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ROS-modulable Injectable Hydrogels via Dual Enzymatic-triggered Reactions for Chronic Wound Healing Application

초록/요약

The treatment of chronic wounds is a complex and challenging process, mainly due to the intricate pathological conditions that often associated within these wounds. One critical aspect in the healing process is the balance of reactive oxygen species (ROS), which play an essential role in tissue repair. At basal levels, ROS work as regulators for cellular activities that promote wound healing. In contrast, excessive ROS levels result in heavy oxidative stress, leading to further complications such as infection, cellular dysfunction, and microvascular damage, which hinder the wound healing process. Consequently, substantial research has focused on chronic wounds treatment through ROS-modulation strategies, with dual enzyme systems emerging as a promising approach. Due to their high selectivity, rapid reaction rate, and excellent biocompatibility, these systems offer effective regulation of ROS levels, while providing additional properties, such as antibacterial, oxygen supply, enhance cellular proliferation, all of which contribute to accelerated wound healing. To further optimize therapeutic effects, the dual enzyme systems are incorporated into hydrogels – a biomaterial capable of mimicking the functions of extracellular matrix (ECM). This combination makes dual enzyme hydrogels a highly comprehensive platform for the treatment of chronic wounds. In this dissertation, we based on the dual enzymes including glucose oxidase (GOx) and horseradish peroxidase (HRP), combined with other bioactive agents to develop a multifunctional hydrogel dressing for chronic wound healing application. In chapter 2, we developed an in situ forming hydrogel capable of releasing H2O2 for an extended time. The hydrogel was formed via HRP-mediated cross- linking method, with glucose oxidase-coated calcium peroxide nanoparticles (CaO2@GOx NPs) serving as a sustained H2O2 generator. It was hypothesized that, H2O2 released from the hydrolysis of CaO2 and subsequent glucose oxidation by GOx would support hydrogel formation while prolonging the duration of H2O2 release resulting hydrogel matrix. The results showed that, the hydrogel fabricated through HRP/CaO2@GOx demonstrated rapid formation, tunable mechanical strength, and a favorable 3D microenvironment that supported fibroblast viability and proliferation more effectively than other hydrogel formulations using only HRP/H2O2 or HRP/CaO2/GOx. Furthermore, the HRP/CaO2@GOx-crosslinked hydrogel enhanced the angiogenic activities of endothelial cells, evidenced by in vitro tube formation and in ovo testing using the chicken eggs membrane model, indicating its potential for supporting wound vascularization in biomedical applications. In chapter 3, we focused on extending the multifunctionality of GOx/HRP system by incorporating with tannic acid (TA) in a hyaluronic acid (HA)-based hydrogel network, which aimed to address the excessive glucose and ROS levels present in diabetic wounds. While the glucose conversion activity of GOx makes it a suitable candidate for mitigating the hyperglycemia condition, this reaction also generates H2O2, which may worsen the elevated ROS levels in the wound. To overcome this limitation, HRP and TA was utilized as H2O2-scavenger, with TA served as a phenolic substrate for the HRP/H2O2 oxidation reaction. It was found that, when incubated in the high glucose environment, this hydrogel (termed HTG hydrogel) was able to reduce glucose concentration from 400 to 200 mg/dL, and H2O2 generated in the process was effectively scavenged by HRP/TA reaction. In addition, HTG hydrogels also exhibited 70% scavenging effect against free radicals, including DPPH and •OH. Finally, under hyperglycemic conditions, HTG hydrogels facilitated the proliferation and migration of human dermal fibroblasts, highlighting its potential to accelerate diabetic wound healing through effective control of glucose and ROS levels. Overall, the dual-enzyme hydrogel systems presented in this thesis demonstrate a versatile and effective approach for chronic wound treatment, offering new pathways and opportunities for therapeutic applications in wound care.

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목차

Chapter 1. General introduction 1
1. Wounds and wound healing process 2
1.1. Wounds and classification of wounds 2
1.2. Wound healing process 3
2. ROS and the role of ROS in wound healing 7
2.1. Overview of ROS 7
2.2. The dual roles of ROS in wound healing 8
2.3. ROS-modulation approaches to chronic wound healing 11
3. ROS-modulation hydrogels for wound healing 17
3.1. Hydrogel for wound healing application 17
3.2. Hydrogels with ROS-modulation properties 20
4. Dual enzyme-triggered reaction for multifunctional hydrogels 25
5. Overall objectives 28
Reference 30
Chapter 2. Glucose Oxidase-Coated Calcium Peroxide Nanoparticles as an Innovative Catalyst for In Situ H2O2-Releasing Hydrogels. 41
1. Introduction and objectives 42
2. Experimental section 45
2.1. Materials 45
2.2. Synthesis of glucose oxidase-coated calcium peroxide nanoparticles (CaO2@GOx NPs) 45
2.3. Characterization of CaO2@GOx NPs 46
2.4. Preparation and rheological analysis of HRP/CaO2@GOx-catalyzed hydrogels 47
2.5. In vitro release behaviors of H2O2 from hydrogels 48
2.6. Cell studies 48
2.7. Statistical analysis 50
3. Results and discussions 51
3.1. Synthesis and characterization of CaO2@GOx NPs 51
3.2. Preparation and rheological analysis of HRP/CaO2@GOx-catalyzed hydrogels 53
3.3. In vitro release behaviors of H2O2 from hydrogels 55
3.4. Effect of H2O2-releasing hydrogels on proliferative and migrative activities of fibroblasts 56
3.5. Angiogenic activities of HRP/CaO2@GOx-catalyzed hydrogels 59
4. Conclusion 62
References 63
Chapter 3. Injectable Multifunctional Hyaluronan based Hydrogel Suppressing the Excessive Glucose and Reactive Oxygen Species in Diabetic Wound Treatment 67
1. Introduction and objective 68
2. Experimental section 73
2.1. Materials 73
2.2. Synthesis and characterization of hyaluronic acid – vinyl sulfone (HA-VS) and hyaluronic acid – thiol (HA-SH) 73
2.3. Fabrication and characterization of HA hydrogels 75
2.4. Glucose and ROS regulation properties of multifunctional HA hydrogels (HTG hydrogels) 77
2.5. Cell studies 80
2.6. Statistical analysis 82
3. Results and discussion 83
3.1. Synthesis and characterization of HA derivatives 83
3.2. Preparation and characterization of thiol-ene cross-linked HA hydrogels 84
3.3. Glucose-lowering and ROS-scavenging capacities of HTG hydrogels 88
3.4. Effect of HTG hydrogels on the proliferation and migration of human dermal fibroblast under hyperglycemic condition 93
3.5. The antibacterial properties of HTG hydrogels 97
4. Conclusions 99
References 100
Chapter 4. Overall conclusion 107

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