Method for preparing antimicrobial coating composition, antimicrobial coating composition and use thereof for imparting antimicrobial properties to substrate surfaces

Abstract

The present invention relates to a method for preparing an antimicrobial coating composition, comprising the following steps: a. providing an aqueous dispersion comprising graphite, at least one oxidizing agent and optionally at least one antimicrobial agent; b. mixing the aqueous dispersion from step a at a mixing speed equal to or greater than 1000 rpm to subject the dispersion to high shear homogenization to obtain an antibacterial aqueous dispersion comprising graphene oxide; c. mixing the aqueous antimicrobial dispersion with at least one film-forming agent to obtain an antimicrobial coating composition. The present invention also relates to a composition obtainable by the above method and its use for imparting antimicrobial properties to a substrate surface.

Description

Technical Field

[0001] The present invention relates to a method for preparing an antimicrobial coating composition, an antimicrobial coating composition and use thereof for imparting antimicrobial properties to a substrate surface. Background Art

[0002] The present invention relates to a method for preparing an antimicrobial coating composition, an antimicrobial coating composition and use thereof for imparting antimicrobial properties to a substrate surface.

[0003] It is known that a considerable number of infections occur through contact between human subjects and surfaces infected with bacteria or viruses, such as surfaces of consumer product containers, transport vehicles, furniture, public spaces, equipment, clothing, or surfaces of medical devices (prostheses, catheters, bandages, etc.). In fact, these surfaces contain trace compounds that are nutritious for microorganisms (sugars, phosphorus, oils, etc.), which favor the proliferation of bacterial colonies and the adhesion of viral particles.

[0004] In order to reduce the risk of infection, it is known that surfaces exposed to possible microbial contamination are coated with antimicrobial coatings, in particular paints, which act by preventing the adhesion of microorganisms or by reducing their proliferation through a cytotoxic effect (biocidal effect).

[0005] Antimicrobial coatings are generally obtained by applying a composition (usually in liquid form) to a coating surface. Typically, the coating composition contains: (1) at least one film former (also known as a binder), (2) a volatile component (e.g., a solvent), (3) a pigment, and (4) an additive. In antimicrobial coating compositions, the antimicrobial effect is generally achieved by incorporating one or more additives having antimicrobial properties into the composition.

[0006] The antimicrobial efficacy of the coating depends, among other factors, on the compatibility of the antimicrobial agent used with the other components of the coating composition, in particular with the film former. Furthermore, from a manufacturing point of view, it is important that the antimicrobial agent can be easily dispersed in the coating composition and that the resulting dispersion is sufficiently stable over time, i.e., it does not undergo phase separation or precipitation phenomena over a sufficiently long period of time.

[0007] In the prior art, graphene-based materials, in particular graphene in an oxidized form (graphene oxide) are known and used as antimicrobial agents in antimicrobial coating compositions. An example of such a use of graphene is the commercial product "Dr.Wall" of Graphene.CA, a water-based acrylic paint containing a combination of graphene and TiO2.

[0008] Further examples of using graphene as an antimicrobial agent in coating compositions are described in Graphene Oxide Coatings as Tools to Prevention Microbial Biofilm Formation on Medical Device by Cacaci M. et al. (2019), in: Donelli G. (eds) Advances in Microbiology, Infectious Diseases and Public Health. Advances in Experimental Medicine and Biology, Vol. 1282. Springer, Cham. https: / / doi.org / 10.1007 / 5584_2019_434.

[0009] A limitation of the graphene-containing coating compositions known from the prior art is the limited antimicrobial efficacy of the coatings obtainable in terms of reduced antimicrobial action and the duration of this action over time.

[0010] In view of the above-mentioned prior art, there is therefore a perceived need for antimicrobial coating compositions, particularly antimicrobial coating compositions containing graphene oxide as an antimicrobial agent, which overcome the disadvantages of compositions known in the prior art.

[0011] In particular, it is desirable to have coating compositions which have a more effective and longer lasting antimicrobial effect than prior art compositions.

[0012] It would also be desirable to have an antimicrobial coating composition that could be easily, quickly and economically prepared. Summary of the invention

[0013] The Applicant has now discovered that this and other objects (which will be better described below) can be achieved by subjecting an aqueous dispersion of graphite to an oxidation and exfoliation process under certain operating conditions, which allows obtaining an aqueous dispersion of graphene oxide that, in addition to being homogeneous and stable over time, can also be easily mixed with a film-forming agent in a wide range of weight ratios. The aqueous dispersion of graphite contains at least one oxidizing agent (e.g., hydrogen peroxide) and is subjected to a high shear homogenization step (equal to or greater than 1000 rpm), during which exfoliation of the graphite occurs, forming graphene nanosheets, accompanied by oxidation of the graphene to graphene oxide (hereinafter also referred to as GO alone).

[0014] The coating composition obtained from the aforementioned aqueous dispersion of graphene oxide can be applied to a substrate surface to form a coating film, the antimicrobial effect of which is excellent in biocidal efficacy and more durable than the antimicrobial effect of compositions known in the prior art.

[0015] It has also been observed that the antimicrobial effect of the coating can be increased by including one or more conventional antimicrobial agents in the graphene oxide-containing coating composition.

[0016] Therefore, according to a first aspect, the present invention relates to a method for preparing an antimicrobial coating composition according to claim 1 .

[0017] According to a second aspect, the present invention relates to an antimicrobial coating composition according to claim 13 obtainable by the above process.

[0018] According to a third aspect, the present invention relates to the use of an antimicrobial coating composition according to claim 14 for imparting antimicrobial properties to a substrate.

[0019] According to a fourth aspect, the present invention relates to a method of imparting antimicrobial properties to a substrate according to claim 15 .

[0020] The dependent claims define additional features of the above-mentioned aspects of the invention.

[0021] The features and advantages of the method according to the invention will become more apparent from the following description.The description of the embodiments and the following examples are provided for the sole purpose of illustrating the invention and should not be construed in any sense as limiting the scope of protection defined by the appended claims.

[0022] The limits and numerical ranges expressed in the present specification and claims also include the mentioned numerical value or values. In addition, all values ​​or subranges of limits or numerical ranges should be understood to be explicitly included, just as if they were explicitly mentioned.

[0023] The compositions according to the present invention may "comprise," "consist of," or "consist essentially of" the essential and optional components described in the specification and the appended claims. The expression "consisting essentially of" means that the composition or component may include additional ingredients, but only to the extent that the additional ingredients do not materially change the basic characteristics of the composition or component.

[0024] For the purpose of this specification and the appended claims, the terms graphene and graphene oxide are to be understood according to the definitions reported in ISO / TS80004-13:2017 (Nanotechnologies-Vocabulary-Part 13:Graphene and related two-dimensional (2D) materials). In particular, in addition to single-layer graphene, the term graphene also includes materials formed by 2 to 10 graphene superposition layers, such as double-layer graphene, "few-layer graphene" consisting of 3 to 10 graphene layers, and graphene nanosheets consisting of graphene superposition layers, with a thickness ranging from 1 nm to 3 nm and a lateral size of 100 nm to 100 micrometers.

[0025] For the purpose of this specification and the appended claims, the term "antimicrobial agent" refers to a substance capable of inactivating any biochemical function of microorganisms (understood as micron-sized organisms such as fungi, bacteria and viruses).

[0026] For the purpose of the specification and the appended claims, the molecular weight of a polymeric substance is expressed as the average molecular weight MW as determined by gel permeation chromatography (GPC).

[0027] According to the present invention, the method for preparing the antimicrobial coating composition comprises the following steps in sequence:

[0028] a. providing an aqueous dispersion comprising graphite, at least one oxidizing agent and optionally at least one antimicrobial agent;

[0029] b. high shear homogenization of the aqueous dispersion from step a by mixing the dispersion at a mixing speed equal to or greater than 1000 rpm to obtain an antimicrobial aqueous dispersion comprising graphene oxide;

[0030] c. mixing the aqueous antimicrobial dispersion comprising graphene oxide with at least one film former to obtain an antimicrobial coating composition.

[0031] The graphite used in step a is preferably high surface area graphite (HSAG) having a high degree of crystalline order in the structural layer. Preferably, the surface area of ​​the graphite measured by ASTM D 6556 method is between 200 and 500 m 2 / g range.

[0032] Preferably, the graphite has a turbostratic structure with a relatively low number of stacked layers, for example 30 to 40 (about 35). Preferably, the lateral dimensions of the graphite layers are about 300-400 nm. For example, the dimensions can be determined as described in Biomacromolecules 2017, 18, 3978-3991.

[0033] Graphite preferably has a carbon content equal to or greater than 99 wt. %. For example, the chemical composition of graphite determined by elemental analysis may be as follows: carbon (99.5% w / w), hydrogen (0.4% w / w), nitrogen 0.1% (w / w).

[0034] The concentration of graphite in the aqueous dispersion of step a is preferably in the range of 0.1% to 10%, more preferably in the range of 0.5% to 5%, even more preferably in the range of 0.8% to 2%, the above percentages being weight percentages relative to the weight of the dispersion.

[0035] In step a, the aqueous graphite dispersion comprises at least one oxidizing agent to oxidize the graphene produced by exfoliation to graphene oxide.

[0036] In the absence of any particular limitation, the oxidant may be selected from those commonly used in the prior art for preparing graphene oxide. In one embodiment, the oxidant is selected from oxygen, hydrogen peroxide, air in the presence of potassium hydroxide, nitric acid, tert-butyl peroxide, meta-chloroperbenzoic acid, ozone, sulfuric acid, permanganate ions (e.g., KMnO4), chromate ions (K2Cr2O7), hypochlorite ions, and mixtures thereof.

[0037] In one embodiment, the oxidizing agent is selected from the group consisting of oxygen, hydrogen peroxide, air in the presence of potassium hydroxide, tert-butyl peroxide, meta-chloroperbenzoic acid, ozone, chromate ions (K2Cr2O7), hypochlorite ions, and mixtures thereof.

[0038] In a preferred embodiment, the oxidizing agent is selected from the group consisting of oxygen, hydrogen peroxide, tert-butyl peroxide, m-chloroperbenzoic acid, ozone, chromate ions (K2Cr2O7), hypochlorite ions, and mixtures thereof.

[0039] In one embodiment, the oxidizing agent is preferably hydrogen peroxide (H2O2), optionally mixed with acetic acid.

[0040] Generally, adding acetic acid to H2O2, either together or separately, can provide several advantages, such as:

[0041] 1) Transferring acidic protons for salifying the amino groups of chitosan;

[0042] 2) Increase the "charge potential" (Z potential) of the mixture to increase its stability.

[0043] In addition, acetic acid combined with H2O2 can form peracetic acid, which is very active as an oxidant and antimicrobial agent.

[0044] Typically, hydrogen peroxide is used in the form of an aqueous solution containing H2O2, the concentration of H2O2 expressed as a percentage of the weight of H2O2 relative to the weight of the solution, ranging from 0.5% to 30%, preferably from 2% to 20%, even more preferably from 5% to 15%. Optionally, the oxidizing solution may also contain acetic acid, preferably in a molar ratio of H2O2:CH3COOH ranging from 30:0.01, preferably 20:0.05, even more preferably 10:0.1.

[0045] Preferably, the water of the aqueous dispersion is demineralized water and / or industrial and / or drinking water. Preferably, the conductivity of the demineralized water is in the range of 30 μS / cm to 100 μS / cm, preferably in the range of 45 μS / cm to 50 μS / cm.

[0046] Preferably, the electrical conductivity of the industrial and / or drinking water is in the range of 3000 μS / cm to 100 μS / cm, preferably in the range of 500 μS / cm to 50 μS / cm.

[0047] Preferably, water is present in the antimicrobial dispersion composition in an amount ranging from 70% to 98%, preferably 80% to 95%, more preferably 88% to 92%, the above percentages being by weight relative to the weight of the antimicrobial coating composition.

[0048] High shear homogenization (step b) refers to mixing the graphite dispersion at a mixing speed equal to or greater than 1,000 rpm, preferably equal to or greater than 2,000 rpm, more preferably equal to or greater than 3,000 rpm.

[0049] Preferably, the mixing speed is equal to or less than 10,000 rpm, preferably equal to or less than 9,000 rpm, and preferably equal to or less than 6,000 rpm.

[0050] In one embodiment, the mixing speed is in the range of 4000 rpm to 9000 rpm, more preferably in the range of 5000 rpm to 7000 rpm.

[0051] High shear homogenization can be achieved with conventional commercially available devices such as rotor-stator mixers. These mixers include a high-speed (typically 10 to 50 m-s-1) mixing element (rotor) and a stationary element (stator) positioned close to each other so that the gap between the rotor end and the stator wall is very narrow, typically 100 microns to 3 mm.

[0052] The duration of the high shear homogenization step is preferably in the range of 1 to 24 hours, more preferably in the range of 5 to 10 hours, even more preferably in the range of 8 to 9 hours.

[0053] At the end of step b, an aqueous dispersion comprising graphene oxide in the form of nanosheets is obtained.

[0054] Preferably, the antimicrobial coating composition comprises graphene oxide in an amount ranging from 0.1% to 10%, preferably in the range of 0.5% to 5%, more preferably in the range of 0.8% to 2%, the percentages being by weight relative to the weight of the antimicrobial coating composition.

[0055] The dispersion of graphene oxide is homogeneous and stable, and no phase separation or precipitation is observed over a relatively long time (at least one month) under the conditions of room temperature and atmospheric pressure.

[0056] The graphene oxide dispersion is compatible with the addition of one or more film formers, i.e. it can be mixed with one or more film formers in a wide range of concentrations. The film former has the function of promoting the formation and adhesion of the coating film on the surface of the substrate to be made antimicrobial.

[0057] For the purposes of the present invention, conventional film formers may be used, such as polymeric resins conventionally used in preparing coating compositions, varnishes, paints, enamels, and the like.

[0058] Non-limiting examples of film formers that can be used for the purpose of the present invention are: acrylic resins, vinyl resins, styrene resins, alkyd resins, epoxy resins, polyester resins, polyvinyl acetate resins, and combinations thereof.

[0059] In the antimicrobial coating composition, the film former is preferably present in an amount in the range of 1% to 99%, more preferably in the range of 1.5% to 75%, even more preferably in the range of 3% to 50%, the percentages being weight percentages relative to the weight of the antimicrobial coating composition.

[0060] Preferably, steps ac are performed at a temperature in the range of 25°C to 90°C, more preferably in the range of 35°C to 85°C, even more preferably in the range of 55°C to 80°C.

[0061] Preferably, steps ac are carried out at an absolute pressure in the range of 0.5 to 2 bar, more preferably in the range of 0.8 to 1.2 bar, even more preferably at atmospheric pressure.

[0062] In a preferred embodiment, the antimicrobial coating composition includes at least one additional antimicrobial agent in addition to graphene oxide.

[0063] In another preferred embodiment, the antimicrobial coating composition comprises at least two additional antimicrobial agents in addition to graphene oxide.

[0064] The antimicrobial agent can be selected, for example, from: quaternary ammonium salts; polyethylene glycols with a molecular weight in the range of 200-12,000 g / mol; polysaccharides with antimicrobial properties, preferably chitosan, galactan, mannan and laminarine; metal ions with antimicrobial properties, preferably silver ions, sodium ions and zinc ions; chlorinated isothiazoles; and mixtures thereof.

[0065] In a particularly preferred embodiment, the at least one antimicrobial agent comprises: a quaternary ammonium salt, preferably a benzalkonium chloride salt; a polyethylene glycol having a molecular weight in the range of 200-12,000 g / mol; and silver ions.

[0066] The quaternary ammonium salt can be selected from quaternary ammonium salts containing benzyl groups and having hydrocarbon chains of different lengths (e.g., benzalkonium chloride, benzethonium chloride, benzalkonium bromide). Preferably, the quaternary ammonium salt has the following general formula I

[0067]

[0068] in:

[0069] - R1 and R2 are independently an alkyl group containing 1 to 10 carbon atoms, preferably 1 to 5, even more preferably 1 to 2;

[0070] - R3 is an alkyl group containing 1 to 10 carbon atoms, preferably 1 to 5, even more preferably 1 to 2;

[0071] - n is an integer from 1 to 20, preferably from 6 to 15, even more preferably from 8 to 12;

[0072] -X represents a halogen counterion selected from fluorine, chlorine, bromine, iodine, preferably chlorine and bromine, even more preferably chlorine.

[0073] In one embodiment, the quaternary ammonium salt has a polymeric structure. Examples of such polymeric salts are polydiallyldimethylammonium halide compounds having the following general formula II

[0074]

[0075] in:

[0076] - R1 and R2 are alkyl groups containing 1 to 10 carbon atoms, preferably 1 to 5, even more preferably 1 to 2;

[0077] -n represents the number of repeating units in the polymer structure;

[0078] - n is an integer from 100 to 3000, preferably from 500 to 2500, even more preferably from 1000 to 2000;

[0079] -X represents a halogen counterion selected from fluorine, chlorine, bromine, iodine, preferably chlorine and bromine, even more preferably chlorine.

[0080] The molecular weight Mw of the salt of formula II is preferably from 20,000 g / mol to 1,000,000 g / mol, preferably from 80,000 to 600,000 g / mol, more preferably from 200,000 to 350,000 g / mol.

[0081] The molecular weight Mw of the polyglycol (preferably polyethylene glycol) useful as an antimicrobial agent is 200 to 12,000 g / mol, preferably 250 to 6,000 g / mol, more preferably 300 to 3,000 g / mol.

[0082] The polysaccharides with antimicrobial properties which can be used for the purposes of the present invention are preferably selected from the group consisting of chitosan, galactan, mannan, laminarin and mixtures thereof. The molecular weight Mw of these compounds is preferably between 50,000 g / mol and 500,000 g / mol, more preferably between 150,000 and 350,000 g / mol, even more preferably between 190,000 and 310,000 g / mol.

[0083] The metal ions having antimicrobial properties are preferably selected from the group consisting of silver ions, sodium ions, zinc ions and copper ions, more preferably silver ions, sodium ions and copper ions. Preferably, these ions are present in the antimicrobial coating composition in the form of corresponding metal salts (e.g. nitrates, chlorides, acetates and sulphodiazines). Examples of sulphodiazines are compounds having the following general formula III

[0084]

[0085] in:

[0086] - R1 is equal to hydrogen or an alkyl group containing 1 to 10 carbon atoms, preferably 1 to 5, even more preferably a methyl group;

[0087] -M is selected from silver, zinc and sodium,

[0088] -n is 1 or 2, depending on the valence of the metal counterion M.

[0089] The metal salt may be used as such in the form of an aqueous solution or a solution in a water-soluble solvent based on an alkanolamine (eg ethanolamine) or a diamine (eg ethylenediamine).

[0090] Preferably, in order to obtain an aqueous dispersion that is more stable over time, the metal ions are used in combination with at least one polysaccharide, for example a polysaccharide of the type described above. Preferably, the polysaccharide is added to the solution containing the metal ions in an amount ranging from 0.2% to 5%, preferably from 0.3% to 4%, even more preferably from 0.5% to 2%, the above percentages being by weight relative to the weight of the solution.

[0091] The chlorinated isothiazole is preferably selected from the group consisting of: 5-chloro-2-methyl-2H-isothiazol-3-one and 2-methyl-2H-isothiazol-3-one.

[0092] The isothiazolinone compound may be selected, for example, from compounds having the following general formula IV

[0093]

[0094] in:

[0095] - R1 is equal to hydrogen or an alkyl radical containing 1 to 10 carbon atoms, preferably 1 to 5, even more preferably a methyl radical; preferably, the isothiazolinone compound is a benzisothiazolinone.

[0096] Typically, the antimicrobial agent used in combination with graphene oxide is present in the antimicrobial aqueous dispersion in a total amount ranging from 0.1% to 20%, more preferably 0.5% to 10%, the above percentages being weight percentages relative to the total weight of the antimicrobial aqueous dispersion.

[0097] Preferably, the weight ratio of the antimicrobial agent to the total weight of graphene oxide in the coating composition is in the range of 0.01 to 5, more preferably 0.05 to 2, even more preferably 0.08 to 0.15.

[0098] For the formulation of the coating composition, the antimicrobial agent can be used pure or in the form of an aqueous solution, the concentration of the aqueous solution is expressed as a percentage of the weight of the antimicrobial agent relative to the weight of the solution, preferably in the range of 20% to 90%, preferably in the range of 30% to 80%, even more preferably in the range of 40% to 60%.

[0099] In the case of metal salts, the concentration of the aqueous or water-soluble solvent solution of the antimicrobial agent, expressed as a percentage of the weight of the metal salt relative to the weight of the solution, is preferably in the range of 0.1% to 3%, preferably in the range of 0.2% to 2%, even more preferably in the range of 0.3% to 1.5%.

[0100] In the case of polysaccharides, in particular chitosan, the concentration of the aqueous solution of the antimicrobial agent, expressed as a percentage of the weight of the polysaccharide relative to the weight of the solution, is preferably in the range of 0.3% to 3%, preferably in the range of 0.5% to 2%, even more preferably in the range of 0.8% to 1.5%.

[0101] In a preferred embodiment, the solution containing the polysaccharide or the metal salt is acidified with glacial acetic acid, and the amount of glacial acetic acid, expressed as a percentage by weight relative to the weight of the polysaccharide solution, is in the range of 1% to 3%, preferably in the range of 0.5% to 2%, and even more preferably in the range of 0.1% to 1%.

[0102] In a preferred embodiment, the antimicrobial coating composition comprises one or more antimicrobial agents selected from the group consisting of benzalkonium chloride salts, chitosan, and silver ions.

[0103] Preferably, the antimicrobial coating composition contains all three of the above antimicrobial agents.

[0104] This combination of antimicrobial agents allows for a coating composition that has antimicrobial efficacy against a wide variety of microorganisms.

[0105] The antimicrobial agent may be added indiscriminately to the aqueous dispersion containing graphite in step a, to the dispersion containing graphene oxide obtained in step b, or to both. The antimicrobial agent may be added in the form of an aqueous solution. When two or more antimicrobial agents are present, they may be added together or separately, each antimicrobial agent being able to be dosed in one or more equal portions. After the addition of the antimicrobial agent, the resulting dispersion is preferably subjected to a high shear homogenization treatment, for example under the conditions of step b above.

[0106] The antimicrobial coating composition according to the present invention optionally comprises conventional additives of the type commonly used in the formulation of coating compositions, such as colorants (pigments or dyes), solvents, coalescing agents, surfactants, thickeners, rheology modifiers, compatibilizers, and the like.

[0107] The graphene oxide (GO) of the present invention is an "edge oxidized" graphene oxide (EOGO), and when it is present in other ingredients (such as antimicrobial agents, film formers, acetic acid) used in the composition of the present invention, it does not undergo any chemical reduction, functionalization, grafting, carboxylation, formation of composites / complexes, etc.

[0108] The only possible interactions, if any, are non-covalent.

[0109] The method for preparing an antimicrobial coating composition according to the present invention can be carried out using conventional apparatuses and equipment known to those skilled in the art.

[0110] In general, the components of the aqueous dispersion prepared in step a may be mixed in any order. In some cases, it may be preferred to add two or more antimicrobial agents separately from each other at a later time to avoid possible interactions between these agents. This is the case, for example, with the addition of benzalkonium chloride and silver nitrate, whose interaction can induce precipitation of AgCl.

[0111] It has also been observed that polysaccharides, in particular chitosan, can act as promoters of the graphite exfoliation process, while quaternary ammonium salts, in particular benzalkonium chloride salts, can act as intercalants between graphene layers (e.g. few-layer graphene). Therefore, in a preferred embodiment, the exfoliation step is carried out in the presence of at least one polysaccharide, preferably chitosan. In another preferred embodiment, the quaternary ammonium salt, preferably benzalkonium chloride salt, is added to the dispersion after the graphite has been at least partially exfoliated.

[0112] Antimicrobial coating compositions according to the present invention can be used for imparting antimicrobial properties to substrate surfaces. For this reason, the composition can be applied with a technology suitable for depositing a liquid coating composition on the substrate surface, which is generally used in the paint industry, such as by spraying, dipping or by a brush. After application, the liquid phase contained in the coating composition evaporates to form a film that is suitably cured and dried on the substrate. Usually, the evaporation of the liquid phase is achieved by being exposed to air at room temperature or at a higher temperature. In order to reduce the time of drying and forming an antimicrobial coating, the coating composition can be dried in an oven.

[0113] Examples of substrates that can be coated with the antimicrobial coating composition of the present invention are surfaces of plastic, metal, wood, concrete, stone, polycarbonate, plexiglass, PVC, latex, ceramic, and the like.

[0114] The coating compositions according to the invention may also be applied to pre-coated substrates, for example those pre-coated with varnishes, paints, lacquers and other types of coatings.

[0115] The antimicrobial efficacy of the composition according to the present invention can be measured as the reduction in the total number of viable microorganisms in contact with the antimicrobial coating.

[0116] For purposes of the present invention, antimicrobial efficacy may be determined, for example, by the ASTM E2180-18 method.

[0117] Generally, the coating compositions of the present invention can be used to combat potentially pathogenic microorganisms present in the environment.

[0118] Microorganisms that the antimicrobial coating composition is effective against include fungi, algae, bacteria and viruses. Examples of fungi are: Aspergillus niger and Penicillium funiculosum. Examples of bacteria are: Gordonia amicus, Pseudomonas resinovorans, Pseudomonas formosanus, Pseudomonas resinovorans and Escherichia coli.

[0119] Other pathogenic bacteria against which the coating compositions of the present invention may be effective are: Listeria monocytogenes, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhimurium, Streptococcus mutans, Staphylococcus epidermidis, Vibrio harveyi and Enterococcus faecalis.

[0120] The coating composition is also effective against viruses. Viruses are small infectious agents (from 0.02 μm to a maximum of 1 μm) consisting of biological matter that cannot survive or reproduce autonomously except within a host cell utilizing the functional mechanisms of the host cell. Examples of viruses are: Coronaviruses, in particular the Sars-Cov2 virus.

[0121] For the purposes of the present invention, the antiviral efficacy of the coating composition can be determined, for example, according to ISO 21702:2019 standard "Measurement of antiviral activity on plastics and other non-porous surfaces". DETAILED DESCRIPTION

[0122] In order to further understand the features of the present invention, the following embodiments are provided below.

[0123] Example

[0124] 1. Materials

[0125] The graphite used was high surface area graphite (HSAG) Nano 27 from Asbury Graphite Mills, Inc. (Asbury, NJ, USA). The graphite has the following properties:

[0126] -Surface area 250m 2 / g,

[0127] - Chemical composition of elemental analysis (US standard test sieves): carbon 99.82%, ash 0.18%, moisture 0.97%;

[0128] -The number of stacking layers is equal to about 50.

[0129] Two different commercially available water-based products, both based on acrylic resins, were used as film formers:

[0130] A. Adesital GS primer from ADESITAL SpA, Italy (20 wt. % solid residue);

[0131] B. San Marco primer from San Marco Group SpA, Italy (containing the following antimicrobial compounds: 0.005%-0.01% 1,2-benzisothiazol-3(2H)-one; 0.00015%-0.0015% of a mixture of 5-chloro-2-methyl-2H-isothiazol-3-one and 2-methyl-2H-isothiazol-3-one).

[0132] For comparison, a coating was made with the commercial composition "Dr. Wall" (Graphene. CA, USA).

[0133] 2. Antimicrobial efficacy test

[0134] The antimicrobial efficacy of the coating obtained with the above composition was tested according to ASTM E2180-18 method.

[0135] The antiviral efficacy was tested according to ISO 21702:2019 “Measurement of antiviral activity on plastics and other non-porous surfaces” with some modifications as described below.

[0136] For the above tests, each coating composition (including the composition used for comparative purposes) was deposited on a microscope slide that had been previously scratched (rubbed with sandpaper), flamed, sterilized with ethanol and coated with the same film former present in the coating composition. For each sample, two depositions were performed per day (6 hours apart) for three consecutive days in an aspirator hood.

[0137] Prior to antimicrobial testing, adhesion of the coating to the slides was verified by repeatedly washing the slide surface with demineralized water and wiping with an absorbent cloth.

[0138] 2.1ASTM E2180-18 test (antimicrobial efficacy)

[0139] The test according to ASTM E2180-18 was conducted on four different bacterial species (two of which were Gram-positive and two of which were Gram-negative) belonging to risk level 1 (non-hazardous to human health) to verify the antimicrobial efficacy of the coating against bacteria with two different cell wall structures. The 4 bacterial strains used were: Gordonia amicus and Microbacterium oxydans (Gram-positive); Pseudomonas formosanus and Pseudomonas resinovorax (Gram-negative).

[0140] The antimicrobial efficacy was determined by evaluating the reduction in the number of viable bacteria in samples with an antimicrobial coating compared to a control sample without coating according to the following method. Approximately 10 of each strain suspended in 100 μl of sterile physiological aqueous solution were added. 8 The number of viable cells (CFU) deposited on the coated glass slide surface. After drying for several minutes under a fume hood, the bacteria were allowed to contact the coating for an exposure time of 5 hours at room temperature. At the end of exposure, the glass slide was introduced into a sterile screw-cap tube containing 10 ml of saline and vortexed vigorously for 5 minutes to wash the biomass from the coating surface. The resulting bacterial suspension was then transferred to the surface of a culture dish containing an agar medium, and the surviving microorganisms were counted by serial dilution (1:10) of the bacterial suspension.

[0141] The inoculated plates were left at room temperature for a period of not less than 1 week. After this period, the plates were checked and colony counts (CFU) were performed for the most appropriate dilution for the purpose.

[0142] The same method was applied to control samples consisting of slides coated only with a film former (Primer A or B) corresponding to the film former present in the antimicrobial coating composition.

[0143] The results are expressed as:

[0144] - The difference between the Log10 of the bacterial count at time 0 and the Log10 of the bacterial count measured 5 hours later;

[0145] - The percentage of viable bacteria surviving compared to the control sample;

[0146] - Percentage of bacterial kill, measured as %(kill) = 100 - %(live bacteria).

[0147] 2.2ISO 21702:2019 test (antiviral efficacy)

[0148] The test according to ISO 21702:2019 was performed on samples inoculated with the "SARS-CoV-2" virus, the cause of COVID-19.

[0149] Cell culture

[0150] Vero E6 cells (monkey kidney epithelial cells) were maintained in DMEM medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamine, 100 units / ml penicillin, and 100 μg / ml streptomycin.

[0151] Isolation of SARS-CoV-2 from nasal swabs

[0152] SARS-CoV-2 was isolated from 500 μl of nasal swabs and inoculated onto Vero cells at 80% confluence; after incubation for 3 h at 37°C and 5% CO2, the inoculum was removed and the cells were incubated in culture medium for 72 h until obvious cytopathic effect (CPE) appeared.

[0153] Quantification of viral copy numbers in the supernatant was performed by quantitative real-time PCR (qRT-PCR) as described in “World Health Organization, WHO. Technical guidance for coronavirus disease (COVID-19): Laboratory tests for 2019-nCoV in humans. CDC Real-time RT-PCR Panel for detection of 2019-novel coronavirus (January 28, 2020).

[0154] Following the manufacturer's instructions, PEG was used to concentrate the novel coronavirus and 10 1 -10 9 The complete nucleotide sequence of the isolated SARS-CoV-2 strain has been deposited in Gen Bank, NCBI (Accession No. GeneBank: MT748758)

[0155] For each sample, an antiviral test area consisting of a square of (25±2) mm×(25±2) mm was selected on the slide.

[0156] Prior to the experiment, each sample was sterilized by exposure to UV radiation under a laminar flow hood for 20 min to eliminate any potential bacterial contamination and then placed in a Petri dish.

[0157] The test was carried out in the following manner. A virus suspension (9×10 5 PFU / ml) is placed on a sample of the material to be tested. The virus inoculum is covered with a 25×25 mm membrane and the sample is incubated at 25° C. for 2 to 4 hours.

[0158] At the end of the 2 to 4 hour exposure, 10 ml of SCDLP broth was added to each sample and then the plaque assay was performed.

[0159] Plaque assays were performed in 6-well plates by assessing the presence of virus in SCDLP medium recovered from culture dishes. For each treatment, three serial dilutions 1-10 were prepared in complete medium of Vero E6 cells and 0.4 ml of each dilution was added to the cell monolayer in duplicate. After 2 hours, the inoculum was removed, the cells were washed with 2 ml of medium and covered with 0.3% agarose dissolved in complete medium. After incubation for 48 hours at 37°C with 5% CO2, the cells were fixed with 4% formaldehyde, the agarose layer was removed and washed, and then stained with methylene blue. Plaques were counted and the results were expressed as plaque forming units (PFU) / ml.

[0160] At time 0, immediately after virus sedimentation, 10 ml of SCDLP broth was added to some samples and residual infectivity was assessed by plaque assay.

[0161] Determination of virus infectivity

[0162] For each sample, determine the infectivity of the recovered virus using the following formula:

[0163] N=(14×C×D×V) / A

[0164] in:

[0165] N is per cm 2 The infectivity of the virus recovered from the sample;

[0166] C is the average number of plaques counted in two wells of replicates;

[0167] D is the dilution factor of the counting well;

[0168] V is the volume of SCDLP broth added to the sample, in ml;

[0169] A is the surface area of ​​the covering film, in cm 2 count.

[0170] Calculation of antiviral activity

[0171] The antiviral activity was calculated using the following equation:

[0172] R=Ut-At

[0173] in:

[0174] R is antiviral activity;

[0175] Ut is the mean logarithm of the number of plaques (in PFU / cm) of the reference samples (primer only, 2 or 4 hours later). 2 count);

[0176] At is the average logarithm of the number of plaques (in PFU / cm2) of the samples treated with the coating composition according to the present invention after 2 or 4 hours. 2 count).

[0177] 3. Coating composition

[0178] The coating compositions were prepared using a Silverson L5M-A rotor-stator homogenizer equipped with an AISI 316 stainless steel head, 290 mm long and 57 mm maximum diameter. The apparatus comprises a glass or steel container of variable volume (500-2000 ml) in which the liquid dispersion to be homogenized is loaded to obtain an antimicrobial aqueous dispersion containing graphene oxide.

[0179] An aliquot (approximately 15 g) of the above antimicrobial dispersion containing graphene oxide was then mixed with the desired amount of film former A or B to obtain the final antimicrobial coating composition.

[0180] Comparison 1 : Preparation of slides with Adesital GS primer

[0181] Deposit an appropriate amount of Adesital GS Primer onto a microscope slide prepared as described in step 2 above.

[0182] Comparison 2 : Preparation of slides with San Marco primer

[0183] Deposit an appropriate amount of San Marco primer onto a microscope slide prepared as described in step 2 above.

[0184] Contrast 3 : Has Doctor Preparation of glass slides

[0185] An appropriate amount of a commercial coating composition "Dr. Wall" containing graphene and titanium oxide was deposited on a microscope slide previously prepared as described in step 2 above.

[0186] Contrast 4 : Preparation of HSAG graphite mixture and Adesital GS commercial primer (28PN / 20 / 1)

[0187] The appropriate amount of HSAG graphite described in point 1 was mixed at 50% by mass with 50% by mass of Adesital primer. The dispersion thus obtained was mixed by magnetic stirring for about 6 hours and then deposited on a microscope slide prepared as described in point 2 above.

[0188] Embodiment 1: Synthesis of dispersion 01 (015PN / 20 / 1 reference bactericidal test, using Adesital primer; 021PN / 20 / 1 reference bactericidal test, using San Marco primer)

[0189] In a reactor with a volume of 500 ml, graphite Nano 27 (HSAG) sold by Asbury Carbons (5.1 g), 30% weight / weight hydrogen peroxide Sigma Aldrich (111.5 g), demineralized water (201.1 g), solid silver nitrate Sigma Aldrich (0.5 g) and glacial acetic acid Sigma Aldrich (3.4 g) were charged respectively.

[0190] At the end of this step, the Silverson mixer is immersed in the reactor and the stirring speed is subsequently set to 5000 rpm. The system is kept under these conditions for 2 hours by monitoring the increase in the autogenous temperature, which reaches a value of about 70°C. By maintaining stirring, a second equal portion of hydrogen peroxide (40.1 g) is added while mixing for another 2 hours. Once this period has passed, a mixture of chitosan (0.43 g), demineralized water (50.1 g) and glacial acetic acid (0.09 g) is added. The stirring conditions are maintained for another two hours at an autogenous temperature value of about 60°C. At the end of this period, a mixture of benzalkonium chloride salt (5.1 g) dissolved in water (5.1 g) is added. The whole mixture is stirred for another two hours until an aqueous dispersion based on graphene oxide is obtained, which is used for subsequent bactericidal tests.

[0191] After adding an amount of Adesital primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was then deposited on a microscope slide as described in point 2. Once the nanodispersion was deposited on the slide, it was left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface. The slide thus obtained did not release the deposited material and was therefore considered suitable for the bactericidal test.

[0192] In the different formulations, after adding a quantity of San Marco primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was subsequently deposited on a microscope slide as described in point 2.

[0193] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0194] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0195] Example 2 : Synthesis of dispersion 02 (016PN / 20 / 1 with reference bactericidal test of Adesital primer; 022PN / 20 / 1 with reference bactericidal test of San Marco primer)

[0196] In a reactor with a volume of 500 ml, graphite Nano 27 (HSAG) sold by Asbury Carbons (5.1 g), 30% w / w hydrogen peroxide Sigma Aldrich (112.2 g), demineralized water (200.4 g) and glacial acetic acid Sigma Aldrich (6.84 g) were charged respectively.

[0197] At the end of this step, the Silverson mixer is immersed in the reactor and the stirring speed is subsequently set to 5000 rpm. The system is kept under these conditions for 2 hours by monitoring the increase in the autogenous temperature, which reaches a value of about 70°C. By maintaining stirring, a second equal portion of hydrogen peroxide 30% weight / weight Sigma Aldrich (40.4 g) is added, in which 0.50 g of solid silver nitrate Sigma Aldrich is dissolved. Mixing is carried out for another two hours at 5000 rpm. Once this period has passed, a mixture of chitosan (0.85 g), demineralized water (50.4 g) and glacial acetic acid (0.43 g) is added. The stirring conditions are maintained for another two hours at an autogenous temperature value of about 60°C. At the end of this period, a mixture of benzalkonium chloride salt (5.1 g) dissolved in water (5.1 g) is added. The whole mixture is stirred for another two hours until an aqueous dispersion based on graphene oxide is obtained, which is used for subsequent bactericidal tests.

[0198] After adding an amount of Adesital primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was then deposited on a previously prepared microscope slide, as described in point 2. Once the nanodispersion was deposited on the slide, it was left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface. The slide thus obtained did not release the deposited material and was therefore considered suitable for carrying out bactericidal tests.

[0199] In a different preparation, after adding a quantity of San Marco primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was then deposited on a previously prepared microscope slide as described in point 2.

[0200] Once the nanodispersion was deposited on the glass slide, it was left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface. The glass slide thus obtained did not release the deposited material and was therefore considered suitable for bactericidal testing.

[0201] Example 3 : Synthesis of dispersion 03 (017PN / 20 / 1 reference bactericidal test, using Adesital primer; 023PN / 20 / 1 reference bactericidal test, using San Marco primer)

[0202] In a reactor (volume = 500 mL) were charged: graphite Nano27 (HSAG) sold by Asbury Carbons (4.2 g), 30% w / w hydrogen peroxide Sigma Aldrich (111.3 g), demineralized water (198.9 g) and glacial acetic acid Sigma Aldrich (6.80 g).

[0203] At the end of this step, the Silverson mixer is immersed in the reactor and the stirring speed is subsequently set to 5000 rpm. The system is kept under these conditions for 1.5 hours by monitoring the increase in the autogenous temperature, which reaches a value of about 70°C. By maintaining stirring, a second equal portion of hydrogen peroxide 30% weight / weight Sigma Aldrich (40.4 g) is added, in which 0.43 g of solid silver nitrate Sigma Aldrich is dissolved. Mixing is continued at 5000 rpm for another two hours. Once this period has passed, a mixture of chitosan (2.55 g), demineralized water (49.3 g) and glacial acetic acid (2.55 g) is added. The stirring conditions are maintained for another two hours at an autogenous temperature value of about 60°C. At the end of this period, a mixture of benzalkonium chloride salt (4.25 g) dissolved in water (4.25 g) is added. The entire mixture is stirred for another two hours until an aqueous dispersion based on graphene oxide is obtained, which is used for subsequent bactericidal tests.

[0204] After adding an amount of Adesital primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was then deposited on a previously prepared microscope slide, as described in point 2. Once the nanodispersion was deposited on the slide, it was left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface. The slide thus obtained did not release the deposited material and was therefore considered suitable for carrying out bactericidal tests.

[0205] In a different preparation, after adding a quantity of San Marco primer equal to 5% by weight of the whole mixture, the nanodispersion was stirred for a period of 3 hours. At the end of this time, the product was then deposited on a previously prepared microscope slide as described in point 2.

[0206] Once the nanodispersion was deposited on the glass slide, it was left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface. The glass slide thus obtained did not release the deposited material and was therefore considered suitable for bactericidal testing.

[0207] Example 4: Synthesis of dispersion 04 (024PN / 20 / 1 preparation number; 033PN / 20 / 1 bactericidal test number at the following times: 25% (033PN / 20 / 1-3, 50% (033PN20 / 1-2, 30% (36PN / 20 / 1-4, 95% (36PN20 / 1-2 / first inoculum), 95% (36PN / 20 / 1-2 / second inoculum), 95% (36PN / 20 / 1-2 / third inoculum), 50% (36PN20 / 1-3 first inoculum), 50% (36PN20 / 1-3 second inoculum), 50% (36PN20 / 1-3 third inoculum)

[0208] In a 500 ml reactor, Nano 27 graphite (5 g) sold by Asbury Carbons (HSAG), demineralized water (200 g), and a mixture consisting of chitosan Sigma Aldrich (0.50 g), demineralized water (49.5 g), and glacial acetic acid Sigma Aldrich (0.06 g) were charged respectively.

[0209] At the end of this step, the Silverson mixer is immersed in the reactor and the stirring speed is subsequently set to 5000 rpm. The system is kept under these conditions for 1.5 hours by monitoring the increase in the autogenous temperature, which reaches a value of about 70° C. By maintaining stirring, a first equal portion of 30% weight / weight hydrogen peroxide Sigma-Aldrich (112.0 g) is added while maintaining stirring. At the end of this step, it is mixed for about one hour and then a second equal portion of 30% weight / weight hydrogen peroxide Sigma-Aldrich (40.0 g) is added, in which 0.50 g of silver nitrate Sigma-Aldrich and glacial acetic acid Sigma-Aldrich (3.8 g) are dissolved. Mixing is continued for another 1.5 hours at 5000 rpm. Once this period has passed, a mixture of chitosan (0.5 g), demineralized water Sigma Aldrich (49.5 g) and glacial acetic acid Sigma Aldrich (0.04 g) is added. The stirring conditions are maintained for another two hours at an autogenous temperature value of about 60° C. At the end of this period, a mixture of benzalkonium chloride salt (5 g) dissolved in water (5 g) was added. The whole mixture was stirred for another two hours until an aqueous dispersion based on graphene oxide was obtained, which was used for the subsequent bactericidal tests.

[0210] This nanodispersion was divided into three different equal parts, to which were added three different amounts of Adesital GS primer, equal to 5%, 50% and 70% by weight of the whole mixture. The solution thus obtained was stirred with a magnetic stirrer for a period of 3 hours. At the end of this time, the products were subsequently deposited on previously prepared microscope slides, as described in point 2.

[0211] The same slides obtained by depositing the formulations containing 5% and 50% of primer were subjected to a second inoculation test which surprisingly confirmed the positive results obtained with the first inoculation in terms of bactericidal activity. This result suggests that the bactericidal efficacy of the slides coated with the coating composition according to the invention can be attributed not only to the phenomenon of slow release of the bactericide from the coating composition, but also to the phenomenon of physical contact between the coating and the pathogens. This hypothesis was confirmed by receiving a third inoculum with the samples from the test with the second inoculum after a week of immersion in water, changing the same water every day.

[0212] The samples thus obtained were then inoculated a third time and the antibacterial test was performed again. The results are reported in Table 2.

[0213] Example 5 : Synthesis of Dispersion 05 (032PN / 20 / 1 Preparation Number; 40PN / 20 / 1-1 Sterilization Test Number)

[0214] In a reactor of 500 ml volume, Nano 27 graphite (HSAG) sold by Asbury Carbons (4.7 g), demineralized water (306.2 g), 30% weight / weight hydrogen peroxide Sigma Aldrich (160.14 g) were charged respectively.

[0215] At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 5000 rpm.The system was maintained under these conditions for 3 hours by monitoring the increase in the autogenous temperature, which reached a value of approximately 70°C.

[0216] At the end of this time, an aqueous dispersion based on graphene oxide was obtained, which was used for subsequent bactericidal tests.

[0217] After addition of an amount of Adesital GS primer equal to 5% by weight of the whole mixture, the nanodispersion is stirred for a period of 3 hours. At the end of this time, the product is then deposited on a previously prepared microscope slide as described in point 2.

[0218] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0219] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0220] Example 6 : Synthesis of Dispersion 06 (034PN / 20 / 1 Preparation Number; 40PN / 20 / 1-2 Sterilization Test Number)

[0221] In a reactor of 500 ml volume were charged: Nano 27 graphite (HSAG) sold by Asbury Carbons (4.24 g), demineralized water (306.2 g), 30% w / w hydrogen peroxide Sigma Aldrich (160.14 g) containing silver nitrate Sigma Aldrich (0.47 g).

[0222] At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 5000 rpm.The system was maintained under these conditions for 3 hours by monitoring the increase in the autogenous temperature, which reached a value of approximately 70°C.

[0223] At the end of this time, an aqueous dispersion based on graphene oxide was obtained, which was used for subsequent bactericidal tests.

[0224] After addition of an amount of Adesital GS primer equal to 5% by weight of the whole mixture, the nanodispersion is stirred for a period of 3 hours. At the end of this time, the product is then deposited on a previously prepared microscope slide as described in point 2.

[0225] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0226] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0227] Example 7 : Synthesis of dispersion 07 (039PN / 20 / 1 preparation number; 40PN / 20 / 1-3 sterilization test number)

[0228] In a reactor of 500 ml volume, Nano 27 graphite (HSAG) sold by Asbury Carbons (4.0 g) and demineralized water (252 g) were charged respectively. At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 5000 rpm. The system was kept under these conditions for 0.5 hours by monitoring the increase in the autogenous temperature, which reached a value of approximately 70° C.

[0229] Then a mixture consisting of benzalkonium chloride salt (4.2 g) dissolved in demineralized water (4.2 g) was added.

[0230] The dispersion thus obtained was mixed for a further two hours at a temperature of about 70° C. At the end of this time, 30% w / w hydrogen peroxide Sigma Aldrich (136 g) was added and stirring was continued for a further 3 hours to obtain an aqueous dispersion based on graphene oxide for subsequent bactericidal tests.

[0231] After addition of an amount of Adesital GS primer equal to 5% of the whole mixture, the nanodispersion is stirred for a period of 3 hours. At the end of this time, the product is then deposited on previously prepared microscope slides as described in point 2.

[0232] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0233] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0234] Subsequent tests were conducted to evaluate the biocidal efficacy using the same method as above, using Adesital GS primer in an amount equal to 75% by weight of the entire mixture.

[0235] Example 8 : Synthesis of Dispersion 08 (041PN / 20 / 1 Preparation Number; 40PN / 20 / 1-4 Sterilization Test Number)

[0236] In a reactor of 500 ml volume, Nano 27 graphite (HSAG) sold by Asbury Carbons (4.0 g) and demineralized water (220 g) were charged respectively. At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 5000 rpm. The system was kept under these conditions for 0.5 hours by monitoring the increase in the autogenous temperature, which reached a value of approximately 70° C.

[0237] Then a mixture of chitosan (0.4 g), demineralized water Sigma Aldrich (39.6 g) and glacial acetic acid Sigma Aldrich (0.04 g) was added.

[0238] The dispersion thus obtained was mixed for a further two hours at a temperature of about 70° C. At the end of this time, 30% w / w hydrogen peroxide Sigma Aldrich (136 g) was added and stirring was continued for a further 3 hours to obtain an aqueous dispersion based on graphene oxide for subsequent bactericidal tests.

[0239] After addition of an amount of Adesital GS primer equal to 5% by weight of the whole mixture, the nanodispersion is stirred for a period of 3 hours. At the end of this time, the product is then deposited on a previously prepared microscope slide as described in point 2.

[0240] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0241] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0242] Example 9 : Synthesis of Dispersion 09 (Bactericidal Test No. 048 / PN / 20 / 1, performed with 5% and 75% primer)

[0243] In a reactor of 500 ml volume, Nano 27 graphite (HSAG) sold by Asbury Carbons (4.0 g) and demineralized water (256 g) were charged respectively. At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 5000 rpm. The system was kept under these conditions for 0.5 hours by monitoring the increase in the autogenous temperature, which reached a value of approximately 70° C.

[0244] Next, polyethylene glycol Sigma Aldrich (4 g) was added and stirred for 2 hours at a temperature close to 70° C. After this period, 136 g of 30% w / w hydrogen peroxide Sigma Aldrich was added and stirring was continued for another three hours. In this way, an aqueous dispersion based on graphene oxide was obtained for subsequent bactericidal tests.

[0245] After addition of an amount of Adesital GS primer equal to 5% by weight of the whole mixture, the nanodispersion is stirred for a period of 3 hours. At the end of this time, the product is then deposited on a previously prepared microscope slide as described in point 2.

[0246] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0247] The slides thus obtained did not release the deposited material and were therefore considered suitable for bactericidal testing.

[0248] Subsequent tests were conducted to evaluate the biocidal efficacy using the same method as above, using Adesital GS primer in an amount equal to 75% by weight of the entire mixture.

[0249] Example 10 : Synthesis of Dispersion 10 (Bactericidal Test No. 068 / PN / 20 / 1, performed using 5% of two different primers)

[0250] In a reactor of 1000 ml volume, the following were charged: Nano 27 graphite (HSAG) sold by Asbury Carbons (9 g), demineralized water (312 g), polyethylene glycol-400 Sigma Aldrich (9 g) and 0.9 g of silver nitrate sold by Sigma Aldrich. At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 6000 rpm. The system was kept under these conditions for 1 hour by monitoring the increase in the autogenous temperature, which reached a value of approximately 60° C.

[0251] Once this time had elapsed, demineralized water (306 g) was added via a dropping funnel over approximately 20 minutes, and the entire reaction mixture was then continued to be mixed for an additional hour.

[0252] At the end of this step, another equal portion of 30% weight / weight hydrogen peroxide Sigma Aldrich (306 g) was added again via the dropping funnel. The system was always kept under stirring for a further hour until a clear aqueous solution containing chitosan Sigma Aldrich (1.8 g), demineralized water (207 g) and acetic acid (0.45 g) was added via the dropping funnel. The system thus obtained was kept under stirring for a further hour until a mixture consisting of benzalkonium chloride Sigma Aldrich (9 g) and 45 g of demineralized water was then added via the dropping funnel.

[0253] The mixture thus obtained was kept under stirring for a further hour until an aqueous dispersion based on graphene oxide was obtained for the subsequent bactericidal tests.

[0254] After addition of an amount of Adesital GS primer equal to 5% by weight of the whole mixture, the nanodispersion is stirred in aliquots for a period of 3 hours. At the end of this time, the product is then deposited on previously prepared microscope slides as described in point 2.

[0255] In another case, 5% by weight of San Marco primer, relative to the total mixture, was added to a second aliquot of the nanodispersion and stirred for a period of 3 hours. At the end of this time, the product was then deposited on a previously prepared microscope slide as described in point 2.

[0256] Once the nanodispersion was deposited on the glass slide, it was allowed to dry for about 12 hours to facilitate fixation of the nanodispersion on the glass surface.

[0257] The slides thus obtained did not release the substances deposited thereon and were therefore considered suitable for bactericidal testing.

[0258] Embodiment 11 : Synthesis of Dispersion 11 using conventional stirring (without Silverson)

[0259] By conventional stirring using mechanical blades, Example 10 was completely repeated. The results obtained at the end of the process resulted in an unstable mixture being obtained, which caused phase separation.

[0260] Therefore, the mixture could not be subjected to the antibacterial test.This result shows the importance and effectiveness of high shear mixing (Silverson) in obtaining stable graphene oxide dispersions compared to conventional mixing using mechanical blades or magnetic stirrers.

[0261] Example 12 : Synthesis of Dispersion 12 (086 / PN / 20 / 1 Bactericidal Test No., Using 5% San Marco Primer)

[0262] In a reactor of 2000 ml volume, the following were charged: graphite Nano 27 (HSAG) sold by Asbury Carbons (13 g), demineralized water (385 g), hydrogen peroxide Sigma Aldrich 30% weight / weight (65 g), polyethylene glycol-400 Sigma Aldrich (6.5 g), acetic acid (6.5 g), silver nitrate sold by the company Sigma-Aldrich (1.3 g), chitosan Sigma Aldrich (1.9 g). At the end of this step, a Silverson mixer was immersed in the reactor and the stirring speed was subsequently set to 6000 rpm. The system was kept under these conditions for 1 hour by monitoring the increase in the autogenous temperature, which reached a value of approximately 60° C.

[0263] At the end of this step, another equal portion of 30% weight / weight hydrogen peroxide Sigma Aldrich (299 g) premixed with acetic acid (6.5 g) was added via a dropping funnel. The system was kept under stirring for another 2 hours until a clear aqueous solution containing chitosan Sigma Aldrich (4.5 g), demineralized water (413.6 g), acetic acid (6.5 g), benzalkonium chloride Sigma-Aldrich (13 g) and 30% weight / weight hydrogen peroxide Sigma-Aldrich (78 g) was added via a dropping funnel. The mixture thus obtained was kept under stirring for another 3 hours until an aqueous dispersion based on graphene oxide was obtained, which was used for subsequent bactericidal and antiviral tests.

[0264] After adding a pre-diluted amount (20% by weight of demineralized water) of San Marco primer, the aliquot of the nanodispersion was stirred for a further 3 hours in a Silverson mixer. The amount of pre-diluted primer added to the nanodispersion was 5% by weight relative to the mass of the entire nanodispersion. At the end of the mixing period, the product thus obtained was deposited by airbrush on a previously prepared microscope slide, as described in point 2.

[0265] At the end of the coating step of the glass slides, they were left to dry for about 12 hours to facilitate the fixation of the nanodispersion on the glass surface.

[0266] The slides thus obtained did not release the deposited substances and were therefore considered suitable for conducting tests to evaluate the antibacterial and antiviral properties.

[0267] For the antiviral tests, slides coated with San Marco primer only were also prepared to allow for a correct comparison with samples coated in the above manner (95% "Dispersion 12" + 5% San Marco primer, diluted with 20% H2O).

[0268] The compositions of Examples 1-10 and 12 are shown in Table 1-3.

[0269] Table 1

[0270]

[0271] Table 2

[0272]

[0273]

[0274] Table 3

[0275]

[0276] Antibacterial test results

[0277] The test results of the bactericidal properties of the formulations reported in the examples are listed in Table 4 below, which shows the following information:

[0278] The number of bacterial cells that survive exposure;

[0279] The difference between the Log10 of the bacterial count at time 0 and the Log10 of the bacterial count after 5 hours of measurement; this value provides an indication of how many orders of magnitude the number of viable bacteria has fallen compared to the control sample (Adesital GS or San Marco primer).

[0280] The percentage of viable bacteria compared to the viable bacteria of the control sample;

[0281] Percentage of bacteria killed

[0282] Table 4

[0283]

[0284]

[0285]

[0286] The first control sample consisted of a slide covered with Adesital primer alone, which returned values ​​consistent with the inoculation values. Therefore, this material was considered the reference blank for comparing the bactericidal effects of all preparations.

[0287] Commercial product Doctor Significant bactericidal activity was shown only in samples exposed to light [Doctor (light)]. The total bacterial count was reduced by only one order of magnitude, resulting in a kill rate of 95% of the negative control value. Similar results were obtained using commercial reference products [compressed Directa Plus graphene and powdered graphite].

[0288] Samples containing only graphene (40PN / 20 / 1-1(H2O2) and graphene plus chitosan [40PN / 20 / 1-4(H2O2+chitosan)] showed a 2-order-of-magnitude reduction compared to the negative control, i.e., a higher efficacy than the commercial products of the state of the art. Samples with graphene plus silver nitrate showed a 3-order-of-magnitude reduction.

[0289] The samples containing silver chloride, chitosan and benzalkonium chloride [16; 17; 21; 22; 23, 36PN / 20 / 1-2 (95%); 36PN / 20 / 1-3 (50%)] determined and prepared according to Examples 01, 02, 03 and 04 showed the maximum biocidal activity that the measurement system employed was able to detect: at the first count dilution (10 -2 ) had no measurable viable colonies, thus, this number was significantly lower than the 2.04*10 found in the negative control. 8 99 viable cells out of 10 cells. The reduction was at least 6 orders of magnitude, with a killing rate of more than 99.99995135%.

[0290] The correlation between the biocidal effect and the percentage of dispersion 04 used relative to the primer is also clear: samples containing equal to or less than 50% by weight of dispersion 04 [33PN / 20 / 1-3 (25%); 33PN / 20 / 1-2 (50%); 36PN / 20 / 1-4 (30%)] show lower biocidal activity than samples containing 95% of the same mixture, except for sample [36PN / 20 / 1-3 (50%)], which still shows the maximum performance. In the samples with low concentrations (25%, 33PN / 20 / 1-3), a reduction of 3 orders of magnitude (equivalent to 99.89189189%) of viable microorganisms is still obtained.

[0291] The second inoculation of samples that had been used and cleaned [36PN / 20 / 1-2 (95%) "Second Inoculum" and 36PN / 20 / 1-3 (50%) Second Inoculum] showed a slight reduction in biocidal activity, which could be quantified as only an order of magnitude less than the first used sample, achieving 99.999% kill.

[0292] Tests conducted on Examples 06, 07 and 08 [40PN / 20 / 1-2 (H2O2 + Ag)]; 40PN / 20 / 1-3 (H2O2 + BAC); 40PN / 20 / 1-4 (H2O2 + chitosan)] showed that dispersion 04 of graphene oxide in the presence of benzalkonium chloride (BAC) ensured a very high level of reduction in the live bacterial load even in the absence of silver nitrate and chitosan.

[0293] The greatest biocidal activity (greater than 6 orders of magnitude) was also obtained for dispersion 10, where graphene oxide was used in combination with polypropylene glycol [48PN / 20 / 1 (PEG)].

[0294] Another positive result in terms of antimicrobial properties was found in the case of Example 12, which showed a biocidal activity greater than six orders of magnitude.

[0295] Results of antiviral trials

[0296] The antiviral efficacy of the composition of Example 12 was tested according to ISO 21702:2019 standard.

[0297] Table 5 summarizes the results of antiviral plaque assays performed after 2 or 4 h of incubation with SARS-CoV-2 virus.

[0298] Table 5

[0299]

[0300]

[0301] 1 N is per cm 2 The infectivity of the virus recovered from the sample;

[0302] 2 R is antiviral activity

[0303] 3 Values ​​obtained by analyzing dilutions of 1:10 and 1:100. All cells in the 1:1 dilution were dead.

[0304] Table 6 shows the results of the antiviral plaque assay performed at time 0, compared to the results obtained after 2 hours of incubation.

[0305] Table 6

[0306]

[0307] 1 N is per cm 2 The infectivity of the virus recovered from the sample;

[0308] 2 R is antiviral activity

[0309] 3 Values ​​obtained by analyzing dilutions of 1:10 and 1:100. All cells in the 1:1 dilution were dead.

[0310] The results showed that the slides coated with "Dispersion 12" and the slides coated with "Primer SanMarco" alone showed antiviral activity against SARS-CoV-2 after 2 hours and 4 hours.

Claims

1. A method for preparing an antimicrobial coating composition, comprising the following steps in sequence: a. providing an aqueous dispersion comprising water, graphite and at least one oxidizing agent, wherein the oxidizing agent is selected from the group consisting of oxygen, hydrogen peroxide, tert-butyl peroxide, meta-chloroperbenzoic acid, ozone, chromate ions, hypochlorite ions and mixtures thereof, wherein the concentration of graphite in the aqueous dispersion is in the range of 0.1 wt % to 10 wt % relative to the weight of the dispersion; b. high shear homogenization of the aqueous dispersion from step a by mixing the dispersion using a rotor-stator mixer at a mixing speed equal to or greater than 2000 rpm, up to 10,000 rpm, to obtain a stable antimicrobial aqueous dispersion comprising graphene oxide as an antimicrobial agent; c. mixing the stable aqueous antimicrobial dispersion comprising graphene oxide with at least one film-forming agent to obtain an antimicrobial coating composition, wherein the film-forming agent is apt to promote the formation and adhesion of a coating film of the composition on a substrate surface; wherein the at least one film former is selected from the group consisting of acrylic resins, vinyl resins, styrene resins, alkyd resins, epoxy resins, polyester resins, polyvinyl acetate resins, and mixtures thereof; wherein the amount of the at least one oxidizing agent is in the range of 0.5 wt % to 30 wt % relative to the weight of the aqueous dispersion; wherein the aqueous dispersion of step a comprises at least one antimicrobial agent other than the graphene oxide, wherein the antimicrobial agent is selected from: a quaternary ammonium salt; a polyethylene glycol having a molecular weight in the range of 200-12,000 g / mol; a polysaccharide having antimicrobial properties; a metal ion having antimicrobial properties; Chlorinated isothiazoles; isothiazolinones; and mixtures thereof.

2. The method according to claim 1, wherein the aqueous dispersion of step a comprises at least two antimicrobial agents other than the graphene oxide.

3. The method according to claim 1, wherein the polysaccharide is selected from the group consisting of chitosan, galactan, mannan and laminarin.

4. The method according to claim 1, wherein the metal ions are selected from the group consisting of silver ions, sodium ions, zinc ions and copper ions.

5. The method according to claim 1 or 2, wherein the antimicrobial agent comprises at least: a quaternary ammonium salt; a polyethylene glycol having a molecular weight in the range of 200-12,000 g / mol; a polysaccharide having antimicrobial properties; and silver ions.

6. The method according to claim 5, wherein the quaternary ammonium salt is selected from benzalkonium chloride salts.

7. The method according to claim 5, wherein the polysaccharide is selected from chitosan.

8. The method of claim 1, wherein the at least one oxidizing agent is hydrogen peroxide, optionally mixed with acetic acid.

9. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises graphene oxide in an amount ranging from 0.1% to 10%, the percentages being by weight based on the weight of the antimicrobial coating composition.

10. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises graphene oxide in an amount ranging from 0.5% to 5%, the percentages being by weight based on the weight of the antimicrobial coating composition.

11. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises graphene oxide in an amount ranging from 0.8% to 2%, the percentages being by weight based on the weight of the antimicrobial coating composition.

12. The method according to claim 1 or 2, wherein the aqueous dispersion of step a comprises at least one oxidizing agent in an amount ranging from 2% to 20%, the percentage being by weight relative to the weight of the aqueous dispersion.

13. The method according to claim 1 or 2, wherein the aqueous dispersion of step a comprises at least one oxidizing agent in an amount ranging from 5% to 15%, the percentage being by weight relative to the weight of the aqueous dispersion.

14. The method of claim 1 or 2, wherein the aqueous antimicrobial dispersion contains an antimicrobial agent in a total concentration ranging from 0.1% to 20%, the above percentages being weight percentages relative to the total weight of the aqueous antimicrobial dispersion.

15. The method of claim 1 or 2, wherein the aqueous antimicrobial dispersion contains an antimicrobial agent in a total concentration ranging from 0.5% to 10%, the above percentages being weight percentages relative to the total weight of the aqueous antimicrobial dispersion.

16. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises at least one film former in an amount ranging from 1% to 99%, the percentages being by weight based on the weight of the antimicrobial coating composition.

17. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises at least one film former in an amount ranging from 1.5% to 75%, the percentages being by weight based on the weight of the antimicrobial coating composition.

18. The method of claim 1 or 2, wherein the antimicrobial coating composition comprises at least one film former in an amount ranging from 3% to 50%, the percentages being by weight based on the weight of the antimicrobial coating composition.

19. An antimicrobial coating composition obtainable by the method according to any one of claims 1 to 18.

20. Use of the antimicrobial coating composition according to claim 19 for imparting antimicrobial properties to a substrate.

21. A method for imparting antimicrobial properties to a substrate surface, comprising: - depositing the antimicrobial coating composition according to claim 19 on at least one surface of a substrate; - Evaporating the liquid phase of the coating composition to obtain an antimicrobial coating film adhered to the surface of the substrate.

Images