臺灣博碩士論文加值系統

Acidic pH value and oxidative stress caused by excess production of reactive oxygen species (ROS) are two of the most typical inflammatory markers. Each of them has received extensive interest as a target stimulus in inflammation. Curcumin (CUR), a member of ginger family, has been used as an antioxidant in the treatment of chronic inflammatory diseases. In this study, we developed self-assembled, pH-responsive N-palmitoyl chitosan nanoparticles with a Cy3 moiety (Cy3-NPCS NPs) to encapsulate a ROS-sensitive thioketal polymer (PPADT) and curcumin, and delivered them to lipopolysaccharide (LPS)-stimulated macrophages to mitigate regional inflammation. The PPADT-CUR-Cy3-NPCS (PCCN) NPs with sensitivity to both pH and ROS showed great inhibitory effects on ROS production, such as hydrogen peroxide (H2O2) and nitric oxide (NO‧). Based on the Förster Resonance Energy Transfer (FRET) effect, we further intracellularly monitored the drug releasing process, and hence better understood the correlation between the FRET effect and CUR release. From our result, the immune-responsive PCCN NPs are potent for both probing and curing of ROS-related diseases.

Abstract I
Table of Contents II
List of Figures V
List of Tables VIII

Chapter 1. Introduction 1
1-1. Inflammation and Reactive Oxygen Species (ROS) 1
1-1-1. Inflammation 1
1-1-2. Reactive Oxygen Species (ROS) 1
1-2. Curcumin (CUR) 2
1-3. Stimuli-responsive NPs 4
1-4. N-palmitoyl chitosan (NPCS) 4
1-5. Poly-(1,4-phenyleneactone dimethylene thioketal) (PPADT) 5
1-6. Förster Resonance Energy Transfer (FRET) Effect 6
1-8. Basic concept of this study 9

Chapter 2. Materials and Methods 12
2-1. Materials 12
2-2. Synthesis of NPCS and preparation of fluorescent NPCS 12
2-3. Preparation and characterization of Cy3-NPCS NPs 13
2-4. Synthesis of PPADT 13
2-5. Degradation of PPADT in ROS environment 14
2-6. Preparation and characterization of PPADT-CUR-Cy3-NPCS (PCCN) NPs 14
2-7. MD simulations 15
2-8. FRET measurements 16
2-9. Drug in vitro release study 16
2-10. Cytotoxicity assay 17
2-11. Hydrogen peroxide (H2O2) scavenging and inhibition effect of PCCNs 17
2-12. Nitric oxide (NO) scavenging and inhibition effect of PCCNs 18
2-13. Intracellular monitoring/imaging CUR release using FRET 19

Chapter 3. Results and Discussion 20
3-1. Synthesis of NPCS and Cy3-NPCS 20
3-2. Synthesis of PPADT 21
3-3. Degradation of PPADT in ROS environment 23
3-4. Characterization of Cy3-NPCS and PCCN NPs 24
3-5. TEM images 25
3-6. MD simulations 26
3-7. FRET measurements 28
3-8. Drug in vitro release profile 30
3-9. Cytotoxicity assay 31
3-10. Hydrogen peroxide (H2O2) scavenging and inhibition effect of PCCNs 33
3-11. Nitric oxide (NO) scavenging and inhibition effect of PCCNs 34
3-12. Intracellular monitoring/imaging CUR release using FRET 36

Chapter 4. Conclusions 38

References 39

References
1.Steen, K.; Steen, A.; Reeh, P., A dominant role of acid pH in inflammatory excitation and sensitization of nociceptors in rat skin, in vitro. The Journal of Neuroscience 1995, 15 (5), 3982-3989.
2.Kim, S.; Park, H.; Song, Y.; Hong, D.; Kim, O.; Jo, E.; Khang, G.; Lee, D., Reduction of oxidative stress by p-hydroxybenzyl alcohol-containing biodegradable polyoxalate nanoparticulate antioxidant. Biomaterials 2011, 32 (11), 3021-3029.
3.Simon, H. U.; Haj-Yehia, A.; Levi-Schaffer, F., Role of reactive oxygen species (ROS) in apoptosis induction. Apoptosis 2000, 5 (5), 415-418.
4.Kirkinezos, I. G.; Moraes, C. T., Reactive oxygen species and mitochondrial diseases. Seminars in Cell &; Developmental Biology 2001, 12 (6), 449-457.
5.Lee, H.; Lee, K.; Kim, I.-K.; Park, T. G., Fluorescent Gold Nanoprobe Sensitive to Intracellular Reactive Oxygen Species. Advanced Functional Materials 2009, 19 (12), 1884-1890.
6.Kim, S.; Seong, K.; Kim, O.; Kim, S.; Seo, H.; Lee, M.; Khang, G.; Lee, D., Polyoxalate Nanoparticles as a Biodegradable and Biocompatible Drug Delivery Vehicle. Biomacromolecules 2010, 11 (3), 555-560.
7.Aggarwal, B. B.; Sung, B., Pharmacological basis for the role of curcumin in chronic diseases: an age-old spice with modern targets. Trends in Pharmacological Sciences 2009, 30 (2), 85-94.
8.Hatcher, H.; Planalp, R.; Cho, J.; Torti, F.; Torti, S., Curcumin: From ancient medicine to current clinical trials. Cellular and Molecular Life Sciences 2008, 65 (11), 1631-1652.
9.Jagetia, G.; Aggarwal, B., “Spicing Up” of the Immune System by Curcumin. Journal of Clinical Immunology 2007, 27 (1), 19-35.
10.Sahu, A.; Kasoju, N.; Bora, U., Fluorescence Study of the Curcumin−Casein Micelle Complexation and Its Application as a Drug Nanocarrier to Cancer Cells. Biomacromolecules 2008, 9 (10), 2905-2912.
11.Yang, S. C.; Bhide, M.; Crispe, I. N.; Pierce, R. H.; Murthy, N., Polyketal Copolymers: A New Acid-Sensitive Delivery Vehicle for Treating Acute Inflammatory Diseases. Bioconjugate Chemistry 2008, 19 (6), 1164-1169.
12.Yoshitomi, T.; Suzuki, R.; Mamiya, T.; Matsui, H.; Hirayama, A.; Nagasaki, Y., pH-Sensitive Radical-Containing-Nanoparticle (RNP) for the L-Band-EPR Imaging of Low pH Circumstances. Bioconjugate Chemistry 2009, 20 (9), 1792-1798.
13.Choi, S.-W.; Zhang, Y.; Xia, Y., A Temperature-Sensitive Drug Release System Based on Phase-Change Materials. Angewandte Chemie International Edition 2010, 49 (43), 7904-7908.
14.Roos, A.; Klee, D.; Schuermann, K.; Höcker, H., Development of a temperature sensitive drug release system for polymeric implant devices. Biomaterials 2003, 24 (24), 4417-4423.
15.Wilson, D. S.; Dalmasso, G.; Wang, L.; Sitaraman, S. V.; Merlin, D.; Murthy, N., Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines. Nat Mater 2010, 9 (11), 923-928.
16. Broaders, K. E.; Grandhe, S.; Fréchet, J. M. J., A Biocompatible Oxidation-Triggered Carrier Polymer with Potential in Therapeutics. Journal of the American Chemical Society 2011, 133 (4), 756-758.
17.Rehor, A.; Hubbell, J. A.; Tirelli, N., Oxidation-Sensitive Polymeric Nanoparticles. Langmuir 2005, 21 (1), 411-417.
18.Mahmoud, E. A.; Sankaranarayanan, J.; Morachis, J. M.; Kim, G.; Almutairi, A., Inflammation Responsive Logic Gate Nanoparticles for the Delivery of Proteins. Bioconjugate Chemistry 2011, 22 (7), 1416-1421.
19.Jeong, S.; Choi, S. Y.; Park, J.; Seo, J.-H.; Park, J.; Cho, K.; Joo, S.-W.; Lee, S. Y., Low-toxicity chitosan gold nanoparticles for small hairpin RNA delivery in human lung adenocarcinoma cells. Journal of Materials Chemistry 2011, 21 (36), 13853-13859.
20.Montembault, A.; Viton, C.; Domard, A., Rheometric Study of the Gelation of Chitosan in Aqueous Solution without Cross-Linking Agent. Biomacromolecules 2005, 6 (2), 653-662.
21.Chiu, Y.-L.; Chen, S.-C.; Su, C.-J.; Hsiao, C.-W.; Chen, Y.-M.; Chen, H.-L.; Sung, H.-W., pH-triggered injectable hydrogels prepared from aqueous N-palmitoyl chitosan: In vitro characteristics and in vivo biocompatibility. Biomaterials 2009, 30 (28), 4877-4888.
22.Shukla, A. K.; Verma, M.; Singh, K. N., Superoxide Induced Deprotection of 1,3-Dithiolanes: A Convenient Method of Dedithioacetalization. ChemInform 2004, 35 (49), 1748-1752.
23.Jares-Erijman, E. A.; Jovin, T. M., FRET imaging. Nat Biotech 2003, 21 (11), 1387-1395.
24.Lee, S.; Park, K.; Kim, K.; Choi, K.; Kwon, I. C., Activatable imaging probes with amplified fluorescent signals. Chemical Communications 2008, (36), 4250-4260.
25.Roy, R.; Hohng, S.; Ha, T., A practical guide to single-molecule FRET. Nat Meth 2008, 5 (6), 507-516.
26.Medintz, I. L.; Clapp, A. R.; Mattoussi, H.; Goldman, E. R.; Fisher, B.; Mauro, J. M., Self-assembled nanoscale biosensors based on quantum dot FRET donors. Nat Mater 2003, 2 (9), 630-638.
27.Chen, K.-J.; Chiu, Y.-L.; Chen, Y.-M.; Ho, Y.-C.; Sung, H.-W., Intracellularly monitoring/imaging the release of doxorubicin from pH-responsive nanoparticles using Förster resonance energy transfer. Biomaterials 2011, 32 (10), 2586-2592.
28.Chiu, Y.-L.; Chen, S.-A.; Chen, J.-H.; Chen, K.-J.; Chen, H.-L.; Sung, H.-W., A Dual-Emission Förster Resonance Energy Transfer Nanoprobe for Sensing/Imaging pH Changes in the Biological Environment. ACS Nano 2010, 4 (12), 7467-7474.
29.Nelson MT, H. W., Gursoy A, Dalke A, Kale LV, Skeel RD, et al. , NAMD: a parallel, object oriented molecular dynamics program. Int J Supercomput Appl High Perform Comput 1996, 10, 251-268.
30.Brooks, B. R.; Bruccoleri, R. E.; Olafson, B. D.; States, D. J.; Swaminathan, S.; Karplus, M., CHARMM: A program for macromolecular energy, minimization, and dynamics calculations. Journal of Computational Chemistry 1983, 4 (2), 187-217.
31.Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera—A visualization system for exploratory research and analysis. Journal of Computational Chemistry 2004, 25 (13), 1605-1612.
32.Lide DR, F. H., CRC handbook of chemistry and physics, vol 8. Boca Raton: CRC Press 1995, 45-46.