-J.; Teng, H., Synergistic effect of oxygen and nitrogen functionalities for graphene-based quantum dots used in photocatalytic H2 production from water decomposition. Nano Energy 2015, 12, 476-485.
[75] Guo, B.; Liu, Q.; Chen, E.; Zhu, H.; Fang, L.; Gong, J. R., Controllable N-doping of graphene. Nano Lett. 2010, 10, 4975-4980.
[76] Nairi, V.; Medda, S.; Piludu, M.; Casula, M. F.; Vallet-Regì, M.; Monduzzi, M.; Salis, A., Interactions between bovine serum albumin and mesoporous silica nanoparticles functionalized with biopolymers. Chem. Eng. J. 2018, 340, 42-50.
[77] Eda, G.; Lin, Y. Y.; Mattevi, C.; Yamaguchi, H.; Chen, H. A.; Chen, I. S.; Chen, C. W.; Chhowalla, M., Blue photoluminescence from chemically derived graphene oxide. Adv. Mater. 2010, 22, 505-509.
[78] Qu, D.; Zheng, M.; Du, P.; Zhou, Y.; Zhang, L.; Li, D.; Tan, H.; Zhao, Z.; Xie, Z.; Sun, Z., Highly luminescent S, N co-doped graphene quantum dots with broad visible absorption bands for visible light photocatalysts. Nanoscale 2013, 5, 12272-12277.
[79] Mothilal, K.; Inbaraj, J. J.; Chignell, C. F.; Gandhidasan, R.; Murugesan, R., Photosensitisation with naphthoquinones and binaphthoquinones: EPR spin trapping and optical studies-formation of semiquinone radical and reactive oxygen species on photoillumination. J. Photochem. Photobiol., A 2004, 163, 141-148.
[80] Matuszak, Z.; Reszka, K. J.; Chignell, C. F., Reaction of melatonin and related indoles with hydroxyl radicals: EPR and spin trapping investigations. Free Radical Biology and Medicine 1997, 23, 367-372.
[81] Fayad-Kobeissi, S.; Ratovonantenaina, J.; Dabiré, H.; Wilson, J. L.; Rodriguez, A. M.; Berdeaux, A.; Dubois-Randé, J.-L.; Mann, B. E.; Motterlini, R.; Foresti, R., Vascular and angiogenic activities of CORM-401, an oxidant-sensitive CO-releasing molecule. Biochem. Pharmacol. 2016, 102, 64-77.
[82] Shih, C.-Y.; Huang, W.-L.; Chiang, I.-T.; Su, W.-C.; Teng, H., Biocompatible hole scavenger–assisted graphene oxide dots for photodynamic cancer therapy. Nanoscale 2021, 13, 8431-8441.