Bacterial strains used in this study were Staphylococcus aureus ATCC25923 and Pseudomonas aeruginosa ATCC27853, both from clinical sample, supplied from the internal laboratory of the Istituto Zooprofilattico Sperimentale del Mezzogiorno. The bacterial strains were diluted with saline to reach a concentration of 150 × 10⁶ CFU/ml. The turbidity of the suspension was measured with a 600 nm densitometer (Lambda-25 spectrophotometer, Perkin-Elmer, USA) according to the Mc Farland 0.5 standard. The bacterial suspension was diluted to the concentration of 15 × 10⁻⁹ CFU/ml.

Various volumes of liposome-vehiculated ozonated oil were added (400 μl, 200 μl, 100 μl and 50 μl) to 100 μl of bacterial suspension, both of Staphylococcus aureus and Pseudomonas aeruginosa and incubated at 37˚C for 10 minutes. This time was chosen to simulate as closely as possible the conditions that occur after applying one drop only in one eye.

After that, 100 μl of the four solutions thus prepared (inoculum A, inoculum B, inoculum C and inoculum D), were seeded on blood Agar plates for Staphylococcus aureus and Agar nutrient for Pseudomonas aeruginosa. Furthermore, for each bacterial strain, a positive control (only Staphylococcus aureus and only Pseudomonas aeruginosa) and a negative control only consisting of liposome-vehiculated ozonated oil were added, in order to rule out any contamination. The Staphylococcus aureus plates were then incubated overnight at 37˚C. While the Pseudomonas aeruginosa ones were incubated at 25˚C for 48 hours.

The cytotoxicity of liposome-vehiculated ozonated oil was analysed at the University of Campania “Luigi Vanvitelli”, Department of Precision Medicine.

The HaCaT epidermal keratinocyte cell line (ATCC, USA) was grown in Dulbecco’s Modified Eagle Medium (Euroclone) with the addition of 10% foetal bovine serum (FBS) (Euroclone), 2 mM of L-glutamine (Euroclone) and antibiotics (100 U/ml penicillin, 100 g/ml streptomycin) (Euroclone).

The MTT assay [3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide] [10] (Sigma) was used to establish cell viability after treatment with the eye drops. The HaCaT were plated at a confluence of 6 × 10⁴ cells per point, in 24-well plates, and treated, in triplicate, with three different amounts of the ozonated oil solution in liposomes at different concentrations (50 µl, 100 µl and 200 µl) three times a day for 3 days. The treatment with the eye drops was performed for 10 min, after which the pharmaceutical product was removed from the plate and the cells were incubated again with fresh medium.

After 72 hours of treatment, the MTT solution was added at a concentration of 0.5 mg/ml, and incubated for 3 hours at 37˚C. The formazan crystals were finally solubilised in DMSO (Sigma) and absorbance was read with TECAN M-200 at a wavelength of 570 nm.

The cells were counted with a 0.5% trypan blue solution in a Burker chamber.

The cell images were captured with Cytation^TM 5 Cell Imaging Multi-Mode Reader (BioTeK).

The bactericide activity for Staphylococcus aureus at the bacterial concentration of 150 CFU/ml was 80% with 400 µl of liposome-vehiculated ozonated oil, 76% with 200 µl of liposome-vehiculated ozonated oil, 54% with 100 µl of liposome-vehiculated ozonated oil, 46% with 50 µl of liposome-vehiculated ozonated oil (Figure 1(a), Figure 1(b) and Figure 2). The bactericide action against Pseudomonas aeruginosa was lower. Indeed no bactericide activity was highlighted at the bacterial concentration of 150 CFU/ml but at the concentration of 15 CFU/ml. The bactericide activity for Pseudomonas aeruginosa was 60% with 400 µl of liposome-vehiculated ozonated oil, 46% with 200 µl of liposome-vehiculated ozonated oil, 28% with 100 µl of liposome-vehiculated ozonated oil, with 50 µl of liposome-vehiculated ozonated oil growth exceeded 400 CFU (~1%) (Figures 3(a)-(c) and Figure 4).

In order to assess the toxicity of ozonated oil in liposomes, the ophthalmic solution was tested on normal epithelial keratinocyte cells, HaCaT. The cell viability test by means of MTT assay [10] made it possible to observe that ozonated oil in liposomes does not inhibit cell growth at all, as shown in Figure 5(a). On the contrary, a quantitative assessment, obtained by cell count, shows how 100 µl and 200 µl of

ozonated oil solution in liposomes is able to promote cell growth after 72 hours of treatment compared to the control (Figure 5(b)). The data are supported by visual analysis via Cytation^TM 5 Cell Imaging Multi-Mode Reader (BioTeK) (Figure 5(c)).