Loading of Oregano Oil in Natural Nanogel and Preliminary Studies on Its Antiviral Activity on Betacoronavirus 1

Figures

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  Figure 1. Histogram of the average size (according to DLS analysis) (a) and TEM (b) of the oregano oil-loaded nanogel.

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  Figure 2. FTIR spectra of oregano oil (black), empty chitosan–albumin nanogel (blue) and oregano oil-loaded chitosan–albumin nanogel (magenta) registered in KBr pellets.

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  Figure 3. XRD pattern of oregano oil (black), empty chitosan–albumin nanogel (blue) and oregano oil-loaded nanogel (magenta).

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  Figure 4. In vitro release profile of oregano oil from the developed nanogel in phosphate buffer medium (pH = 7.4).

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  Figure 5. Cytotoxicity of OrO-NG on MDBK cells after 90 h of incubation: (a) in vitro cytotoxicity of OrO-NG and NG on MDBK cells after 90 h of incubation—the grey column represents the untreated control which is the same for OrO-NG and NG; (b) dose-response curve used for calculation of the median inhibitory concentration (IC50) of OrO-NP on MDBK cells after 90 h of incubation presented in Table 2; (c) response surface analysis (RSA) for IC50 = const; (d) RSA for m = const. Legend: OrO-NG—oregano oil-loaded nanogel; NG—empty nanogel; Fa—cytotoxic effect; Exp. Data—experimental data; m—hillslope.

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  Figure 6. Antiviral activity of OrO-NG on bovine coronavirus after 90 h of treatment: (a) direct treatment with or without preincubation—MDBK cells, treated with BCoCV as follows: adding of the virus and OrO-NG or NG together to the cell culture without or after 1 h preincubation of the virus with OrO-NG or NG, the grey column represents the untreated control and the black column represents cells incubated with BCoV; (b) pretreatment—incubation of the cultures with 10 µg/mL OrO-NG or NG before inoculation with the virus and adding of 5, 10, 15 or 20 µg/mL OrO-NG or NG after removing the virus; treatment—incubation of the cells with BCoV followed by treatment with OrO-NG or the corresponding concentrations of NG, the grey column represents the untreated control and the black column represents cells incubated with BCoV; (c) Antiviral activity of OrO-NG on BCoV—survival curve; (d) response surface analysis based on the applied concentration (dose) and the hillslope for IC50 = const; (e)—response surface analysis based on the dose and IC50 value for m = const. Legend: OrO-NG—oregano oil-loaded nanogel; NG—empty nanogel; Fa—cytotoxic effect; Exp. Data—experimental data; m—hillslope.

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  Figure 7. Cytopathic effect of bovine coronavirus on MDBK cells with and without OrO-NG treatment—200× magnification. Legend: (a) BcoV, bovine coronavirus; (b) 15 μg/mL OrO-NG; (c) 20 μg/mL OrO-NG; (d) 25 μg/mL OrO-NG; (e) 30 μg/mL OrO-NG; (f) Control cells without virus and treatment.

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  Figure 8. Digital droplet PCR for enumeration of viral particles in OrO-NG treated samples and the virus control—dilution of the cDNA. Legend: (a) Number of generated droplets per sample; (b) Histogram of the positive and negative droplets in each sample—the fluorescent positive droplets (blue) are separated on the histogram from the non-fluorescent negative droplets (grey) through the threshold line (pink color) generated by the data analysis with the QuantaSoft® Software of the ddPCR device.