One-Step Physical and Chemical Dual-Reinforcement with Hydrophobic Drug Delivery in Gelatin Hydrogels for Antibacterial Wound Healing

Abstract

Gelatin-based bioadhesives, especially methacrylated gelatin (GelMA), have emerged as superior alternatives to sutureless wound closure. Nowadays, their mechanical improvement and therapeutic delivery, particularly for hydrophobic antibiotics, have received ever-increasing interest. Herein, a reinforced gelatin-based hydrogel with a hydrophobic drug delivery property for skin wound treatment was reported. First, photosensitive monomers of N'-(2-nitrobenzyl)-N-acryloyl glycinamide (NBNAGA) were grafted onto GelMA via Michael addition, namely, GelMA-NBNAGA. Second, gelation of the GelMA-NBNAGA solution was accomplished in a few seconds under one step of ultraviolet (UV) light irradiation. Multiple effects were realized simultaneously, including chemical cross-linking initiated by lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), physical cross-linking of uncaged dual hydrogen bonding, and hydrophobic drug release along with o-NB group disintegration. The mechanical properties of the dual-reinforcement hydrogels were verified to be superior to those only with a chemical or physical single-cross-linked network. The hydrophobic anticancer doxorubicin (DOX) and antibiotic rifampicin (Rif) were successfully charged into the hydrogels, separately. The in vitro antimicrobial tests confirmed the antibacterial activity of the hydrogels against Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. The in vivo wound-healing assessment in mice further assured their drug release and efficacy. Therefore, this NBNAGA-modified GelMA hydrogel has potential as a material in skin wound dressing with a hydrophobic antibiotic on-demand delivery.

Scheme 1. Schematic Illustrations of the Preparation of Drug-Loaded Hydrogels and the Process of Wound Healing

(A) Schematic for the synthesis of GelMA and GelMA-NBNAGA. (B) Gelation of the GelMA-NBNAGA solution containing drugs in situ by chemical and physical dual-cross-linking network under irradiation of UV light. (C) Process of wound healing with antibiotic hydrogel treatment.

Figure 1

(a) ¹H NMR spectra and (b) GPC traces recorded for gelatin, GelMA, and GelMA-NBNAGA. (c) Time-dependent UV–vis absorbance spectra recorded for GelMA-NBNAGA (0.08%, w/v) in PBS upon irradiation with a 365 nm LED light (30 mW/cm²) for varying durations (0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, and 5 min).

Figure 2

Mechanical characterization of the hydrogels. (a) Formation kinetics recorded from dynamic time–sweep rheological analysis of GelMA-NBNAGA/LAP, GelMA-NBNAGA, and GelMA/LAP hydrogels, respectively. (b) Gel points of different hydrogels. (c) Final storage modulus G′ of different hydrogels after being stored at 4 °C for 12 h.

Figure 3

Pore characteristics, swelling ratios, and in vitro degradation properties. (a) Representative SEM images, (b) pore size characterization, (c) swelling ratios in PBS, and (d) degradation properties in PBS of different hydrogels.

Figure 4

Adhesion properties of different hydrogels. (a) Mechanism of the adhesive study of lap shear, wound closure, and peeling adhesion used porcine skin for the study. (b) Shear strength, (c) adhesive strength, and (d) peeling strength of hydrogels and the commercially available fibrin glue.

Figure 5

In vitro release profiles and release extent after 400 min incubation of DOX (a) and Rif (b) from the drug-loaded GelMA-NBNAGA/LAP hydrogel under varying temperatures.

Figure 6

In vitro antibacterial analysis of the hydrogels. Relative bacterial viability of (a) E. coli and (b) S. aureus incubated with or without the Rif-loaded GelMA-NBNAGA/LAP hydrogel (Rif@gel). Also shown inside are their Images of agar plates with E. coli and S. aureus suspensions.

Figure 7

(a) Representative pictures of control, GelMA-NBNAGA/LAP, GelMA-NBNAGA, and GelMA/LAP hydrogels taken by a confocal laser microscope after staining with a Live/Dead Kit for 1, 3, and 5 days. (b) OD value and (c) relative cell viability of different groups at 450 nm after incubating with a CCK-8 Kit.

Figure 8

In vivo assessment of hydrogels for wound healing. (a) Digital images showing temporal development of wounds, (b) wound size in mice, and (c) wound-healing rates after treatment with and without the Rif-loaded GelMA-NBNAGA/LAP hydrogel (Rif@gel) on days 0, 3, 6, 9, and 12. (d) H&E staining and (e) Masson’s trichrome staining of the wound section on day 18.