發炎是身體對免疫系統對刺激如感染或刺激的反應。在正常生理條件下，發炎是一種保護機制;然而，慢性炎症破壞身體的平衡，導致各種疾病如關節炎，糖尿病，癌症和炎症性腸病。發炎與高含量的活性氧（ROS）有關。 ROS是自由基，包括超氧自由基，過氧化氫，羥基自由基等。在病理生理條件下，ROS顯著增加，導致嚴重的氧化應激，引起發炎。據報導，一氧化氮（NO）具有很強的消滅ROS的能力。然而，體內NO的半衰期只有2-6秒。本研究的目的是開發一種NO載體，一種含有DETA-NONOate和癸酸鈉的PLGA微球。製備這種微球體並以加載NO前趨物（DETA-NONOate），並且能夠保護NO免受環境的降解。為了釋放NO，將微粒置於pH 6.8（模擬發炎部位的pH）。此時，質子擴散到微球體內並與DETA-NONOate反應產生NO氣泡。該研究中的癸酸鈉作為表面活性劑，其中癸酸鈉的疏水末端會朝向NO氣泡，藉以穩定結構、提供空間位阻，並逹到保護NO氣泡免受由身體中的血紅蛋白等蛋白質引起的降解的功能，從而延長其半衰期。因此，我們預計本研究中製備的微球可以延長NO的半衰期，並且還可以抑制發炎反應。

Inflammation is the body’s response of the imune system to stimulus such as infection or irritation. Under normal physiological condition, inflammation is a kind of protective mechanism; however, the chronic inflammation destroys the balance of the body, leading to various diseases such as arthritis, diabetes, cancer and inflammatory bowel disease. Inflammation is associated with high level of reactive oxygen species (ROS). ROS is free radicals including superoxide radicals, hydrogen peroxide, hydroxyl radical and so on. Under pathophysiological condition, ROS increase significantly, result in serious oxidative stress and causing inflammation. Nitric oxide (NO) is reported that it has great ability to quench ROS. However, the half-life of NO in vivo is only 2-6 seconds. The purpose of this study was to develop a NO carrier, a kind of PLGA microsphere that contained DETA-NONOate and sodium decanoate. This microspheres were made to load the NO precursor (DETA-NONOate) and were able to protect the NO from the degradation of environment. For the release of NO, the microparticles were put in pH 6.8 (mimicking pH of inflammatory site). At this time, protons diffused inside the particle and reacted with DETA-NONOate to generate NO bubbles. Sodium decanoate in this study acted as a surfactant in which the hydrophobic end of sodium decanoate toward NO bubbles, in order to stabilize structure, provide steric hindrance and protect NO bubbles from the degradation caused by proteins such as hemoglobin in the body, thus extending its half-life. Therefore, we expect that the microspheres prepared in this study can prolong the half-life of NO, and also inhibit the inflammation

Contents

Abstract i
Contents ii
List of Figures iv
Chapter 1: Introduction 1
1.1. Introduction of inflammation 1
1.2. The potential treatment of nitric oxide in anti-inflammation. 1
1.3. Limit of nitric oxide therapy 1
1.4. Purpose of the research 2
1.5. Experiment design 5
Chapter 2: Materials and methods 6
2.1. Characteristic of microsphere 6
2.1.1. Microsphere marterials 6
2.1.2. The preparation of microsphere 6
2.1.3. Morphology of particles 7
2.1.4. The release of nitric oxide from particles 7
2.1.5. The detection of sodium decanoate 7
2.1.6. The stability of particles 7
2.1.7. Micelle formation detection 7
2.1.8. Anti-degradation activity 8
2.1.9. Antioxidant activity 8
2.2. Cell experiment 8
2.2.1. Cell culture 8
2.2.2. Cell viability 8
2.2.3. Anti-oxidant ability 8
2.2.4. Anti- inflammatory ability 9
Chapter 3: Result and discussion 10
3.1. Characteristic of microsphere 10
3.1.1. Morphology of particles 10
3.1.2. The release profile of nitric oxide from particles 10
3.1.3. The detection of sodium decanoate 11
3.1.4. Particle stability 12
3.1.5. Micelle formation detection 13
3.1.6. Anti-degradation activity 14
3.1.7. Anti oxidant capacity 16
3.2. Cell experiment 17
3.2.1. Cell viability 17
3.2.2. Anti-oxidant ability 20
3.2.3. Anti-inflammatory ability 22
Chapter 4: Conclusion 23
References 24

[1] Petersen, O. H., Spät, A., & Verkhratsky, A. (2005). Introduction: reactive oxygen species in health and disease.
[2] Sharma, J. N., Al-Omran, A., & Parvathy, S. S. (2007). Role of nitric oxide in inflammatory diseases. Inflammopharmacology, 15(6), 252-259.
[3] Gill, S. S., & Tuteja, N. (2010). Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant physiology and biochemistry, 48(12), 909-930.
[4] Katsuyama, K., Shichiri, M., Marumo, F., & Hirata, Y. (1998). NO inhibits cytokine-induced iNOS expression and NF-κB activation by interfering with phosphorylation and degradation of IκB-α. Arteriosclerosis, Thrombosis, and Vascular Biology, 18(11), 1796-1802.
[5] Zhao, L., Guo, X., Wang, O., Zhang, H., Wang, Y., Zhou, F., ... & Ji, B. (2016). Fructose and glucose combined with free fatty acids induce metabolic disorders in HepG2 cell: A new model to study the impacts of high‐fructose/sucrose and high‐fat diets in [6] Fehrenbacher, J. C., Vasko, M. R., & Duarte, D. B. (2012). Models of Inflammation: Carrageenan‐or Complete Freund's Adjuvant (CFA)–Induced Edema and Hypersensitivity in the Rat. Current Protocols in Pharmacology, 5-4.
[7] Cai, C., Chen, Y., Zhong, S., Ji, B., Wang, J., Bai, X., & Shi, G. (2014). Anti-inflammatory activity of N-butanol extract from Ipomoea stolonifera in vivo and in vitro. PloS one, 9(4), e95931.
[8] Durgashivaprasad, E., Mathew, G., Sebastian, S., Reddy, S. M., Mudgal, J., & Nampurath, G. K. (2014). Novel 2, 5-disubstituted-1, 3, 4-oxadiazoles as anti-inflammatory drugs. Indian journal of pharmacology, 46(5), 521.
[9] Jang, M., Jeong, S. W., Cho, S. K., Ahn, K. S., Lee, J. H., Yang, D. C., & Kim, J. C. (2014). Anti-inflammatory effects of an ethanolic extract of guava (Psidium guajava L.) leaves in vitro and in vivo. Journal of medicinal food, 17(6), 678-685.
[10] Gomaa, A. A., Elshenawy, M. M., Afifi, N. A., Mohammed, E. A., & Thabit, R. H. (2009). Dual effect of nitric oxide donor on adjuvant arthritis. International immunopharmacology, 9(4), 439-447.
[11] Liao, Guojian, Qing Liu, and Jianping Xie. (2013). Transcriptional analysis of the effect of exogenous decanoic acid stress on Streptomyces roseosporus. Microbial cell factories, 12(1), 1.
[12] Liu, W. F., Ma, M., Bratlie, K. M., Dang, T. T., Langer, R., & Anderson, D. G. (2011). Real-time in vivo detection of biomaterial-induced reactive oxygen species. Biomaterials, 32(7), 1796-1801.
[13] Kanabus, M., Fassone, E., Hughes, S. D., Bilooei, S. F., Rutherford, T., O’Donnell, M., ... & Rahman, S. (2016). The pleiotropic effects of decanoic acid treatment on mitochondrial function in fibroblasts from patients with complex I deficient Leigh syndrome. Journal of inherited metabolic disease, 39(3), 415-426.
[14] Wallace, J. L. (2005). Nitric oxide as a regulator of inflammatory processes. Memorias do Instituto Oswaldo Cruz, 100, 5-9.
[15] Pacher, Pal, Joseph S. Beckman, and Lucas Liaudet. (2007). Nitric oxide and peroxynitrite in health and disease. Physiological reviews, 87(1), 315-424.
[16] Kobayashi, Y. (2010). The regulatory role of nitric oxide in proinflammatory cytokine expression during the induction and resolution of inflammation. Journal of leukocyte biology, 88(6), 1157-1162.
[17] Hiramoto, K., Ohkawa, T., Oikawa, N., & Kikugawa, K. (2003). Is nitric oxide (NO) an antioxidant or a prooxidant for lipid peroxidation?. Chemical and pharmaceutical bulletin, 51(9), 1046-1050.
[18] Shan, L., Wang, B., Gao, G., Cao, W., & Zhang, Y. (2013). L-Arginine supplementation improves antioxidant defenses through L-arginine/nitric oxide pathways in exercised rats. Journal of Applied Physiology, 115(8), 1146-1155.
[19] Wink, D. A., Miranda, K. M., Espey, M. G., Pluta, R. M., Hewett, S. J., Colton, C., Grisham, M. B. (2001). Mechanisms of the antioxidant effects of nitric oxide. Antioxidants and redox signaling, 3(2), 203-213.
[20] Patel, R. P., Levonen, A. L., Crawford, J. H., & Darley-Usmar, V. M. (2000). Mechanisms of the pro-and anti-oxidant actions of nitric oxide in atherosclerosis. Cardiovascular research, 47(3), 465-474.
[21] Chiueh, C. C. (1999). Neuroprotective properties of nitric oxide. Annals of the New York Academy of Sciences, 890(1), 301-311.
[22] Violi, F., Marino, R., Milite, M. T., & Loffredo, L. (1999). Nitric oxide and its role in lipid peroxidation. Diabetes/metabolism research and reviews, 15(4), 283-288.
[23] Dengler, T., Kellner, S., & Fürst, G. (1988). Quantitative determination of sodium-octanoate in human serum albumin preparations. Transfusion Medicine and Hemotherapy, 15(6), 273-274.
[24] Kelm, M. (1999). Nitric oxide metabolism and breakdown. Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1411(2), 273-289.
[25] Hakim, T. S., Sugimori, K., Camporesi, E. M., & Anderson, G. (1996). Half-life of nitric oxide in aqueous solutions with and without haemoglobin. Physiological measurement, 17(4), 267.
[26] Dominiak, A., Wilkaniec, A., Jęśko, H., Czapski, G. A., Lenkiewicz, A. M., Kurek, E., ... & Adamczyk, A. (2017). Selol, an organic selenium donor, prevents lipopolysaccharide-induced oxidative stress and inflammatory reaction in the rat brain. Neurochemistry International.
[27] Jeong, Y. H., Park, J. S., Kim, D. H., Kang, J. L., & Kim, H. S. (2017). Anti-inflammatory mechanism of lonchocarpine in LPS-or poly (I: C)-induced neuroinflammation. Pharmacological research, 119, 431-442.
[28] Nurhasni, H., Cao, J., Choi, M., Kim, I., Lee, B. L., Jung, Y., & Yoo, J. W. (2015). Nitric oxide-releasing poly (lactic-co-glycolic acid)-polyethylenimine nanoparticles for prolonged nitric oxide release, antibacterial efficacy, and in vivo wound healing activity. International journal of nanomedicine, 10, 3065.
[29] Stevenson, S. A., & Blanchard, G. J. (2006). Investigating internal structural differences between micelles and unilamellar vesicles of decanoic acid/sodium decanoate. The Journal of Physical Chemistry B, 110(26), 13005-13010.
[30] Andrade-Dias, C., Lima, S., Teixeira-Dias, J. J., & Teixeira, J. (2008). Why do methylated and unsubstituted cyclodextrins interact so differently with sodium decanoate micelles in water?. The Journal of Physical Chemistry B, 112(48), 15327-15332.
[31] Namani, T., & Walde, P. (2005). From decanoate micelles to decanoic acid/dodecylbenzenesulfonate vesicles. Langmuir, 21(14), 6210-6219.
[32] Merta, J., Torkkeli, M., Ikonen, T., Serimaa, R., & Stenius, P. (2001). Structure of cationic starch (CS)/anionic surfactant complexes studied by small-angle X-ray scattering (SAXS). Macromolecules, 34(9), 2937-2946.
[33] Rodríguez-Pulido, A., Casado, A., Munoz-Ubeda, M., Junquera, E., & Aicart, E. (2010). Experimental and theoretical approach to the sodium decanoate− dodecanoate mixed surfactant system in aqueous solution. Langmuir, 26(12), 9378-9385.
[34] Pryor, W. A., & Squadrito, G. L. (1995). The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide. American Journal of Physiology-Lung Cellular and Molecular Physiology, 268(5), L699-L722.
[35] Sun, S., Zhang, H., Xue, B., Wu, Y., Wang, J., Yin, Z., & Luo, L. (2006). Protective effect of glutathione against lipopolysaccharide-induced inflammation and mortality in rats. Inflammation Research, 55(11), 504-510.
[36] Coleman, J. W. (2001). Nitric oxide in immunity and inflammation. International immunopharmacology, 1(8), 1397-1406.
[37] Murphy, T. F. (2006). The role of bacteria in airway inflammation in exacerbations of chronic obstructive pulmonary disease. Current opinion in infectious diseases, 19(3), 225-230.
[38] Dean, H. G., Bonser, J. C., & Gent, J. P. (1989). HPLC analysis of brain and plasma for octanoic and decanoic acids. Clinical chemistry, 35(9), 1945-1948