Title Page
Contents
ABSTRACT 20
CHAPTER 1. INTRODUCTION 22
1.1. Protein-based biomaterials 23
1.2. Applications of protein-based biomaterials 25
1.2.1. Anti-aging 25
1.2.2. Drug Delivery System (DDS) 30
CHAPTER 2. Materials & Methods 38
2.1. Section I: Bombyx mori derived peptide conjugated with Caffeic acid for anti-aging applications 39
2.1.1. Synthesis of Caffeic acid-APPPKK 39
2.1.2. Cell culture and viability assay 40
2.1.3. Detection of reactive oxygen species (ROS) production 41
2.1.4. DPPH assay 42
2.2. Section II: PGC-1α protein derived peptide conjugated with Gallic acid for anti-aging applications 42
2.2.1. Synthesis of (gallic acid) 2 -KTPPTTP 42
2.2.2. Cell cultures and viability assays 44
2.2.3. Determination of antioxidant activity 45
2.2.4. Mitochondrial membrane potential assay 45
2.2.5. Elastase inhibitory activity assay 46
2.2.6. Real-time quantitative PCR (RT-qPCR) 46
2.2.7. Statistical analysis 47
2.3. Section III: pH-responsive albumin nanoparticles & DDS application 47
2.3.1. Synthesis of bovine serum albumin nanoparticles (BNPs) 47
2.3.2. Loading of the drug into nanoparticles 48
2.3.3. Synthesis of FCDBNPs 48
2.3.4. Characterization of FCDBNPs 49
2.3.5. In vitro drug release studies 49
2.3.6. Combination therapy 50
2.3.7. RT-qPCR 50
2.3.8. Western blot analysis 51
2.3.9. Confocal laser scanning microscopy analysis 51
2.3.10. Zebrafish husbandry and exposure to folic-acid nanoparticles 52
2.3.11. Statistical analysis 52
2.4. Section IV: Hypoxia-responsive albumin nanoparticles & DDS application 53
2.4.1. Preparation of the RP/CA/PHNPs 53
2.4.2. Characterization of the RP/CA/PHNPs 54
2.4.3. Cell cultures, cell viability tests, and confocal imaging 54
2.4.4. Penetration of drug and growth inhibition of HeLa cell spheroids 55
2.4.5. In vivo antitumor analysis 55
2.4.6. Western blot analysis 56
2.4.7. Statistical analysis 57
CHAPTER 3. Results & Discussion 60
3.1. Section I: Bombyx mori derived peptide conjugated with Caffeic acid for anti-aging applications 61
3.1.1. Introduction 61
3.1.2. Characterization studies of Caffeic acid-APPPKK 62
3.1.3. Cell viability assay 67
3.1.4. Antioxidant activity and long-term stability of Caffeic acid- APPPKK 69
3.1.5. Summary 74
3.2. Section II: PGC-1α protein derived peptide conjugated with Gallic acid for anti-aging applications 75
3.2.1. Introduction 75
3.2.2. Synthesis and conjugation of gallic acid with peptide 76
3.2.3. Cell viability assays 79
3.2.4. Antioxidant activity of synthesized peptides 81
3.2.5. Effect of synthesized peptides on the mitochondria membrane potential 84
3.2.6. Elastase inhibitory activity 86
3.2.7. Expression of type I collagen, MMP-1, and PGC-1α genes 88
3.2.8. Summary 91
3.3. Section III: pH-responsive albumin nanoparticles & DDS application 92
3.3.1. Introduction 92
3.3.2. Preparation and characterization of FCDBNPs 93
3.3.3. In vitro drug release from FCDBNPs 98
3.3.4. In vitro combination therapy studies 100
3.3.5. Effect of Ce6-mediated photodynamic therapy on apoptosis 108
3.3.6. Effect of FCDBNPs on zebrafish larvae 113
3.3.7. Summary 116
3.4. Section IV: Hypoxia-responsive albumin nanoparticles & DDS application 117
3.4.1. Introduction 117
3.4.2. Preparation and characterization of the RP/CA/PHNPs 120
3.4.3. In vitro combination therapy 124
3.4.4. In vitro drug distribution 130
3.4.5. Antitumor potential of RP/CA/PHNPs in vivo 138
3.4.6. Summary 143
CHAPTER 4. Conclusion & Perspectives 144
4.1. Conclusions 145
REFERENCES 147
Table 1. List of the primer sets 58
Table 2. List of antibodies 59
Table 3. Characterization of FCDBNPs. 97
Figure 1. Advantages and applications of protein-based biomaterials 24
Figure 2. Mechanism of skin aging 27
Figure 3. Design of drug delivery system 32
Figure 4. HPLC spectrum of Caffeic acid-APPPKK. 63
Figure 5. MALDI-TOF spectrum of synthesized peptides (A) and (B) MS² mass spectrum of [M + H]+, (C) and (D) MS² mass spectrum of [M + 2H]2+, (E) LC-...[이미지참조] 64
Figure 6. FT-IR spectrum of caffeic acid, APPPKK and Caffeic acid-APPPKK. 65
Figure 7. Cytotoxicity of human dermal fibroblast cells treated with caffeic acid, APPPKK and Caffeic acid-APPPKK at different concentrations (12.5, 25, 50 and... 68
Figure 8. Determination of the antioxidant activity of Caffeic acid, APPPKK and Caffeic acid-APPPKK, (A) ROS generation, (B) DPPH radical antioxidant... 72
Figure 9. Cell viability of HaCaT cells (human skin keratinocytes) stimulated with (a) control untreated cells, (b) only UVB, (c) cells treated with 100 μM of... 73
Figure 10. Synthetic step and sequence confirmation of peptides using quadrupole-time of flight (Q-TOF) mass spectrometry: (A) detailed synthetic... 78
Figure 11. Cell viability of a) HaCaT cells and b) dermal fibroblasts with gallic acid and peptide conjugates treatment. 80
Figure 12. (A) Effect of gallic acid and peptide conjugates on radical scavenging activity using DPPH assays, (B) Long-term stability testing using DPPH assays... 83
Figure 13. Measurement of the mitochondrial membrane potential using JC-1 dye in a) HaCaT cells and b) dermal fibroblasts. Asterisks indicate statistically... 85
Figure 14. Measurement of the elastase activity in dermal fibroblasts at gallic acid and peptide concentrations of 100 µM. 87
Figure 15. Gene expression analysis in dermal fibroblasts using RT-qPCR: a) Type I collagen, b) MMP-1, and c) PGC-1α. 90
Figure 16. The detailed synthetic strategy of FCDBNPs 94
Figure 17. Characterization of synthesized NPs (A) Normalized fluorescence spectrum (λex=400 nm), (B) FT-IR spectrum, (C) DLS data of BNPs and...[이미지참조] 95
Figure 18. Cumulative drug release profile of DOX from FCDBNPs at different pHs 99
Figure 19. Chemo-photodynamic combination therapy of FCDBNPs and FCDBNPs + Laser (10 min, 15 mW/cm²) in COS7 and HeLa cells, (B)... 101
Figure 20. (A) Cell viability of FCBNPs, (B) Temperature changes during laser irradiation in HeLa cells at 250 µg/mL of FCDBNPs, (C) Laser irradiation effect... 102
Figure 21. Targeting of the folate receptor using FA, Confocal fluorescence images of FCDBNPs-treated (A) COS7, (B) HeLa, (C) HeLa cells pretreated... 104
Figure 22. Determination of cellular uptake of FCDBNPs in HeLa cells by confocal image analysis. 107
Figure 23. FCDBNPs induce oxidative damage in HeLa cells (A) Fluorescence image assay of HeLa cells, (B) Intracellular ROS measurement using DCF-DA,... 110
Figure 24. Gene and protein expression analysis in HeLa cells (A) RT-qPCR and (B and C) Protein quantification and western blot analysis. (D) Apoptosis... 111
Figure 25. Cytotoxic effect of FCDBNPs in zebrafish. (A) Bright images of zebrafish, i) negative control, ii) 250 µg/mL of FCDBNPs, iii) 250 µg/mL of... 115
Figure 26. Anticancer strategy for RP/CA/PHNPs. 119
Figure 27. The detailed synthetic strategy of RP/CA/PHNPs. 122
Figure 28. Characterization of RP/CA/PHNPs. (A) SEM and TEM images of RP/CA/PHNPs, (B) absorbance spectra, (C) FT-IR analysis, (D) zeta potential,... 123
Figure 29. (A) Cytotoxic effect of RP/HNPs in COS7 and HeLa cells, (B) Free Ce6 cytotoxicity (µg/mL), (C) COS7 cell viability analysis with RP/CA/PHNPs... 125
Figure 30. In vitro cytotoxicity of RP/CA/PHNPs. (A) Cytotoxicity of nanoparticles under normoxic conditions (µg/mL) and (B) cytotoxicity of nanoparticles under hypoxic conditions (µg/mL). Filled bars represent no laser irradiation, and striped bars represent... 126
Figure 31. Confocal images and DCF-DA quantification analysis. (A‒D) In vitro Z-section confocal images and (E) quantification analysis of DCF-DA... 129
Figure 32. Effects of the cellular environment and the presence of an azo group on the release of Nile red dye from nanoparticles. 131
Figure 33. HeLa spheroid characterization. (A) Bright images of HeLa spheroid, (B) Area and diameter of HeLa spheroid, (C) Fluorescence images of DAPI and... 133
Figure 34. Penetration of the nanoparticles into HeLa spheroids. RP/C/PHNPs and RP/CA/PHNPs were incubated with spheroids for 4 h and washed.... 134
Figure 35. Penetration activity of the nanoparticles in HeLa spheroids. RP/CA/PHNPs were incubated for 4 h and washed. Incubated for an additional... 136
Figure 36. Growth inhibition of avascular HeLa spheroids. (a) Bright images of HeLa spheroids, (b) area measurement, (c) maximum Feret diameter... 137
Figure 37. Antitumor activity effect of the synthesized nanoparticles evaluated against a HeLa-xenograft mice model. (A) Tumor growth inhibition, (B)... 140
Figure 38. Molecular characterization was done using western blot analyses and normalized with β-actin levels. (A) The protein levels of various markers... 141