Ionic complexation improves wound healing in deep second-degree burns and reduces in-vitro ciprofloxacin cytotoxicity in fibroblasts

Abstract

The development of new treatments capable of controlling infections and pain related to burns continues to be a challenge. Antimicrobials are necessary tools, but these can be cytotoxic for regenerating cells. In this study, antibiotic-anesthetic (AA) smart systems obtained by ionic complexation of polyelectrolytes with ciprofloxacin and lidocaine were obtained as films and hydrogels. Ionic complexation with sodium alginate and hyaluronate decreased cytotoxicity of ciprofloxacin above 70% in a primary culture of isolated fibroblasts (p < 0.05). In addition, the relative levels of the proteins involved in cell migration, integrin β1 and p-FAK, increased above 1.5 times (p < 0.05) with no significant differences in cell mobility. Evaluation of the systems in a deep second-degree burn model revealed that reepithelization rate was AA-films = AA-hydrogels > control films > no treated > reference cream (silver sulfadiazine cream). In addition, appendage conservation and complete dermis organization were achieved in AA-films and AA-hydrogels. Encouragingly, both the films and the hydrogels showed a significantly superior performance compared to the reference treatment. This work highlights the great potential of this smart system as an attractive dressing for burns, which surpasses currently available treatments.

Figure 1

Effect of PE_L and PE_H on CV% of HDF cells after 24 h of treatment. The CV% was calculated from Eq. (1). Values are expressed as the means ± their standard errors (n = 8). Differences between the treatments were evaluated with ANOVA and Tukey's post-test (p < 0.05), with different capital letters above the bars indicating significant differences. The dotted line determines the cell viability limit allowed by ISO 10993 5 (CV ≥ 70%). Plotted with GraphPad Prism v.7.00 software (GraphPad, USA, https://www.graphpad.com/).

Figure 2

Cell viability of HDFs after 24 h of treatment with (A) Cip₃ and its complexes with PE_L or PE_H, and (B) Cip₃₀ and its complexes with PE_L or PE_H. The CV% was calculated according to Eq. (1). Values are expressed as the means ± their standard errors (n = 8). Differences between the treatments were evaluated by ANOVA and Tukey's post-test (p < 0.05), with different capital letters above the bars indicating significant differences. The dotted line determines the cell viability limit allowed by ISO 10993 5 (CV ≥ 70%). Plotted with GraphPad Prism v.7.00 software (GraphPad, USA, https://www.graphpad.com/).

Figure 3

Effect of CB, SA, SH, or PE-Cip complexes on cell migration. (A) Representative images of wound healing assays performed on HDF cells treated as indicated. An open furrow was generated by scratching confluent cells using a pipette tip, and cells images were obtained at the initial time and after 24 h. (B) The distance between furrow edges in control or treated cells of three independent experiments was measured and presented graphically as the percentage of the initial distance (0 h) according Eq. (2); p < 0.05 compared to control cells. Different capital letters above the bars indicating significant differences. Plotted with GraphPad Prism v.7.00 software (GraphPad, USA, https://www.graphpad.com/).

Figure 4

Effect of Cip, PE or PE-Cip complexes on integrin β1 and p-FAK protein expression. (A) Protein expression was analyzed by Western blot assays from cell lysates (100 µg/lane) of HDF-treated cells electrophoresed on a 7.5% SDS-PAGE and transferred to a nitrocellulose filter. Filters were incubated with anti-integrin β1, anti-p-FAK or anti-α-tubulin antibodies. Representative blot of three independent experiments with similar results is shown. Cropped blots from different parts of the same gel are separated by white spaces. (B, C) The bar graphs represent the densitometric quantification of protein levels in treated cells normalized to α-tubulin of three separate experiments compared to the corresponding normalized protein levels in control cells defined as 1 (mean ± SEM). Differences in treatments were evaluated using ANOVA with Dunnett's post-test *p < 0.05; **p < 0.01. Plotted with GraphPad Prism v.7.00 software (GraphPad, USA, https://www.graphpad.com/).

Figure 5

Representative photomicrographs of burn wounds for the NT group and after treatment with R-cream, AA-films, C-films or AA-hydrogel at days 7, 14, and 28 post wounding. H&E (× 100, scale bars: 100 μm). (A) Absent epidermis, (B) epidermis in regeneration, (C) continuous epidermis, (D) hemorrhagic appendages, (E) inflammatory cells, (F) dermal disorganization, (G) fibrous dermis, (H) appendages (hair follicles or glands), (I) differentiated papillary and reticular dermis.

Figure 6

Epidermis scores were recorded as relative frequency of cases/week as: ( 1) Absent, ( 2) discontinuous and ( 3) continuous.

Figure 7

Dermis scores were recorded as relative frequency of cases/week as: ( 1) Complete dermal disorganization, ( 2) Reticular recovery, papillary disorganization, ( 3) Recovery of reticular and papillary dermis, ( 4) Normal reticular dermis with papillary recovery, ( 5) Completely normal dermis.

Figure 8

Macroscopic healing versus time: burn wound area (% with respect to initial burned area) after treatments calculated according to Eq. (3) and representative photographs. References: AA-films, C-films, AA-hydrogel, R-cream and NT group. Scale bars are 1 cm; *significantly smaller than the NT group; **significantly larger than the NT group. Plotted with GraphPad Prism^® v.7.00 software (GraphPad, USA, https://www.graphpad.com/).

Figure 9

Biopsy location. The wounds were divided into four parts with two imaginary lines. Biopsies were taken clockwise from the center of each part (according to day 0) on days 0, 7, 14, and 21. Thus, each animal was its own control, and in this way, we managed to reduce the number of experimental animals used, according to the Guide for the Care and Use of Experimental Animals.