Potent Inhibition of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by photosensitizers

The pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has exploded since December 2019, and causes more than 2 million death with more than 95 million people infected as of Jan. 21th, 2021 globally1,2. Angiotensin-converting enzyme 2 (ACE2), expressed in the lungs, arteries, heart, kidney, intestines, and nasal epithelium3, has been shown to be the primary entry point targeted by the surface spike protein of SARS-CoV-2. Currently, no proven antiviral treatment for SARS-CoV-2 infection is available. In this study, we screened a number of photosensitizers for photodynamic viral inactivation, and found compounds pentalysine β-carbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with half-maximal effective concentrations (EC50) values at nanomolar level. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

no proven antiviral treatment for SARS-CoV-2 infection is available. In this study, we screened a number of photosensitizers for photodynamic viral inactivation, and found compounds pentalysine βcarbonylphthalocyanine zinc (ZnPc5K) and chlorin e6 (ce6) potently inhibited the viral infection and replication in vitro with half-maximal effective concentrations (EC50) values at nanomolar level. Such viral inactivation strategy is implementable, and has unique advantages, including resistance to virus mutations, affordability compared to the monoclonal antibodies, and lack of long-term toxicity.

Background
Firstly, we established a facile pseudoviral entry assay. In this system, the pseudovirus containing the spike (S) protein of SARS-CoV-2 and EGFP protein encapsulated on HIV viral capsid (Fig.1a)  Then, we screened a number of representative photosensitizers for their antiviral effects using the pseudoviral entry assay. The photosensitizers (Fig.1c) were mixed with the pseudoviruses at the indicated concentrations and illuminated with a light-emitting diode (LED) light source to a light dose of 0.48 J/cm 2 , then the mixture was incubated with ACE2-293T cells for 48 hr, followed by image analysis on a high-content imager. Two photosensitizers (Fig.1c, ZnPc5K and ce6) were identi ed to completely abolish the infection of the pseudoviruses to the host cells at doses of 1.0 μM and with LED light illumination ( Fig.1c). Importantly, these two photosensitizers did not affect the cell proliferation, adhesion, and morphology at this concentration even under the light illumination (Fig.S1b). As a control, the photosensitizers did not affect the infection under the otherwise identical condition in the absence of LED light illumination (Fig.S1c). Furthermore, we measured the inhibitory potencies of the two photosensitizers, and identi ed the EC50 values of 6.2 nM and 177 nM for ZnPc5K and ce6, respectively ( Fig.1c). Meanwhile, the 90% effective concentrations (EC 90 ) values were 114 nM and 515 nM for ZnPc5K and ce6, respectively (Fig.1c). We then studied if the photodynamic effect could inactivate the pseudovirus inside the ACE2-293T cells. We incubated the pseudoviruses with the ACE2-293T cells and photosensitizers for 4 hr to allow viral infection to the cells, followed by LED illumination (0.48 J/cm 2 ). The cells were allowed to continue to grow for 2 days, followed by image analysis on a high-content imager. The results showed that both photosensitizers reduced the green uorescence signal of the cells in a dose dependent manner (Fig.1d). For ZnPc5K, the pseudoviral uorescence signal was reduced bỹ 40% at a dose of 740 nM, while only about 20% reduction of pseudoviral uorescence signal for ce6 was observed. Moreover, both photosensitizers showed little toxicity to the infected host cells as detected by nucleus stain (Fig.S1d).
To further con rm the antiviral e ciency of the two compounds, we used live SARS-CoV-2 to test the antiviral activity in vitro. The photosensitizers (ZnPc5K or ce6) were incubated with SARS-CoV-2 viruses, followed by LED light illumination (0.48 J/cm 2 ). The mixture was then allowed to infect the Vero cells for 2 days, and the inactivation rates were evaluated by quanti cation of viral copy numbers in the cell supernatant via quantitative RT-PCR (qRT-PCR) and further con rmed with immuno uorescence staining ( Fig.2a, b). Similar results with the pseudoviral entry assay were found. ZnPc5K and ce6 e ciently were shown almost no phototoxicity at concentration lower than 5 μM (Fig.2c). Next, we studied the photodynamic effect on the viruses in the host cells. The live virus and photosensitizers were mixed and added to the Vero cells for 4 hr incubation at 37ºC. The cell-containing mixtures were illuminated with LED once and allowed to continue to grow for 44 hr before qRT-PCR viral quanti cation. The results showed that ZnPc5K and ce6 reduced the virus load by 60% and 10%, respectively, at 50 nM concentration (Fig.S3). The ce6 required higher concentration (100 nM) to reduce the virus load by 50%. The host cells maintained normal morphology during photodynamic treatment (Fig.S3), suggesting no toxicity at these concentrations. Cytotoxicity was further studied on broblast (HELF cells) and ACE2-293T cell lines, and ZnPc5K showed no phototoxicity at concentration lower than 5 μM (Fig.S4), while ce6 showed no phototoxicity at concentration lower than 1 μM (Fig.S4).
Here we demonstrate that photodynamic method is possible to inactivate SARS-CoV-2 outside and within the host cells without toxicity. Such speci city of photosensitizer is likely due to the detoxifying capability of the cells, but not the virus. The cells contain a number of enzymes like superoxide dismutase and catalase, which can remove the reactive oxygen species (ROS) generated by the photosensitizers, making them more resilient to the insults from photosensitizers. ZnPc5K showed high antiviral e cacy while maintaining no toxicity to cells. This photosensitizer has low toxicity (acute oral toxicity > 5000 mg/kg) with no irritation to eyes or skins, and causes no mutagenesis to cells 4 . It has been demonstrated to be effective in antibacterial and anti-tumor cells in many studies. Generally, phthalocyanine type compound is quite safe and has been used as a color dye for out ts and underwear in fabric industry for over a century. Phthalocyanine type compound has also been used as an anti-tumor drug (Photosense®) for cancer treatment in Ukraine since 1990s 5  Phthalocyanine was also approved by FDA as Indirect Additives used in food contact substances. A surprised nding from this work is the high potency of ZnPc5K to inactivate virus with an EC 50 of 177 nM, much higher than the typical micromolar range potencies for antimicrobial or antitumor applications of photosensitizers [9][10][11] . A number of studies found that photosensitizer-like structure with large ring-like organic compound, including methylene blue 12  The current method of virus inactivation in our study has several advantages. First, this method works independent of viral sequences, and resistant to virus mutations. Photosensitizers use ROS to damage viral envelope proteins and/or nucleic acids. Such mechanism is most likely not sensitive to the mutations of SARS-CoV-2, which has been reported, e.g., D614G of the spike protein 17 . Second, the debris or the fragments generated from the virus by ROS could stimulate host immune defense. Third, photodynamic method to inactivate virus can be more affordable than other therapeutics like monoclonal antibody, which can be an important factor for preventive use at home. Other advantages of photodynamic inactivation include the lack of long-term toxicity, the ability to remove virus in a very short time, less damage to adjacent tissues, and high repeatability without viral resistance.
In summary, we demonstrated the high potencies (nM of EC50) of photosensitizers in the inactivation of SARS-CoV-2 virus. Importantly, the method can also reduce the virus load inside the human host cells with a large safety margin to cells.

Chemicals and reagents
ZnPc5K was synthesized accordingly to our previously published procedure 18 . ce6 was from Cayman Chemical. Polybrene was from Sigma-Aldrich, USA. Puromycin was from Invitrogen, USA. Oligo nucleotides were synthesized by Sangon Biotech, China.

Cells and Viruses
293T and Vero cells negative for mycoplasma were obtained from ATCC and cultured at 37°C with 5% CO 2 in Dulbecco's Modi ed Eagle medium (DMEM) supplemented with 10% FBS (Gibco) and 1x penicillin- Pseudovirus cellular inactivation assay was carried out by mixing SARS-CoV-2 pseudovirus with serially diluted photosensitizers. The mixture was added into ACE2-293T cells, and 4 hr later illuminated by LED with a power of 4mW/cm 2 for 2 min. After incubation in CO 2 incubator for 48 hr, images (EGFP for virus and Hoechst33342 for nucleus stain) were taken by Operetta High Content Imaging System to detect EGFP intensity.
An automated analysis method was used based on Operetta CLS™ System. To measure bioactivity of SARS-CoV-2 pseudovirus, we calculated the sum of EGFP uorescent intensity for pseudovirus extracellular inactivation assay and the mean of EGFP uorescent intensity for pseudovirus cellular inactivation assay. For such analysis, we rst de ned nucleus with Hoechst-staining images, then cytoplasm with bright eld images, and nally calculated the EGFP uorescent intensity by going through all EGFP spots inside cells. The virus inactivation percentage was calculated by subtracting the intensity of EGFP of each group from the control of no photosensitizer, and divided by the control uorescence. Error bars represent the SD of triplicates in one experiment.
Evaluation of antiviral activities of the photosensitizers using the live virus Viral RNAs were extracted from the samples using the QIAamp RNA Viral Kit (Qiagen, Heiden, Germany), and qRT-PCR was performed using a commercial kit (Genrui-bio) targeting the S and N genes. The specimens were considered positive if the Ct value was less than 38.0, and negative if the results were undetermined. Specimens with a Ct higher than 38 were repeated. The specimen was considered positive if the repeat results were the same as the initial result and between 38 and 40. If the repeat Ct was undetectable, the specimen was considered negative.
Immuno uorescence assay 19 Vero cells were xed in 4% formaldehyde at 48th h.p.i. Then cells were permeabilized in 0.5% Triton X-100, blocked in 5% BSA in PBS, and then probed with the plasma of this patient or healthy control at a dilution of 1:500 for 1 h at room temperature. The cells were washed three times with PBS and then incubated with either goat anti-human IgG conjugated with Alexa uor 488 at a dilution of 1:500 for 1 h (Invitrogen). The cells were then washed and stained with Hoechest33342 (Invitrogen) to detect nuclei. Fluorescence images were obtained and analyzed using EVOS FL Auto Imaging System (Invitrogen).

CCK-8 assay
The cell viability/cytotoxicity was measured by Cell Counting Kit-8 (CCK-8) (Meilunbio). Brie y, the cells were seeded in ViewPlate-96-well plate (Perkin Elmer), and treated with serially diluted photosensitizers, followed by LED illumination with a power of 4mW/cm 2 for 2 min at 4 hr later. After incubation in CO2 incubator for 24 hr, 10 μl of CCK-8 solution was added to each well, and the 96-well plate was incubated at 37 °C for 1 hr. Normal DMEM with CCK-8 solution and the cells with no photosensitizer served as the blank control and normal control, respectively. The cell viability was calculated according to the product manufacturer's instructions using the OD450nm measured on a microplate reader (SpectraMax® i3x, Molecular Devices). All doses were done at triplicates.