Application of a novel thermal/pH-responsive antibacterial paeoniflorin hydrogel crosslinked with amino acids for accelerated diabetic foot ulcers healing

Abstract

Diabetic foot ulcers (DFUs), a severe and common complication of diabetes, present significant treatment challenges due to the limitations of conventional dressings, such as poor mechanical properties, bioactivity, and limited functionality, which hinder fast and effective wound healing. To address these issues, we developed a novel natural amino acid-based hydrogel loaded with paeoniflorin (PF@PNMA1) and comprehensively evaluated its properties and functions. The nanogel particles (NGs) were synthesized via emulsion polymerization using N-isopropylacrylamide (NIPAM), methacrylic acid (MAA), and chemically modified arginine (MArg). The poly(NIPAM-co-MAA) (PNM) and poly(NIPAM-co-MAA-co-MArg) (PNMA) gels were prepared by functionalizing the NGs with glycidyl methacrylate (GMA). The different concentrations of amino acids were added to explore the optimal mechanical properties of the gel. Through the rheological measurement, we found that PNMA1 gel has good ductile properties with a critical strain up to about 63 %. At the same time, we also verified its antibacterial activity and found that the viability of bacteria decreased to 47.46 % after 3 h. Preliminary tests using network pharmacology and molecular docking confirmed the therapeutic potential of PF for DFUs. The PF@PNMA1 gel demonstrated excellent biocompatibility, and in vivo experiments revealed its effectiveness in promoting angiogenesis and wound healing. After 10 days, the wound healing rate was 25.6 % higher than that of the control group. The PF@PNMA1 shows great potential as an effective therapy for DFUs treatment.

Keywords: Amino acids; Crosslinker; Diabetic foot ulcers; Paeoniflorin; Wound dressing.

Graphical abstract

Scheme 1

Schematic of the design, synthesis, and applications of gels. (A) Synthesis of PNM/PNMA nanogel particles and their response to conditioned stimuli. (B) Virtual screening of PF and preparation of PF@PNMA1 gel. (C) Characterization of PF@PNMA1 gel and validation in animal models.

Fig. 1

TEM images of the(A) PNM, (B) PNMA0.5, (C) PNMA1, and (D) PNMA1.5 NGs and size distributions obtained by measuring at least 100 particles (from a to d). The scale bar is 100 nm. (E) Particle sizes variations of the PNM and PNMA systems at different pH and temperature values. (F) Swelling ratio (Q) of PNM and PNMA NGs at different pH and temperature values. (G) GMA functionalization.

Fig. 2

(A) Scanning electron microscopy (SEM) images of freeze-dried PNM and PNMA gels at pH 7.4, 5 wt%, and 37 °C; The scale bar is 10 μm. (B) Statistical analysis of the pore sizes of the PNM and PNMA gels. (C) The frequency-sweep test was conducted at 1 % strain for the PNM and PNMA gels prepared at 37 °C. (D) The strain-sweep test was conducted at 1 Hz for the PNM and PNMA gels prepared at 37 °C. (E) The frequency-sweep test was conducted at 1 % strain for the PNMA1 gel prepared at different temperatures. (F) The strain-sweep test was conducted at 1 Hz for the PNMA1 gel prepared at different temperatures. (G) Critical strain (${\gamma }^{\ast }$) for the PNM and PNMA gels under different conditions. (H, I) Gelation time at different temperatures (Taking PNMA1 gel as an example, there is a consistent trend across different groups).

Fig. 3

(A) Inhibition of S. aureus by PNM and PNMA gels, photo of colonies of 3 h and 10 h. (B) Statistical analysis of colony data, ∗P < 0.05, ∗∗∗∗P < 0.0001. (C) Adhesion properties of the PNMA1 gel on porcine skin tissues. The scale bar is 5 mm.

Fig. 4

Preliminary confirmation of PF for the treatment of DFUs and in vitro bioassay evaluation of the PNMA1 gel. (A) In vitro release of PF from PNMA1. (B–C) Effects of PF, PNMA1 and PF@PNMA1 on L929 cells and HUVECs, (ns, not significant). (D) HUVECs tube formation experiments. The scale bar is 100 μm. (E–F) Nodes and total length of HUVEC tube formation, ∗P < 0.05, ∗∗P < 0.01. (G) The wound healing of different formulations. The scale bar is 100 μm. (H) Wound healing rate of L929 cells, ∗P < 0.05, ∗∗∗P < 0.001.

Fig. 5

(A) Animal model construction. (B) Blood glucose curve in SD rats after streptozotocin modeling. (C) Promotion of diabetic wound healing in rats by the control and PNMA1/PF@PNMA1 groups, recorded from wound images over 10 d. (D) Healing rate curve of diabetic wound in SD rats, ∗P < 0.05, ∗∗P < 0.01. (E) Results of HE and Masson staining. (F) Results of CD31 fluorescent staining.