Antibacterial plastic

Abstract

The bioplastic additives and antibacterial plastics include polyphenols and organic polymers. The polyphenols may be phenols, polyphenols, bioflavenoids, flavonoids, tannins, coumarins, lagmans, quinones, stibene, or curcuminoid stenins. The organic polymers may be chitosan, chitin, cellulose, and keratin. In one advantageous embodiment, the polyphenol is hesperidin and the organic biopolymer is chitosan.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to plastics or polymers, and in particular to antimicrobial plastics or polymers.

[0002] Various surfaces and the materials that make up surfaces are potential breeding grounds for microorganisms such as bacteria and fungi. Additionally, various materials and surfaces may prove to be suitable environments for the survival of viruses.

[0003] The global SARS-CoV-2 pandemic has created a great demand for new technologies and products with antimicrobial properties. Because many consumer products contain some form of plastic or plastic material, plastic additives with antimicrobial properties would be beneficial, including additives that target infectious viral strains like rhinovirus, influenza, SARS, and SARS-CoV-2 (COVID-19), as well as dangerous bacterial strains like E. coli, staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA), and methicillin-sensitive Staphylococcus aureus (MSSA).

[0004] Recently, agents containing antibacterial and antiviral additives have been incorporated into various materials, including plastics. However, the effectiveness of conventional antibacterial agents, including antiviral additives for plastics, is limited. For example, common antibacterial additives currently in use are metals such as zinc and ionic silver, thiazoles, and halogenated compounds. While these additives are effective, they have downsides due to their environmental toxicity. In particular, the current SARS-CoV-2 crisis has raised major concerns that the overuse of these materials and the subsequent dangerous accumulation of these materials in the environment could result in serious long-term exposure consequences.

[0005] There has been a notable lack of innovation in this field regarding microbial-resistant plastics, polymers, and plastic surfaces. For example, the most commonly used antimicrobial plastic additives to date have been metals such as silver, zinc, and copper. The only recent advances have been the simple combination of these metals with themselves or with other ionic or non-reactive compounds. This can be illustrated by the following examples: silver / zinc / copper, silver / citric acid, silver sulfadiazine, silver phosphate, silver ceramic, silver palladium, silver platinum, silver zeolite, silver copper, silver / glass, etc. These combinations are routinely repeated by substituting zinc or copper metal ions for silver.

[0006] Another current limitation in the field of bioplastics (e.g., antimicrobial plastics) is the method of antimicrobial delivery. Current applications of antimicrobial agents rely on integrated migration, where the additive is mixed into the plastic material and simply migrates or diffuses toward the surface of the material due to the inherent incompatibility gradient between the antimicrobial additive and the polymer substrate. This technique and mode of action is not only inefficient, but also creates weaknesses and structural instability in the plastic material itself due to mechanical destabilization of the structural polymerization.

[0007] A further limitation in the field of bioplastics is their inherent acute and chronic toxicity. The main toxicity concerns with the current state-of-the-art technology are not directed at humans, but rather focus on the impacts on aquatic systems from landfill runoff and the potential for disruption to marine systems. Current technologies, silver, zinc, copper, arsenic, triclosan, thiazoles, arsenic, and oxybisphenoxarsine (OBPA), show significant marine toxicity potential.

[0008] Another limitation in this field is thermal degradation. Most of the antimicrobial compounds currently used have thermal decomposition constants close to those of the matrix polymer itself. This is a major drawback, since as the temperature of the bioplastic increases, the mobility of the additive also increases, affecting and increasing the migration rate. This factor significantly impacts the lifetime of the additive, reducing its usefulness.

[0009] Another important limitation is the lack of sophisticated or targeted efficacy. Currently used antibacterial compounds operate by a very simple mechanism. Their antibacterial activity works by interacting with and paralyzing key metabolic enzymes upon exposure. This one-sided and simplistic approach limits their efficacy, duration, and application, especially against non-enveloped microorganisms such as rhinoviruses. A much more sophisticated and multifaceted approach is needed to create improved antibacterial agents. Summary of the Invention

[0010] The present invention relates to bioplastics, including bioplastic materials and / or plastic additives, which, when combined with plastics, including polymers, have antimicrobial properties, including antiviral properties. These properties include, but are not limited to, antiviral properties that create an environment inhospitable to viruses, such as SARS-CoV-2. The additives of the present invention result in plastics that create an environment inhospitable to microorganisms, including viruses, thereby preventing microbial growth and viral persistence.

[0011] In one aspect, the present invention relates to an antimicrobial polymeric additive comprising a polyphenol and an organic polymer. In one advantageous aspect, the polyphenol is hesperidin. In another advantageous aspect, the organic polymer is selected from the group consisting of chitosan (also called chitosene), chitin, cellulose, and keratin.

[0012] In another aspect of the invention, the aforementioned antimicrobial polymers may be added to one or more of the polymers, chitosan and hesperidin.

[0013] In yet another alternative, the polyphenol is 1-3% hesperidin and the organic biopolymer is 1-5% chitosan.

[0014] Without being limited to a specific mechanism of antibacterial activity, polyphenols such as hesperidin form a surface structure similar to a "poison chip" that is inhospitable to microorganisms. For example, when the polyphenols of the present invention are added to a plastic polymer or plastic, the material has antibacterial properties. Therefore, the antibacterial polymer of the present invention, which includes a polyphenol and an organic polymer, can be incorporated as a surface layer, thereby forming an antibacterial surface.

[0015] The polyphenols and organic biopolymers can also be added to paints or formulated into plastics used in medical devices such as implants and bandages.

[0016] In various alternatives, the polyphenol or phenolic compound is hesperidin or any phenol / polyphenol including bioflavenoids, flavinoids, tannins, coumarins, lagmanes, quinones, stibenes, or curcuminoids. [Brief explanation of the drawings]

[0017] The invention will now be described with reference to the following drawings.

[0018] FIG. 1 is a graph comparing microbial growth on various materials according to the present invention.

[0019] FIG. 2 is a graph comparing microbial growth of various materials according to the present invention.

[0020] FIG. 3 is a graph comparing microbial growth of various materials according to the present invention.

[0021] FIG. 4 is a graph comparing microbial growth of various materials according to the present invention.

[0022] FIG. 5 is a graph comparing microbial growth of various materials according to the present invention.

[0023] FIG. 6 is a graph comparing microbial growth of various materials according to the present invention.

[0024] FIG. 7 is a graph comparing microbial growth of various materials according to the present invention.

[0025] Figure 8 is a graph comparing microbial growth on various materials according to the present invention.

[0026] The antimicrobial additives of the present invention are incorporated into plastics to form a bioplastic, a unique combination of materials that is inhospitable to microorganisms. The materials of the present invention are stable and have no known toxicity, making them particularly suitable for use in a variety of materials where antimicrobial properties are desired.

[0027] Without being bound by a specific mechanism of antimicrobial effect, the combination of various additive materials creates a physical structure that proves inhospitable to microorganisms. At the microscopic level, the bioplastics of the present invention have tiny spears within their polymer matrix. These spears or tips can act to actively pierce the cell walls of bacteria (microorganisms). For example, if the organic polymer is chitosan, the tips of the chitosan can function as spears to pierce the cell walls of living microorganisms.

[0028] In one form of antimicrobial polymer, the polymer includes a polyphenol and an organic biopolymer. The organic biopolymer can be chitosan, and the polyphenol can be hesperidin. Advantageously, the chitosan forms fine tips within the polymer's plastic matrix, which serve several functions. First, the chitosan tips are believed to dissolve or perforate microbial membranes. Second, the chitosan tips actively deliver the antimicrobial agent, i.e., hesperidin, to the microbial cell wall. This delivery method is an improvement over methods previously known in the art that rely on more passive transfer. This active nature significantly extends the kill time against microorganisms.

[0029] An advantage of using hesperidin as the polyphenol and chitosan as the organic polymer is that both are stable, with chitosan having a melting point of 290°C and hesperidin having a melting point of 250°C. Another advantage of hesperidin is that it has both thermal and radiation protection properties. These properties improve the thermal and electromagnetic (UVA, UVB, etc.) properties or characteristics of the plastic. Hesperidin also stabilizes thermal degradation due to its antioxidant properties.

[0030] Furthermore, when the antimicrobial polymer comprises chitosan, the chitosan enhances the antimicrobial effect by actively dissolving the antimicrobial film, and also serves as a delivery vehicle for the antimicrobial agent hesperidin, which itself functions as an antimicrobial agent.

[0031] Hesperidin inhibits microbial activity through a multifaceted approach. First, hesperidin inhibits biofilm formation, particularly B-lactamase. Second, hesperidin inhibits viral replication, including polymerase, helicase, and ATPase. Third, hesperidin reduces the autoinduction (aggression) of microbial agents through a direct effect on quorum sensing by inhibiting acyl-homoserine lactase.

[0032] Chitosan is commercially produced by the assimilation of chitin, a structural component of the exoskeletons of crustaceans such as crabs and shrimp, and the cell walls of fungi. The chemical structure of chitin consists of two N-acetylglucosamine units, which repeat to form long chains with β-(1→4) linkages, resulting in the following structure: JPEG2025528436000001.jpg68129

[0033] The chemical structure of chitosan is shown below. JPEG2025528436000002.jpg59115 [Example]

[0034] The present invention is now described with reference to experiments that demonstrate the antimicrobial properties of the bioplastics and / or bioplastic additives of the present invention.

[0035] Experiment 1

[0036] Experimental data

[0037] Plastic samples containing polystyrene were created for testing for SARS-CoV-2 in a Biosafety 3 facility. A control sample was created using polystyrene as the base. Two "test" articles / platforms were also created. The test articles consisted of a polystyrene base and varying concentrations of chitosan and hesperidin. The two formulations tested were as follows: Formulation (1) = Polystyrene (93%), Chitosan (4.66%), Hesperidin (2.33%) Formulation (2) = Polystyrene (97%), Chitosan (2%), Hesperidin (1%)

[0038] The samples were sent to a Biosafety 3 facility for testing for SARS-CoV-2. Testing procedures were specified according to ISO 22196. The samples were tested at both the high and low viral ranges. The high viral range was 2.5 x 10 5And the low range is 2.5×10 3 It was.

[0039] result

[0040] While control samples showed only a slight reduction in virus, test samples demonstrated excellent antiviral activity. Plastics coated with 93% / 4.66% / 2.33% polystyrene / chitosan / hesperidin and 97% / 2% / 1% polystyrene / chitosan / hesperidin reduced SARS-CoV-2 by more than 1 log(10)-fold (90% reduction) in 30 minutes and approximately 2 log(10)-fold (99% reduction) in 2 hours. The antibacterial curves for the test articles against SARS-CoV-2 showed continued logarithmic reduction even after 6 hours.

[0041] The results show an incredibly fast antibacterial effect, with a 90% reduction in viral load within 30 minutes. It's important to note that this 30-minute period is the earliest a sample should be initially inoculated and analyzed in the laboratory. This is consistent with the plastic immediately starting to kill the virus due to its high antibacterial activity at this 30-minute point.

[0042] The rapid antibacterial effect demonstrated in this study is due to the sophisticated synergistic mechanisms employed in the innovative use of chitosan and hesperidin, creating an effective microscopic mechanistic tool that far exceeds the functionality of the one-dimensional approaches described in the current state-of-the-art.

[0043] Experiment 2

[0044] the purpose

[0045] Measure the anti-SARS-CoV-2 efficacy of bioplastic-coated plastics.

[0046] Experimental Overview

[0047] 2.5×10 5 or 2.5 x 10 3 TCID50 of SARS-CoV-2 was applied to the provided coupons in a volume of 400 μl. The material was transferred to an incubator maintained at 250°C and incubated for various periods of time. For 30-second treatments, the material was left on the benchtop in a biosafety cabinet for the appropriate period of time.

[0048] The coupons were incubated at room temperature for 30 seconds, 30 minutes, 2 hours, and 6 hours. The deposited virus suspension was collected, and the coupons were washed three times to a total volume of 1 ml. The collected suspension was diluted to various concentrations and plated on Vero E6 cells to measure TCID50.

[0049] The TCID50 of all recoveries was compared with the TCID50 obtained with control material provided by GRDG.

[0050] Coating / surface to be tested Control PS PS / SLS (3%) PS / SLS (7%) PS / SAP (3%) PS / SAP (7%) PS / SAB (3%) PS / SAB (7%) PS / Chitosan / Hesperidin (93% / 4.66% / 2.33%) PS / Chitosan / Hesperidin (97% / 2% / 1.0%)

[0051] The dose of virus on a surface High dose – 2.5×10 5 TCID50 Low dose – 2.5×10 3 TCID50

[0052] Abbreviation SLS: Sodium lauryl sulfate SAB: Strontium Aluminate Blue SAP: Strontium Aluminate Purple Chi: Chitosan Hesp: Hesperidin PS: Polystyrene

[0053] result

[0054] The experimental data results are shown in the graphs in Figures 1-8.

[0055] Experimental Considerations

[0056] No significant antiviral effect was observed in any sample after 30 seconds of exposure. Furthermore, the high and low inoculation doses (2.5 × 10 5 TCID50 or 2.5 x 10 3 No significant antibacterial effect was observed in either TCID50 or TCID50 within 30 seconds.

[0057] Furthermore, in the PS / SLS and PS / SAB samples, the high inoculum size (2.5 × 10 5 TCID50) or low inoculum (2.5 × 10 3 No significant antiviral effect was observed in either TCID50.

[0058] For the PS / Chi / Hesp ​​samples (both concentrations), the high inoculum (2.5 × 10 5 A significant antiviral effect (p<0.05) was observed at low inoculum doses (TCID50). A trend toward an antiviral effect was observed at low inoculum doses, but this did not reach statistical significance (p=0.08).

[0059] For the PS / SLS and PS / SAB samples, the high inoculum size (2.5 × 10 5 TCID50) or low inoculum (2.5 × 10 3 No significant antiviral effect was observed in either TCID50.

[0060] In the PS / Chi / Hesp ​​samples (both concentrations), the high inoculum (2.5 × 10 5 TCID50) and low inoculum (2.5 × 10 3 A significant antiviral effect was observed in both TCID50 and TCID50 (p<0.001).

[0061] In the PS / SLS (3%) and PS / SAB samples, the high inoculum (2.5 × 10 5 TCID50) or low inoculum (2.5 × 10 3 No significant antiviral effect was observed in either the PS / SLS (7%) or PS / SLS (7%) samples (TCID50). However, a slight decrease in viral titer was observed in the PS / SLS (7%) samples (p<0.05).

[0062] In the PS / Chi / Hesp ​​samples (both concentrations), the high inoculum (2.5 × 10 5 TCID50) and low inoculum (2.5 × 10 3 Significant antiviral effects were observed in both TCID50 and TCID50 (p<0.001).

[0063] Test limitations

[0064] These tests (6 samples) were performed using a protocol according to ISO 22196. Therefore, the starting SARS-CoV-2 concentration was 2.5 x 10 per 1 inch x 1 inch surface area in a 400 μl volume. 5 The incubation is carried out at 25°C and a relative humidity of approximately 50%.

[0065] All samples contained a lower concentration of virus (2.5 × 10 3 This low concentration of virus does not comply with ISO standards.

[0066] Plastics coated with PS / chitosan / hesperidin 93% / 4.66% / 2.33% and PS / chitosan / hesperidin 97% / 2% / 1% maintained a 1 log kill rate of SARS-CoV-2 in 30 minutes. 10more than double (90% reduction), and approximately 2 log 10 2x reduction (99% reduction).

[0067] Plastic coated with 7% PS / SLS retained SARS-CoV-2 at approximately 1 log in 6 hours. 10 2-fold reduction (90% reduction).

[0068] The following is a summary of the test conditions:

[0069] (Testing the antiviral properties of bioplastics)

[0070] the purpose Initial screening to determine the potential antiviral properties of target materials (bioplastics) using SARS-CoV-2 (WA1 / USA-2020) as the target microorganism. A reduction of at least 1 log in TCID50 titer is considered for further characterization (based on ISO 22196-SARS-CoV-2).

[0071] subject Control material / plastic or film (1 tube, 3 replicates) Coated or treated plastic or film (12 rolls, repeated 3 times)

[0072] summary The material was wiped with 70% ethanol for at least 3 minutes of contact time. A predefined SARS-CoV-2 titer (2.5 × 10) was measured. 3 and 2.5 x 10 5400 μl of medium containing PFU / ml of WA1 / USA-2020 was applied (in triplicate) to a 1-inch x 1-inch area of ​​each type of material in a B2 biosafety cabinet in a certified BSL-3 laboratory. The film / material was covered with a thin film and incubated in a humidified chamber at 25°C for 30 seconds, 30 minutes, 6 hours, and 24 hours. The material was then washed three times with 300 μl of medium and harvested. 100 μl of the harvested medium (stock and 1:10, 1:100, and 1:1000 dilutions) was applied to Vero E6 cells. After 72 hours of incubation, the viable virus titer was counted using a TCID50 assay. This test was repeated three times. A 1-log reduction in virus titer compared to the inoculum and control material was considered significant in this initial screening.

[0073] material Sixty samples of 3% and 7% control material were tested at both high and low concentrations.

[0074] SARS-CoV-2 Infectivity Assay / TCID50 General Protocol - Modified from the MJN Sutton Lab 1. Prepare a 20 mg / mL stock solution in DMSO. Aliquot and store at -20°C. 2. 24 hours prior to infection, seed the desired cell type into a 24-well or 96-well plate. Place in a 37°C CO2 incubator. 3. On the day of infection, prepare a compound working solution of the desired concentration and dilute the virus stock solution to the desired MOI. 4. At -2 hours, 0 hours, or +2 hours, add virus suspension, treatment solution, or a mixture of the two, as appropriate, to individual wells. 5. The virus is incubated with the cells for 1 hour at 37°C and the virus suspension is aspirated. 6. The medium is replaced with treatment compound or plain medium, and the cells are incubated for the desired period of time. 7. After incubation, collect the supernatant from each well and centrifuge to purify. 8. For 24-well TCID50 assay, seed Vero E6 cells 24 hours before the assay. Place in a 37°C CO2 incubator. 9. Dilute the stock solution 10-fold in steps to 10 -10 Dilute to 10. Aspirate the old medium from the Vero E6 cells and add 900 μL / well of fresh medium containing 2% FBS. 11. The lowest concentration of the supernatant sample (10 -10 ), add 100 μL of each sample to four labeled Vero E6 wells. Successively, add 100 μL of each sample to the corresponding wells in order from the lowest concentration sample to the highest concentration sample. 12. Include untreated wells as negative controls. 13. Incubate at 37°C for 72 hours, read the cytopathic effect, and calculate the TCID50 value.

[0075] Experiment 3

[0076] Samples 1-8 were tested for antibacterial activity. Samples 1-8 are as follows: Sample 1 - Untreated paper; Sample 2 - Coated paper - Strontium aluminate blue 3%; Sample 3 - Coated paper - SLS 2%; Sample 4 - Coated paper - Chitosan 3%; Sample 5 - Coated paper - Hesperidin 1.29%; Sample 6 - Coated paper - Chitosan 1.5% / Hesperidin 1%; Sample 7 - SBSC untreated control; and Sample 8 - Cytotoxicity optimization.

[0077] ISO 21702 specifies a method for assessing the antiviral activity of non-porous surfaces.

[0078] Each product was tested in clean (no additional soil) testing conditions. JPEG2025528436000003.jpg238163JPEG2025528436000004.jpg234162JPEG2025528436000005.jpg161162

[0079] ISO 21702:2019 – Determination of antiviral activity on plastics and other non-porous surfaces

[0080] The ISO 21702 method is used to evaluate the antiviral efficacy of non-porous products. Testing can incorporate various exposure times, soils, virus types, and other variables depending on the test criteria or the specific needs of the product. The most common test conditions employ a standard method protocol requiring 24 hours of exposure to the test material, depending on the product's intended use. The test virus is prepared and titered prior to testing. The resulting inoculum is then used to expose the test material to the virus.

[0081] ISO 21702 Optimization of Intrinsic Cytotoxicity

[0082] Antiviral testing requires the use of host cells for the growth and enumeration of virus concentrations. Viral infection requires that the host cells be viable and biologically intact. Successful infection by the virus results in replication and eventual loss of the host cell through lysis. This process provides a means of measuring the virus. Host cell cytotoxicity due to carryover of chemicals from the test sample can affect the biological viability of the host cells and interfere with processes necessary after the cells are exposed to the virus. This interference is generally considered the inherent cytotoxicity of the test sample.

[0083] The test is performed by incubating the test sample with a neutralizing recovery solution for 30 seconds, and then exposing host cells to eight concentration levels of the recovery solution. Intrinsic cytotoxicity is determined by a decrease in cell culture viability.

[0084] Project List 1. Untreated Paper Baseline (32) 2. Coated paper - Strontium aluminate blue 3% (24 times) 3. Coated paper - SLS 2% (24 times) 4. Coated paper - Chitosan 3% (24 times) 5. Coated Paper - Hesperidin 1.29% (24 repeats) 6. Coated paper - Chitosan 1.5% / Hesperidin 1% (24 times) 7. SBSC-untreated control 8. Optimization of Cytotoxicity

[0085] test

[0086] Inoculum preparation

[0087] A suspension of virus with a known titer is prepared at least 10 6 Prepare to a concentration of TCID50 / ml. Do not use viruses that have been passaged more than 10 times from the original seed culture.

[0088] Experimental conditions

[0089] After inoculation, the samples are incubated at 25°C + / - 1°C (unless otherwise specified). This incubation is done to prevent the inoculum from drying out while in contact with the test surface. After the incubation period, the virus is recovered in neutralizing medium and diluted for culture.

[0090] Recovery of virus from test specimens

[0091] Two time points are established for each test item: Immediately after inoculation, the inoculated sample is placed in a vial, 10 ml of the selected neutralizer solution is added, vortexed, and the wash is collected.

[0092] A second collection is made after the intended incubation period (24 hours), after which the sample is placed in a vial containing 10 ml of neutralizer solution and vortexed.

[0093] After neutralizing the test samples, aliquots of the samples are withdrawn and used to determine the infectious titer after each incubation period.

[0094] Preparation of virus inoculum

[0095] Virus is prepared by growing the desired host cell culture monolayer to approximately 90% confluency. The cell culture is then washed and inoculated with the prepared virus inoculum. The cells are maintained at 35-37°C in a 5% CO2 atmosphere and observed for viral replication, indicated by cytotoxic effects. The cell culture is harvested within 4-7 days, and cell debris is removed by centrifugation at 1 k × g for 5 minutes. The virus culture is then serially cultured and titered according to the TCID50 method.

[0096] TCID50 cell plate

[0097] Cell plates are prepared for use in measuring virus concentration. In a 96-well 200 μl plate, approximately 3 μl of host cells specific for the virus are added. 5 The cells are inoculated at a concentration of 100 μl / ml. The plates are incubated in complete growth medium appropriate for the cell line. Once the cells have settled, they are ready to be inoculated with the virus. The plates are washed twice with PBS, and then 100 μl of virus maintenance medium appropriate for the virus being tested is added. The cell plates are now ready to be inoculated with the harvested test samples.

[0098] Inoculation of test specimens

[0099] The test materials are inoculated by pipetting 0.2 ml of the desired inoculum preparation onto the surface of the test material. The inoculum solution is then covered with a piece of parafilm pre-cut to approximately 40 mm x 40 mm -F / -2 mm. The parafilm is gently pressed so that the test inoculum spreads to the edges but does not exceed the boundaries of the sandwiched surface. Duplicates of each sample are placed in sterile Petri dishes, which are then placed in separate containers for incubation.

[0100] Keeping inoculated test specimens warm

[0101] Viral inoculum

[0102] The test materials are kept at a temperature of 25 + / - 1°C in a closed, moist environment with a relative humidity of at least 90%.

[0103] Inoculum recovery

[0104] Virus recovery

[0105] After the incubation period, the samples were removed from the incubator, and neutralizing medium was added directly to the samples and triturated three to four times. The total amount of neutralizing medium added was 10 ml. The neutralizing medium was recovered, and 1 ml of the solution was added to a 96-well 2 ml sample block in a column-specific manner, allowing for sample processing by serial dilution. Gel filtration was performed on samples that tested positive in the intrinsic cytotoxicity test. Gel filtration was performed by recovering the neutralizing solution from the sample and passing 1 ml of the solution through a 2 ml prepared gel filtration bed.

[0106] Virus quantification

[0107] Virus counts are determined using a serial endpoint dilution process according to the TCID50 method. The sample collection block contains 1 ml of neutralized inoculum in column 1 of an eight-column 96-well block. The 96-well block is prepared by placing 900 μl in each well of columns 2 through 8. After the sample is added to column 1, a 1 / 10 serial dilution is performed by removing 100 μl from column 1 and pipetting it into column 2, followed by trituration and mixing. This process is continued with each subsequent column until a total of seven serial dilutions have been performed.

[0108] The diluted samples are then used to inoculate prepared 96-well cell plates. For 96-well blocks, each well is added to two 96-well cell plate sample wells. This is done by pipetting 100 μl of the diluted, recovered virus sample into the prepared cell plate, for a total volume of 200 μl in the plate. Once all wells have been transferred to the cell plates, the plates are incubated at 35°C and 5% CO2 for 2–6 days, depending on the characteristics of the progression of viral cytopathic cytopathic events (CPE). After the incubation period, the plates are removed from the incubator, fixed, and stained to identify CPE compared to intact cell monolayers. The CPE per well is counted and used to determine the TCID50 based on the representative dilution factor exhibited by the CPE for each virus sample.

[0109] reagent Dulbecco's Modified Eagle's Medium (DMEM; EM-1) Soybean Casein Lecithin Polysorbate 80 Medium (SCDLP) Phosphate-buffered saline (PBS) Formaldehyde solution (3.7%) Crystal Violet (0.5%) fetal bovine serum Virus maintenance medium trypsin Ethylenediaminetetraacetic acid solution (EDTA) Deionized Laboratory RO Water

[0110] Test Organism (by Method) (Stock ID / Lot Number) ISO 21702:2019 – Determination of antiviral activity on plastics and other non-porous surfaces Human coronavirus (OC43); VR-1558 70035458 (Betacoronavirus 1; strain: OC43)

[0111] Sample preparation

[0112] Each test article was prepared according to the requirements of the analytical method.

[0113] Each test sample was prepared in triplicate for each time point.

[0114] The samples were cut to approximately 50 x 50 mm where available. Sample variability was adjusted as required for standard testing. Differences in sample properties were recorded in the summary report.

[0115] The test sample is ideally flat and non-hydrophobic, and the surface of the sample can be layered with an inoculum.

[0116] calculation

[0117] The recovered virus inoculum was subjected to end-point dilution using serial log10 dilution factors. Based on the end-point dilution results, TCID50 (Spearman-Karber, modified by MA Ramakrishnan) was used to measure the concentration of the inoculated virus and obtain the CTE of the host cells. This represents the mean end-point dilution of the host cell monolayer exhibiting CTE.

[0118] Log10 50% endpoint dilution = -[(total number of CTE wells / total number of replicate dilutions) + 0.5] × Log dilution factor R = -[Total CTE / Replicate counts per dilution) + 0.5] × Log dilution factor R = Log 50% endpoint dilution Total CTE is the number of viable viruses (cells / cm) recovered from untreated test specimens immediately after inoculation. 2 ) is the average of the base 10 logarithms of Replicate counts per dilution are the number of replicate wells inoculated at each dilution. Log dilution factor is the dilution factor used in each serial dilution (usually 10-fold or log10(10)=1).

[0119] Antiviral activity value R = U(t24) - C(t24) R = Antiviral activity value C(t24) = mean of the base 10 logarithms of the three infectious titer values ​​after 24 hours from untreated material U(t24) = the mean of the base 10 logarithms of the three infectious titer values ​​after the contact time (24 hours) with the treated (test) sample

[0120] statistical methods

[0121] Replicate data are used for calculations according to the Spearman-Karber method, without further statistical analysis.

[0122] The results of this experiment are summarized in Tables 1 to 6 below.

[0123] Table 1 : JPEG2025528436000006.jpg236121

[0124] Table 2 : JPEG2025528436000007.jpg165166

[0125] Table 3 JPEG2025528436000008.jpg165157

[0126] While the present invention has been described above in connection with preferred embodiments thereof, those skilled in the art will recognize that variations and modifications can be made to these preferred embodiments without departing from the scope and spirit of the invention.

Claims

1. Antimicrobial polymers, including polyphenols and organic biopolymers.

2. 2. The antimicrobial polymer of claim 1, wherein the polyphenol is hesperidin.

3. 10. The antimicrobial polymer of claim 1, wherein the organic biopolymer is selected from the group consisting of chitosan, chitin, cellulose, and keratin.

4. 4. The antimicrobial polymer of claim 3, comprising, by weight percentage, 1-5% of the organic biopolymer chitosan and 1-3% of the polyphenol hesperidin.

5. 10. The antimicrobial polymer of claim 1, further comprising one or more polymers selected from the group consisting of polyethylene terephthalate (PET), high density polyethylene (HDPE), polyvinyl chloride (PVC), low density polyethylene (LDPE), polypropylene (PP), and polystyrene (PS).

6. 6. The antimicrobial polymer of claim 5, comprising, by weight percentage, 1-5% of the organic biopolymer chitosan and 1-3% of the polyphenol hesperidin.

7. 7. The antimicrobial polymer of claim 6, wherein the one or more polymers are present in a weight percent of 92 to 98%.

8. 6. The antimicrobial polymer of claim 5, comprising, by weight percent, 93-97% polystyrene, 2-5% chitosan of the organic biopolymer, and 1-3% hesperidin of the polyphenol.

9. 6. The antimicrobial polymer of claim 5, comprising, by weight percent, 93% polystyrene, 4.66% of the organic biopolymer chitosan, and 2.33% of the polyphenol hesperidin.

10. 6. The antimicrobial polymer of claim 5, comprising, by weight percent, 97% polystyrene, 2% of the organic biopolymer chitosan, and 1% of the polyphenol hesperidin.

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