Photodynamic disinfection of SARS-CoV-2 clinical samples using a methylene blue formulation

Catarina S Lobo, Paulo Rodrigues-Santos, Dina Pereira, Jisette Núñez, João C D Trêpa, David Lopes Sousa, Jorge Vaz Lourenço, Maria Filomena Coelho, Luis Pereira de Almeida, José Saraiva da Cunha, Luis G Arnaut, Catarina S Lobo, Paulo Rodrigues-Santos, Dina Pereira, Jisette Núñez, João C D Trêpa, David Lopes Sousa, Jorge Vaz Lourenço, Maria Filomena Coelho, Luis Pereira de Almeida, José Saraiva da Cunha, Luis G Arnaut

Abstract

The amplitude of the coronavirus disease 2019 (COVID-19) pandemic motivated global efforts to find therapeutics that avert severe forms of this illness. The urgency of the medical needs privileged repositioning of approved medicines. Methylene blue (MB) has been in clinical use for a century and proved especially useful as a photosensitizer for photodynamic disinfection (PDI). We describe the use of MB to photo-inactivate SARS-CoV-2 in samples collected from COVID-19 patients. One minute of treatment can reduce the percentage inhibition of amplification by 99.99% under conditions of low cytotoxicity. We employed a pseudotyped lentiviral vector (LVs) encoding the luciferase reporter gene and exhibiting the S protein of SARS-CoV-2 at its surface, to infect human ACE2-expressing HEK293T cells. Pre-treatment of LVs with MB-PDI prevented infection at low micromolar MB concentrations and 1 min of illumination. These results reveal the potential of MB-PDI to reduce viral loads in the nasal cavity and oropharynx in the early stages of COVID-19, which may be employed to curb the transmission and severity of the disease.

Trial registration: ClinicalTrials.gov NCT04615936.

Keywords: Antimicrobial photodynamic disinfection; Methylene blue; Photodisinfection; Photodynamic inactivation; Pseudotyped lentiviral vector; SARS-CoV-2.

Conflict of interest statement

On behalf of all authors, the corresponding author states that there is no conflict of interest.

© 2022. The Author(s), under exclusive licence to European Photochemistry Association, European Society for Photobiology.

Figures

Fig. 1
Fig. 1
Design of the PDI of virus. a SARS-CoV-2 in clinical samples. b SARS-CoV-2 pseudotyped lentiviral vectors (LVs) encoding for luciferase and exhibiting the S protein at the surface. VTM viral transport medium
Fig. 2
Fig. 2
PDI of three independent clinical samples. a MB-1:20 and 103.2 J (i.e., 4 min illumination); b MB-1:20 and 25.8 J (i.e., 1 min illumination); c MB-1:100 and 25.8 J (i.e., 1 min illumination). V = virus only; V + L = virus and light for 4 or 8 min; V + MB1:20 = virus and MB-1:20; V + L + MB1:20 = virus and light and MB-1:20; V + L + MB1:100 = virus and light and MB-1:100; Controls: virus alone or in the presence of MB without light, or in the presence of light but without MB
Fig. 3
Fig. 3
Toxicity of the MB formulation towards HaCaT cells after 5 min of incubation, in the dark or exposed to 25.8 J at 670 nm. CTR untreated cells, L light
Fig. 4
Fig. 4
Infection of HEK-293 T-hACE2 cells with SARS-CoV-2 pseudotyped lentivirus (LV). a Cell viability. b Ratio between chemiluminescence intensity and cell viability in %. C: cells with the addition of PBS; V: addition of LVs; MB1: [MB] = 3.2 µM; MB2: [MB] = 16 µM; L: light of 25 J

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