CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority from Australian Provisional Patent Application No. 2021904263, filed on 24 December 2021, the contents of which are incorporated herein by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to compositions which may be formulated as coating compositions. The compositions may be used as antipathogenic compositions, antipathogenic films, or antipathogenic coatings. The compositions disclosed herein may be applied across multiple industries including, but not limited to: infrastructure, construction, food processing/packaging, manufacturing and/or medical applications.

BACKGROUND

COVID-19 has irreversibly changed the way we live highlighting the risk of disease and societal impacts caused by respiratory viruses such as SARS-CoV-2. Furthermore, with the growth of antimicrobial resistance, society needs innovative solutions to ensure that people can continue to live safe and healthy lives. In the absence of drugs or vaccines, it is human behaviour that becomes the primary weapon to control the spread of disease. Prevention of infection through inactivation of pathogens on surfaces is crucial to ongoing control of disease transmission. It will also play an important role in the restoration of business confidence and reducing the risk to public health due to surface contamination.

The global pandemic associated with the coronavirus SARS-CoV-2 and the looming threat of a global health crisis associated with antimicrobial resistance have both highlighted the need for anti -pathogenic technologies. Despite the availability of agents with both antimicrobial and antiviral properties, simultaneous reduction of the infectivity of both microorganisms is limited by their different mechanisms of action.

The ability of pathogens, such as the SARS-CoV-2 virus, to remain on surfaces such as stainless steel and plastic for hours, as well as the ability of bacterial biofilms to display inherent resistance against antimicrobial agents have created immense challenges for the healthcare system and the demand for anti-infective surfaces. The U.S. Centers for Disease Control and Prevention notes that people can be infected with SARS-CoV-2 through contact with surfaces. CSIRO’s Australian Centre for Disease Preparedness (ACDP)’s research on SARS-CoV-2 survivability found that the virus can survive for up to 28 days on common surfaces including banknotes, glass (such as mobile phone screens), and stainless steel. Additionally, surface transmission in hotel quarantine has already been attributed by health authorities to have sparked a cluster in Adelaide’s northern suburbs which led to South Australia’s lockdown in November 2020.

Routine sanitisation and disinfection of surfaces to reduce the spread of pathogens, for example hospital-related bacteria have been implemented. However, increased use of common disinfectants products containing quaternary ammonium compounds, hydrogen peroxide, bleach and alcohols may cause detrimental effects on human health and the environment. The repeated application of disinfectant processes may be labour intensive, with efficacy waning over a limited period, such as over minutes as opposed to hours. Moreover, the costs associated with routine cleaning of the surfaces are high and may not guarantee their complete decontamination. Anti-infective surfaces where antipathogenic agents (such as antiviral/antibacterial agents) are released over time would reduce the cost of routine sanitisation by decreasing the survival of pathogens for longer periods.

SARS-CoV-2 is one of many pathogens causing problems around the globe. The synthesis and application of materials and coatings that could assist in reducing or eliminating the presence of one or more pathogens could potentially reduce the number of people being affected by infections, and potentially prevent serious illnesses.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each of the appended claims.

SUMMARY

Disclosed herein is a composition capable of forming an antipathogenic film or coating, the composition comprising: (i) at least one essential oil; (ii) at least one metal species; and (iii) at least one cationic species.

Disclosed herein is a method of controlling, preventing and/or reducing the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof, the method comprising applying a composition as defined herein, to at least a portion of a surface of an article. Disclosed herein is the use of a composition as defined herein, to control, prevent and/or reduce the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof.

Disclosed herein is the use of a composition as defined herein in the formation of an antipathogenic composition, film or coating.

Disclosed herein is the use of a composition as defined herein as an antifouling composition, film or coating.

Disclosed herein is a composition comprising a combination of compounds selected from: (i) at least one essential oil; (ii) at least one metal species; and (iii) at least one cationic species. The composition may also use a polymer to provide increased antipathogenic activity (for example antibacterial, antiviral and/or antifungal activity,) potentially via a synergistic combination of combined compounds, based on the pathogen or groups of pathogens that need to be controlled or eliminated.

Herein “pathogen” refers to a microorganism that can potentially cause a disease or illness in an organism, and includes: a virus, bacterium, protozoan, prion, viroid, or fungus.

"Antipathogenic" or “antipathogen” as used herein, refers to molecules and/or compositions that kill and/or prevent fouling by microorganisms including: bacteria, yeast, fungi, mycoplasma, and viruses.

Herein the term "antipathogenic activity" is defined as an activity that kills or inhibits the growth of microorganisms including, but not limited to bacteria, viruses, parasites, and fungi.

It will be understood that a reference to the term “antipathogenic”, “antipathogenic agent”, “antipathogenic compound” and the like herein encompasses, the antipathogenic/antipathogenic agent/antipathogenic compound and, where permitted, all derivatives, isomeric forms, racemates, amorphous forms, crystalline forms, solvates, acceptable salts, solvates of said salts, and prodrugs thereof in isolation from one another as well as mixtures.

Herein the composition may comprise a polymer base. The polymer base may provide a medium that allows the composition to adhere to at least a portion of an article, comprising or composed of a material, such as wood, metal and/or plastics. The polymer base may also act as a vehicle for the components of the composition (for example (i) at least one essential oil; (ii) at least one metal species; and (iii) at least one cationic species), to move through the article (for example a substrate) and onto one or more surfaces. The polymer base may comprise one or more film formers. In one embodiment, the components of a composition described herein are activated to control, reduce and/or eliminate the concentration of at least one pathogen, once the polymer base is in contact with an aqueous solution or solvent. The aqueous solution may, for example, be the sweat of a person contacting the composition, such as from the hand of a subject contacting the composition disposed on an article, such as a door handle. The composition may therefore have a “start stop” mechanism, wherein the composition controls, reduces and/or eliminates one or more pathogens whilst an appropriate concentration of water and/or an appropriate solvent is provided. This mode of action may preserve the longevity of the composition, and in one embodiment, extend the antipathogenic activity of the composition. This mechanism may also minimise the overuse of antipathogenic compounds, when not necessary, reducing the chance of developing any associated resistance to one or more pathogens, and/or its overall environmental impact. In one example, the antipathogenic compounds contained in a composition disposed on an article become mobile and active when wiped down by a wet cloth during normal sanitation process.

The compositions may also be able to be tailored for specific applications and/or the materials that the compositions are to be applied. For example, the type and concentration of the components. The thickness of any composition, for example a composition comprising a polymer base may be controlled, for example by applying multiple layers of the same composition after an earlier applied composition has been applied. This may be appropriate in manufacturing settings, wherein equipment can be coated with the antipathogenic composition, film or coating, to create a protective barrier when in contact with disease causing materials.

The composition may be tailored using different polymer bases. These polymer bases can be in the form of commercially available products. For example, a paint formulation used in an air conditioning unit, may comprise the antipathogenic formulation (being i) at least one essential oil, ii) at least one metal species, and iii) at least one cationic species), thereby creating a purified surface or air flow.

The composition may also be tailored so numerous application techniques are viable, the composition could be applied as a liquid, a paste or aerosol. For example, the composition may be sprayed onto a surface through a manual mechanism such as a spray bottle activated by a hand trigger. In large-scale manufacturing settings, it may also be spray coated by a high-pressure nozzle onto products before they are sent to market. Large scale mixing processes of the composition into the bulk of a material may also be used for high volume manufacturing. In addition, the components of the composition can be tailored to target specific pathogens or groups of pathogens. For example, the composition may be tailored with a group of compounds displaying the highest efficacy for one or more pathogens. During a pandemic, or outbreak, the composition may be adapted to provide efficacy for the pandemic pathogen, and potentially adapted again to account for variants of said pathogens.

The composition may be tailored so that it can be removed with a solvent, optionally removed to re-apply a fresh amount of the composition after an appropriate period of use/application has elapsed. This may be useful in construction mould remediation settings, where continued application of the composition may create layers too thick for purpose. The removability of this layer allows a fresh coat to be added without a build-up of previous coatings.

The compositions described herein may be used to potentially reduce the risk for acquiring infectious diseases through surface transmission, for example in environments where care is provided to vulnerable residents such as aged care facilities or hospitals, as well as in high-density environments. A single application of a composition described herein may control, reduce and/or eliminate one or more bacteria, viruses and/or fungi.

The composition may be used on medical devices to be implanted inside the human body. Wherein the antipathogenic composition, film or coating, has a biocompatible nature, it may be used on medical devices like catheters that are inserted into the body, which require a high level of antipathogenic activity over long periods of time.

The compositions described herein may display efficacy for an extended period, for example the compositions may display an efficacy against one or more pathogens or microbes for 30 or more days. The compositions may display an efficacy of 30 or more days during continuous use during this period, for example without the need for a further application of the same or different composition. An instance of this is that it may be used in the food industry for packaging of agricultural food products to be shipped to consumers. For example, an anti-pathogenic layer on food packaging that continuously kills for multiple days may allow it to reduce mould growth and extend the shelf-life of food.

The compositions described herein may provide advantages such as saving on costs for a business or an organisation by assisting in the simplification of sanitation processes, for example by relieving staff of the need to disinfect as often. This may save on the amount of hours and material needed to disinfect commonly touched surfaces and the costs associated with this time and effort. The composition may prophylactically kill pathogens, reducing the need for other surfaces to be as frequently disinfected via other means. For example, from the application of a chemical or the use of wipes. This may save on the amount of hours and material needed to disinfect commonly touched surfaces, reducing excess waste and improving its environmental friendliness, for example in high-density environments.

The compositions may provide higher public safety outcomes. By reducing the amount of hours for disinfection, this may reduce the risk for parties such cleaners acquiring infectious diseases from areas such as high-density environments. The compositions herein may therefore provide a means to improve safety, whilst also potentially saving on labour intensive cleaning processes.

The compositions described herein may provide one or more of the following advantages:

• an acceptable, high or sufficiently high pathogen kill rate

• a broad spectrum with regards to the variety of pathogens killed or the reduction in number of said pathogens

• the safety of the composition and its ingredients in regards to its application and/or use on an article or surface thereof

• longevity in relation to antimicrobial activity and/or general physical stability, especially when applied to high contact articles or high contact surfaces

• the ability to tailor the compositions for application to a specific surface or article, or required application

• the speed at which the composition can be applied and/or to dry and/or

• an advantageous price per unit or application.

It will be appreciated that the embodiments of each aspect of the present disclosure may equally be applied to each other aspect, mutatis mutandis.

BRIEF DESCRIPTION OF DRAWINGS

Whilst it will be appreciated that a variety of embodiments disclosed herein may be utilised, in the following, described herein are a number of examples with reference to the following drawings:

Figure 1 - Schematic of the antipathogen coating containing the three- components including an essential oil, a metal compound and a cationic polymer. Figure 2 - Activity of different compounds evaluated using the zone of inhibition test against Staph, aureus. The compounds tested were: A) polymeric formulation (control); B) AgCu nanoparticles; C) copper oxide (Cu(II)O); D) cinnamaldehyde; E) carvacrol; F) tea tree oil; G) s-poly-L-lysinc (a-PL); and H) PHMB.

Figure 3 - Antibacterial activity of different compounds evaluated using the zone of inhibition test against P. aeruginosa. The compounds tested were: A) polymeric formulation (control); B) AgCu nanoparticles; C) copper oxide (Cu(II)O); D) cinnamaldehyde; E) carvacrol; F) tea tree oil; G) a-PL; and H) PHMB.

Figure 4 - Graph of crystal violet test of biofdm mass vs combination of two antipathogenic compounds against Staph, aureus.

Figure 5 - Graph of crystal violet test of biofdm mass vs combination of two antipathogenic compounds against P. aeruginosa.

Figure 6 - Graph of biofdm experiment using aluminium substrates. Logarithmic reduction of the cfu/mL obtained from the viable counting of the samples. Both Staph, aureus and P. aeruginosa were evaluated.

Figure 7 - Pictures of 96 well plate biofdm experiment using three components combination. Agar plates labelled with number one used Staph, aureus while number two represent P. aeruginosa. Combination 1: A 1-2) control samples - polymeric fdm; Bl-2) Ag-Cu/Cinnamaldchydc/s-PL. Combination 2: Cl-2) Ag- Cu/Cinnamaldehyde/PHMB; DI -2) Cu(II)O/Cinnamaldehyde/8-PL; El -2) Cu(II)O/Cinnamaldehyde/PHMB .

Figure 8 - Graph of Log (cfu/mL) vs A) Control samples - polymeric fdm. B) Ag-Cii/cinnamaldchydc/s-PL. C) Ag-Cu/cinnamaldehyde/PHMB. D) Cu(II)O/cinnamaldehyde/ s-PL. E) Cu(II)O/cinnamaldehyde/PHMB, for Staph, aureus and P. aeruginosa.

Figure 9 - Graph of antiviral infectivity assay for a group of antipathogenic components vs OC43.

Figure 10 - Graphs of antiviral infectivity assay for a group of antipathogenic components vs RV-A1 on A - a logarithmic scale; and B - a linear scale. DESCRIPTION OF EMBODIMENTS

Terms and Definitions

With regards to the definitions provided herein, unless stated otherwise, or implicit from context, the defined terms and phrases include the provided meanings. Unless explicitly stated otherwise, or apparent from context, the terms and phrases below do not exclude the meaning that the term or phrase has acquired by a person skilled in the relevant art. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed invention, because the scope of the invention is limited only by the claims. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

All publications discussed and/or referenced herein are incorporated herein in their entirety, unless described otherwise.

Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Throughout this disclosure, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or groups of compositions of matter. Thus, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context clearly dictates otherwise. For example, reference to “a” includes a single as well as two or more; reference to “an” includes a single as well as two or more; reference to “the” includes a single as well as two or more and so forth.

Those skilled in the art will appreciate that the disclosure herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the examples, steps, features, methods, compositions, formulations, and processes, referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.

The term “and/or”, e.g., “X and/or Y” shall be understood to mean either “X and Y” or “X or Y” and shall be taken to provide explicit support for both meanings or for either meaning.

Unless otherwise indicated, the terms “first,” “second,” etc. are used herein merely as labels, and are not intended to impose ordinal, positional, or hierarchical requirements on the items to which these terms refer. Moreover, reference to a “second” item does not require or preclude the existence of lower-numbered item (e.g., a “first” item) and/or a higher-numbered item (e.g., a “third” item).

As used herein, the phrase “at least one of’ or “one or more of’ when used with a list of items, means different combinations of one or more of the listed items may be used and only one of the items in the list may be needed. The item may be a particular object, thing, or category. In other words, “at least one of’ means any combination of items or number of items may be used from the list, but not all of the items in the list may be required. For example, “at least one of item A, item B, and item C” may mean item A; item A and item B; item B; item A, item B, and item C; or item B and item C. In some cases, “at least one of item A, item B, and item C” may mean, for example and without limitation, two of item A, one of item B, and ten of item C; four of item B and seven of item C; or some other suitable combination.

It is to be appreciated that certain features that are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any sub-combination.

Throughout the present specification, various aspects and components of the disclosure can be presented in a range format. The range format is included for convenience and should not be interpreted as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range, unless specifically indicated. For example, description of a range such as from 1 to 5 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 5, from 3 to 5 etc., as well as individual and partial numbers within the recited range, for example, 1, 2, 3, 4, 4.5, and 5, unless where integers are required or implicit from context. This applies regardless of the breadth of the disclosed range. Where specific values are required, these will be indicated in the specification.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Throughout this specification, the term "consisting essentially of is intended to exclude elements which would materially affect the properties of the claimed composition, although may include elements that do not materially affect properties. The terms "comprising", "comprise" and "comprises" herein are intended to be optionally substitutable with the terms "consisting essentially of, "consist essentially of, "consists essentially of, "consisting of, "consist of and "consists of, respectively, in every instance.

“Article”, as used herein, refers to an object or substrate, comprising one or more materials, on which a composition as described herein, optionally in the form of a coating, is applied, or is incorporate on or within, or on which the composition is immobilised.

Herein "coating", refers to any temporary, semi-permanent or permanent layer, or layers, treating or covering a surface, or a portion thereof of an article. The coating may be a chemical modification of at least a portion of the underlying article or may involve the addition of new materials to at least a portion of an article. It includes any increase in thickness to the substrate or change in surface chemical composition of at least a portion of the article. A coating may be applied as a liquid, for example as a paint or spray, and solidified into a solid coating. The coating may be applied as and/or comprise a single layer of a composition (and may take the form of a film), or the coating may be applied as and/or comprise a plurality of layers of one or more compositions. The plurality of layers may be the same or comprise different compositions. In one embodiment the coating comprises a single composition. The coating may be contiguous over a surface of an article, or may be present on at least a portion of a surface of an article.

Herein “film” refers to a material, including a coating, deposited or used in thin sections or layer form. The layer may be contiguous over a surface of an article, or may be present on at least a portion of a surface of an article.

Herein the % may relate to v/v, w/w or w/v, unless specifically described.

Herein, unless indicated otherwise, the term “about” encompasses a 10% tolerance in any value or values connected to the term.

Herein an “essential oil” may comprise any one or more oils, said oils which may be water insoluble, or substantially water-insoluble. One or more essential oils may be soluble in one or more of: an alcohol, an ether, an organic solvent, such as a hydrocarbon, and mixtures thereof. In one embodiment the solubility of one or more essential oil may be less than about 5, 4, 3, 2 1, or 0.5 % by weight in water at 25 °C or, optionally, less than about 0.1%.

Herein, the one or more essential oils may be volatile aromatic oils which may be synthetic or may be derived from plants by distillation, expression or extraction, and may carry the odour or flavour of the plant from which they are obtained. One or more essential oils may provide antiseptic activity. Examples of essential oils include but are not limited to: citra, thymol, menthol, methyl salicylate (wintergreen oil), eucalyptol, carvacrol, camphor, anethole, carvone, eugenol, isoeugenol, limonene, osimen, n-decyl alcohol, citronel, a-salpineol, methyl acetate, cloves, citronellyl acetate, methyl eugenol, cineol, linalool, ethyl linalaol, safrole, vanillin, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil, laurel oil, cedar leaf oil, gerianol, verbenone, anise oil, bay oil, benzaldehyde, bergamot oil, bitter almond, chlorothymol, cinnamic aldehyde, citronella oil, eucalyptus oil, guaiacol, tropolone derivatives such as hinokitiol, lavender oil, mustard oil, phenol, phenyl salicylate, pine oil, pine needle oil, sassafras oil, spike lavender oil, storax, thyme oil, tolu balsam, terpentine oil, and mixtures thereof.

In certain embodiments, the essential oils are selected from the group consisting of thymol ((CHs CHCeHs CH OH, also known as isopropyl-m-cresol), eucalyptol (CioHisO, also known as cineol), menthol (CHsCeH^Cs^OH), also known as hexahydrothymol), methyl salicylate (CeEEOHCOOCHs, also known as wintergreen oil), isomers of each of these compounds, and combinations of two or more thereof.

The present disclosure considers salts of essential oils or components of essential oils such as the salts of monoterpenoid compounds. An example of a monoterpenoid compound is thymol. Thus, the present disclosure contemplates salts of thymol as acceptable salts. For example, an acceptable salt of thymol is the berberine ammonium cation salt of thymol and, vice versa, an acceptable salt of berberine is the thymol phenolate salt of berberine. It will be recognised that such a salt may display a combination of the biological activity possessed by the berberine ammonium cation and the biological activity possessed by the thymol phenolate counter anion.

Compositions

Disclosed herein is a composition which may be used for forming an antipathogenic composition, fdm or coating, the composition comprising:

(i) at least one essential oil

(ii) at least one metal species and

(iii) at least one cationic species.

Herein the composition comprises at least one essential oil. In one embodiment the at least one essential oil may be or an extract of: cloves, thyme, fennel, caraway, peppermint, lemon myrtle, tea tree, thymol, carvacrol, eugenol, menthol, terpineol, carvone, citral, arborvitae, Asteraceae, Bergamot, cajuput, Cassia, Cedarwood, Cinnamon, Citron, clary sage, coriander, Eucalyptis, fennel, frankincense, lavender, lemon, lemongrass, Manuka, oregano, Perilla, rosemary, white fir, wild orange, wintergreen, ylang, or mixtures thereof.

In one embodiment the at least one essential oil may be selected from: cinnamaldehyde, tea tree oil, carvacrol, or mixtures thereof.

In one embodiment, one or more essential oils may be present in a range of about 0.05% v/v to about 10% v/v, or about 0.1 % v/v to about 5% v/v , or about 0.5 % v/v to about 3% v/v ; or in a range of about 0.05% w/v to about 10% w/v, or about 0.1 % w/v to about 5% w/v, or about 0.5% w/v to about 3% w/v; or in a range of about 0.05% w/w to about 10% w/w, or about 0.1% w/w to about 5% w/w, or about 0.5% w/w to about 3% w/w.

In one embodiment, one or more essential oils may be present in an amount of about, or at least about: 0.05% v/v, 0.1%, v/v, 0.5% v/v, 1% v/v, 1.5% v/v, 2% v/v, 2.5% v/v, 3% v/v, 3.5% v/v, 4% v/v, 4.5% v/v, 5% v/v, 5.5% v/v, 6% v/v, 6.5% v/v, 7% v/v, 7.5% v/v, 8% v/v, 8.5% v/v, 9% v/v, 9.5% v/v, or 10% v/v. In another embodiment, one or more essential oils may be present in an amount of about, or at least about: 0.05% w/v, 0.1%, w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w/v, 7.5% w/v, 8% w/v, 8.5% w/v, 9% w/v, 9.5% w/v, or 10% w/v. In another embodiment, one or more essential oils may be present in an amount of about, or at least about: 0.05% w/w, 0.1%, w/w, 0.5% w/w, 1% w/w, 1.5% w/w, 2% w/w, 2.5% w/w, 3% w/w, 3.5% w/w, 4% w/w, 4.5% w/w, 5% w/w, 5.5% w/w, 6% w/w, 6.5% w/w, 7% w/w, 7.5% w/w, 8% w/w, 8.5% w/w, 9% w/w, 9.5% w/w, or 10% w/w.

Herein the composition comprises at least one metal species. In one embodiment, the at least one metal species comprises at least one elemental metal.

In another embodiment, at least one metal species, or all the metal species, may be replaced with at least one transition element, for example selenium, or a salt, oxide or complex thereof.

Herein the metal species may be or comprise one or metals (for example as a mixture or elements and/or compounds, or alloys). In one embodiment, the at least one metal species comprises at least one metal selected from: copper, silver, titanium aluminium, cobalt, gold, lead, magnesium, molybdenum, nickel, tin, zinc, zirconium, compounds thereof, mixtures thereof, and alloys thereof. In one embodiment, at least one metal species is an oxide. Examples of oxides include, but are not limited to: copper oxide, titanium oxide, aluminium oxide, cobalt oxide, magnesium oxide, nickel oxide, tin oxide, zinc oxide and mixtures thereof.

In one embodiment the at least one metal species is a salt.

In one embodiment the at least one metal species is a complex.

In one embodiment, at least one metal species is a mixture of metals.

In one embodiment, at least one metal species is an alloy.

The at least one metal species may be used in any form known in the art. In one embodiment, the at least one metal species may be in the form of a powder. In another embodiment the at least one metal species may be in the form of a particle. In yet another embodiment the at least one metal species may be colloidal. The at least one metal species may be in the form of a coated particle, for example a metal particle comprising a mixture of metals, for example silver and copper.

The at least one metal species may comprise particles on the nanoscale. For example, the at least one metal species may be a particle with a mean diameter of about, or at least about: 50 nm to about 100 nm. The at least one metal species may have a mean diameter of about, or at least about: 1 nm, 5 nm, 10, nm, 15 nm, 20 nm, 25 nm, 30 nm, 35 nm, 40 nm, 45 nm, 50 nm; 55 nm; 60 nm; 65 nm; 70 nm; 75 nm; 80 nm; 85 nm; 90 nm; 95 nm; or 100 nm.

In one embodiment, one or more metal species may be present in a range of about 0.05% v/v to about 10% v/v, or about 0.1 % v/v to about 5% v/v , or about 0.5 % v/v to about 3% v/v ; or in a range of about 0.05% w/v to about 10% w/v, or about 0.1 % w/v to about 5% w/v, or about 0.5% w/v to about 3% w/v; or in a range of about 0.05% w/w to about 10% w/w, or about 0.1% w/w to about 5% w/w, or about 0.5% w/w to about 3% w/w.

In one embodiment, one or more metal species may be present in an amount of about, or at least about: 0.05% v/v, 0.1%, v/v, 0.5% v/v, 1% v/v, 1.5% v/v, 2% v/v, 2.5% v/v, 3% v/v, 3.5% v/v, 4% v/v, 4.5% v/v, 5% v/v, 5.5% v/v, 6% v/v, 6.5% v/v, 7% v/v, 7.5% v/v, 8% v/v, 8.5% v/v, 9% v/v, 9.5% v/v, or 10% v/v. In another embodiment, one or more metal species may be present in an amount of about, or at least about: 0.05% w/v, 0.1%, w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w/v, 7.5% w/v, 8% w/v, 8.5% w/v, 9% w/v, 9.5% w/v, or 10% w/v. In another embodiment, one or more metal species may be present in an amount of about, or at least about: 0.05% w/w, 0.1%, w/w, 0.5% w/w, l% w/w, 1.5% w/w, 2% w/w, 2.5% w/w, 3% w/w, 3.5% w/w, 4% w/w, 4.5% w/w, 5% w/w, 5.5% w/w, 6% w/w, 6.5% w/w, 7% w/w, 7.5% w/w, 8% w/w, 8.5% w/w, 9% w/w, 9.5% w/w, or 10% w/w.

Herein the composition comprises at least one cationic species.

Examples of cationic species include, but are not limited to:

(i) quaternary ammonium compounds, such as those in which one or two of the substituents on the quaternary nitrogen has from 8 to 20, for example from 10 to 18 carbon atoms, such as an alkyl group, which may optionally be interrupted by an amide, ester, oxygen, sulfur, or heterocyclic ring, while the remaining substituents have a lower number of carbon atoms, for instance from 1 to 7, and are optionally alkyl, for instance methyl or ethyl, or benzyl. Examples of such compounds include benzalkonium chloride, dodecyl trimethyl ammonium chloride, benzyl dimethyl stearyl ammonium chloride, hexadecyltrimethyl ammonium bromide, benzethonium chloride (diisobutyl phenoxyethoxyethyl dimethyl benzyl ammonium chloride) and methyl benzethonium chloride;

(ii) pyridinium and isoquinolinium compounds, including hexadecylpyridinium chloride and alkyl isoquinolinium bromides;

(iii) pyrimidine derivatives such as hexetidine (5-amino-l,3-bis(2-ethylhexyl)- 5 -methyl -hexahydropyrimidine) ;

(iv) amidine derivatives such as hexamidine isethionate (4,4'-diamidino-a,co- diphenoxy-hexane isethionate);

(v) bispyridine derivatives such as octenidine dihydrochioride (N,N'[l,10- decanediyldi-1 (4H)-pyridinyl-4-ylidene]-bis (1-octanamine) dihydrochioride);

(vi) guanides, for example, mono-biguanides such as p- chlorobenzylbiguanide and N'(4-chlorobenzyl)-N" -(2,4-dichlorobenzyl) biguanide, poly(biguanides) such as polyhexamethylene biguanide hydrochloride; and/or

(vii) N"-acyl amino acid alkyl esters and salts; and

(viii) mixtures thereof.

In one embodiment, one or more cationic species may be present in a range of about 0.05% v/v to about 10% v/v, or about 0.1 % v/v to about 5% v/v , or about 0.5 % v/v to about 3% v/v ; or in a range of about 0.05% w/v to about 10% w/v, or about 0.1 % w/v to about 5% w/v, or about 0.5% w/v to about 3% w/v; or in a range of about 0.05% w/w to about 10% w/w, or about 0.1% w/w to about 5% w/w, or about 0.5% w/w to about 3% w/w.

In one embodiment, one or cationic species may be present in an amount of about, less than about, or at least about: 0.05% v/v, 0.1%, v/v, 0.5% v/v, 1% v/v, 1.5% v/v, 2% v/v, 2.5% v/v, 3% v/v, 3.5% v/v, 4% v/v, 4.5% v/v, 5% v/v, 5.5% v/v, 6% v/v, 6.5% v/v, 7% v/v, 7.5% v/v, 8% v/v, 8.5% v/v, 9% v/v, 9.5% v/v, or 10% v/v. In another embodiment, one or more cationic species may be present in an amount of about, less than about, or at least about: 0.05% w/v, 0.1%, w/v, 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w/v, 7.5% w/v, 8% w/v, 8.5% w/v, 9% w/v, 9.5% w/v, or 10% w/v. In yet another embodiment, one or more cationic species may be present in an amount of about, less than about, or at least about: 0.05% w/w, 0. 1%, w/w, 0.5% w/w, 1% w/w, 1.5% w/w, 2% w/w, 2.5% w/w, 3% w/w, 3.5% w/w, 4% w/w, 4.5% w/w, 5% w/w, 5.5% w/w, 6% w/w, 6.5% w/w, 7% w/w, 7.5% w/w, 8% w/w, 8.5% w/w, 9% w/w, 9.5% w/w, or 10% w/w.

In one embodiment the cationic species is a cationic polymer. The cationic polymer may be a homopolymer or a copolymer. In one embodiment the cationic polymer is a homopolymer. In another embodiment the cationic polymer is a copolymer, for example a statistical, gradient or block copolymer.

The cationic polymer may comprise one or more monomers selected from: lysine, hexamethylene guanidine, imidazolium, piperidinium, quaternary ammonium, ethylenimine, 2-dimethy(aminoethyl) methacrylate, chitosan, and mixtures thereof.

In one embodiment a polymer of lysine is used. Examples of lysine polymers include polymers of L-lysine and D-lysine. The lysine polymers may be in the a or s form. The lysine polymer may be: a-poly(L-lysine), a-poly(D-lysine), 8-poly (L-lysine), e-poly(D-lysine), or a mixture thereof. In one embodiment the cationic polymer is 8- poly (L-lysine).

The cationic polymer may be a salt known in the art. For example, the cationic polymer may be a salt selected from: a halo salt such as a chloride salt, a sulfate salt, a nitrate salt, or a mixture thereof.

In one embodiment, one or more cation polymers may be present in a range of about 0.05% v/v to about 10% v/v, or about 0.1 % v/v to about 5% v/v , or about 0.5 % v/v to about 3% v/v ; or in a range of about 0.05% w/v to about 10% w/v, or about 0.1 % w/v to about 5% w/v, or about 0.5% w/v to about 3% w/v; or in a range of about 0.05% w/w to about 10% w/w, or about 0.1% w/w to about 5% w/w, or about 0.5% w/w to about 3% w/w.

In one embodiment, one or cationic polymers may be present in an amount of about, less than about, or at least about: 0.5% v/v, 1% v/v, 1.5% v/v, 2% v/v, 2.5% v/v, 3% v/v, 3.5% v/v, 4% v/v, 4.5% v/v, 5% v/v, 5.5% v/v, 6% v/v, 6.5% v/v, 7% v/v, 7.5% v/v, 8% v/v, 8.5% v/v, 9% v/v, 9.5% v/v, or 10% v/v. In another embodiment, one or more cationic polymers may be present in an amount of about, less than about, or at least about: 0.5% w/v, 1% w/v, 1.5% w/v, 2% w/v, 2.5% w/v, 3% w/v, 3.5% w/v, 4% w/v, 4.5% w/v, 5% w/v, 5.5% w/v, 6% w/v, 6.5% w/v, 7% w/v, 7.5% w/v, 8% w/v, 8.5% w/v, 9% w/v, 9.5% w/v, or 10% w/v. In yet another embodiment, one or more cationic polymers may be present in an amount of about, less than about, or at least about: 0.5% w/w, l% w/w, 1.5% w/w, 2% w/w, 2.5% w/w, 3% w/w, 3.5% w/w, 4% w/w, 4.5% w/w, 5% w/w, 5.5% w/w, 6% w/w, 6.5% w/w, 7% w/w, 7.5% w/w, 8% w/w, 8.5% w/w, 9% w/w, 9.5% w/w, or 10% w/w.

The composition may further comprise an organic solvent. The organic solvent may be selected from: alkylacetates, arylacetates, alcohols (including primary, secondary and/or tertiary alcohols), carboxylic acids, ethers, ketones, alkanes, hydrocarbons (aliphatic, nitrated and chlorinated hydrocarbons), amine, esters, terpenoids, and mixtures thereof. For example, the organic solvent may be selected from: butyl acetate, ethyl acetate, ethanol, methanol, isopropanol, diacetone alcohol, acetone, ethanol, acetone-water mixtures, propanol, butanol, tert-butyl alcohol, diethyl ether, tetrahydrofuran, methyl ethyl ketone, benzyl alcohol, ethyl acetate-ethanol mixtures, glycol mixtures, acetic acid, lactic acid, propionic acid, diethylene glycol butyl ether, diethylene glycol ethyl ether, diethylene glycol methyl ether, ethylene glycol butyl ether, ethylene glycol ethyl ether, ethylene glycol methyl ether, dioxane, cyclohexanone, amyl alcohol, sec-butyl alcohol, iso-butyl carbinol, diacetone alcohol, citronellol, and mixtures thereof.

In one embodiment the composition described herein further comprises a polymer base.

The polymer base may comprise one or more of: a polymer, an organic solvent, a plasticizer, a dye or pigment, an adhesive, a bulking agent, a thickening agent, emulsion polymers, and mixtures thereof.

In one embodiment, the polymer base comprises at least one polymer, which may be a homo polymer or a copolymer. In another embodiment the polymer is suitable for forming a fdm. For example, the composition may comprise a polymer and forms a fdm after initially being applied to at least a portion of a surface on an article in a non-solid form, for example as a spray or as a liquid.

The polymer base may comprise one or more polymers, for example: a polymer comprising cellulose, a polyurethane, polyethylene terephthalate, an epoxy resin, a vinyl ester, a phenolic polymer, an acrylic ester, an acrylate homopolymer or copolymer, a latex paint, a polyester, and mixtures thereof.

In one embodiment at least one polymer comprises a cellulose, optionally selected from one or more of: nitrocellulose, cellulose acetate, cellulose acetate butyrate, ethyl cellulose, alkyd resin, and mixtures thereof.

The polymer base may comprise one or more organic solvents, optionally selected from: alkylacetates, arylacetates, alcohols (including primary, secondary and/or tertiary alcohols), carboxylic acids, ethers, ketones, alkanes, hydrocarbons (aliphatic, nitrated and chlorinated hydrocarbons), amine, esters, terpenoids, and mixtures thereof. The organic solvent may be optionally selected from: butyl acetate, ethyl acetate, ethanol, methanol, isopropanol, diacetone alcohol, acetone, ethanol, acetone-water mixtures, propanol, butanol, tert-butyl alcohol, diethyl ether, tetrahydrofuran, methyl ethyl ketone, benzyl alcohol, ethyl acetate-ethanol mixtures, glycol mixtures, acetic acid, lactic acid, propionic acid, diethylene glycol butyl ether, diethylene glycol ethyl ether, diethylene glycol methyl ether, ethylene glycol butyl ether, ethylene glycol ethyl ether, ethylene glycol methyl ether, dioxane, cyclohexanone, amyl alcohol, sec-butyl alcohol, iso-butyl carbinol, diacetone alcohol, citronellol, and mixtures thereof.

The polymer base may comprise one or more plasticizers, optionally selected from: phthalates, dibutyl phthalate, a citrate, tributyl acetyl citrate, ethyl toluene sulfonamide (ethyl tosylamide), n-cyclohexyl para-toluene sulfonamide, glycerol, a glycol, glycol ethers, glycol esters, hydrogenated castor oil or an epoxidized oil, polyesters, polybutylenes, aliphatic polyurethanes (optionally with a molecular wright of about 2000 to about 5000), toluene sulfonamide urea formaldehyde, y-butyral lactone, n- butylphthalimide/isopropylphthalimide, ethylhexyl diphenyl phosphate, wool fat derivatives, chlorinated paraffins, glyceryl triacetate, camphor, sucrose acetate isobutyrate (SAIB), and mixtures thereof.

The polymer base may comprise one or more dyes known in the art and mixtures thereof.

The polymer base may comprise one or more adhesives, optionally in the form of a polymer, optionally selected from: tosylamide-formaldehyde resin, alkyd resin or another polymer known in the art, and mixtures thereof. The polymer base may comprise one or more thickening agents and/or bulking agents, optionally selected from: fluorphlogopite (for example synthetic fluorphlogopite) which comprises magnesium aluminium silicate and potassium, an aluminium borosilicate, for example calcium aluminium borosilicate, stearalkonium bentonite, stearalkonium hectorite, and mixtures thereof.

In one embodiment, the polymer base is formulated as a nail polish.

In another embodiment, the composition may comprise one or more additives, for example an additive optionally selected from: iodine, benzalkonium chloride, graphene oxide, and mixtures thereof.

Herein the compositions may or may not be cross-linked. In one embodiment the composition is cross-linked. In another embodiment the composition is not cross-linked. One potential advantage obtained from the crosslinking of a composition, film or coating is the stability of the resulting materials, for example crosslinking can reduce the solubility of the coating in comparison to similar compositions which are not crosslinked. In addition, the cross-linked nature of a coating may increase the chemical resistance of the composition. The application of a cross-linked polymer to a surface of an article, may also yield a surface which has an increased heat tolerance, decreased permeability, improved abrasion resistance and/or extend the life of the article.

In one embodiment the composition may be removed from an article, or portion thereof, by the application of at least one solvent optionally selected from: alkylacetates, arylacetates, alcohols (including primary, secondary and/or tertiary alcohols), carboxylic acids, ethers, ketones, alkanes, hydrocarbons (aliphatic, nitrated and chlorinated hydrocarbons), amine, esters, terpenoids, and mixtures thereof. For example, the composition may be removed by the application of an organic solvent optionally selected from: butyl acetate, ethyl acetate, ethanol, methanol, isopropanol, diacetone alcohol, acetone, ethanol, acetone-water mixtures, propanol, butanol, tert-butyl alcohol, diethyl ether, tetrahydrofuran, methyl ethyl ketone, benzyl alcohol, ethyl acetate-ethanol mixtures, glycol mixtures, acetic acid, lactic acid, propionic acid, diethylene glycol butyl ether, diethylene glycol ethyl ether, diethylene glycol methyl ether, ethylene glycol butyl ether, ethylene glycol ethyl ether, ethylene glycol methyl ether, dioxane, cyclohexanone, amyl alcohol, sec-butyl alcohol, iso-butyl carbinol, diacetone alcohol, citronellol, and mixtures thereof.

The composition may be a synergistic composition. As used herein, the term "synergistic" refers to a greater than additive effect that is produced by a combination of compounds selected from: (i) at least one essential oil; (ii) at least one metal species; and (iii) at least one cationic species, which exceeds the effect that would otherwise result from use of one or more of (i) to (iii).

Substrate/Article

Herein an article defined herein could be a substrate. Herein the substrate could be an inert surface or it could comprise functional groups on at least one surface thereon, which can be used to anchor or immobilise a composition such as a coating.

The composition, film or coating may be immobilised on a variety of different substrates. Examples of suitable substrate materials include, but are not limited to: metals, ceramics, glass, polymers (such as plastics), materials of biological origin, woven and non-woven fibres, inert materials such as silicon, and combinations thereof. In one embodiment the substrate is selected from the group consisting of: plastics, metals, ceramics, woven materials, silicon materials, and combinations thereof

The composition, film or coating may utilise premade polymers that are covalently attached to the surface and cross-linked. Multifunctional cross-linkers allow both attachment to the surface and crosslinking between the components of polymer, which can provide the low fouling properties.

Suitable metallic materials which may act as substrates include, but are not limited to: metals and alloys based on titanium, such as unalloyed titanium (ASTM F67) and titanium alloys, such as ASTM Fl 108, Ti-6A1-4V ELI (ASTM F136), Nitinol (ASTM F2063), nickel titanium alloys, and thermo-memory alloy materials; stainless steel (such as ASTM F138 and F139), tantalum (such as ASTM F560), palladium, zirconium, niobium, molybdenum, nickel-chrome, or certain cobalt alloys including Stellite, cobaltchromium (such as Vitallium, ASTM F75 and Wrought cobalt-chromium (ASTM F90)), and cobalt-chromium-nickel alloys (such as ELGILOY® and PHYNOX®).

Suitable ceramics which may act as substrates include, but are not limited to: oxides, carbides, or nitrides of the transition elements such as titanium oxides, hafnium oxides, iridium oxides, chromium oxides, aluminium oxides, and zirconium oxides. Silicon based materials, such as silica, may also be used.

Suitable polymer substrates include, but are not limited to: polystyrene and substituted polystyrenes, polyalkylenes, such as polyethylene and polypropylene, poly(urethane)s, polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, polyesters (for example DACRON®), polysiloxanes, poly -ethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoate)s, polyfluorocarbons (for example polytetrafluoroethylene), polyether ether ketone (PEEK), parylene based polymers, dopamine based polymers, hydrogen cyanide derived polymers (aminomalononitrile), silicones, epoxy resins, polyalkenes, phenolic resins, aromatic polyamides, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitrile, biopolymers such as polysaccharides and natural latex copolymers thereof, and combinations thereof.

The composition, film or coating may also be immobilised on to industrial or residential or other common substrates to prevent mildew, bacterial contamination, and in other applications where it is desirable to prevent fouling, such as: marine applications (ship hull coatings), fuel tanks, oil pipelines, industrial piping, pharmaceutical equipment, drug delivery devices such as inhalers, contact lenses, dental implants, coatings for in vivo sensors, textiles such as hospital drapes, gowns, or bedding, ventilation conduits, doorknobs, devices for separations, such as membranes for microbial suspension, biomolecule separation, protein fractionation, cell separation, waste water treatment, water purification, bioreactors, and food processing.

These compositions or coatings may also be used to coat and thus treat surfaces of fibres, particulates and films for the applications of textiles, additives, electric/optical appliances, packaging materials and colorants/inks.

Applications

In one embodiment, the composition is formulated to form a film or coating. The composition may be applied in any form known in the art, on to at least a portion of a surface of an article. For example, the composition may be formulated to be applied as a spray, as a paint, as a liquid, or incorporated into a device such a sleeve for a door handle, an adhesive cover, a protective covering, such as a film.

In one embodiment, the composition is formulated to provide an antipathogenic composition, film or coating. In another embodiment, the composition is suitable for controlling, preventing and/or reducing the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof.

In one embodiment, the composition is an antibacterial, antifungal, and/or antiviral composition, film or coating.

In one embodiment, the composition is an antibacterial, antifungal, and/or antiviral composition, film or coating, and controls, reduces and/or eliminates at least one pathogen, and displays an efficacy for the at least one pathogen for about, or at least about: 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, or 60 days. In one embodiment, the composition is an antibacterial composition, film or coating. In another embodiment, the composition is an antibacterial composition, film or coating suitable for controlling, preventing and/or reducing the concentration of a bacteria selected from, but not limited to: gram-negative bacteria, gram-positive bacteria, and mixtures thereof. In yet another embodiment, the composition is an antibacterial composition, film or coating suitable for controlling, preventing and/or reducing the concentration of a bacteria selected from: Pseudomonas aeruginosa, Staphylococcus aureus, and mixtures thereof.

In one embodiment, the composition is an antifungal composition, film or coating.

In one embodiment, the composition is suitable for controlling, preventing and/or reducing the concentration of a fungus, mildew, mould and mixtures thereof.

In one embodiment, the composition is an antiviral composition, film or coating. In another embodiment, the composition is an antiviral composition, film or coating suitable for controlling, preventing and/or reducing the concentration of a virus selected from: an enveloped virus, a non-enveloped virus, and mixtures thereof. In yet another embodiment, the composition is an antiviral composition, film or coating suitable for controlling, preventing and/or reducing the concentration of a virus selected from: SARS-CoV-2, or a surrogate of SARS-CoV-2, and mixtures thereof.

The Therapeutic Goods Administration of Australia recognises Murine Hepatitis Virus MHV-1 as a surrogate for COVID-19.

In one embodiment the composition is formulated as an antifouling composition, film or coating.

In one embodiment the composition is formulated for an in vivo application, for example a coating for a medical device such as a catheter or an implant.

In one embodiment, the composition is antipathogenic, optionally in the form of a coating, wherein the efficacy of the composition provides a reduction in the concentration of at least one pathogen, in a time scale of less than about, or about: 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 120 minutes, or 180 minutes. A reduction in the concentration of at least one pathogen may be a reduction of about, or at least about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9%, of the original concentration of the pathogen.

In one embodiment, the composition is an antipathogenic composition, optionally in the form of a film or coating (for example as shown in Figure 1), wherein the antibacterial, antifungal and/or antiviral efficacy of the composition provides a reduction in the concentration of at least one pathogen (for example a bacteria, virus and/or fungus), in a time scale of less than about, or about: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 90 minutes, 120 minutes, or 180 minutes. A reduction in the concentration of a pathogen, such as one or more bacteria, virus and/or fungus, may be a reduction of about, or at least about: 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.5%, or 99.9%, of the original concentration of the pathogen.

The composition described herein may be formulated to be applied to at least a portion of a surface of an article. The article may comprise or may be composed of a material selected from: wood, metal, plastic, and mixtures thereof.

The compositions described herein may comprise one or more film formers. Herein a polymer base may comprise one or more film formers. A film former is a compound (or a mixture of compounds), that is capable of leaving a cohesive film or coating on a surface. The film or coating may be pliable. One or more film formers may comprise a cellulose and/or one or more polymers. Examples of polymers include, but are not limited to: acrylate copolymers, for example those obtained by emulsion polymerisation, for example, acrylate/Ci2-22 methacrylate copolymers polymerised from monomers of methacrylic acid, methyl methacrylate, butyl acrylate, and cetyl-eicosiyl methacrylate; and PPG-17/isophorone diisocyanate (IPDI)/dimethylol propionic acid (DMPA) copolymer; polyurethane polymer dispersions, for example Polyurethane-34 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, hexamethylene diisocyanate, ethylene diamine, N-(2-aminoethyl)- 3 aminoethane sulphonic acid; Polyurethane -32 which is a sodium salt of polymerized monomers of polytetramethylene glycol, hexamethylene diisocyanate, (IPDI), ethylene diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid; Polyurethane-35 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, dicyclohexylmethane diisocyanate, ethylene diamine, N-(2-aminoethyl)- 3 aminoethane sulphonic acid; and Polyurethane-48 which is a sodium salt of polymerized monomers of adipic acid, 1,6-hexandiol, neopentyl glycol, isophorone diisocyanate, isophorone diamine, N-(2-aminoethyl)-3-aminoethanesulphonic acid; vinylpyrrolidone (VP) and polyvinylpyrrolidone (PVP) crosspolymers, copolymers and/or interpolymers such as hydrolyzed wheat protein/PVP crosspolymers; maltodextrin/VP copolymer; butylated/PVP copolymer; VP/polycarbamylpolyglycol ester; VP/dimethylaminoethylmethacrylate/polycarbamyl polyglycol ester; and VP/dimethiconylacrylate/ polycarbamyl polyglycol ester; and mixtures thereof.

Herein the composition may comprise one or more polyester copolymers, which may be fdm formers, such as those derived from diols, for example branched alkylene diols, for example, neopentyl glycol, and alkane diacids, such as adipic acid, alkane polyacids, or alkane or aryl acid anhydrides, such as trimellitic anhydride.

Herein the composition may form a film. The composition may be in the form of a coating. In one embodiment the components of the composition for a homogenous mixture, or substantially homogenous mixture that can be applied to an article, or a portion thereof (such as a one or more surfaces of the article). In one embodiment the components of the composition can be applied to an article for forming a film.

Herein a film or coating formed from a composition defined herein, may be formed by a polymerisation reaction and/or drying.

Herein the composition may be formulated for use or application on a “high contact surface” or “high contact article”. Herein the high contact surface” or “high contact article” may be a surface that is touched repeatedly by one individual or a plurality of different individuals, and more particularly a surface that is touched repeatedly in a short space of time, such as within 30 minutes, 10 minutes, 5 minutes or sub 1 minute intervals. Examples of such high contact surfaces include, but are not limited to door handles, petrol bowser handles, touchscreens or touchpads, particularly including those in public areas, such as payment terminals, ATMs, elevator buttons, light switches, kiosk / vending machine input buttons, or similar, keyboards, user input devices, such as a mouse, the like. However, it will be appreciated that this is not essential and the techniques described herein can be applied to a wide range of different contact surfaces and/or articles.

The composition may be formulated so that it can be applied to at least a portion of a surface, for example a high contact surface.

The composition may be formulated so that it can be applied to at least a portion of a surface of an article selected from: a table, a door, a door handle, or a mixture thereof. For example, the composition may be formulated to be applied to at least a portion of a surface of an article as a liquid composition, such as a paint or as a spray, or another form such as a paste, gel or an aerosol, which then dries to form a solid, for example a film. The composition (for example initially in the form of a liquid), may be formulated to dry in about, or less than about: 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or 60 minutes.

In one embodiment, the composition is formulated to form a fdm or coating in a time in a range of about 1 minute to about 60 minutes, for example: about 1 minute to about 50 minutes, about 1 minute to about 40 minutes, about 1 minute to about 30 minutes, about 1 minute to about 20 minutes, about 1 minute to about 15 minutes, about 1 minute to about 10 minutes, or about 1 minute to about 5 minutes.

Disclosed herein is a method of controlling, preventing and/or reducing the concentration of at least one pathogen, optionally selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof, the method comprising applying the composition as defined herein to at least a portion of a surface of an article.

Also disclosed herein is the use of a composition as defined herein, to control, prevent and/or reduce the concentration of at least one pathogen, optionally selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof.

Also disclosed herein is the use of a composition as defined herein, in the formation of an antipathogenic film.

Also disclosed herein is the use of a composition as defined herein, as an antifouling composition, film or coating.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the above-described embodiments, without departing from the broad general scope of the present disclosure. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

EXAMPLE EMBODIMENTS

The present disclosure may rely on one or more of the following example embodiments.

1. A composition capable of forming an antipathogenic composition, film or coating, the composition comprising: at least one essential oil; at least one metal species; and at least one cationic species.

2. The composition of example embodiment 1, where the composition is in the form of a film or a coating.

3. The composition of example embodiment 1 or example embodiment 2, further comprising a polymer base, wherein the polymer base comprises one or more polymers and optionally one or more film formers. 4. The composition of example embodiment 3, wherein the one or more polymers are suitable for forming a film.

5. The composition of any one of example embodiments 2 to 4, wherein the polymer base comprises: a polymer comprising: a cellulose, a polyurethane, polyethylene terephthalate, an acrylate homopolymer or copolymer, and mixtures thereof.

6. The composition of example embodiment 5, wherein the cellulose is selected from one or more of: nitrocellulose, cellulose acetate, cellulose acetate butyrate, ethyl cellulose, and mixtures thereof.

7. The composition of any one of the preceding example embodiments, wherein the at least one essential oil is selected from or an extract of: cloves, thyme, fennel, caraway, peppermint, lemon myrtle, tea tree, thymol, carvacrol, eugenol, menthol, terpineol, carvone, citral, or mixtures thereof.

8. The composition of any one of the preceding example embodiments, wherein the at least one essential oil is selected from: cinnamaldehyde, tea tree oil, carvacrol, or mixtures thereof.

9. The composition of any one of the preceding example embodiments, wherein the at least one metal species comprises at least one elemental metal.

10. The composition of any one of the preceding example embodiments, wherein the at least one metal species comprises at least one metal selected from: copper, silver, titanium aluminium, cobalt, gold, lead, magnesium, molybdenum, nickel, tin, zinc, zirconium, compounds thereof, salts thereof, oxides thereof, complexes thereof, mixtures thereof, and alloys thereof.

11. The composition of any one of the preceding example embodiments, wherein the at least one metal species is a salt.

12. The composition of any one of example embodiments 1 to 10, wherein the at least one metal species is an oxide.

13. The composition of any one of example embodiments 1 to 10, wherein the at least one metal species is an oxide selected from: copper oxide, titanium oxide, and mixtures thereof.

14. The composition of any one of example embodiments 1 to 10, wherein the at least one metal species is a mixture of metals.

15. The composition of any one of example embodiment 1 to 10, wherein the at least one metal species is an alloy. 16. The composition of any one of the preceding example embodiments, wherein the at least one metal species is a powder.

17. The composition of any one of the preceding example embodiments, wherein the at least one metal species is a particle or a colloid.

18. The composition of any one of the preceding example embodiments, wherein the at least one metal species is a coated particle, optionally a metal particle comprising a mixture of metals.

19. The composition of any one of the preceding example embodiments, wherein at least one metal species is replaced with a transition element, or a salt, oxide or complex thereof.

20. The composition of any one of the preceding example embodiments, wherein the at least one metal species is a particle with a mean diameter of about 50 nm to about 100 nm.

21. The composition of any one of the preceding example embodiments, wherein the cationic species is a polymer.

22. The composition of any one of the preceding example embodiments, wherein the cationic species is a cationic polymer and comprises one or more monomers selected from: lysine, hexamethylene guanidine, and mixtures thereof.

23. The composition of any one of the preceding example embodiments, wherein the cationic species is a cationic polymer and cationic polymer is a salt, optionally a chloride salt.

24. The composition of any one of the preceding example embodiments, wherein the cationic species is a cationic polymer and cationic polymer is a homopolymer.

25. The composition of any one of example embodiments 1 to 23, wherein the cationic species is a cationic polymer and the cationic polymer is a copolymer.

26. The composition of any one of the preceding example embodiments, wherein one or more of: the at least one essential oil; the at least one metal species and/or the at least one cationic species, is present in a range of about 0.05% w/w to about 10% w/w.

27. The composition of any one of the preceding example embodiments, wherein the composition further comprises an organic solvent selected from: alkylacetates, arylacetates, alcohols (including primary, secondary and/or tertiary alcohols), carboxylic acids, ethers, ketones, alkanes, hydrocarbons (optionally aliphatic, nitrated and chlorinated hydrocarbons), amine, esters, terpenoids, and mixtures thereof. 28. The composition of the preceding example embodiments, further comprising an organic solvent selected from: butyl acetate, ethyl acetate, ethyl alcohol, isopropanol, diacetone alcohol, acetone, and mixtures thereof.

29. The composition of any one of the preceding example embodiments, wherein the polymer base further comprises one or more of: an organic solvent, a plasticizer, a dye or pigment, an adhesive, a bulking agent, a thickening agent, and mixtures thereof.

30. The composition of example embodiment 29, wherein the organic solvent is selected from: alkylacetates, arylacetates, alcohols, ethers, ketones, alkanes, hydrocarbons, and mixtures thereof.

31. The composition of example embodiment 29 or example embodiment 30, wherein the organic solvent is selected from: butyl acetate, ethyl acetate, ethyl alcohol, isopropanol, diacetone alcohol, and mixtures thereof.

32. The composition of any one of example embodiments 29 to 31, wherein the plasticizer is selected from: phthalates, dibutyl phthalate, a citrate, tributyl acetyl citrate, ethyl toluene sulfonamide (ethyl tosylamide), n-cyclohexyl para-toluene sulfonamide, glycerol, a glycol, glycol ethers, glycol esters, hydrogenated castor oil or an epoxidized oil, polyesters, polybutylenes, aliphatic polyurethanes (optionally with a molecular wright of about 2000 to about 5000), toluene sulfonamide urea formaldehyde, y-butyral lactone, n-butylphthalimide/isopropylphthalimide, ethylhexyl diphenyl phosphate, wool fat derivatives, chlorinated paraffins, glyceryl triacetate, camphor, sucrose acetate isobutyrate (SAIB), and mixtures thereof.

33. The composition of any one of example embodiments 29 to 32, wherein the adhesive is a polymer, optionally selected from tosylamide-formaldehyde resin.

34. The composition of any one of example embodiments 29 to 33, wherein the bulking agent and/or the thickening agent is selected from: fluorphlogopite, an aluminium borosilicate, optionally calcium aluminium borosilicate, stearalkonium bentonite, stearalkonium hectorite, and mixtures thereof.

35. The composition of any one of the preceding example embodiments, wherein the polymer base is formulated as a nail polish.

36. The composition of any one of the preceding example embodiments, wherein the composition is formulated as a liquid or aerosol to form a film or coating.

37. The composition of any one of the preceding example embodiments wherein the composition is formulated for slow release, optionally over a period of about, or at least about: 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, 110 days, 115 days, or 120 days.

38. The composition of any one of the preceding example embodiments wherein the composition is formulated for the slow release of one or more of (i) to (iii), over a period of about, or at least about: 1 day, 2 days, 3 days, 4 days, 5 days, 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 45 days, 50 days, 55 days, 60 days, 65 days, 70 days, 75 days, 80 days, 85 days, 90 days, 95 days, 100 days, 105 days, 110 days, 115 days, or 120 days.

39. The composition of any one of the preceding example embodiments wherein the composition is formulated such that the composition displays efficacy against at least one pathogen following the application of an aqueous solvent.

40. The compositions of example embodiment 39, wherein the aqueous solvent comprises sweat.

41. The composition of any one of the preceding example embodiments, wherein the composition is formulated to be applied as a spray, as a paint, as a liquid, a paste, a gel or an aerosol.

42. The composition of any one of the preceding example embodiments, wherein the composition is in the form of an antipathogenic film or coating.

43. The composition of any one of the preceding example embodiments, wherein the composition is suitable for controlling, preventing and/or reducing the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof.

44. The composition of any one of the preceding example embodiments, wherein the composition is an antibacterial composition, film or coating.

45. The composition of any one of the preceding example embodiments, wherein the composition is an antifungal composition, film or coating

46. The composition of example embodiment 45, wherein the composition is suitable for controlling, preventing and/or reducing the concentration of a fungi, mildew, mould or a mixture thereof.

47. The composition of any one of the preceding example embodiments, wherein the composition is an antibacterial composition, film or coating suitable for controlling, preventing and/or reducing the concentration of a bacteria selected from: Gram-negative bacteria, Gram-positive bacteria, and mixtures thereof. 48. The composition of any one of the preceding example embodiments, wherein the composition is an antibacterial composition, fdm or coating suitable for controlling, preventing and/or reducing the concentration of a bacteria selected from: Pseudomonas aeruginosa, Staphylococcus aureus, and mixtures thereof.

49. The composition of any one of the preceding example embodiments, wherein the composition is an antiviral composition, fdm or coating.

50. The composition of any one of the preceding example embodiments, wherein the composition is an antiviral composition, fdm or coating suitable for controlling, preventing and/or reducing the concentration of a virus selected from: an enveloped virus, a non-enveloped virus, and mixtures thereof.

51. The composition of any one of the preceding example embodiments, wherein the composition is an antiviral composition, fdm or coating suitable for controlling, preventing and/or reducing the concentration of a virus selected from: SARS-CoV-2, or a surrogate thereof, influenza, and mixtures thereof.

52. The composition of any one of the preceding example embodiments, wherein the composition is an antibacterial, antifungal, and/or antiviral composition, fdm or coating.

53. The composition of any one of the preceding example embodiments, wherein the composition is formulated as an antifouling composition, fdm or coating.

54. The composition of any one of the preceding example embodiments, wherein the composition is an antipathogenic composition, fdm or coating which displays efficacy against at least one pathogen for at least about: 5, 10, 15, 20, 25 or 30 days.

55. The composition of any one of the preceding example embodiments, further comprising at least one other component, optionally selected from: iodine, graphene oxide, and mixtures thereof.

56. The composition of any one of the preceding example embodiments, wherein the composition is formulated to be applied to at least a portion of a surface of an article.

57. The composition of example embodiment 56, wherein the article comprises or is composed of a material selected from: wood, metal, plastic, and mixtures thereof.

58. The composition of any one of the preceding example embodiments, wherein the composition is formulated to be applied to at least a portion of a surface of an article selected from: a table, a door, a door handle, or a mixture thereof.

59. The composition of any one of the preceding example embodiments, wherein the composition is formulated to be applied to at least a portion of a surface of an article as a liquid composition, solid composition or gaseous composition. 60. The composition of example embodiment 59, wherein the liquid composition, solid composition or gaseous composition, is formulated to dry in less than about: 5, 10, 15, 20, 25, or 30 minutes.

61. The composition of any one of the preceding example embodiments, wherein the composition is formulated for an in vivo application.

62. The composition of any one of the preceding example embodiments, wherein the composition is formulated for an in vivo application for a medical device, optionally selected from a catheter or an implant.

63. A coating or fdm comprising the composition of any one of the preceding example embodiments.

64. A method of controlling, preventing and/or reducing the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof, the method comprising applying the composition of any one of example embodiments 1 to 62, to at least a portion of a surface of an article.

65. Use of a composition of any one of example embodiments 1 to 62, to control, prevent and/or reduce the concentration of at least one pathogen selected from: bacteria, fungi, mould, mildew, algae, viruses, and mixtures thereof.

66. Use of a composition of any one of example embodiments 1 to 62 in the formation of an antipathogenic composition, fdm or coating.

67. Use of a composition of any one of example embodiments 1 to 62, as an antifouling composition, fdm or coating.

EXAMPLES

Example 1 - Antimicrobial Activity

Materials

A commercial nail polish formulation was used to incorporate compounds with anti-pathogen properties and to produce coatings. Poly E-L-lysine HC1 (MW 3,500- 5,000Da) and polyhexamethylene guanidine obtained from Biosynth Carbosynth®. Cinnamaldehyde, carvacrol, copper (II) oxide (nanopowder <50nm particle size (TEM)), silver and copper nanoparticles were acquired from Sigma-Aldrich. Tea tree oil was purchased from Thursday Plantation. Silver-coated nanoparticles were obtained from Nanoshel containing copper 80% silver 20%, size 80-100 nm, purity 99.9%. Nutrient agar, Mueller-Hinton broth and sterile fdter paper were purchased form ThermoFisher Scientific (Oxoid). Aluminium discs were laser cut to various sizes and were used as substrates in the different assays.

Bacterial Strains and Growth Conditions

Gram-negative Pseudomonas aeruginosa ATCC 27853 and Gram-positive Staphylococcus aureus ATCC 25923 strains were employed. Bacterial stocks were prepared in 60% glycerol and stored at -80 °C until needed. All surfaces were sterilized with UV prior to the bacterial assay.

Coatings Fabrication

For 2.5% two or three component mixtures, 25 mg of each component and 300 pL ethyl acetate were added to a vial per mb of the polymer formulation before thorough mixing with a spatula. For the 5% single component systems 50 mg of each component and 200 pL of ethyl acetate were added to a vial per mb of polymer paint mixture. This was mixed thoroughly with a spatula. The compositions were painted on the sides and edges, or bottom and sides of various substrates of 96 well plates. If the paint became overly viscous a few drops of ethyl acetate was added to allow the composition to be applied. Samples were painted within an aura 2000 M.A.C. biohazard cabinet. After allowing at least 3 hours or overnight dry, samples were UV treated within the cabinet for 20 minutes, turned over then UV treated again.

X-ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) analysis was used to characterize the surface chemistry (surface elemental composition) of the coatings. XPS analysis was performed using an AXIS Ultra-DUD spectrometer (Kratos Analytical Utd, U.K.) equipped with a monochromated Al-Ka X-ray source at a power of 180 W (12 mA, 15 kV). An internal electron flood gun was used to compensate for sample charging during irradiation. All elements presented were identified from low resolution survey spectra (acquired at a pass energy of 160 eV). The atomic concentrations of the detected elements were calculated using integral peak intensities and the sensitivity factors supplied by the manufacturer. Data processing was performed using CasaXPS processing software version 2.3.21(Casa Software Utd., Teignmouth, United Kingdom).

Antimicrobial Susceptibility Assays

Zone of inhibition assay

Bacterial stocks were streaked onto nutrient agar plates to be used as working stock. After 24 hours incubation at 37 °C, a single colony from the streaked plate was used and inoculate in 10 mL of Mueller-Hinton broth (MHB) which was incubated overnight (180 rpm) at 37 °C. This culture was further diluted in MHB to obtain 10 ⁶ colony-forming units per mL (cfu mL-1). Cultures were spread evenly onto agar plates using a sterile swab. Sterile fdter paper discs (6 mm 0 - Oxoid antimicrobial susceptibility test discs) previously coated with mixtures of the polymeric formulation and individual antimicrobial compounds at 5% concentration were gently pressed onto the surface of the agar plates. Discs coated with the polymeric fdm alone were used as controls. Plates were then inverted and incubated for approximately 24hours at 37 °C and the diameter of the inhibition zones was measured in mm, including the diameter of the disc.

Crystal violet assay

The bottom and walls of the wells in a 96 well plate were painted with a combination of two antimicrobial compounds and the polymeric formulation and dry overnight inside the biohazard cabinet. One hundred microlitres of the diluted bacterial suspensions (106 cfu mL ^-1 ) were pipetted into each well and incubate for 24 hours at 37 °C with gentle agitation (75 rpm). After overnight incubation, the cell suspensions were carefully removed, and the wells were rinsed twice with 110 pl of PBS per well to remove non-adherent cells. To quantify biofdm biomass formed on the surface of the coatings, the plates were heat-fixed in a 60 °C oven for 1 hour and then stained with 100 pl of 1% (w/v) crystal violet for 10 minutes. The crystal violet was removed, and the plates were washed three times by immersing them in cold water to remove excess stain. Excess water after washing was removed by gently tapping the plates against paper towels. One hundred microliters of 95% ethanol were added to each well and the plate was incubated at room temperature for 10 minutes. One hundred microliters of solution from each well were transferred into a new plate. The biomass of biofilm from each well was determined by reading the optical density using a BioTek plate reader at 600 nm.

Biofilm experiments

For biofilm experiments, bacterial stocks were streaked onto nutrient agar plates to be used as working stock. After 24 hours incubation at 37 °C, a single colony from the streaked plate was used and inoculate in 10 mL of MHB which was incubated overnight (180 rpm) at 37 °C. For the biofilm test, aluminium disks (6 mm 0 laser cut aluminium round discs) with both sides coated with the specific antipathogen combination were incubated for 24 hours at 37 °C with 50 pL of bacteria solution (106 cfu mL ^-1 ). After incubation samples were gently washed 3 times using sterile phosphate buffered saline (PBS) to remove any planktonic cell. The samples were individually transferred to a sterile Eppendorf tube containing ImL of sterile PBS. Samples were vortexed (3 times as 30 seconds) and sonicated for 10 minutes to detach bacterial biofdm from the surface. After sonication, samples were vortex again for 30 seconds. The suspensions obtained were serially diluted and aliquots of 20 pL were plated on nutrient agar plates for viable counts.

Results and Discussion

Homogeneous mixtures of individual compounds and the polymeric formulation were fabricated. Filter papers were coated with each mixture and the antimicrobial activity was evaluated via the zone of inhibition test.

In vitro antibacterial activity of each individual compound against both Grampositive and Gram negative bacteria was assessed via the zone of inhibition test (Figure 2 and Figure 3). The cationic polymers PHMB and s-PL showed antibacterial effects on both bacteria (Figure 2G-H and Figure 3G-H). Structural characteristics of the external layer of Gram-negative bacteria particularly its thickness and composition (lipoprotein, lipid bilayer and endotoxin) confer more protection against antimicrobial agents when compared to Gram-positive bacteria. Essential oils such as cinnamaldehyde and carvacrol were effective against Staph, aureus with cinnamaldehyde being extremely effective (Figure 2D) with the biggest zone of inhibition of all compounds (~25 mm).

The antibacterial activity observed from the metal nanoparticles in the zone of inhibition test could be attributed to the lack of diffusion of the nanoparticles from the fdter paper disc to the agar plate or the limited solubility of the ions and other species released by the nanoparticles into the agar plate. The diffusivity of nanoparticles with sizes greater than 10 nm in culture media is expected to be in the order of 10 ^-11 m ² /s, which is lower compared to the diffusion of antibiotics commonly employed in this test.

For a second antimicrobial assay, the effect of the combination of two antimicrobial compounds was evaluated using both a semi-quantitative standard biofdm assay based on crystal violet staining (Figure 4 and Figure 5) and a quantitative biofdm 96 well-plate experiment (Figure 7). For the semi -quantitative analysis, crystal violet was incorporated after 24 hours bacterial incubation in a 96 well plate where each well was individually coated with the respective antimicrobial combination at a final concentration of 5%, 2.5% of each compound (Figure 4 and Figure 5). Optical density results showed a significant dropping in biofdm biomass growth for combinations containing cinnamaldehyde, copper oxide, silver and copper nanoparticles which was consistent for both Gram-positive and Gram-negative bacteria. The samples that showed lower biofdm mass were then employed in a 96 well plate biofdm test. To evaluate the ability of the coatings to resist microbial biofilm formation, coated samples were subjected to a 96 well plate biofilm experiment where viable counts were assessed. Aluminium substrates of 6 mm diameter were painted on both sides each sample with different combinations of two antimicrobial compounds (Figure 6). Samples were incubated with both Gram-positive and Gram-negative bacteria for 24 hours. Then, samples were washed to remove any non-adherent planktonic bacteria followed by detachment of bacterial biofilm in PBS. Aliquots of the solution were diluted and plated on agar to evaluate bacterial viability. Colony counting of viable bacteria was attempted showing a greater reduction in the viability of Staph, aureus when combinations of cinnamaldehyde and either copper oxide or silver-coated copper nanoparticles were employed (Figure 6). Higher antibacterial activity of cinnamaldehyde against Staph, aureus compared to other bacterial strains including Gram-negative Escherichia coli has been reported. The biofilm formation by Staph, aureus has been described to be dominated by the production of polysaccharide intercellular adhesin (PIA) synthesized by icaADBC encoded proteins including the biofilm-associated protein (Bap) which is crucial for adherence and biofilm development. A study using methicillin resistant Staph, aureus (MRSA) reported that the expression of the gene SarA (a regulator of the Bap protein) can be critically affected by the presence of cinnamaldehyde at subminimum inhibitory concentrations. In terms of copper nanoparticles, although their exact mechanism of action is still unclear, some studies have proposed cytoplasm membrane damage due to the affinity of copper nanoparticles to amines and carboxyl groups present at the cell surface of some microorganisms.

From the zone of inhibition data showing the effectiveness displayed by the cationic polymers against P. aeruginosa and considering the ambiguity in the results from the cationic combinations in the crystal violet test mentioned above, a three- component antimicrobial system including the cationic polymers was evaluated. Table 1 presents the three-component combination coating for this set of experiments.

Table 1. Three components combination coatings The three component system was then evaluated via a biofilm experiment similar to what was previously conducted with the two component coatings. All combinations showed significant activity against both Staph. Aureus and P. aeruginosa, providing a 5- log and 6-log reduction respectively (Figure 7 and Figure 8). P. aeruginosa is commonly observed during colonization and infection of for example chronic ulcers, studies in this area have suggested the use of PHMB due to its proteolysis resistance properties. This cationic polymer has been widely employed in hospitals, industries and homes as a result of its antibacterial and antiviral properties. The ability of PHMB to penetrate bacterial membrane leading to bacterial death has commonly been accepted as its mechanism of action. PHMB has been incorporated into for example polydimethylsiloxane (PDMS) films where inhibition of E. coli, Staph, aureus and Staph, epidermis among other strains has been reported at different loading concentrations (0.1, 0.3, 0.5% (w/w)). Despite the low cytotoxicity against L929 cells of the PHMB loaded samples at concentration of 0.1% in this study, other reports have revealed the likelihood of PHMB to enter mammalian cells. For the specific case of s-PL. electrostatic adsorption as a result of its cationic nature has been the recognized mechanism of action. s-PL has been widely used in food applications but recently its uses have been expanded into the medical research and industrial areas. For biomedical applications, s-PL has been grafted to methacrylamide for the fabrication of hydrogels which showed a 3 -log reduction on the counts of P aureginosa in vitro. Similarly, s-PL was cross-linked with catechol for the fabrication of antibacterial paint for medical devices. The coatings at the highest concentration of s-PL reported a 99.99% reduction in both Gram-positive and Gramnegative bacteria as well as the ability to inhibit biofilm formation for one week. When the antimicrobial properties of s-PL were evaluated against different strains of P. aeruginosa, 3 -log reduction in the viability was reported when concentrations greater than two times the minimum inhibitory concentrations were employed. Here, s-PL was reported to hold the highest microbial selectivity over mammalian cells compared to other cationic polymers such as linear polyethylenimine (LPEI), cc-poly-D-lysine (PDL) and cc-poly-L-lysine (PLL), among others.

The surface of the three compounds combination was analysed by XPS. Coatings comprised of individual and two component combinations were prepared. The XPS survey spectra of individual compounds and their combinations including cinnamaldehyde, copper oxide, s-PL and PHMB indicated the presence of oxygen, nitrogen and carbon (Table 2) mostly related to the polymeric formulation being the main component of the coating. Traces of silicon, a common contamination in XPS were observed. Interestingly, copper presence was not detected in any of the samples, it is assumed that the dry condition of the analysis did not allow copper ions to migrate to the surface as when wet conditions are employed.

Table 2. XPS analysis of individual, two and three components combination

Example 2 - Antiviral efficacy

The antimicrobial compositions were tested for the inactivation of human coronavirus OC43 (Infectivity Assay 1 - component antiviral testing) and to extend the testing of the antimicrobial composition on a panel of virus consisting of OC43, 229E and RVA1 (Infectivity Assay 2 - antiviral testing on a range of viruses). Infective Assay 1 - Antiviral Testing Using OC43

Materials and Methods

Products used:

• Single component Cu(II)O

• Single component cinnamaldehyde

• Single component lysine

• Single component PHMB

• Two component Cu(II)O and cinnamaldehyde

• Two component Cu(II)O and lysine

• Two component cinnamaldehyde and lysine

• Control nail polish labelled as C

• Three component Cu(II)O, cinnamaldehyde and lysine

• Three component Cu(II)O, cinnamaldehyde and PHMB - Labelled as PB

Protocol and Total Number of Samples

25 pL OC43 or control was applied to each material in sterile 24 well tissue culture plates. Test samples were incubated for one hour (class II BSC, lid of the plate left on).

The harvest was one hour post virus addition/media addition, virus was collected in 975 pL of TCID virus infectivity assay media (EMEM 1% FCS) and immediately transferred to a sterile 1 ,7mL tube. All samples were stored at -80 °C upon harvest, until infectivity assay.

The remaining virus inoculum was stored at -80 °C.

Results

Figure 9 shows TCID values for OC43 infectious virus after exposure to the tested compounds. PB and PHMB component showed toxicity to MRC-5 cells. The toxicity of all other component was negligible. Of the single components, the viral infectivity of OC43 was significantly lower in the cinnamaldehyde and PHMB treated discs (p<0.0005), in comparison to baseline. There was no significant reduction of OC43 infectivity for Cu(II)O and lysine treated discs after 60 minutes, however the combination of Cu(II)O with lysine resulted in a significant reduction of infectious OC43 in comparison to untreated discs (p<0.005). Similarly, infectious virus was reduced for discs treated with Cu(II)O combined with cinnamaldehyde (p<0.0005), but the reduction was comparable to cinnamaldehyde alone. Maximum reduction in infectious virus was observed for the double component cinnamaldehyde and lysine, as well as the LY and PB treated discs (p<0.0005). For these three components, the reduction of infectious OC43 was greater than 99.9% in comparison to baseline (Table 3). In Table 3, the reduction was calculated based on averages using the following formula: 100* (baseline

- tested component)/baseline. A negative value corresponds to a reduction of infectious virus in the tested compound, in comparison to baseline.

Table 3: Percentage of reduction of OC43 infectious virus components after one hour exposure to tested components. Infectivity Assay 2- Antiviral Testing OC43, 229E and RVA1

Materials and Methods

Products used:

• Control Nail Polish labelled as C

• Three component Cu(II)O, cinnamaldehyde and lysine - Vials labelled as LY

• Three component Cu(II)O, cinnamaldehyde and PHMB - Vials labelled as PB

Protocol

25 pL of 229E, OC43 or RV lb diluted as follow was applied to SF/baseline discs in sterile 24 well tissue culture plates. Note - updated timing'. Test samples were incubated for 30 min, 1 hour (class II BSC, lid of the plate left on):

. 229E at 4x 10 ⁵ TCID50/mL (diluted in EMEM 1% FCS to give lx 10 ⁴ TCID50/25 pL) = repeat from previous experiment

. OC43 at 4x 10 ⁷ TCID50/mL (diluted in EMEM 1% FCS to give lx 10 ⁶ TCID50/25 pL) = other virus with higher titre to increase range of antiviral testing

. RVlB 4x 10 ⁷ TCID50/mL (diluted in DMEM 1% FCS to give lx 10 ⁶ TCID50/25 pL)

• Control wells 25uL of 1 % EMEM or 1 % DMEM was added to assess cell toxicity of the materials. Half the samples were assayed in MRC-5 (as done for coronavirus infection) and half the samples were assayed in RD ICAM (as done for rhinovirus infection)

For the harvest, at the specified time post virus addition, virus were collected in 975 pL of TCID virus infectivity assay media (EMEM 1% FCS or 1% FCS DMEM ) and immediately transferred to a sterile 1.7mL tube. All samples were stored at -80 °C upon harvest, until infectivity assay.

The remaining virus inoculum were stored at -80 °C.

Results

Figure 10 shows TCID values for OC43 infectious virus after exposure to the tested compounds and Table 4 shows corresponding reduction percentage in comparison to baseline. A cell toxicity was observed for PB treated discs in RD-ICAM cells. The toxicity of the LY component was negligible. There was no significant reduction of RV infectivity for the treated disc in comparison to baseline at 30 minutes of exposure. After one hour, the viral infectivity assay showed a significant reduction in infectious RV-A1 both for the LY (*: p<0.05; 60% reduction to baseline) and PB (**: p<0.001; 63% reduction to baseline) treated discs in comparison to untreated discs.

In Table 4, the reduction was calculated based on averages using the following formula: 100* (baseline - tested component)/baseline. A negative value corresponds to a reduction of infectious virus in the tested compound, in comparison to baseline. On the contrary, a positive value corresponds to an increase in infectious virus in the tested compounds, in comparison to baseline. Baseline at 1 hour post infection was compared to baseline infectious virus at 30 min of exposure. One technical replicate was excluded for the LY compound at 1 hour.

Table 4: Percentage of reduction of RV-A1 infectious virus after exposure to LY or PB compounds for 30 minute or one hour