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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Pavlík, Vojtěch (Contipro, a.s.)


"Introduction: Many refractory chronic wounds are infected. Hyaluronan chloramide (HA-Cl) is a novel derivative that combines pro-regenerative hyaluronan and antimicrobial chlorine. While the synthesis and stability of HA-Cl were described recently1, its oxidative activity, efficacy against bacteria, and safety in wounds remain to be evaluated.

Methodology: The rate of substrate oxidation mediated by HA-Cl was investigated by means of a tetramethylbenzidine (TMB) colorimetric assay. The amount of HA that resulted from HA-Cl oxidation was analyzed using LC-MS/MS chromatography. The antimicrobial effects of HA-Cl were evaluated in Staphylococcus aureus using both planktonic bacteria (a modified disk diffusion method and growth inhibition in liquid media), as well as bacterial biofilm (Calgary biofilm device). The biocompatibility of HA-Cl was investigated in mouse excisional 6-mm wounds. HA-Cl in the form of nanofibrous textile was applied onto freshly created wounds and reapplied every 2-3 days during redressings until harvesting the wounds on day 14. Wound healing was assessed by means of histology and qRT-PCR. The numbers and proportions of blood cells were assessed using a haemoanalyzer. In vivo observations were complemented by assessing human blood haemolysis induced by HA-Cl in vitro.

Results: HA-Cl exerted slower substrate (BSA) oxidation than that of NaOCl, and chloramine T (CAT) as assessed with the TMB assay. The oxidation rate of HA-Cl was markedly increased by NaI, which served as a catalyzer. The analysis of HA-Cl degradation products showed its conversion to pure hyaluronan. The slower substrate oxidation mediated by HA-Cl prolonged its antimicrobial effect in liquid culture. When 106 CFU/ml S. aureus was reinoculated into the same culture media, bacteria were able to grow only in the samples treated previously by NaOCl, while no growth was observed in the samples containing HA-Cl. HA-Cl efficiently eradicated bacterial biofilm.
Aimed at investigating the safety of HA-Cl in vivo, the nanofibers containing HA-Cl were applied into mouse excisional wounds. Wound healing in the treated animals was normal when compared to controls in terms of granulation, inflammation, and epithelization (p>0.05). Blood profiles of the HA-Cl-treated animals did not deviate from those of untreated controls. In addition, HA-Cl, unlike NaOCl and CAT, did not cause haemolysis of blood in vitro (p<0.05).

Conclusions: HA-Cl is a safe, biocompatible, and efficient antiseptic, which is suitable for wound healing. It can be manufactured as sheets of nanofibers, which are conveniently applied topically. After HA-Cl is reduced in wounds, the resulting hyaluronan may further promote wound healing.

1. Buffa, R. et al., Carbohydr Polym 250:116928"

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