*4.5. Cytotoxicity Assay of the Formulations*

The cytotoxicity of the tested formulations was estimated by MTT assay in PK and Vero cell lines [32,33]. A monolayer of cells (1 × 104 cells/well) grown in 96-well plates was treated with different concentrations of tested formulations (from 0 to 2000 μg/mL) and cultured at 37 ◦C in a CO2-incubator for six days; untreated cells were used as controls. Then 20 μL of MTT solution (5 mg/mL) (methylthiazolyltetrazolium bromide, Sigma, Saint-Louis, MO, USA) was added in each well followed by incubation at 37 ◦C for one hour. The MTT solution was removed, and 150 μL/well of isopropanol was added. Optical density (OD) was measured at 540 nm using an ELISA microplate reader (Labsystems Multiskan RC, Vantaa, Finland) with a reference absorbance at 620 nm. The viability of the cells was calculated as (ODt)/(ODc) × 100%, where ODt and ODc correspond to the absorbance of treated and control cells, respectively. Cytotoxicity was expressed as 50% cytotoxic concentration (CC50) of the tested formulation that reduced the viability of treated cells by 50% compared with control cells. Experiments were performed in triplicate and repeated three times.

#### *4.6. Antiviral Activity ASSAY of Formulations*

The antiviral activity of formulations against TBEV and HSV-1 was evaluated using cytopathic effect (CPE) inhibition assay in PK and Vero cells, respectively. The overnight monolayer of cells grown on 96-well plates (1 × 104 cells/well) was infected with 100 <sup>μ</sup>l/well of serial dilutions of virus suspension (10−1–10−8) and simultaneously treated by formulations (100 μL/well in triplicate) of different concentrations (from 0 to 400 μg/mL) for one hour at 37 ◦C. After virus absorption, the virus–formulations mixture was removed; the cells were washed, and a maintenance medium with 1% FBS was added. The plates were kept at 37 ◦C in CO2-incubator for six days for TBEV or for three days for HSV-1 until CPE appeared. The antiviral activity was determined by the difference of the viral titers between treated infected cells and untreated infected cells and expressed as the inhibition rate (IR, %), using the formula [27]: IR = (1 − *T*/*C*) × 100, where *T* is the antilog of the formulations-treated viral titers and *C* is the antilog of the control (without formulations) viral titers. The concentration of formulation that reduced the virus-induced CPE by 50% was determined as the 50% inhibitory concentration (IC50). The selectivity index (SI) was calculated as the ratio of CC50 to IC50. Experiments were repeated three times.

## *4.7. Time-of-Formulation-Addition assay*

PK and Vero cells were grown in 96-well plates (1 × <sup>10</sup><sup>4</sup> cells/well). An infectious dose of both TBEV and HSV-1 was of 100 TCID50/mL, tested formulations were used at a concentration of 20 μg/mL, and acyclovir was used at a concentration of 10 μg/mL. The plates were kept at 37 ◦C in CO2-incubator for six days for TBEV or for three days for HSV-1 until 80–90% CPE was observed in viruses control compared with cells control.


Antiviral activity of formulations was assessed by MTT test, and the viral inhibition rate (IR, %) was calculated according to the formula [34], IR = (ODtv − ODcv)/(ODcd − Odcv) × 100, where ODtv represents the OD of cells infected with virus and treated with the test formulation; ODcv corresponds to the OD of the untreated virus-infected cells, and ODcd is OD of control (untreated and noninfected) cells.
