Ultrasmall Antioxidant Copper Nanozyme to Enhance Stem Cell Microenvironment for Promoting Diabetic Wound Healing

Abstract

Purpose: Stem cell therapy is a promising approach for treating chronic diabetic wounds. However, its effectiveness is significantly limited by the high oxidative stress environment and persistent inflammation induced by diabetes. Strategies to overcome these challenges are essential to enhance the therapeutic potential of stem cell therapy.

Methods: Cu_5.4O ultrasmall nanoparticles (Cu_5.4O-USNPs), known for their excellent reactive oxygen species (ROS) scavenging properties, were utilized to protect adipose-derived stem cells (ADSCs) from oxidative stress injury. In vitro experiments were conducted to evaluate the viability, paracrine activity, and anti-inflammatory capabilities of ADSCs loaded with Cu_5.4O-USNPs under oxidative stress conditions. In vivo experiments in diabetic mice were performed to assess the therapeutic effects of Cu_5.4O-USNP-loaded ADSCs on wound healing, including their impact on inflammation, collagen synthesis, angiogenesis, and wound closure.

Results: ADSCs treated with Cu_5.4O-USNPs showed significantly enhanced viability, paracrine activity, and anti-inflammatory properties under oxidative stress conditions in vitro. In diabetic mice, Cu_5.4O-USNP-loaded ADSCs reduced inflammatory responses in wound tissues, promoted collagen synthesis and angiogenesis, and accelerated diabetic wound healing. These findings suggest that Cu_5.4O-USNPs effectively mitigate the adverse effects of oxidative stress and inflammation, enhancing the therapeutic efficacy of ADSCs.

Conclusion: This study presents a simple and effective approach to improve the therapeutic potential of stem cell therapy for diabetic wounds. By incorporating Cu_5.4O-USNPs, the antioxidative and anti-inflammatory capabilities of ADSCs are significantly enhanced, offering a promising strategy for ROS-related tissue repair and chronic wound healing.

Keywords: ADSCs; Cu5.4O-USNPs; inflammatory environment; stem cell therapy.

Scheme 1

Cu5.4O-USNPs were integrated into ADSCs to enhance their survival, paracrine signaling, and anti inflammatory properties under oxidative stress conditions, resulting in significant therapeutic benefits.

Figure 1

(A) TEM image of Cu_5.4O-USNPs. (B) Particle size distribution of Cu_5.4O-USNPs determined from TEM image analysis (n=300). (C) In vitro H₂O₂ scavenging efficiency of Cu_5.4O-USNPs (2 mm H₂O₂). (D) Cell viability of ADSCs loaded with different concentrations (0–7000 ng/mL) of Cu_5.4O-USNPs. (E) Cell viability of ADSCs loaded with different concentrations (0–6000 ng/mL) of Cu_5.4O-USNPs. (F) Cell viability of ADSCs loaded with different concentrations (0–1000 ng/mL) of Cu_5.4O-USNPs. ***P<0.001, NS P>0.05.

Figure 2

(A) Cell viability and IC₅₀ of ADSCs after 24 hours of stimulation with 0–200 μM H₂O₂. (B) Cell viability of Cu_5.4O-ADSCs at different concentrations after 24 hours of stimulation with 100 μM H₂O₂. (C and D) Microscopic scratch images of different treatment groups at three time points: 0h, 24h, and 48h. (E) Representative images of different treatment groups after DCFH-DA staining under a fluorescence microscope. (F) Intracellular ROS content of different treatment groups obtained by flow cytometry. (G) Average fluorescence intensity of different groups under a fluorescence microscope. (H) Relative fluorescence intensity of different treatment groups obtained from flow cytometry results. *P<0.05,** P<0.01, NS P>0.05.

Figure 3

(A) VEGF secretion levels of ADSCs and Cu_5.4O-ADSCs at 24h, 48h, and 72h under normal conditions. (B) VEGF secretion levels of ADSCs and Cu_5.4O-ADSCs at 24h, 48h, and 72h under oxidative stress conditions. (C) Schematic diagram of the co-culture system of RAW264.7 and ADSCs. (D) TNF-α levels secreted by different treatment groups. (E) IL-10 levels secreted by different treatment groups. ** P<0.01,***P<0.001, NS P>0.05.

Figure 4

(A) Wound healing process and (B) wound closure rate of diabetic mice in different treatment groups. ** P<0.01.

Figure 5

(A) Histological sections of wound sites stained with H&E on postoperative days 7 and 14 in different treatment groups of diabetic mice. (B and C) Histological sections of wound sites stained with Masson’s trichrome on postoperative days 7 and 14 in different treatment groups of diabetic mice. Blue represents collagen fibers, and red represents muscle fibers. *P<0.05,** P<0.01,***P<0.001, NS P>0.05.

Figure 6

(A) Immunohistochemical staining of Ki67 in wound tissues of diabetic mice from different treatment groups on postoperative day 14. (B) Quantitative analysis of Ki67 positive expression areas in diabetic mice wounds under different treatment conditions. (C) Immunohistochemical staining of CD31 in wound tissues of diabetic mice from different treatment groups on postoperative day 14. (D) Quantitative analysis of CD31 positive expression areas in diabetic mice wounds under different treatment conditions. ** P<0.01,***P<0.001.

Figure 7

(A) Immunohistochemical staining of IL-10 in wound tissues of diabetic mice from different treatment groups on postoperative day 14. (B) Quantitative analysis of IL-10 positive expression areas in diabetic mice wounds under different treatment conditions. (C) Immunohistochemical staining of TNF-α in wound tissues of diabetic mice from different treatment groups on postoperative day 14. (D) Quantitative analysis of TNF-α positive expression areas in diabetic mice wounds under different treatment conditions. *P<0.05,** P<0.01,***P<0.001, NS P>0.05.