Antiviral Effects of Ophthalmic Formulations

The effect of Oftasecur and Visuprime on HSV-1 infection was evaluated by co-treatment, cell and viral pretreatment and post-treatment via plaque reduction assays in infected cells. In the co-treatment assay, the eye drops at the selected volumes and the viral suspension at a density of 2 × 10³ plaque-forming units/mL (PFU/mL) were simultaneously inoculated on the cell monolayer in DMEM without FBS for 1 h at 37 °C. In cell pre-treatment, cell monolayers were first exposed to ophthalmic solutions for 1 h at 37 °C and then infected with the viral suspension at 2 × 10³ PFU/mL in DMEM without FBS for 1 h at 37 °C. In virus pretreatment, the viral suspension at 2 × 10⁴ PFU/mL was exposed to formulations for 1–10–30 min and 1 h, diluted 1:10 in DMEM without FBS, and used to infect cell monolayers, for 1 h. Lastly, in post-treatment, cells were first infected with the viral suspension at the density of 2 × 10³ PFU/mL in DMEM without FBS for 1 h at 37 °C; then, they were washed and treated with the formulations at the selected volumes for 1 h. Regarding CTRL+, melittin (5 µM) was applied in the co-treatment and virus pretreatment, dextran-sulfate (1 µM) in cell pretreatment, and aciclovir (5 µM) in post-treatment, whereas uninfected cells constituted CTRL−. After the viral adsorption time, the cell monolayer was washed twice with 1 × PBS and covered with a culture medium supplemented with 5% carboxymethylcellulose. After 48 h, Vero CCL-81 cells were fixed with 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) and stained with 0.5% crystal violet (Sigma-Aldrich, St. Louis, MO, USA). The plaques were counted and related to the coincident ones at the CTRL− to obtain the percentage of viral inhibition [31 (link)]. Lastly, the virus pretreatment assay was also confirmed using GFP-engineered HSV-1, exposing to 12.5–3.12 µL of Oftasecur and 6.25–1.56 µL of Visuprime for 1 h, prior to cell infection. Bright-field and fluorescent images were acquired by the Nikon ECLIPSE Ti2-U fluorescence microscope (Nikon Europe B.V., Amsterdam, The Netherlands) after 48 h of exposure. The fluorescence intensity was gained by Cytation 5 plate reader (Cytation 5, BioTek, Milan, Italy).