Tetrahedral framework nucleic acids/hyaluronic acid-methacrylic anhydride hybrid hydrogel with antimicrobial and anti-inflammatory properties for infected wound healing

Abstract

Bacterial resistance and excessive inflammation are common issues that hinder wound healing. Antimicrobial peptides (AMPs) offer a promising and versatile antibacterial option compared to traditional antibiotics, with additional anti-inflammatory properties. However, the applications of AMPs are limited by their antimicrobial effects and stability against bacterial degradation. TFNAs are regarded as a promising drug delivery platform that could enhance the antibacterial properties and stability of nanodrugs. Therefore, in this study, a composite hydrogel (HAMA/t-GL13K) was prepared via the photocross-linking method, in which tFNAs carry GL13K. The hydrogel was injectable, biocompatible, and could be instantly photocured. It exhibited broad-spectrum antibacterial and anti-inflammatory properties by inhibiting the expression of inflammatory factors and scavenging ROS. Thereby, the hydrogel inhibited bacterial infection, shortened the wound healing time of skin defects in infected skin full-thickness defect wound models and reduced scarring. The constructed HAMA/tFNA-AMPs hydrogels exhibit the potential for clinical use in treating microbial infections and promoting wound healing.

Fig. 1

Preparation and characterization of HAMA/tFNA-GL13K hydrogel. a Schematic diagram of the preparation of HAMA/tFNA-GL13K hydrogel. b Analysis of Polypropylene Acyl Amine Gel Electrophoresis (PAGE) showed the effective synthesis of tFNA and tFNA-GL13K with the appropriate tFNA/GL13K ratio. c Particle size measurement by DLS and ζ potential measurement by ELS of tFNA and t-GL13K. d Atomic force microscopy evaluation. Scale bars = 500 nm. e TEM images of tFNA (yellow triangles) and tFNA-GL13K (red triangles). Scale bar = 100 nm. f Optical photos of the hydrogel before and after photocuring. g SEM images demonstrating the porous structure of hydrogel. Scale bar = 1 mm (white) and 300 μm (orange). h Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) of HAMA and HAMA/tFNA-GL13K hydrogel. i Variations of the storage modulus (G′) and the loss modulus (G″) with oscillation frequency of the hydrogels. T-GL13K is the abbreviation of tFNA-GL13K

Fig. 2

Antimicrobial activity of HAMA/tFNA-GL13K hydrogel on E. coli and S. aureus in vitro. a Images of E. coli and S. aureus incubated on agar plates with different hydrogels. b Count bacteria of CFU of E. coli and S. aureus. Superscripts a–d indicate significant differences for CFU between different treatment groups (P < 0.05). c Growth curves of E. coli and S. aureus in different hydrogels treatment. n = 3 independent samples. Data are presented as mean ± SD. Error bars represent SD. T-GL13K is the abbreviation of tFNA-GL13K

Fig. 3

The distribution of tFNA-GL13K in HaCaT cells. a Distribution of Cy5-tFNAs, FITC-GL13K and Cy5- FITC-t-GL13K complex in HaCaT cells (Cy5: tFNAs: red; FITC: yellow; nucleus: blue; cytoskeleton: green). Scale bars = 20 μm. b Flow cytometric analysis of distribution of Cy5-tFNAs, FITC-GL13K and Cy5- FITC-t-GL13K complex in HaCaT cells. Superscripts a–d indicating significant differences (P < 0.05)

Fig. 4

The effect of HAMA/tFNA-GL13K hydrogel on the biological behavior of HaCaT cells. a CCK-8 assays to detect the cytotoxicity of hydrogels on HaCaT cells. Superscripts a–d indicating significant differences (P < 0.05). b Images of scratch tests on HaCaT cells treated with different hydrogels at 0 h, 4 h, 8 h, 12 h and 24 h. Scale bars = 200 μm. Statistical analysis of scratch tests was marked with superscripts a–e indicating significant differences (P < 0.05). c Quantification of ROS in HaCaT cells. d Quantification of expression of inflammatory factors (IL-1β, IL-G, TNF-α, and p65) in HaCaT cells. Superscripts a–e indicating significant differences (P < 0.05). Data are presented as the mean ± SD (n = 3). T-GL13K is the abbreviation of tFNA-GL13K

Fig. 5

Evaluation of HAMA/tFNA-GL13K hydrogel in vivo for skin repair and wound healing in general observation. a Flow chart of the experiment in vivo. b Representative images of the wound healing process in 5 groups at the indicated time point. c Wound area of 5 groups on the 7^th and 14^th day. Superscripts a–c indicating significant differences (P < 0.05). d Wound healing rate of diverse groups (P < 0.000 1, ****)

Fig. 6

Evaluation of HAMA/tFNA-GL13K hydrogel in vivo for skin repair and wound healing in microscopic observation. a Images of wound tissue after H&E and Masson staining on the 7^th day. Scale bars = 200 μm. b Images of wound tissue after H&E and Masson staining on the 14^th day. Scale bars = 4 mm (black) and 200 μm (white) (The unhealed area was denoted by black and white dotted lines). c Quantitative analysis of the unhealed dermis area in five groups. d Quantitative analysis of epidermal thickness in five groups. e Quantitative analysis of collagen volume fraction. Superscripts a–d indicating significant differences (P < 0.05). Data are presented as the mean ± SD (n = 3). T-GL13K is the abbreviation of tFNA-GL13K