Title Page
ABSTRACT
Contents
CHAPTER 1. INTRODUCTION 16
1.1. Soultion plasma process 16
1.2. Application of alginate and its derivatives 23
1.3. Application of chitosan and its derivatives 28
1.4. Silver nanoparticles (AgNPs): synthesis and application 31
1.5. Nanceria (nCe): synthesis and application 33
1.6. Purposes and significance of the investigation 35
CHAPTER 2. MATERIALS AND METHODS 37
2.1. Culture media and culture conditions 37
2.2. Synthesis of alginate/AgNPs via SPP 37
2.3. Degradation of alginate via SPP 38
2.4. Synthesis of oligo-alginate (OA)/nanoceria (nCe) via SPP 38
2.5. Synthesis of oligo-chitosan (OC)/nanoceria (nCe) via SPP 41
2.6. UV-Vis spectrophotometry 41
2.7. 3,5-dinitrosalicylic acid (DNS) assay 44
2.8. Fourier-transform infrared spectroscopy (FT-IR) analysis 44
2.9. X-ray photoelectron spectroscopy (XPS) 44
2.10. Dynamic light scattering (DLS) & zeta-potential (ZP) analysis 45
2.11. Scanning electron microscopy (SEM)/energy dispersive spectroscopy (EDS) 45
2.12. Transmission electron microscopy (TEM) 45
2.13. Antioxidant activity assays 45
2.13.1. DPPH radical scavenging assay 46
2.13.2. Hydroxyl radical scavenging assay 46
2.13.3. Superoxide radical scavenging assay 47
2.13.4. Hydrogen peroxide radical scavenging assay 47
2.14. Assays of antimicrobial activities 47
2.14.1. Minimal inhibitory concentration (MIC) assay 47
2.14.2. Agar diffusion assay 48
2.14.3. Anti-biofilm assay 48
2.15. Cytotoxicity assay 49
2.15.1. WST-1 assay 49
2.15.2. MTT assay 50
2.16. Extraction of RNA 50
2.17. Synthesis of cDNA 51
2.18. qRT-PCR 51
CHAPTER 3. RESULTS AND DISCUSSIONS 54
3.1. Synthesis and characterization of alginate/AgNPs using Ag electrodes via SPP 54
3.1.1. Confirmation of alginate/AgNPs synthesis 54
3.1.2. Chemical structural modification in alginate/AgNPs 56
3.1.3. Dispersion property of the alginate/AgNPs biocomposites 58
3.1.4. Structure of the alginate/AgNPs biocomposites 61
3.1.5. Efficacy of alginate/AgNPs as antimicrobial agents 65
3.1.6. Cytotoxicity of the alginate/AgNPs biocomposites 67
3.2. Characterization of OA proliferating the animal cell growth 71
3.2.1. Proliferation of the HEK29 cells by OA 71
3.2.2. Effect of OA on the expression of the genes related to cell cycle and division in the HEK29 cells 71
3.3. Synthesis and characterization of the OA/nCe biocomposites 73
3.3.1. Confirmation of the OA/nCe synthesis 73
3.3.2. Chemical structure analysis of the OA/nCe biocomposites 77
3.3.3. Elemental states of the OA/nCe biocomposites 80
3.3.4. DLS and zeta-potential of the OA/nCe biocomposites 80
3.3.5. Structure of the OA/nCe biocomposites 82
3.3.6. Antioxidant activities of the OA/nCe biocomposites 87
3.3.7. Cytotoxicity of the OA/nCe biocomposites 91
3.3.8. Anti-biofilm activity of the OA/nCe biocomposites 93
3.4. Characterization of the OC/nCe biocomposites 95
3.4.1. Confirmation of the OC/nCe biocomposite synthesis 95
3.4.2. Chemical structure analysis of the OC/nCe biocomposites 95
3.4.3. Elemental states of the OC/nCe biocomposites 99
3.4.4. DLS and zeta-potential of the OC/nCe biocomposites 99
3.4.5. Shape and size of the OC/nCe biocomposites 104
3.4.6. Antioxidant activities of the OC/nCe biocomposites 107
3.4.7. Cytotoxicity of the OC/nCe biocomposites 110
3.4.8. Anti-biofilm activity of the OC/nCe biocomposites 110
3.4.9. Comparison of OA/nCe and OC/nCe biocomposites for their antioxidant and anti-biofilm activities 113
CHAPTER 4. CONCLUSIONS 116
REFERENCES 118
국문초록 131
Table 1. Primers used for qRT-PCR 53
Table 2. Zeta-potentials of the alginate/AgNPs biocomposites 60
Table 3. MIC of the alginate/AgNPs biocomposites against various pathogens 66
Table 4. Zone of inhibition formed by alginate/AgNPs against various pathogens 69
Table 5. Biological functions and the Ct values of the reference genes 74
Table 6. Particle sizes of the OA/nCe biocomposites 83
Table 7. Zeta-potentials of the OA/nCes biocomposites 85
Table 8. Particle sizes of the OC/nCe biocomposites 101
Table 9. Zeta-potentials of the OC/nCes biocomposites 103
Table 10. Anti-biofilm effects of the OA/nCe and OC/nCe biocomposites 115
Figure 1. A model of solution plasma process. There are two interfaces, gas/liquid interface and plasma/gas interface. Solution plasma is located at the center and surrounded by a gas phase,... 17
Figure 2. Schematic representation of solution plasma process. Two metal wire electrodes were placed in a 500 ml volume of Teflon chamber and plasma was discharged by using unipolar pulsed... 18
Figure 3. Applications of silver nanoparticles. 22
Figure 4. Chemical structure of alginate. Alginate is a natural anionic polysaccharide composed of repeated residues of D-mannuronate (M) and L-guluronate (G) with 1, 4-linkage. 24
Figure 5. Applications of alginate. Since alginate is biocompatible, biodegradable, non-antigenic and non-toxicity, it can be used in wound dressings, bone regeneration, neovascularization, protein... 26
Figure 6. Applications of oligoalginate. Alginate oligosaccharides, also known as oligoalginate, has potential to be used in food, pharmaceutical and agricultural industries, since it has excellent... 27
Figure 7. Chemical structure of chitosan. Chitosan is a linear polysaccharide composed of β-(1→4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). 29
Figure 8. Application of chitosan. Since chitosan is a biodegradable natural polymer which is non toxicity and has biocompatibility and biodegradability, it has potential to be used in... 30
Figure 9. Applications of nanoceria. 34
Figure 10. Strategy for the synthesis of the alginate/AgNPs biocomposites. Alginate solution (0.5- 0.75%, w/v) dissolved in dH₂O (300 ml) was discharged with plasma using Ag electrodes for 2, 5, or... 39
Figure 11. Strategy for depolymerization of alginate. Alginate solution (0.5-0.75%, w/v) dissolved in dH2O (300 ml) was discharged with plasma using Ag electrodes for 2, 5, or 7 min at 800 V, 30 kHz. 40
Figure 12. Strategy for the synthesis of the OA/nCe biocomposites. First, 0.75% alginate solution was depolymerized to OA by using SPP for 1 h. After freeze-drying, the OA/nCe biocomposites were synthesized by discharging plasma in the 0.6-0.8% of OA solution containing 1-5 mM of Ce(NO₃)₃·6H₂O. 42
Figure 13. Strategy for the synthesis of the OC/nCe biocomposites. First, 0.5% chitosan solution dissolved in 1N acetic acid was depolymerized to OC by using SPP for 90 min. After freeze-drying, the OC/nCe biocomposites were synthesized by discharging plasma in the 1.0-2.0 % of OC with 1-5... 43
Figure 14. UV-Vis spectroscopy of the alginate/AgNP biocomposites. 0.3-0.5% alginate/AgNPs were discharged at 600-800 v for 1 min (a) and 0.5-0.75% of alginate were discharged at 800 V, 30... 55
Figure 15. FT-IR analysis of the alginate/AgNP biocomposites. Interferogram at 3315, 2932, 1594, 1409, 1085 and 1026 cm-1 corresponds to O-H stretching, C-H stretching, COO- stretching...[이미지참조] 57
Figure 16. Zeta-potential analysis of the alginate/AgNP biocomposites. The zeta-potential values of 0.75% alginate, alginate/AgNPs (0.75%, 2 min), alginate/AgNPs (0.75%, 5 min) and alginate/AgNPs (0.75%, 7 min) refers to -54.10 ± 1.70, -75.78 ± 1.69, -72.86 ± 0.59 and -75.11 ± 1.57 mV, respectively. 59
Figure 17. SEM analysis of the alginate/AgNP biocomposites. Substantial scaffolds of biocomposites were observed in 0.75% alginate/AgNPs discharged for 2 (a), 5 (b) and 7 (c,d) min. In the... 62
Figure 18. EDS analysis and mapping of the alginate/AgNP biocomposites. Overall portion of Ag was increased as the discharging time goes longer and the atomic percentage was increased from 0 to 1.04, 1.40 and 1.57 % at 2 min (a), 5 min (b) and 7 min (c), respectively. 63
Figure 19. TEM analysis of the alginate/AgNP biocomposites. Overall, spherical AgNPs well-dispersed in matrixes were observed in 0.75% alginate/AgNPs discharging for 2 min (a), 5 min (b), or... 64
Figure 20. Agar diffusion assay of the alginate/AgNP biocomposites. Growth zone of inhibitions were formed against both gram-(-) and gram-(+) bacteria, and among the biocomposites, alginate/AgNPs (0.75%, 5 min) formed largest clear zone, indicating that it is the most effective antimicrobial agents. 68
Figure 21. Cell viability assay of the alginate/AgNP biocomposites. against HEK293 cells. No siginificant cytotoxicity was observed in a range of 0-125 μg/ml of biocomposites. However,... 70
Figure 22. Effect of OA on proliferation of HEK293 cells. OA derived from 0.5% (w/v) alginate solution by plasma discharge for 60 min promoted growth of the cells for 136.41 ± 2.44 % when... 72
Figure 23. Gene expression of cell-division related genes in the HEK293 cells. The gene expression was increased to 84, 88, 136, 165, 130, and 122% of the BCL2, BCL2L1, c-Myc, MCM-2,... 75
Figure 24. UV-Vis spectra of OA. Unique peaks were absorbed in the range of 270-271.5 nm of wavelength, implying that OA was synthesized. As discharging time is increased, the absorbance gets higher and the alginate solution turned to vivid yellow color (a). In the DNS analysis (b), the... 76
Figure 25. UV-Vis spectra of the OA/nCe biocomposites. In the 0.6% OA (a) and 0.8% OA (b) with 1, 3, or 5 mM Ce(NO₃)₃·6H₂O, peaks were observed at 260-273 nm, indicating the presence of OA and unique peaks were observed at 362-368 nm, indicating the synthesis of nCe. Among them, 0.8%... 78
Figure 26. FT-IR analysis of the OA/nCe biocomposites. Peaks were observed at 3500 cm-1 indicating O-H bonds; 1599 cm-1, COO- (asymmetric stretching); 1410 cm-1, COO- bonds (symmetric...[이미지참조] 79
Figure 27. High-resolution of XPS spectra of the OA/nCe biocomposite. Survey scan XPS spectrum of CeO₂ with 0.8 OA/5 nCe (a) and high resolution XPS spectra of Ce 3d of CeO₂ (b) shows the... 81
Figure 28. DLS analysis of the OA/nCe biocomposites. 68.0 ± 8.0, 236.4 ± 16.3, 327.8 ± 8.4 and 347.5 ± 12.7 nm of size diameter was measured at 0.8% OA, 0.8 OA/1nCe, 0.8 OA/3 nCe and 0.8... 83
Figure 29. Zeta-potential analysis of the OA/nCe biocomposites. -32.2 ± 1.5 mV, -40.3 ± 1.1 mV, -40.1 ± 0.9 mV and -45.1 ± 1.3 mV of zeta-potential value was obtained at 0.8% OA with 0, 1, 3, or 5 mM nCe, respectively 84
Figure 30. SEM, EDS analysis and mapping of the OA/nCe biocomposites. 3D scaffolds were observed in SEM analysis (b) and Ce compound well-distributed in biocomposites were observed in... 86
Figure 31. TEM analysis and size distribution of nCes in OA/nCe. Uniformly sized cubical nCes were observed in the OA/nCe biocomposites (a) and. 50% of nCes were in the range of 21-30 nm... 88
Figure 32. Antioxidant activities of the OA/nCe biocomposite. Panel (a) represents the scavenging activity of 0.8 OA/5 nCe against DPPH; panel (b), hydroxyl radicals; panel (c), superoxide... 90
Figure 33. Cytotoxicity of the OA/nCe biocomposite. No significant cytotoxicity of OA/nCe was observed and the LD50 value was 1995.3 μg/ml.[이미지참조] 92
Figure 34. Anti-biofilm effects of the OA/nCe biocomposite. About 130.4%, 55.2%, 63.3% and 24.7% of biofilm was formed by addition of 0.8 OA/5 nCe. in E. coli, P. aeruginosa, V. vulnificus and S. aureus. 94
Figure 35. UV-Vis spectra of OC. Unique peaks were absorbed in the range of 287 - 300 nm of wavelength, implying the synthesis of OC. 96
Figure 36. UV-Vis spectra of the OC/nCe biocomposites. In the 1.0 (a) and 2.0%(b) OC with Ce(NO₃)₃·6H₂O, peaks were observed at 257-268 nm and 289-293.5 nm, indicating the synthesis of nCe. Afte r the SPP, the solution turned to dust color, also implying the synthesis of nCe. Among them, 2.0... 97
Figure 37. FT-IR analysis of the OC/nCe biocomposites. The peaks were observed at 3504 cm-1, indicating O-H stretching; 2981 cm-1, C-H stretching; 1645 cm-1, amide I; 1533 cm-1, N-H...[이미지참조] 98
Figure 38. High-resolution of XPS spectra of the OC/nCe biocomposites. Survey scan XPS spectrum of CeO₂ with 2.0 OC/5 nCe (a) and high- resolution XPS spectra of Ce 3d of CeO₂ (b) shows... 100
Figure 39. DLS analysis of the OC/nCe biocomposites. 64.4 ± 3.2, 93.9 ± 19.9, 136.4 ± 34.7 and 229.3 ± 22.2 nm of particle size was measured at 2.0% OC with 0, 1, 3, or 5 mM nCe, respectively. 101
Figure 40. Zeta-potential analysis of the OC/nCe biocomposites. +36.9 ± 0.2 mV, 33.2 ± 0.7 mV, 31.0 ± 0.4 mV and 32.6 ± 0.2 mV of zeta-potential value was obtained at 2.0% OC with 0, 1, 3, or 5 mM nCe, respectively 102
Figure 41. SEM, EDS analysis and mapping of the OC/nCe biocomposites. 3D scaffolds were observed in SEM analysis (b) and Ce compound well-distributed in biocomposites were observed in... 105
Figure 42. TEM analysis and size distribution of nCes in the OC/nCe biocomposite. Cubical nCes with uniform size were observed. The average of diagonal length of nCe was 15.7 ± 5.8 nm and... 106
Figure 43. Antioxidant activities of the OC/nCe biocomposite. Panel a represents the scavenging activity of 2.0 OC/5 nCe against DPPH; panel b, hydroxyl radicals; panel c, superoxide radicals; and... 108
Figure 44. Cytotoxicity of the OC/nCe biocomposite. No significant cytotoxicity of the biocomposite was observed and the LD50 value was 1737.0 μg/ml.[이미지참조] 111
Figure 45. Anti-biofilm effects of the OC/nCe biocomposite. Only 24.4%, 22.5%, 10.3% and 3.6% of biofilm from E. coli, P. aeruginosa, V. vulnificus and S. aureus was formed by addition of 0.8 mg/ml of 2.0 OC/5 nCe. Overall, as the concentration of nCe increases, the anti-biofilm effect of OC/nCe... 112
Figure 46. Antioxidant activities of the OA/nCe and OC/nCe biocomposites. Panel a represents the scavenging activity of biocomposites against DPPH; panel b, hydroxyl radicals; panel c, super... 114