Fabrication of Phenolated Lignin Nanoparticles and Phenolated Lignin-MgO-based Nanocomposite Films for Rapid Wound Healing = 상처 치유를 위한 페놀화 리그닌 나노입자 및 페놀화 리그닌?MgO 기반 나노복합 필름의 제조

다국어 초록 (Multilingual Abstract)

This thesis explores two integrated approaches to lignin valorization for biomedical applications: (i) the comparative synthesis of phenolated lignin nanoparticles, and (ii) the development of bio-based phenolated lignin-MgO-chitosan nanocomposite films for accelerated wound healing.
In the first study, Kraft and Milled wood lignin samples were chemically modified via phenolation to enhance their reactivity and biological performance. Phenolation successfully introduced additional phenolic hydroxyl groups (3.65 mmol g⁻¹ for Kraft lignin and 2.10 mmol g⁻¹ for milled wood lignin), confirmed by ³¹P-NMR and 2D-HSQC-NMR analyses. Nanoprecipitation and solvent-exchange methods were compared to fabricate nanoparticles.
Nanoprecipitation yielded uniform, highly stable, monodisperse particles (90–160 nm, PDI < 0.1, ζ = –26 to –45 mV), while solvent exchange produced larger and less stable aggregates (550–800 nm). Phenolated nanoparticles produced by nanoprecipitation showed strong antioxidant activity (DPPH > 50 µM TE mL⁻¹; ABTS > 200 µM TE mL⁻¹) and complete inhibition of both Staphylococcus aureus and Escherichia coli. Cytotoxicity and
1hemocompatibility tests confirmed high cell viability (> 90 % for human dermal fibroblasts) and < 5 % hemolysis, demonstrating their biosafety for biomedical use.
In the second study, phenolated Kraft lignin nanoparticles were incorporated with chitosan and MgO to fabricate bioinspired nanocomposite films (F0–F10) with multifunctional wound- healing properties. The addition of MgO nanoparticles improved ionic crosslinking and thermal stability, as supported by FTIR and XRD analyses. The optimized film (F10) showed excellent physicochemical performance, including a high swelling ratio (≈ 92 %) and a low water contact angle (≈ 41°), indicating improved hydrophilicity and exudate absorption. F10 also demonstrated strong antioxidant activity (ABTS = 325 ± 8 µg TE mL⁻¹; DPPH = 152 ± 5 µg TE mL⁻¹), high protein adsorption (2.3 g BSA g⁻¹ film), and effective UV shielding. Scratch- wound assays revealed that F10 promoted fast fibroblast migration, achieving nearly 100 % scratch closure within 48 hours, significantly faster than F0 and untreated cells.
In vivo wound healing studies confirmed the strong healing effect of the films. F10 showed the fastest recovery, with 74.1% closure by Day 15, outperforming DuoDerm, F0, and untreated controls. Wound images and measurements showed quicker epithelialization and tissue repair in the F10 group. Together, these results demonstrate that MgO-reinforced phenolated lignin/chitosan films provide a sustainable, multifunctional platform with strong antioxidant, antibacterial, and regenerative effects suitable for future wound-care technologies.

목차 (Table of Contents)

• Contents
• Abstract I
• Contents III
• List of Tables VIII
• List of Figures XI
• List of Abbreviations XV Table of Contents
• 1. Introduction 21
• 1.1 Sustainable Biomass Valorization and the Need for Bio-based Materials 21
• 1.2 Lignin: Structure, Production, and Functional Modification 23
• 1.3 Phenolated Lignin Nanoparticles: Synthesis Approaches and Biomedical Application 25
• 1.4 Lignin/MgO/Chitosan bio-nanocomposite for Accelerated Wound Healing 28
• 1.5 Objectives 30
• 2. Literature review: 32
• 2.1 Renewable and Bio-Based Polymers in Biomedical Applications 32
• 2.2 Lignin as a Renewable Biomaterial 35
• 2.3 Phenolation and Its Importance in Lignin Modification 38
• 2.4 Downstream Techniques for Lignin Nanoparticle Formation 39
• 2.5 Bioactive Films and Composites in Wound Healing 42
• 2.5.1 Functional Synergy of Lignin, MgO, and Chitosan 42
• 2.5.2 Current Gaps in Sustainable Wound Dressing Technologies 43
• 3. Materials and Methods 45
• 33.1 Phenolated lignin nanoparticles with improved stability and bio functionality: A comparative study of nanoprecipitation and solvent exchange fabrication techniques 45
• 3.1.1 Materials 45
• 3.1.2 Phenolation of Kraft lignin and Milled Wood lignin 45
• 3.1.3 Characterization of Phenolated Lignin Samples 47
• 3.1.4 Fabrication of lignin nanoparticles 48
• 3.1.5 Characterization of lignin nanoparticles 49
• 3.1.6 In vitro biological assessments 49
• 3.1.6.1 Radical Scavenging Activity (RSA) by DPPH and ABTS 49
• 3.1.6.2 Antibacterial activity 50
• 3.1.6.3. Cell viability analysis 50
• 3.1.6.4 Hemolysis of the LNPs 51
• 3.1.7 Statistical analysis 52
• 3.2 Bioinspired Phenolated Lignin/Chitosan Hybrid Films Reinforced with MgO Nps for Enhanced Antioxidant, Antibacterial, and Rapid Wound Healing Performance: In Vitro and In Vivo Evaluation. 54
• 3.2.1 Materials 54
• 3.2.2. Phenolation 54
• 3.2.3. Preparation of LNPs 55
• 3.2.4 Synthesis of MgO Nps 55
• 3.2.5 Biofilms Synthesis 55
• 3.2.6 Characterization 59
• 43.2.6.1 Characterization of Phenolated Lignin by 31PNMR and GPC 59
• 3.2.6.2 Characterization of Nanoparticles 60
• 3.2.7 Characterization of Films 60
• 3.2.7.1 Fourier transform infrared spectroscopy (FTIR) 60
• 3.2.7.2 X-ray Diffraction (XRD) 60
• 3.2.7.3 Scanning Electron Microscope (FESEM) and Energy Dispersive X-ray Spectroscopy (EDS) 61
• 3.2.8 Properties of Film Composite 61
• 3.2.8.1 Thermal degradation of Films 61
• 3.2.8.2 Swelling, pH responsiveness, Wettability, and Bacterial Inhibition of films 61
• 3.2.8.3 Bacterial Inhibition by CFU 62
• 3.2.8.4 Antioxidant properties of films using DPPH and ABTS 63
• 3.2.8.5 Protein Adsorption Assay 63
• 3.2.8.6 Optical properties 64
• 3.2.8.7 Tensile Strength 64
• 3.2.9 Biocompatibility of Films 64
• 3.2.9.1 Hemocompatibility 64
• 3.2.9.2 In-vitro blood compatibility test (BCI) 65
• 3.2.9.3 Cytotoxicity on HDF and HACAT cell lines for 24 and 48 hr 66
• 3.2.9.4 In-Vitro Wound Closure (Scratch assay) 68
• 3.2.9.5 In-Vivo Wound Healing 69
• 53.2.10 Statistical Analysis 70
• 4. Results and discussion 71
• 4.1 Phenolated lignin nanoparticles with improved stability and bio functionality: A comparative study of nanoprecipitation and solvent exchange fabrication techniques 71
• 4.1.1 Characteristics of phenolated lignin samples 71
• 4.1.2 Proposed mechanism of phenolation 71
• 4.1.3 Structural features, molecular weight, and morphology. 77
• 4.1.4 Synthesis of SE-LNPs & NP-LNPs and their characterization 90
• 4.1.4. Radical scavenging activity by ABTS and DPPH assays of LNPs 103
• 4.1.5 Evaluation of Antibacterial Potential of LNPs 110
• 4.1.6 Biocompatibility of LNPs 115
• 4.2 Bioinspired Phenolated Lignin–Chitosan Hybrid Films Reinforced with MgO Nanoparticles for Enhanced Antioxidant, Antibacterial, and Rapid Wound Healing Performance: In Vitro and In Vivo Evaluation 123
• 4.2.1 Characterization of Phenolated Lignin GPC and PNMR 123
• 4.2.2 Characterization of Lignin and MgO Nanoparticles 127
• 4.2.3 FTIR and XRD 138
• 4.2.4 SEM EDS 141
• 4.2.5 Radical scavenging activity using DPPH and ABTS 145
• 4.2.6 Swelling and pH responsiveness, hydrophilicity (contact angle), and Bacterial Inhibition 150
• 4.2.7 Thermogravimetric analysis (TGA) 154
• 64.2.8 Protein Adsorption by Bovine Serum Albumin (BSA) 156
• 4.2.9 UV-Vis Spectrophotometer 157
• 4.2.10 Tensile Strength and Mechanical Performance 158
• 4.2.11Cytocompatibility using HDF and HaCat cell lines over 24 and 48 hr 161
• 4.2.12 Hemocompatibility and Blood Clotting Index 164
• 4.2.13 In vitro wound closure on HDFs 167
• 4.2.14 In-vivo wound healing and Hematoxylin and Eosin staining. 170
• 5. Conclusion 177
• 6. References 179