key: cord-0277673-cbdsesom authors: Kaya, Kemal; Khalil, Mohammed I.; Fetrow, Benjamin; Fritz, Hugh; Jagadesan, Pradeepkumar; Bondu, Virginie; Ista, Linnea; Chi, Eva Y.; Schanze, Kirk S.; Whitten, David G.; Kell, Alison M. title: Rapid and Effective Inactivation of SARS-CoV-2 by a Cationic Conjugated Oligomer with Visible Light: Studies of Antiviral Activity in Solutions and on Supports date: 2021-10-19 journal: bioRxiv DOI: 10.1101/2021.10.18.464882 sha: 0ada1eeac52870f557d1f76be07a7faa5b9d6e2c doc_id: 277673 cord_uid: cbdsesom This paper presents results of a study of a new cationic oligomer that contains end groups and a chromophore affording inactivation of SARS-Cov-2 by visible light irradiation in solution or as a solid coating on wipes paper and glass fiber filtration substrates. A key finding of this study is that the cationic oligomer with a central thiophene ring and imidazolium charged groups give outstanding performance in both killing of E. coli bacterial cells and inactivation of the virus at very short times. Our introduction of cationic N-Methyl Imidazolium groups enhances the light-activation process for both E. coli and SARS-Cov-2 but dampens the dark killing of the bacteria and eliminates the dark inactivation of the virus. For the studies with this oligomer in solution at concentration of 1 μg/mL and E. coli we obtain 3 log killing of the bacteria with 10 min irradiation with LuzChem cool white lights (mimicking indoor illumination). With the oligomer in solution at a concentration of 10 μg/mL, we observe 4 logs inactivation (99.99 %) in 5 minutes of irradiation and total inactivation after 10 min. The oligomer is quite active against E. coli on oligomer-coated wipes papers and glass fiber filter supports. The SARS-Cov-2 is also inactivated by the oligomer coated glass fiber filter papers. This study indicates that these oligomer-coated materials may be very useful as wipes and filtration materials. The World Health Organization (WHO) has declared the novel coronavirus outbreak of 2019 to be a global pandemic. To date, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 229 million people globally, and claimed more than 4 million deaths in the world. 1 Several vaccines have been developed that are very effective against SARS-CoV-2. Although more than six billion doses of the vaccines have been administered, a high percentage of the world population has been reluctant to take the vaccines, despite scientific evidence of their effectiveness. It appears that getting a sufficient fraction to be vaccinated and thus obtain "herd" immunity is difficult and likely impossible for the current SARS-CoV-2 and future viral pathogens. The virus is known to spread via respiratory droplets 2 , close contact with infected individuals, and by viral contamination of frequently touched surfaces. 3 As such, government and health authorities have urged the use of personal protective equipment (PPE), such as masks, the avoidance of indoor congregation, and disinfection of surfaces. 4 While mask wearing has been shown to mitigate the spread of Covid-19, there is also much resistance to the wearing of masks and closure of schools and popular venues such as theaters, restaurants and businesses. There is a clear need for antimicrobial agents that inactivate SARS-CoV-2 and minimize its spread via surface contact and air. Several recent works have shown new methods for disinfecting surfaces against coronavirus infections. 5 In a study published in 2020, Poon, Ducker, et al. reported Cu2O bound with polyurethane rapidly reduce virus titer and prevent re-infection for periods of days to weeks. 6 Other studies indicate the virus can be inactivated but in many cases the components are volatile and cannot provide lasting disinfection. 4 Currently, there are few treatment options and very few long-lasting disinfectants available for inactivating the virus before it can spread and infect humans. While masks and protective clothing and "social distancing" may offer some protection, their use has not always halted or slowed the spread. Our group has studied oligomers of poly-phenylene ethynylene 7 , 8 , 9 as potent antimicrobial agents 10,11,12 against bacteria, fungi and viruses. 13, 14, 15, 16 Specifically, we have found that the non-enveloped viruses attacking E. coli exhibit potent antiviral activity under both light-activation and dark treatment with several cationic oligomers and polyelectrolytes. 14 For both dark and light-activation the level of inactivation reaches several logs. 14 We have recently developed a series of antimicrobial reagents that are effective against SARS-CoV-2 in aqueous suspensions when activated by ultraviolet or visible light. 16 These materials include a series of cationic poly-phenylene-ethynylenes and polythiophenes and smaller cationic and anionic oligomers. 16 In contrast to our earlier findings these materials only exhibited virus inactivation under visible or uv light irradiation. 16 We attributed the lack of dark antiviral application to the viral envelope of the SARS-Cov-2. While it appears that the dark activity involving membrane penetration or destruction cannot occur with these materials, they must engage in a non-damaging ground state docking process that enables singlet oxygen formation from the excited triplet of the oligomer or polymer and subsequent penetration of singlet oxygen into the virus core. Subsequent to publishing our first paper we have focused our attention to developing stronger oligomeric antimicrobials, particularly on their speed of inactivation 17, 18, 19 and employment in other formats. 20, 21, 22 Herein, we report the development of a new oligomer (1) that we have found to very rapidly and effectively inactivate SARS-CoV-2 in aqueous solutions with visible light where these materials are soluble and in combination with scaffolds in filtration trials which offer the potential for water and air disinfection processes. In this paper we discuss a method of destroying, inactivating, reducing the infectivity of, or otherwise inhibiting the activity of SARS-CoV-2 by irradiation of 1 with visible light. In follow up to our initial studies, we tested new oligomers and polymers with SARS-CoV-2 and found that two of five materials tested with visible light activation ("cool white" light tubes from LuzChem) were very effective in inactivating the virus with these lamps, which are described as a good model for interior lighting by artificial light sources. 16 In designing new oligomers for potential applications against SARS-CoV-2 we considered that the simplest oligomers we found effective were an end-only OPE (EO-OPE-1) where the chromophore and charged groups may be easily tuned by straight-forward synthetic methods. We designed and synthesized two OPEs substituted with a central thiophene ring and potent but different charged end groups. While we could synthesize each of these oligomers from a common dibromo derivative, we found that the DABCO derivative 2 was impractical for use due to its insolubility in water. However, oligomer 1 is readily soluble in water and easy to use. Gram-negative E.coli were investigated under cool white light irradiation at 1 μg/mL and 10 μg/mL concentrations in order to minimize potential inner filter effect 23,24 and for different times (5, 10 and 30 min). Log reduction or "log kill" was determined by serial dilution assay where colony forming units were quantified. The results are summarized in Figure 1 and Table 1 . Noticeably, for all control experiments, there is no killing activity in dark (control dark) and no killing activity from the light that is used (control light). When 10 μg/mL concentration of the compound was used, there is very little killing under dark conditions. However, compound 1 is extremely reactive against E. coli and exhibits enhanced antimicrobial activity when it is irradiated with cool white light. We have observed 4 log kills after 5 min irradiation (SI Figure S1 ) and 6 log kills for the OPE family that shows 5 log killing enhancement with light irradiation. Interestingly, when we have run the biocidal experiment at 1 μg/mL concentration for 5, 10 and 30 min, dark reactivity of compound 1 has disappeared. Having demonstrated effective antimicrobial activity of 1 in solution, we decided to coat commercial wipes paper and glass fiber filters with 1 and test them for biocidal activity. These materials were coated with 1 and the coated materials were cut so that the final effective concentration of 1-10 μg/mL was used for biocidal experiments. A piece of glass fiber (3 x 6 cm 2 , 127 mg) was cut and immersed into 50 mL of conjugated cationic oligomer solution (100 μg/mL) for 12 hrs. After soaking, the filter was removed from solution and dried at room temperature for 3 days. UV Vis spectra was used to determine how much compound was absorbed by glass fiber. Because of the high absorbance of compound 1 at 100 μg/mL, this solution was diluted to 10 μg/mL and UV spectra were taken (SI Figure S2 ). Considering the dilution, calculations were done accordingly in order to determine the amount of compound 1 that is absorbed by glass fiber. To test whether or not coated glass fiber is releasing 1 into water or PBS, coated glass fiber was immersed into milliQ water and PBS. There was no detectible release by UV into either water or PBS. The antibacterial activity of both coated samples shows 5 logs of inactivation, 1 log less inactivation than in solutions of 1. When the materials were coated, it was found that there was no release of 1 by rinsing of the materials with water or PBS. Thus, we can conclude that 1 is effective against bacteria while adsorbed to the support and that the inactivation is not due to released 1 in the solution. For the studies of 1 vs SARS-CoV-2, we used a procedure similar to that described above. However only samples of 1 in solution added to aqueous suspensions of SARS-CoV-2 and samples of 1 coated on glass fiber filter mater were studied. Results A stock solution of compound 1 (100 μg/mL) was prepared by dissolving compound 1 in water. A piece of glass fiber filter (3 x 6 cm 2 , 127 mg, HYDAC company)) was cut and immersed into 50 mL of the OPE solution for 12 hrs. After soaking, the filter was removed from solution and dried at room temperature for 3 days. In order to calculate how much compound is absorbed by the glass fiber filter, UV-Vis spectra of the solution were taken before and after immersing and removing the filter (SI Figure S2 ). 0.84 mg of coated glass fiber filter was cut to use for a final oligomer concentration of 10 μg/mL in the SARS-CoV-2 plaque assay as described below to determine antiviral activity. A stock solution of compound 1 (25 μg/mL) was prepared by dissolving the compound in water. A commercial wipe paper sheet (3 x 6 cm, Spontech) was cut and immersed into 30 mL of oligomer solution for 2 hr. After 2 hr, the paper was removed from solution and dried at room temperature for 3 days. In order to calculate how much compound is absorbed by the wipe paper. UV-Vis spectra of the solution were taken before and after immersing paper (SI Figure S3 ). 1.6 mg of coated wipe (2 x 2 mm) was cut for a concentration of 10 μg/mL for subsequent testing. Vero A solution of compound was diluted to 10 μg/mL in DMEM culture media (4% FBS). SARS-CoV-2 was added to compound-media solution at a final concentration of 1x10 5 pfu/mL. Virus was incubated in media supplemented with a volume of RNA/DNAsefree water equal to that used for the compound dilutions served as a negative control. Samples were then incubated at room temperature inside a LuzChem light chamber and exposed to the "cool white" light for the specified time intervals. Infectious virus present in the samples was then quantified by plaque assay. 16 For experiments testing compound-coated filter inactivation, a media solution of SARS-CoV-2 at a concentration of 1x10 5 pfu/mL was added to 1.5 mL tubes containing 1 coated filter at a solution concentration of 10 μg/mL and gently agitated by pipetting up and down multiple times. As a negative control, filters not coated with 1 were incubated with viral preparations and treated similarly. Samples were then incubated at room temperature inside a LuzChem light chamber and exposed to "cool white" light for the specified time intervals. Infectious virus present in the samples was then quantified by plaque assay. 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