Three in One with Dual-Functional Hydrogel of Lactoferrin/NZ2114/LMSH Promoting Staphylococcus aureus-Infected Wound Healing

Abstract

Wound infections caused by Staphylococcus aureus often result in localized suppurative lesions that severely impede the healing process, so it is urgent to develop a dress with efficient antimicrobial and pro-healing functions. In this study, the bifunctional injectable hydrogel lactoferrin (Lf)/NZ2114/lithium magnesium silicate hydrogel (LMSH) was first successfully prepared through the electrostatic interaction method. The physical, biological, and efficacy properties are systematically analyzed with good shear-thinning capacity and biocompatibility. More importantly, it inhibits infection and promotes wound healing in a mouse wound infection model after 14 d treatment, and the bactericidal rate and healing rate were over 99.92% and nearly 100%, respectively. Meanwhile, the massive reduction of inflammatory cells, restoration of tissue structure, and angiogenesis in mice showed the anti-inflammatory and pro-healing properties of the hydrogel. The healed wounds showed thickening with more hair follicles and glands, suggesting that the hydrogel Lf/NZ2114/LMSH (Three in One) could be a better dressing candidate for the treatment of S. aureus-induced wound infections.

Keywords: Lf/NZ2114/LMSH (Three in One); Staphylococcus aureus infection; injectable hydrogel; would dressing.

Figure 1

The photograph, antimicrobial activity and FT–IR spectrum of the injectable hydrogel Lf/NZ2114/LMSH. (a) The photograph of the synthetic injectable hydrogel; (b) Inhibition zone of NZ2114, Lf, 1% LMSH/Lf/NZ2114, 3% HACC/Lf/NZ2114, 3% SA, 1% LMSH; (c) The FT–IR spectra of 1% LMSH, 1% LMSH + Lf, 1% LMSH + NZ2114, 1% LMSH + Lf + NZ2114, 1.5% LMSH + Lf + NZ2114, 3% HACC, 3% HACC + Lf, 3% HACC + NZ2114, 3% HACC + Lf + NZ2114.

Figure 2

The morphology of the synthetic injectable hydrogel. The SEM image of 0.5% LMSH + NZ2114, 1% LMSH + Lf + NZ2114, 1% LMSH + Lf + NZ2114, 3% HACC + Lf + NZ2114, 1% LMSH, 3% HACC, Lf + NZ2114.

Figure 3

The viscosity, modulus, and bactericidal properties of different hydrogel samples. (a) The viscosity of 0.5% Lf/NZ2114/LMSH and 1% Lf/NZ2114/LMSH during sheer increase from 0.01 to 100 s⁻¹; (b) The viscosity of 3% Lf/NZ2114/HACC during sheer increase from 0.01 to 100 s⁻¹; (c) The storage modulus (G′) and loss modulus (G″) of 0.5% Lf/NZ2114/LMSH, 1% Lf/NZ2114/LMSH, and 3% Lf/NZ2114/HACC during strain increase from 0.1% to 1000% at the frequency of 1 Hz; (d,e) The bactericidal rate of 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, Lf + NZ2114, Lf, NZ2114, 1% LMSH, 3% HACC against S. aureus CVCC 546 (n = 3). These (−2, −3, −4, −5) are the number of dilutions. (* p < 0.05, *** p < 0.001).

Figure 4

The biocompatibility of different hydrogel samples. (a,b) The images and hemolysis rate of Lf, Lf + NZ2114, 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC, 0.1% Trix-100; (c) Cytotoxicity of HACAT cells co-cultured with Lf, Lf + NZ2114, 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC. Samples at 1, 2, and 4 days, n = 3; (d) The images of calcein–AM/PI double staining of the HACAT cells that were incubated with 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC for 1, 2 and 4 days. (Scale bar = 100 μm). * p < 0.05, *** p < 0.001.

Figure 5

The body weight, skin load, and wound diagram of mice (n = 3). (a) The weight of mice untreated and treated with Lf + NZ2114, 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC for 0–14 days; (b) The bacteria of skin untreated and treated with Lf + NZ2114, 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC samples for 4 d; (c) The macroscopic images of wounds untreated and treated with 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC samples for 4, 8, 12 and 14 d; (d) The wound healing rate of mice untreated and treated with Lf + NZ2114, 1% Lf/NZ2114/LMSH, 3% Lf/NZ2114/HACC, 1% LMSH, 3% HACC samples for 4, 8, 12, and 14 d. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

HE staining of the wounds without any treatment or treated with 1% Lf/NZ2114/LMSH and 3% Lf/NZ2114/HACC samples for 14 days.

Figure 7

The cytokine secretion without any treatment or treated with 1% Lf/NZ2114/LMSH and 3% Lf/NZ2114/HACC samples for 14 days. (a–c) The expressions of IL-6, VEGF and EGFR were measured by ELISA kit. (d–g) The expressions of IL-6, VEGF, EGFR, and CD31 were measured at RNA level by qPCR.

Figure 8

The immunohistochemistry of without any treatment or treated with 1% Lf/NZ2114/LMSH and 3% Lf/NZ2114/HACC samples for 14 days. CD31 staining of the wounds without any treatment or treated with different hydrogel samples for 14 days. IL-6 staining of the wounds for 14 days. (Red arrows are CD31 binding sites).