Purpose SARS-CoV-2-infected individuals may be asymptomatic, and therefore, the virus is highly contagious. We aimed to develop an agent to control viral replication in the upper respiratory tract and to prevent progression of the disease into the lower airways as well as inter-individual transmission. For this purpose, we investigated the antibacterial and antiviral activities of our novel nanobubble ozonated hyaluronic acid-decorated liposomal (NOHAL) solution, developed by using nanotechnology. Methods The MIC levels of NOHAL solution were determined on blood agar cultures of Staphylococcus aureus (ATCC 6538), Streptococcus pneumoniae (ATCC 49619) and Escherichia coli (ATCC 25922). The in vitro anti-viral activity of NOHAL solution was studied using recombinant SARS-CoV-2 copies of the original virus, grown in Vero cells generated by reverse genetic technology. Human primary lung epithelial cells obtained by bronchoscopy or lung resection were used for cell viability tests using flow cytometry analysis. The cytotoxicity testing was performed using the BALB/c 3T3 (CCL-163) cell line. Skin, oral, nasal and ocular irritation tests were performed using New Zealand albino rabbits, Syrian hamsters, BALB c mice and New Zealand albino rabbits of both sexes. Results Bacterial growth was prevented by NOHAL solution in a time-/dose-dependent manner. In vivo or in vitro experiments did not show any toxicity of NOHAL solution. No cytotoxicity was recorded on cell viability. No skin, oral, nasal or ocular toxicities were recorded. In addition, in a SARS-CoV-2 mouse infection model, NOHAL solution diminished the viral RNA levels effectively in nasopharyngeal and lung samples after its prophylactic intranasal application. Conclusion NOHAL solution has the potential to reduce or prevent the spread of SARS-CoV-2 through the nose and/or oral cavity. The clinical efficacy of this solution needs to be tested in order to determine its efficacy in the early phase of COVID-19.

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Backrounds: In our study, a nanoparticle liposome molecule with patent application number TR2021004032 was used, and the Minimum Inhibitor Concentration (MIC) was found to be 1562 ppm. According to the ASTM F 1980 standard, it has been determined that the nanoparticle liposome solution kept at 37 days and 55 oC in return for one-year stability preserves its effectiveness. Our study aimed to show that the newly developed solution maintains its effectiveness for a long time. Methods: In this study, a nanobubble ozone liposome solution containing 2% ZnCl2 was used. The aging tests were conducted according to the ASTM F 1980 [1] standards. The minimum inhibitory concentration (MIC) level of the nanobubble ozone liposome solution with 2% ZnCl2 was determined as 1.562 ppm for strains of Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922) by the CLSI M07 A9 [2] standard test method. To assess the time-dependent antibacterial effect of the nanobubble liposome solution with 2% ZnCl2, the solution’s efficiency at a concentration of 2000 ppm and for different time intervals was tested on strains of Salmonella enterica subsp. enterica (ATCC® 14028™) and Listeria monocytogenes (ATCC® 7644™). Results: The results showed that the antibacterial activity of the strains of S. enterica subsp. enterica started at the end of the 10th minute and the solution was effective after 30 minutes. For strains of L. monocytogenes, it was observed that the activity started at the end of the 2nd minute and the product was effective after the 10th minute. According to the ASTM F 1980 standards, it was found that the nanobubble ozone liposome solution retained its effectiveness in one-year stability tests. Conclusion: As a result, the nanoparticle liposome solution, a new product, does not lose its stability and effectiveness for a long time, contrary to what is known. Although the half-life of gaseous ozone is as short as 20 minutes, the stability in the nanoparticle liposome solution has been determined as at least one year. Since nanoparticle liposome solution is a natural and slow-release product, nanobubble ozone liposome solution with 2% ZnCl2 may be used as a newly developed agent against contaminations in food processing facilities caused by biofilm-forming microorganisms through the use in disinfections of surfaces that are in direct contact with food products.

Objective: In our study, a nanoparticle liposome molecule with patent application number TR201804452A2 was used, and the Minimum Inhibitor Concentration (MIC) was found to be 1562 ppm. According to the ASTM F 1980 standard, it has been determined that the nanoparticle liposome solution kept at 37 days and 55 oC in return for one-year stability preserves its effectiveness. Our study aimed to show that the newly developed solution maintains its effectiveness for a long time. Methods: CLSI M07-A10 (Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Tenth ed. Approved Standard) standard test method of the nanoparticle liposome solution developed with a technique different from the standard ozonation mechanisms, and antibacterial tests were performed by modifying the contact time and the MIC value of the solution, and its effect on time has been determined. For the stability test of the nanoparticle liposome solution, it was kept at 55 oC for 37 days in return for one-year stability according to the ASTM F 1980 standard. Results: MIC of nanoparticular ozone solution CLSI M07-A10 standard test method for S. aureus (ATCC 25923) and E. coli (ATCC 25922) strains by modifying contact time It was determined as 1.562 ppm. For S. aureus (ATCC 25923), at the end of the first hour, it was determined that the activity started at 2000 and 1750 ppm nanoparticle liposome solution concentration. For E. coli (ATCC 25922) it was determined that the activity started at the 10th minute at 2000 ppm nanoparticular ozone solution concentration. The solution was still effective at the end of one year according to the ASTM F 1980 standard in terms of effectiveness. Conclusions: As a result, the nanoparticle liposome solution, a new product, does not lose its stability and effectiveness for a long time, contrary to what is known. Although the half-life of gaseous ozone is as short as 20 minutes, the stability in the nanoparticle liposome solution has been determined as at least one year. Since nanoparticle liposome solution is a natural and slow-release product, it is thought that it can create a barrier in mucosal membranes in regions such as the nose, throat, eye and ear with solutions to be prepared in appropriate doses thus preventing bacteria from settling. Keywords: Nanoparticle liposomes, ozone, antibacterial efficiency, Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922)