Title：Engineered heterojunction microneedles initiate ROS-mediated “two-hit” mechanism for accelerating impaired wound healing in diabetes

Journal: Cell Biomaterials

Original link：https://doi.org/10.1016/j.celbio.2025.100248

Reporter：Zhenqing Guo-26-master

    Diabetic wound healing is severely impaired by hyperglycemia and bacterial infection. In recent years, bioactive wound dressings have been developed to consume glucose and generate reactive oxygen species (ROS), offering ideal antibacterial and pro-regenerative abilities. However, the generation of antibacterial ROS is glucose-dependent; therefore, a decline in glucose concentration at a later stage of wound healing reduces ROS levels and diminishes antibacterial efficacy. In this study, we propose a “two-hit” strategy: ROS production is initially glucose oxidase (GOx)-dominated during early healing, while the Fenton reaction sustains ROS generation post-glucose depletion. We further design a CNOH/MXene heterojunction (C@M) and composite microneedle system (GC@M): C@M heterojunction is prepared by anchoring MXene (Ti3C2) quantum dots onto alkalinized g-C3N4 (CNOH) nanosheets and then integrated into gelatin methacryloyl microneedles. We found that GC@M microneedles produce ROS through this two-hit mechanism and inhibit bacterial proliferation and survival by triggering lipid peroxidation-related bacterial membrane damage and cell death. In conclusion, this work offers a transformative two-hit hypothesis as a clinically viable solution.

    Diabetes is one of the most prevalent non-communicable chronic diseases worldwide, posing significant challenges to healthcare systems. The latest epidemiological data shows that by 2022, the number of adult diabetes patients had exceeded 820 million, and this figure is expected to keep rising.

    Reactive Oxygen Species (ROS), as a versatile antibacterial and anti-biofilm strategy, has gradually attracted attention. ROS exerts bactericidal effects through multiple mechanisms, including protein denaturation and DNA damage, while enhancing the oxidative stress of microbial populations. The unique hyperglycemic condition of diabetic wounds presents both challenges and opportunities for the development of ROS-mediated antibacterial therapies. Recent advances in the field of nanozymes have highlighted the potential of glucose oxidase (GOx)-mimicking nanomaterials in ROS production. In addition to the activity of GOx-like nanozymes, the Fenton reaction is also one of the important pathways for ROS generation.

    Therefore, the study proposes an innovative "two-hit" ROS generation strategy that synergistically combines enzymatic catalysis with Fenton chemistry. This biphasic approach ensures sustained antibacterial activity through a phase-adaptive mechanism: initial GOx-dominated ROS production occurs in the early healing stage rich in glucose (Glu), followed by Fenton reaction-mediated ROS generation as Glu levels decrease. The strategic integration of these complementary mechanisms leverages the changing wound microenvironment while maintaining therapeutic efficacy throughout the healing process.

    In this study, a two-hit therapeutic strategy for diabetic wound management was developed by combining a novel nanocomposite with advanced microneedle technology. The nanocomposite was synthesized by anchoring Ti3C2 onto alkalized g-C3N4 (CNOH) nanosheets, which was then incorporated into bilayer GelMA microneedles (GC@M). This design utilizes wound Glu dynamics to achieve sustained antibacterial efficacy throughout the healing process, thereby accelerating tissue regeneration. The study systematically characterized the physicochemical properties, bioactivity, and therapeutic performance of GC@M to validate the proposed two-hit strategy. Special emphasis was placed on clarifying the ROS-mediated antibacterial mechanism, focusing on lipid peroxidation (LPO). Overall, this work establishes a dual-effect antibacterial and anti-biofilm approach targeting both intracellular and extracellular ROS, and provides a promising avenue for the treatment of diabetic wounds.

1.Synthesis and Characterization of Nanosheets

    Figure 1 focuses on the basic characterization of nanosheets after synthesis, while demonstrating the enhanced optical properties after assembly into heterojunctions.

2.Performance Evaluation of Materials

    In Figure 2, the optical activity, nanozyme performance, and Fenton-like reaction activity of the materials are mainly evaluated.

3.Preparation and Characterization of Microneedle Hydrogels

   The prepared heterojunction materials are integrated into gelatin methacryloyl microneedles, and the basic characterization and photothermal properties of the hydrogel microneedles are determined.

4.Antibacterial Activity

    E. coli and S. aureus are selected as research objects to evaluate the antibacterial activity and anti-biofilm performance of the materials.

5.Biosafety Assessment

    The biosafety and compatibility of the hydrogel microneedles are evaluated. GC@M and GC@M/N show ideal biocompatibility, meeting the preliminary requirements for Class III medical devices.

6.Diabetic Wound Model with Bacterial Infection

    The regeneration-promoting mechanism of GC@M/N mainly includes: (1) Glucose deprivation, (2) Antibacterial activity, and (3) Anti-biofilm effect. It can significantly promote wound healing in diabetic bacterial biofilm infections through multiple synergistic mechanisms.

7.Antibacterial Mechanism

   The C@M heterojunction combined with near-infrared irradiation (C@M/N) not only generates extracellular reactive oxygen species (ROS) through photocatalytic performance, but also produces intracellular ROS through the release of Ti⁴⁺/Ti³⁺ ions and Fenton-like reactions. In the early stage of diabetic wound healing, the increased local glucose (Glu) level enhances the photocatalytic activity of C@M/N, generating a large amount of extracellular ROS and exhibiting effective antibacterial and anti-biofilm effects. However, with the consumption of Glu, the production of extracellular ROS decreases. In the middle and late stages of diabetic wound healing, more Ti⁴⁺/Ti³⁺ ions are released from C@M/N, and the Glu concentration returns to a lower value. At this stage, Glu deprivation synergizes with the Fenton reaction to trigger an outbreak of intracellular ROS production. Therefore, intracellular ROS replaces the function of extracellular ROS.

    Wound dressings are designed to generate reactive oxygen species (ROS) by consuming glucose, thereby providing ideal antibacterial activity for diabetes management. However, the decline in high glucose levels can restrict ROS production, which in turn impairs antibacterial and pro-regenerative efficacy. This study aims to fabricate a wound dressing with the ability to continuously generate ROS and exert antibacterial effects.Firstly, we propose a "two-hit" strategy: during the early stage of wound healing, the production of ROS is initially dominated by glucose oxidase (GOx), while the Fenton reaction maintains ROS generation after glucose depletion. We also designed a CNOH/MXene heterojunction (C@M). Initially, it consumes glucose to generate ROS through near-infrared (NIR)-activated GOx activity, and then sustains ROS production via Ti-mediated Fenton reaction. The heterojunction was integrated into gelatin methacryloyl microneedles to form a bilayer system (GC@M).Experimental validation confirmed that GC@M accelerates diabetic wound healing through this two-hit mechanism. Further studies revealed that ROS exerts antibacterial effects by triggering lipid peroxidation-related bacterial membrane damage and cell death. This work presents a transformative two-hit mechanism for diabetic wound management.