The Potential of Novel Synthesized Carbon Dots Derived Resveratrol Using One-Pot Green Method in Accelerating in vivo Wound Healing

Abstract

Background: Carbon dots (CDs), a novel nanomaterial, have gained significant attention over the past decade due to their remarkable fluorescence properties, low toxicity, and biocompatibility. These characteristics make them promising in various applications, especially in biomedicine. However, most CDs are currently synthesized using chemical materials, and their biocompatibility falls short of natural compounds. Research on extracting CDs from natural sources is limited, and their potential in biomedicine remains largely unexplored.

Methods: We extracted CDs from resveratrol, a natural plant compound, and enhanced their water solubility using citric acid. Characterization of resveratrol-based carbon dots (RES-CDs) was carried out using various techniques, including UV-Vis, SEM, TEM, FTIR, XRD, and fluorescence spectroscopy. Extensive biocompatibility tests, wound healing assays, cell migration studies, and angiogenesis experiments were conducted using human umbilical vein endothelial cells (HUVEC). In addition, we investigated the biocompatibility and wound healing potential of RES-CDs in an in vivo rat model of inflammation.

Results: RES-CDs exhibited stable yellow-green fluorescence under 365-nanometer ultraviolet light and demonstrated excellent biocompatibility. In wound healing experiments, RES-CDs outperformed resveratrol in terms of cell scratch healing, migration, and tube formation. In a rat skin defect model, RES-CDs promoted wound healing and stimulated the formation of blood vessels and tissue regeneration near the wound site, as evidenced by increased CD31 and VEGF expression.

Conclusion: Resveratrol-derived CDs with enhanced water solubility show superior performance in tissue healing compared to resveratrol. This discovery opens new possibilities for the clinical application of resveratrol-based carbon dots.

Keywords: bio-imaging; biocompatibility; carbon dots; fluorescence; resveratrol; wound healing.

Graphical abstract

Figure 1

(a) TEM images and size distribution histogram of CDs. (b) Image of CDs captured by HRTEM. (c) XRD spectrum of CDs.

Figure 2

(a) FT-IR spectra of CDs. (b) The full XPS spectrum of the CDs. The picture(c) shows the XPS spectrum of C elements in CDs, while (d) represents the spectrum of oxygen elements. (e) CDs and resveratrol were separately dissolved in water. (f) Zeta Potential of CDs.

Figure 3

(a) Pictures of the CDs solution were taken under 365nm and natural light. The absorption peaks, optimal excitation wavelength, and the optimal emission wavelength was assessed using a UV-VIS and fluorescence spectrophotometer. (b) The emission spectra of CDs under different excitation wavelengths. (c) The emission spectra of CDs at different pH levels.

Figure 4

(a) EDU assay was performed to detect the HUVEC cell proliferation. (b) Performance of CDs at different concentrations in the hemolysis assay. (c) CCK-8 cell viability assay was performed after 1, 3, and 5 days of co-culturing HUVEC cells with CDs in vitro. (d) Live HUVEC cell staining with calcein AM dye(green) on Day 1 and 3. The uptake of CDs in HUVEC cells was observed at 12h (e) and 48h (f) by laser confocal microscopy.

Figure 5

The experiments of wound healing include HUVEC cell scratch assay (a), transwell migration assay (b), and tube formation assay (c). (d) The expression of VEGF factor in HUVEC cells. (e) Cell migration area percentage in the scratch assay, (f) and (g) the number of migrated cells in the Transwell migration assay, (h) the total length of formed blood vessels, and (i) the total number of branch points in the angiogenesis assay. *p < 0.05, **p < 0.01, ***p <0.001, ****p <0.0001.

Figure 6

(a) Treatment procedure for SD rat skin defect. (b) Comparison of wound healing between control group and experimental group at different time points. (c) Analysis of wound healing between control group and experimental group.

Figure 7

(a) HE-stained sections of organs from SD rats after intravenous injection of different solutions. (b) Immunohistochemical staining of CD31 in the rat skin defect site. (The brown-black area pointed by the black arrows represents the region around blood vessels expressing the CD31 factor). (c) Analysis of HE-stained sections and epithelial thickness in different treatment groups of skin defects. (d) Analysis of Masson-stained sections and collagen fiber content in different treatment groups of skin defects. **p < 0.01, ***p <0.001.