Ozone-Based Eye Drops Activity on Ocular Epithelial Cells and Potential Pathogens Infecting the Front of the Eye

Roman Paduch, Teresa Urbanik-Sypniewska, Jolanta Kutkowska, Tomasz Chorągiewicz, Anna Matysik-Woźniak, Sandrine Zweifel, Aleksandra Czarnek-Chudzik, Wojciech Załuska, Robert Rejdak, Mario Damiano Toro, Roman Paduch, Teresa Urbanik-Sypniewska, Jolanta Kutkowska, Tomasz Chorągiewicz, Anna Matysik-Woźniak, Sandrine Zweifel, Aleksandra Czarnek-Chudzik, Wojciech Załuska, Robert Rejdak, Mario Damiano Toro

Abstract

Confirmation of the biological effectiveness of new ophthalmic preparations introduced in the market is an important element in maintaining the safety of using this type of medications. This study aimed to investigate the activity of Ozodrop® on human corneal and conjunctival epithelial cells, as well as its antibacterial and antifungal activity. Cytotoxicity analyses of ocular surface epithelial cells were performed in vitro by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) and Neutral Red uptake assays. The level of nitric oxide released by the cells was assessed by the Griess method. The reduction of the DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical by the tested formulation was analyzed. Microbiological tests were also performed. It was found that the Ozodrop® preparation exhibited biological activity, but was less active than the reference antibiotics and the anti-yeast agent. The cytotoxic activity of the Ozodrop® formulation was dependent on the time of cell exposure to it. No toxic effect was observed in the short-term, for up to 3 h. It appeared after 24 h of exposure of the cells to the preparation. The drops showed antioxidant activity in the specified concentration range. They also stimulated the release of nitric oxide, mainly by corneal epithelial cells. The Ozodrop® formulation exhibits biological activity that can be considered useful in the treatment of infections in the front part of the eye.

Keywords: bacteria; cell culture in vitro; conjunctival epithelium; corneal epithelium; ozone-based drops; toxicity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Effect of Ozodrop® preparation on the viability of human corneal epithelium (pRSV-T) in vitro, after 1, 3, and 24 h of incubation. Cell viability was determined by means of neutral red cytotoxicity (NR) assay.
Figure 2
Figure 2
Effect of Ozodrop® preparation on the viability of human conjunctival epithelium (HC0597) in vitro after 1, 3, and 24 h of incubation. Cell viability was determined by means of neutral red cytotoxicity (NR) assay.
Figure 3
Figure 3
Effect of Ozodrop® preparation on metabolic activity of human corneal epithelium (pRSV-T) in vitro, after 1, 3, and 24 h of incubation. Cell metabolic activity was determined by means of an MTT assay.
Figure 4
Figure 4
Effect of Ozodrop® preparation on the metabolic activity of human conjunctival epithelium (HC0597) in vitro, after 1, 3, and 24 h of incubation. Cell metabolic activity was determined by means of an MTT assay.
Figure 5
Figure 5
Nitric oxide (NO) secretion in a culture of human corneal and conjunctival epithelium, during 24 h of incubation with Ozodrop® preparation. The concentration range of starting drops dilution was 0–10%. Analysis was performed using the Griess method. Columns and bars show the mean ± standard deviation (n = 3). * p ≤ 0.05—a culture of corneal or conjunctival epithelial cells treated with Ozodrop® preparation, as compared to a non-treated culture control.
Figure 6
Figure 6
May–Grünwald–Giemsa (MGG) staining of the human corneal epithelial and conjunctival epithelial cells incubated with Ozodrop® formulation for 24 h. (A) Control; (B) Ozodrop® in a 1% concentration; (C) Ozodrop® in a 2.5% concentration; (D) Ozodrop® in a 5% concentration; (E) Ozodrop® in a 7.5% concentration; and (F) Ozodrop® in a 10% concentration. Bar = 20 μm. Magnification 200×.

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