Effects of Usnic Acid to Prevent Infections by Creating a Protective Barrier in an In Vitro Study

Abstract

Nasal sprays are medical devices useful for preventing infection and the subsequent spread of airborne pathogens. The effectiveness of these devices depends on the activity of chosen compounds which can create a physical barrier against viral uptake as well as incorporate different substances with antiviral activity. Among antiviral compounds, UA, a dibenzofuran derived from lichens, has the mechanical ability to modify its structure by creating a branch capable of forming a protective barrier. The mechanical ability of UA to protect cells from virus infection was investigated by analyzing the branching capacity of UA, and then the protection mechanism in an in vitro model was also studied. As expected, UA at 37 °C was able to create a barrier confirming its ramification property. At the same time, UA was able to block the infection of Vero E6 and HNEpC cells by interfering with a biological interaction between cells and viruses as revealed also by the UA quantification. Therefore, UA can block virus activity through a mechanical barrier effect without altering the physiological nasal homeostasis. The findings of this research could be of great relevance in view of the growing alarm regarding the spread of airborne viral diseases.

Keywords: mechanical barrier; nasal spray; usnic acid; virus protection.

Figure 1

Crosslinking analysis by staining. The images reported are an example obtained from each treatment obtained at 40× magnification from four technical replicates.

Figure 2

Analysis of infection of Vero E6 cells with VSV-based pseudovirus SARS-CoV-2 (MOI 0.1) pre-treated with the different UA-based formulations. In (A), the images reported were an example of each stimulation for GFP fluorescence expression obtained at 40× magnification by Paula (Leica DM1000); in (B) the analysis of GFP expression in cells obtained by the image analysis using Image J Software and the results were expressed as mean ± SD (%) compared to infected control cells (0 line) of four independent experiments. In (C), cell viability measured by MTT test after infection and expressed as mean ± SD (%) compared to uninfected control cells (0 line) obtained from four independent experiments. In (D) UA spectrophotometry quantification at 281 nm by Tecan (Infinite 200 Pro Mplex, Tecan, Milan, Italy) and data are expressed as ng/mL ± SD of four independent experiments. Solution 1 = 150 mcg/mL β-CD +HPMC; Solution 2 = 150 mcg/mL UA+ 150 mcg/mL β-CD + 1% HPMC; Solution 3 = 75 mcg/mL UA+ 150 mcg/mL β-CD + 1% HPMC; HEXEDRA+ = 75 mcg/mL UA+ 150 mcg/mL β-CD + 0.1% Tpmix + 1% HPMC. * p < 0.05 vs. Control; ** p < 0.05 vs. Solution 1; ^y p < 0.05 vs. Solution 2; ^yy p < 0.05 vs. Solution 3; ^φ p < 0.05 vs. Infected control.

Figure 3

Analysis of infection of Vero E6 cells with VSV-based pseudovirus SARS-CoV-2 (MOI 0.1) pre-treated with the different UA-based formulations. The images reported were an example of each stimulation for GFP fluorescence expression obtained at 40× magnification by Paula (Leica DM1000). The positive cells were counted analyzing GFP expression in cells by the Image J software and the results were expressed as mean ± SD (%) compared to infected control cells (0 line) of four independent experiments. The abbreviations are the same reported in Figure 2. * p < 0.05 vs. infected control; ** p < 0.05 vs. Solution 1; ^y p < 0.05 vs. Solution 2; ^yy p < 0.05 vs. Solution 3.

Figure 4

Analysis of infection of Vero E6 and HNEpC cells with VSV-based pseudovirus SARS-CoV-2 (MOI 0.1) pre-treated with the different UA-based formulations. The images reported were an example of each stimulation for GFP fluorescence expression obtained at 40× magnification by Paula (Leica DM1000). The positive cells were counted analyzing GFP expression in cells by the Image J software and the results were expressed as mean ± SD (%) compared to infected control cells (0 line) of four independent experiments. The abbreviations are the same reported in Figure 2. * p < 0.05 vs. infected control; ** p < 0.05 vs. Solution 1; ^y p < 0.05 vs. Solution 2.