Antiviral photodynamic therapy: Inactivation and inhibition of SARS-CoV-2 in vitro using methylene blue and Radachlorin

Abstract

Introduction: Recently, the COVID-19 pandemic has spread globally, necessitating the development of new methods for its prevention and treatment. The purpose of this study was to evaluate the antiviral activity of photodynamic therapy (PDT) against SARS-CoV-2 in vitro.

Methods: Vero E6 cells and SARS-CoV-2 isolated in Russia were used for PDT with methylene blue (MB) and Radachlorin. A continuous laser with wavelength λ = 662 nm in doses of 16 J/cm² and 40 J/cm² laser irradiation was used for PDT of a viral suspension and SARS-CoV-2-infected cells. The direct cytopathogenic effect of SARS-CoV-2 was evaluated via light microscopy to calculate the TCID₅₀ in the samples and perform statistical analysis.

Results: Viral suspensions of SARS-CoV-2 that had a TCID₅₀ greater than 10³ were inactivated by PDT in the presence of MB and Radachlorin. Vero E6 cells were protected from 10⁴ TCID₅₀ of SARS-CoV-2 by PDT post infection. The range of protective concentrations was 1.0-10.0 μg/ml and 0.5-5.0 μg/ml for MB and Radachlorin, respectively. Additionally, it was found that MB and Radachlorin also possess significant antiviral activity even without PDT. The 50 % inhibitory concentration (IC₅₀) against 10² TCID₅₀ of SARS-CoV-2 was found to be 0.22 and 0.33 μg/mL with the addition of MB and Radachlorin, respectively, to cells concomitantly with virus, whereas in the case of applying the photosensitizers at 3.5 h post infection, the IC₅₀ was 0.6 and 2.0 μg/mL for MB and Radachlorin, respectively.

Conclusion: PDT shows high antiviral activity against SARS-CoV-2 when combined with MB and Radachlorin in vitro.

Keywords: Antiviral photodynamic therapy; COVID-19; Methylene blue; Radachlorin; SARS-CoV-2.

Fig. 1

Photodynamic inactivation of SARS-CoV-2 in viral suspensions using MB and Radachlorin. Data are the mean ± SEM of 2 independent MTT tests (3 replicates per point). % viable cells = (abs_sample – abs_blank)/(abs_control – abs_blank) x 100. 1. Monolayers (2 × 10⁵ cells in each well) were infected with a mixture containing 10³ TCID₅₀ of SARS-CoV-2, with the addition of MB at a concentration of 10.0 μg/mL, following laser treatment for 40 s at a 16 J/cm² dose; 2. 10³ TCID₅₀ of SARS-CoV-2, 10.0 μg/mL MB, 100 s, 40 J/cm²; 3. 10³ TCID₅₀ of SARS-CoV-2, 10.0 μg/mL MB, without laser treatment; 4. 10³ TCID₅₀ of SARS-CoV-2, 1.0 μg/mL MB, 40 s, 16 J/cm²; 5. 10³ TCID₅₀ of SARS-CoV-2, 1.0 μg/mL MB, 100 s, 40 J/cm²; 6. 10³ TCID₅₀ of SARS-CoV-2, 1.0 μg/mL MB, without laser treatment; 7. 10³ TCID₅₀ of SARS-CoV-2, 5.0 μg/mL Radachlorin, 40 s, 16 J/cm²; 8. 10³ TCID₅₀ of SARS-CoV-2, 5.0 μg/mL Radachlorin, 100 s, 40 J/cm²; 9. 10³ TCID₅₀ of SARS-CoV-2, 5.0 μg/mL Radahlorin, without laser treatment; 10. 10³ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radachlorin, 40 s, 16 J/cm²; 11. 10³ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radachlorin, 100 s, 40 J/cm²; 12. 10³ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radachlorin, without laser treatment; 13. 10³ TCID₅₀ of SARS-CoV-2, laser irradiation, 40 s, 16 J/cm²; 14. 10³ TCID₅₀ of SARS-CoV-2, laser irradiation, 100 s, 40 J/cm²; 15. 10³ TCID₅₀ of SARS-CoV-2, without laser treatment.

Fig. 2

Microphotographs of Vero E6 monolayers* (magnification × 100) 48 h after infection with SARS-CoV-2 and having undergone PDT. * - 3 flasks/images per group. A) Vero E6 cells infected with 10⁵ TCID₅₀ of the SARS-CoV-2 suspension, followed by treatment with 1.0 μg/mL MB and laser irradiation for 100 s, 40 J/cm²; B) 10⁴ TCID₅₀ of SARS-CoV-2, 1.0 μg/mL MB, 100 s, 40 J/cm²; C) 10⁴ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radachlorin, 100 s, 40 J/cm²; D) 10³ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radachlorin, 100 s, 40 J/cm²; E) 10³ TCID₅₀ of SARS-CoV-2, 1.0 μg/mL MB, without laser treatment; F) 10³ TCID₅₀ of SARS-CoV-2, 0.5 μg/mL Radahlorin, without laser treatment; G) 10³ TCID₅₀ of SARS-CoV-2; H) Uninfected control.