Dual role of iodine, silver, chlorhexidine and octenidine as antimicrobial and antiprotease agents

Abstract

Objectives: The majority of human chronic wounds contain bacterial biofilms, which produce proteases and retard the resolution of inflammation. This in turn leads to elevated patient protease activity. Chronic wounds progressing towards closure show a reduction in proteolytic degradation. Therefore, the modulation of protease activity may lead to the faster healing of chronic wounds. Antimicrobials are used to control biofilm-based infection; however, some of them also exhibit the inhibition of matrix metalloproteinases and bacterial proteases. We investigated the antimicrobial agents used in wound healing for their potential to inhibit bacterial and host proteases relevant to chronic wounds.

Methods: Using in vitro zymography, we tested the ability of povidone-iodine, silver lactate, chlorhexidine digluconate, and octenidine hydrochloride to inhibit selected human proteases and proteases from Pseudomonas aeruginosa, Staphylococcus aureus, Serratia marcescens, and Serratia liquefaciens. We investigated penetration and skin protease inhibition by means of in situ zymography.

Results: All the tested antimicrobials inhibited both eukaryotic and prokaryotic proteases in a dose-dependent manner in vitro. The tested compounds were also able to penetrate into skin ex vivo and inhibit the resident proteases. Silver lactate and chlorhexidine digluconate showed an inhibitory effect ex vivo even in partial contact with skin in Franz diffusion cells.

Conclusions: Our in vitro and ex vivo results suggest that wound healing devices which contain iodine, silver, chlorhexidine, and octenidine may add value to the antibacterial effect and also aid in chronic wound healing. Antiprotease effects should be considered in the design of future antimicrobial wound healing devices.

Conflict of interest statement

We declare that VP, MS, MM and VV were employed during the study by a commercial company Contipro that manufactures wound healing products with antimicrobials iodine and octenidine hydrochloride. None of the products was evaluated in the presented study. This does not alter our adherence to PLOS ONE.

Figures

Fig 1. Activity of bacterial proteases with antimicrobials in SDS-PAGE zymography.

(A) A mixture of bacterial proteases was separated on polyacrylamide gels with gelatin and incubated overnight in developing buffer that contained decreasing amounts of povidone-iodine (PVP-I), silver lactate (Ag), chlorhexidine digluconate (Chl), and octenidine hydrochloride (Oct). The values are expressed as relative protease activity related to control (Ctrl) with no added antimicrobial. (B) The activity of eukaryotic proteases similarly incubated with varying concentrations of antimicrobials. In panels A and B, bars represent standard deviation, * denotes p

Fig 2. Activity of bacterial proteases with antimicrobials in fluorogenic assay.

Pure proteases (MMP-2 or trypsin) or single-source proteases from bacterial or eukaryotic media were evaluated in fluorogenic assay with quenched gelatin as a substrate. Reactions were in the presence or absence of different concentrations of silver lactate (A), chlorhexidine digluconate (B), and octenidine hydrochloride (C). Differences in fluorescence increase were related to control without antiseptics. Measurements were repeated two to three times. X-axis is in log10 scale. PA–P. aeruginosa, SL–S. liquefaciens, SM—S. marcescens.

Fig 3. Activity of porcine skin endogenous proteases with antimicrobials.

Whole skin was incubated with the tested compounds in a tube, homogenized, and assessed by means of gelatin zymography (A) or incubated in Franz diffusion cells (dermal side up) and analysed using in situ zymography, as illustrated with representative images (B). Color-inverted images, darker shades correspond to increased proteolysis. Each sample was developed with fluorogenic substrate either at 37°C (the upper sample section of each treatment) or at -20°C (assay control, the lower sample section). Squares show areas of fluorescence intensity measurement, which were evaluated in replicates (n = 5) with image analysis (C). Bars in (A) and (C) show mean protease activity related to PBS control ± SD. * denotes p < 0.05 (Student's one sample t-test to control). The tested compounds were povidone-iodine (PVP-I), silver lactate (Ag), chlorhexidine digluconate (Chl), octenidine hydrochloride (Oct), commercial protease inhibitors (P. Inh.).