WO2013005215A2

WO2013005215A2 - Phmg modified antimicrobial agents

Info

Publication number: WO2013005215A2
Application number: PCT/IL2012/050237
Authority: WO
Inventors: Abraham J. Domb; Stanislav RATNER; Oren Mizrahi; Haim Wilder; Hella Frenkel
Original assignee: Strauss Water Ltd.
Priority date: 2011-07-05
Filing date: 2012-07-05
Publication date: 2013-01-10
Also published as: WO2013005215A3

Abstract

The invention concerns an antimicrobial agent comprising modified polyhexamethylene guanidine (PHMG), products containing it and uses thereof.

Description

PHMG MODIFIED ANTIMICROBIAL AGENTS

TECHNOLOGICAL FIELD

This invention relates to the synthesis and uses of polymers and particles having antimicrobial activity.

BACKGROUND

PHMG is known to be an antimicrobial agent. However, its water solubility limits its applicability to systems which are only briefly exposed to aqueous medium. In order to stabilize and extend its usability, there is a need for modifying its solubility.

REFERENCES

[1] RU2258064

[2] RU2181808

[3] Yudanova et al, Fibre Chemistry, 31(1), pp. 38-43, 1999

[4] Bazaron and Stel'makh, Russian Journal of Applied Chemistry, 81(11), pp. 2021-2025, 2008

[5] Yudanove et al., Pharmaceutical Chemistry Journal, 37(12), pp. 667-670, 2003 [6] Qian et al, Polymer, 49(10), pp. 2471-2475, 2008

[7] US 2003/044377

SUMMARY OF THE INVENTION

The invention generally relates to the use of polymers and particles having antimicrobial activity.

The inventors of the present invention have demonstrated the efficient use of modified polyhexamethylene guanidine (PHMG) as an antimicrobial agent. The use of modified PHMG, used in accordance with the present invention, as an antimicrobial agent has been demonstrated by the inventors to be effective in a variety of applications, as detailed herein.

In one of its aspects, the invention provides an antimicrobial agent comprising modified polyhexamethylene guanidine (PHMG). As used herein the term "antimicrobial agent" refers to a substance or a composition which reduces, eliminates or prevents the presence of bacteria in a liquid medium, in most cases in water. The antimicrobial agent of the present invention comprises polyhexamethylene guanidine, having the following formula

Within the scope of the present invention, the PHMG is considered to be an antimicrobial agent on its own, having active amine groups which are reactive towards microorganisms. However, the PHMG of the invention is modified (namely "modified polyhexamethylene guanidine" or "modified PHMG") in order to reduce its water solubility and enhance its stability in aqueous media. In some embodiments the modified PHMG is substantially insoluble in water (i.e. at least 99%wt of the modified PHMG is insoluble).

In some other embodiments, the modified PHMG has a molecular weight of between about 1,000 and 20,000 g/mole, and in other embodiments between 2,000 and 5,000 g/mole.

The modified PHMG, which may be prepared by a variety of ways, may be selected from a cross-linked PHMG, a complexed PHMG, a grafted PHMG, a surface- associated PHMG, an N-halogenated PHMG, a mixture of PHMG and polyethylene glycol (PEG), and combinations thereof.

According to some embodiments, the modified PHMG may be a cross-linked PHMG comprising PHMG and a cross-linking agent. The term "cross-linking" or any lingual variation thereof, refers in general to a reaction between a polymer (in this case PHMG) and a second chemical entity (i.e. a "cross-linking agent"), which may be another polymer or a chemical compound to which the polymer is reactive. Such a reaction results in a network-like structure, in which each PHMG polymeric chain is chemically bonded to adjacent chains through the cross-linking agent. The cross-linking may for example be achieved by employing appropriate chemical conditions, as are known to a person skilled in the art and demonstrated in the Examples. In a specific example, PHMG having repeating units which bare at least one amine group per unit was cross-linked with a dialdehyde-containing compound.

The cross-linking effectively increases the molecular weight of the PHMG, while binding the PHMG molecules into a complex structure, thereby reducing its solubility in water. Therefore, control of the solubility may be achieved by controlling the degree (or extent) of the cross-linking. In some embodiments, the cross-linked PHMG is at least 0.1% cross-linked. In other embodiments, the cross-linked PHMG is between 1 % and 30% cross-linked.

The degree of cross-linking may be controlled by tailoring the molar ratio between the PHMG and the cross-linking agent. According to some embodiments, the molar ratio of PHMG to the cross-linking agent is between 99.9: 0.1 to 70:30.

In some embodiments, the cross-linking agent is selected from a triamine, a polyetheramine (Jeffamine), poly(vinyl amine), polyallylamine, a diisocyanate or polyisocyanate, a di or polyacyl halide, a dialdehyde or polyaldehyde, a diepoxide or polyepoxide, and poly(aminomethyl methacrylate).

The Jeffamine triamine series and Jeffamines having 3 or more primary amino groups are commercially available from HUNTSMAN CORPORATION, USA.

Isocyanates applicable for cross-linking of PHMG may be phenylene di and tri- isocyanates, triphenylme thane trisocyanate and hexamethylene diisocyanate.

In the context of the invention, acyl-halides can be selected from citroyl chloride, phenylene tricarboxyloyl chloride and poly(acryloyl chloride), while di and polyaldehydes may be selected from glutar aldehyde, polyaldehydes based on oxidized oligo and polysaccharides. Diepoxides of special relevance may be 1,3- butadienediepoxide or 4-vinylcycl-l-cohexene diepoxide.

In other embodiments, the modified PHMG is a complexed PHMG comprising PHMG and a complexing agent.

According to some embodiments, the complexing agent is at least one " poly anion" , namely a polymer whose repeating units bear an anion group. The polyanion may be selected, according to some embodiments, from an acrylic acid polymer, a sulfate containing polymer, alginate, a polymer comprising hyaluronic acid moieties, and homo and copolymers of methacylic or acrylic acid, carboxy methyl cellulose and others. In some embodiments, the polyanion is polysulfonic acid. The PHMG is said to be "complexed" with the complexing agent, resulting from their opposite charges; i.e. the positively charged PHMG is electro-statically associated to the negatively charged polyanion, thereby forming a bulk complex. Such complexation reduces the solubility of PHMG in water.

In some embodiments, the molar ratio between said PHMG and said polyanion is between about 10-90% molar ratio per carboxylic acid versus guanidine groups along the polymer chain.

According to other embodiments of the invention, the modified PHMG is a grafted PHMG, having side chain moieties grafted onto the PHMG backbone. The term "grafted PHMG" refers to branching of the PHMG polymeric backbone, in which the branching consist of side chain moieties which are structurally and/or chemically distinct from the backbone. Such grafting may be achieved, for example, by co- polymerization of PHMG with vinyl derivatives.

According to some embodiments, the side chain moieties that can bind or form PHMG side chains are selected from primary amines, aldehydes, isocyanates and guanidine groups.

The reduction of the water solubility of PHMG by using grafting may be controlled by the length and characteristics of the side chain moieties, as well as the density of grafting along the PHMG backbone. In some embodiments, the distance between two adjacent side chains is between 1 and 10 monomers/PHMG carbons. In other embodiments, the side chain moieties have a molecular weight of between 300 to 3,000 g/mole.

According to further embodiments of the present invention, the modified PHMG is a surface-associated PHMG, the PHMG being associated to at least a portion of an external surface of a particle.

In such embodiments, the PHMG is associated to the surface of the particle in a substantially non-labile association, namely via an association which would not be decomposed or revered under the conditions of use; such an association may be selected from ionic, hydrophobic or amphiphilic association, covalent association, hydrogen bonding, and combinations thereof.

As the association of the PHMG chains to the surface of the particle is non- labile, the PHMG is prevented from solubilizing in water by anchoring the PHMG chains onto the particle surface. The PHMG may be associated with at least a portion of the particle's surface. The "portion" (region) of the particle's surface may be of any size and structure, the portion may be continuous or comprise of several non-continuous sub-regions on the surface. In some embodiments, the surface of the substrate is substantially two- dimensional. In other embodiments, the surface is that of a three-dimensional object. In other embodiments, the at least one portion of the particle's surface is its whole surface.

In some embodiments, said portion is at least 10% of the external surface of the particle.

In other embodiments, the particle is substantially coated with said PHMG.

In other embodiments, the particle has a core of one material and a surface of another material, said surface being associated with said PHMG.

According to some embodiments, the particle may be selected from a substantially spherical particle, a flake, a pellet, a substantially cylindrical particle, or fiber like.

The particles used to modify PHMG may have an average size of between 100 nm and 10,000 nm. For spherical particles, the term "average size" typically refers to the average diameter of the particles. When the particles are of non-spheroid shape, the term refers to the average equivalent diameter of the particle, namely the diameter of an equivalent spherical particle based on the longest dimension of the particle.

In other embodiments, the particle is selected from a silica particle, a polystyrene particle, and a particle having amine groups on its external surface.

Another type of modification of PHMG is N-halogenation. This refers to the formation of halo-amine groups on the PHMG backbone, in which a halogen atom is covalently bound to the amine group of PHMG. When contacted with water, said halogen atom is released as active halogen, such as active bromine or chlorine, thereby providing the antibacterial activity.

Therefore, according to some other embodiments, the modified PHMG is an N- halogenated PHMG.

In some embodiments, the N-halogenated PHMG is between 5 and 60% halogenated. In other embodiments, N-halogenated PHMG is halogenated with bromine or chlorine. In such embodiments, upon contact with water, active bromine or active chlorine is released from said N-halogenated PHMG.

According to some embodiments, the modified PHMG is a mixture of PHMG and polyethylene glycol (PEG). The PEG may have a molecular weight of between 10,000 and 50,000 g/mole. In some embodiments, the PEG has a molecular weight of between 20,000 and 40,000 g/mole.

According to other embodiments, the weight ratio of PHMG to PEG is between 1 :99 and 30:70. In other embodiments, the weight ratio of PHMG to PEG is between 5:95 and 25:75.

The antimicrobial agent of the invention is effective in reducing or eliminating a microorganism population or a biofilm of such microorganisms. As demonstrated herein, the formulations of the invention provide instant and persistent antimicrobial activity against a wide spectrum of microorganisms and specifically against a broad spectrum of bacteria. The term "microorganism " relates herein to single cell (unicellular), cell clusters, or no cell (acellular) organism such as bacteria, fungi, yeast, mold, archaea, protists, viruses and algae.

In some embodiments, the antimicrobial agent of the invention is an antimicrobial preservative, attesting to the ability of the formulations of the invention to suppress microbial growth, reduce microbial infestation, treat products or surfaces to improve product resistance to microbial infestation, reduce biofilm, prevent conversion of bacteria to biofilm, prevent or inhibit microbial infection, prevent spoilage, retard or minimize or prevent quorum sensing, and retard microbial reproduction.

The antimicrobial agent is capable of endowing a product, or the product surface, with a biological resistance to at least one biological effect, which in the absence of such agent would eventually bring about a short-term or long-term damage to the product. In the context of the invention, the antimicrobial agent improves the product's resistance to a certain environmental condition. In some embodiments, the resistance to such a condition is resistance to biofouling.

In some embodiments, the microorganism is a bacteria, being selected, in some embodiments from Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (E. coli), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, Vibrio harveyi and Yersinia pestis.

In other embodiments, the microorganism is a fungus, selected in some embodiments from Absidia corymbifera, Ajellomyces capsulatus, Ajellomyces dermatitidis, Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Aspergillus flavus, Aspergillus fumigates, Aspergillus niger, Blastomyces dermatitidis, Candida albicans, Candida albicans var. stellatoidea, Candida dublinensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida pelliculosa, Candida tropicalis, Cladophialophora carrionii, Coccidioides immitis, Cryptococcus neoformans, Cunninghamella sp., Epidermophyton floccosum, Exophiala dermatitidis, Filobasidiella neoformans, Fonsecaea pedrosoi, Geotrichum candidum, Histoplasma capsulatum, Hortaea werneckii, Issatschenkia orientalis, Madurella grisae, Malassezia furfur, Malassezia furfur complex, Malassezia globosa, Malassezia obtuse, Malassezia pachydermatis, Malassezia restricta, Malassezia slooffiae, Malassezia sympodialis, Microsporum canis, Microsporum fulvum, Microsporum gypseum, Microsporum gypseum complex, Microsporum gypseum, Mucor circinelloides, Nectria haematococca, Paecilomyces variotii, Paracoccidioides brasiliensis, Penicillium marneffei, Phialophora verrucosa, Pichia anomala, Pichia guilliermondii, Pneumocystis jirovecii, Pseudallescheria boydii, Rhizopus oryzae, Rodotorula rubra, Saccharomyces cerevisiae, Scedosporium apiospermum, Schizophyllum commune, Sporothrix schenckii, Stachybotrys chartarum, Trichophyton mentagrophytes, Trichophyton mentagrophytes complex, Trichophyton mentagrophytes, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, Trichophyton verrucosum, Trichophyton violaceum, Trichosporon asahii, Trichosporon cutaneum, Trichosporon cutaneum complex, Trichosporon inkin and Trichosporon mucoides.

According to other embodiments, the microorganism is yeast, being selected, in some embodiments, from Candida albicans, Candida albicans var. stellatoidea, Candida dublinensis, Candida glabrata, Candida guilliermondii, Candida krusei, Candida parapsilosis, Candida pelliculosa, Candida tropicalis, Cryptococcus neoformans, Filobasidiella neoformans, Geotrichum candidum, Issatschenkia orientalis, Malassezia furfur, Malassezia pachydermatis, Pichia anomala, Pichia guilliermondii, Pneumocystis jirovecii, Rodotorula rubra, Trichosporon asahii, Trichosporon cutaneum, Trichosporon inkin and Trichosporon mucoides.

In further embodiments, the microorganism is mold, being selected, in some embodiments, from Absidia corymbifera, Arthroderma benhamiae, Arthroderma fulvum, Arthroderma gypseum, Arthroderma incurvatum, Arthroderma otae, Arthroderma vanbreuseghemii, Aspergillus flavus, Aspergillus fumigates, Aspergillus niger, Cladophialophora carrionii, Coccidioides immitis, Epidermophyton floccosum, Exophiala dermatitidis, Fonsecaea pedrosoi, Hortaea werneckii, Madurella grisae, Microsporum canis, Microsporum fulvum, Microsporum gypseum, Microsporum gypseum, Microsporum gypseum, Mucor c ire inello ides, Nectria haematococca, Paecilomyces variotii, Paracoccidioides brasiliensis, Penicillium marneffei, Pseudallescheria boydii, Rhizopus oryzae, Scedosporium apiospermum, Schizophyllum commune, Sporothrix schenckii, Stachybotrys chartarum, Trichophyton mentagrophytes complex, Trichophyton mentagrophytes, Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, Trichophyton verrucosum and Trichophyton violaceum.

In another one of its aspects, the present invention provides a self-sterilizing product comprising the antimicrobial agent as described herein. The term "self- sterilizing" refers to the ability of the product (or surface thereof) to eliminate or prevent microorganisms' growth and accumulation without the need of additional sterilizing substances.

Products which are contemplated by the invention may be selected from a water filtering medium, a water filter, a water purifier, a water container, a water conduction tube, and others. Another aspect of the invention provides the use of the antimicrobial agent described herein in the manufacturing of a product having a self-sterilizing surface.

A further aspect provides a method for reducing bacterial concentration in water, the method comprising contacting the water with the antimicrobial agent of the invention as herein described.

As used herein, the term "contacting" , or any lingual variation thereof, refers to the bringing together of the liquid to be treated (e.g., water) and the antimicrobial agent embedded or coated onto at least a part of the product's surface in such a way to allow elimination of organisms within the water. The contacting may be, for example, by flowing the water over or through the product, which may be a solid product or a porous product.

Yet a further aspect provides a method for preventing growth of bacteria on a surface being in contact with water for at least a period of time, the method comprising incorporating the antimicrobial agent of the invention onto said surface.

In some embodiments, the product's surface is coated with the antimicrobial agent as described herein.

In other embodiments, the product's surface consists of the antimicrobial agent as described herein.

The invention also provides substrates coated on at least one region with a material comprising the antimicrobial agents of the invention. In some embodiments, the surface coating coats the entire surface of the substrate. In some embodiments, the coating uniformly covers the substrate surface.

The antimicrobial agents are present material impart the antimicrobial properties to the substrate. The surface may be associated with the surface in any way, e.g., Van der Waals forces, ionic bonding, hydrogen bonding, or through a coating linker such as a glue, forming stable coatings that exhibits minimal or no degradation or leaching, e.g., when exposed to an aqueous medium. As such, the coatings comprising the antimicrobial agents in accordance with the invention are safe for use in a variety of daily applications.

In some embodiments, the coating disposed on the substrate surface is provided with the structural functionality of the substrate to carry out the applications disclosed herein. In some embodiments, the substrate is designed for water applications. In other cases, the substrate to be coated is designed for biological uses or devices used in medical applications, such as for assisting a medical procedure, e.g., catheters, stents.

The coated substrates according to the invention may be used for (a) reducing or preventing bacterial infection without the need to use drug materials, e.g., antibiotics, to the end product, (b) reducing the degree of decomposition or degradation of biological material, e.g., blood or biologies, and (c) reducing or preventing the fouling of aqueous media. Thus, the substrates may be any region of a storage container or a delivery system for use in food packaging, food and beverage containers, food and beverage preparation or disposing equipment, blood bags, proteins or pharmaceuticals.

Alternatively, the antimicrobial agents of the invention may be used in the construction of a personal product or an industrial product such as devices used in sporting activities, orthodontic devices, face or breathing masks, pacifiers, contact lenses, adult products, food preparation surfaces, food packaging, reusable water containers, hydration systems, water bottles, computer keyboards, telephones, rental car steering wheels, health club equipment, whirlpool spas and humidifiers to provide antimicrobial properties.

In some embodiments, the products employing an antimicrobial agent according to the invention are selected from filtration devices and water lines, reusable water containers, hydration systems, water bottles and containers.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the disclosure and to see how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:

Fig. 1 depicts the general synthesis scheme of PHMG.

Fig. 2 shows the ^lR NMR spectrum of PHMG.

Fig. 3 shows the FTIR spectrum of PHMG.

Fig. 4 depicts the in-situ synthesis process of cross-linked PHMG using tris(2- aminoethyl)amine.

Fig. 5 depicts the process of cross-linking of PHMG by amines.

Fig. 6 depicts the process of cross-linking PHMG by polyepxides.

Fig. 7 depicts the process of cross-linking PHMG by di or tri-isocyanate. Fig. 8 depicts the process of surface-association of PHMG to polystyrene particles.

Fig. 9 depicts an additional process of surface-association of PHMG to polystyrene particles.

Fig. 10 depicts the process of N-halogenation of PHMG.

Fig. 11 shows the dissolution rate of cylindrical samples of PHMG/PEG mixtures (· PEG 20,000 g/mole;■ PEG 35,000 g/mole).

DETAILED DESCRIPTION OF EMBODIMENTS

The modifications of PHMG according to the invention will now be demonstrated in a non-limiting manner, with reference to the examples.

Synthesis of modified PHMG

Example 1 : Synthesis of PHMG

PHMG polymer was synthesized by polycondensation between guanidine hydrochloride (Mw = 95.53 g/mole) and hexamethylene diamine (Mw = 116.2 g/mole) at 1: 1 molar ratio. Equal molar amounts of hexamethylene diamine and guanidine hydrochloride were added to a round bottom flask and melted at 100°C. After complete melting of the monomers, the temperature was increased to 150-155°C and mechanically-stirred for 10 hours. The temperature was increased to 180-185°C for 3 hours to achieve a white polymeric product with high viscosity. The reaction followed the scheme shown in Fig. 1.

Molecular structure was characterized mainly by NMR and FTIR (Figs. 2 and 3, respectively). A small amount of modified guanidine polymer solution was air dried and then dissolved in D₂0. *H NMR and ¹³C NMR of PHMG were conducted using a BRUKER DPX-300 (300 MHz). In the 1H-NMR spectra, the peaks are: 1.374 (A, -CH₂-), 1.573-1.592 (B, -CH₂-), 2.795 (D, CH₂-NH₂), 3.166-3.23 (C, -CH₂-NH-) and 4.79 (D₂0). The characteristic peaks of PHMG samples FTIR spectra are: 3350 and 3170 (NH), 1650 (C=N) and 1628 cm^"1 (NH₂), which are consistent with the data produced by other investigators.

Table 1 summarizes the results of the OmniSEC Homopolymer method. Mw is the weight average molecular weight (Da), Mn is the number average molecular weight (Da), polydispersity of the sample is Mw/Mn, IV is the intrinsic viscosity (dL/g), Rh is the hydrodynamic radius (nm), and dn/dc is the refractive index increment.

Example 2: In-situ synthesis of cross-linked PHMG with Jeffamine

Cross-linked PHMG was synthesized in-situ by polycondensation between guanidine hydrochloride (Mw = 95.53 g/mole) and hexamethylene diamine (Mw = 116.2 g/mole) at 1 :1 molar ratio in the presence of Jeffamine (Mw~ 5,000 g/mole) as cross-linking agent.

50g of hexamethylene diamine and 41. lg of guanidine hydrochloride were added to a round bottom flask and melted at 100°C, adding the Jeffamine in different molar percentages (0.5%, 1%, 5% and 10%). After complete melting of the monomers, the flask was heated to 150-155°C and mechanically-stirred for 10 hours. The temperature was increased to 180-185°C for 3 hours to achieve a white to yellow water- insoluble polymeric gel with high viscosity.

Example 3: In-situ synthesis of cross-linked PHMG with PEI

Cross-linked PHMG was synthesized in situ by polycondensation between guanidine hydrochloride (Mw = 95.53 g/mole) and hexamethylene diamine (Mw = 116.2 g/mole) at 1: 1 molar ratio in the presence of polyethylene-imine (PEI) (Mw~ 750,000 g/mole) as a cross-linking agent.

50g of hexamethylene diamine and 41. lg of guanidine hydrochloride were added and to a round bottom flask and melted at 100°C, adding the dry PEI to the reaction flask in different molar percentages (10% or 50%). The flask was heated to 150-155°C after complete melting of the monomers and stirred using mechanical stirring for 10 hours. The temperature was increased to 180-185°C for 3 hours to achieve a white to yellow water-insoluble polymeric gel with high viscosity. Example 4: In-situ synthesis of cross-linked PHMG using tris(2-aminoethyl)amine (Fig. 4)

Cross-linked PHMG was synthesized in situ by polycondensation between guanidine hydrochloride (Mw = 95.53 g/mole) and hexamethylene diamine (Mw = 116.2 g/mole) at 1 : 1 molar ratio in the presence of tris(2-aminoethyl)amine as a cross- linking agent.

Equal molar amounts of hexamethylene diamine and guanidine hydrochloride were added to a round bottom flask and melted at 100°C, adding the tris(2- aminoethyl)amine in different molar percentages (0.5%, 1%, 5% and 10%). The flask was heated to 150-155°C after complete melting of the monomers and mechanically- stirred for 10 hours. Increasing the temperature to 180-185°C for 3 hours to achieve to achieve a white to yellow water-insoluble polymeric gel with high viscosity. The polymer structure was determined by NMR and IR.

Example 5: Cross-linking of PHMG by amines (Fig. 5)

PHMG of Mw -2,500 g/mole was reacted with an amine-based cross-linking agent in different molar ratios according to the following procedure.

To a round bottomed glass flask, 15g of PHMG hydrochloride and 100ml DMF were added. The flask was heated to 150-160°C to allow the polymer to melt. The amine-based cross-linking agent (Jeffamine or PEI or tris(2-aminoethyl)amine) was added dropwise in different molar percentages (0.5%, 1%, 5% and 10%). Increasing the temperature to 180-185°C for 3 hours to achieve a white to yellow water-insoluble polymeric gel with high viscosity. The polymer structure was determined by NMR and IR.

Example 6: Cross-linking PHMG by polyepxides (Fig. 6)

Covalent binding of PHMG to the surface was achieved using the reaction of epoxy group with amine of PHMG. PHMG / Epoxy plates were obtained by mixing PHMG with epoxy glue monomers (EP-520 and EPC-520 hardener in 10:3 ratio, respectively) at different concentrations followed by drying in the hood for the weekend. From the FTIR spectra the difference between the epoxy material and after it was mixed with PHMG are clearly shown. The peak in 1240 cm^"1 of C-0 stretch which is typical to the epoxy group does not appear in the reacted product, indicating all epoxy groups reacted. Typical N-H stretch is related to PHMG appear in 3150 cm^"1 and 3325 cm^"1, and at 1635 cm^"1 of N-H bends. Peaks at 1100 cm^"1 can be attributed to C-0 stretch of secondary alcohol, together with broad area above 3300 cm^"1 of O-H stretch, which is not seen due to overlapping of the N-H of PHMG.

Example 7: Cross-linking PHMG by di or tri-isocyanate (Fig. 7)

8g PHMG and 60ml DMF were fed into 3-neck round bottom flask with magnetic stirrer at 135°C under reflux conditions. 900mg 2,4-tolylene diisocyante dissolved in 3ml DMF was added dropwise, causing a color change to light yellow. After 1 hour the DMF was evaporated, then water was added for rinsing. The material obtained was dried in an evaporator, obtaining a yellow-brown particulate solid. The particle size was determined to be between 90-400nm. FTIR results, PHMG and cross- linked PHMG, explorer the appearance of two new peaks; 1667 cm-1 wave number is attributed to an aromatic C-H bend and 1667 cm-1 wave number is attributed to urea bend.

Antimicrobial activity:

HPP plates with 0.5% and 1% w/w of cross-linked PHMG were obtained by injection of the mixture. The mixtures were tested for virucidal and bactericidal efficacy against the E. coli bacteria by immersing samples in bacterial suspensions for predetermined period of time. The microorganisms used to assess biological activity are given in Table 2. Bacterial concentrations on the surface of the samples are provided in Table 3.

Table 2: microorganisms used

Bacteria stitched into

No. of E. coli bacteria on the surface of the

the suspension sample (CFU/surface)

Sample (CFU/cm²)

After 24 hours After 76 hours

After 76 hours

Side l Side 2 Side l Side 2

Cross-linked PHMG

- - 98 45 - 0.5% in HPP

Cross-linked PHMG - - 41 24 - 1% in HPP

HPP (reference) -lxlO³ -lxlO³ -lxlO³ -lxlO³ ~2xl 0⁴

Table 3: bacterial count on the surface of samples

Example 8: Complexation of PHMG with polyacrylic acid

An aqueous solution of 5% wt/v PHMG was mixed with an aqueous solution of 5% polyacrylic acid MW=2,000 g/mole at room temperature. The solution was allowed to mix overnight, during which a complex was formed. Water was removed to form a solid complex where PHMG is slowly dissolved.

Example 9: Surface-association of PHMG to polystyrene particles (Fig. 8)

Step A: Preparation of amino polystyrene beads from chloromethylepolystyrene beads. lOg of chloromethyleted polystyrene beads were transferred to a round bottom flask containing 200ml of acetone and stirred for 2 hrs and then isolated and dried. 5.47g cleaned particles were transferred to a round bottom flask and 28.28 ml of ethylene diamine (0.423 mole) was added and refluxed overnight. The particles were isolated and washed 3 times with 50 ml DDW and dried.

Step B: Connecting PHMG to amino polystyrene beads (from step A). 16g of PHMG (Mw of repeated unit 177 g/mole) were added in 50 ml double-distilled water in a 250 ml flask. After completion of solubilization, 5g of amino polystyrene beads were added and allowed to reflux over night. The particles were isolated by filtration and washed 10 times with 100ml water and dried.

Example 10: Surface-association of PHMG to polystyrene particles (Fig. 9)

Step A: lOg chloromethyleted polystyrene beads (4.513x l0^~3 reactive sites per gram; total of 0.0451 mole reactive sites) were added to 10.5g (0.0902 mol, 2 eqv to reactive sites) hexamethylene diamine dissolved in 100 ml DMF anhydrous and heated to 140°C for 24 hrs. The beads were isolated by filtration and washed 3 times with 50 ml DMF and 5 times with 100 ml double-distilled water.

Step B: the beads were reacted with 0.09 mol guanidine hydrochloride (8.6225g) dissolved in 100 ml DMF anhydrous at 140°C for 24 hrs and isolated, washed and dried.

The same process was repeated three more times to form a chain containing four guanidine groups. Elemental analysis showed the decrease of functional group (CI) with a followed of nitrogen (N) content, see Table 4.

Table 4: elemental analysis results of step 1, N-methylation of polystyrene chlorinated

FTIR spectrum showed the appearance of a new peak in 1667 cm^"1 in the PS- HMDA material compared to PS-C1 spectra. This wave number is attributed to an N-H bend of a primary or a secondary amine, indicating of the presence of amine group presence in the molecule, supporting the element analysis results. Solid C NMR results explorer the typical aromatic shifts at 126.88, 128.53, 127.58 ppm and 137.5/139.87 ppm for the substituted carbon which are assigned to polystyrene. 46.10 ppm of the methylene group in the PS has changed to 53.74 ppm after the reaction. That, and calculated shifts regarding aliphatic C at 29.4, 29.14, 26.98, 26.83 ppm and 49.62 ppm for C-N, which also appear in the obtained spectra of the analyzed materials, indicating on the binding of HMD A to PS.

Example 11 : N-halogenation of PHMG (Fig. 10)

N-bromo PHMG was prepared from the reaction of PHMG with 0.5, 1, 5 and 10 equivalents of aqueous hypodromide (4% w/v in water, pH 6.5) for 8 hours at room temperature. The bromine content was 21-23%w/w as determined by elemental analysis. The polymer showed a higher antibacterial activity compared to native PHMG.

Antimicrobial activity:

HPP plates with 0.5% and 1% w/w of brominated PHMG were obtained by injection of the mixture. The mixtures were tested for virucidal and bactericidal efficacy against the E. coli bacteria by immersing samples in bacterial suspensions for predetermined period of time. The microorganisms used to assess biological activity are given in Table 5. Bacterial concentrations on the surface of the samples are provided in Table 6. Microorganism ATCC number Supplier

E. coli 8739 ATCC

E. coli 15597 ATCC

Table 5: microorganisms used

Table 6: bacterial count on the surface of samples

Example 12: N-halogenation of surface-associated PHMG

The beads containing guanidine chains from Example 10 were immersed in 10 equivalents of aqueous hypobromide for 24 hours. The beads were isolated by filtration and dried to yield yellowish particles with 9.75% w/w bromine as determined by elemental analysis.

Antibacterial activity:

Particles of PHMG and particle-associates N-brominated PHMG were tested for virucidal and bactericidal efficacy against the MS2 phage and E. coli bacteria according Water Quality Assurance Tap Water Source for the developing countries requirements (3 log reduction for E. coli and 3 log reduction for MS2). For this aim, treatment columns with different types of the mediums were tested.

The data of materials used is given in Table 7.

Table 7: technical data of materials The microorganisms used to assess biological activity are given in Table 8.

Table 8: technical data of materials

Particles of PHMG and particle-associates N-brominated PHMG were tested for their antimicrobial activities using the following procedures:

Bactericidal efficacy of different materials into bacterial suspension - Small amount of tested materials (0.005g) was added to 1 ml of E. coli (2xl0³ CFU/ml) and MS2 (2x l0³ PFUml) suspensions. After 30 minutes and 18 hours, bacterial and virucidal concentration in all suspensions was tested. As is evident from the results provided in Tables 9-10, the particle-associates N-brominated PHMG possess strong antimicrobial activity.

Table 9: The Log reduction of E. coli bacteria and MS2 bacteriophages

Table 10: Log reduction of MS2 bacteriophages in different conditions

The results show that the particle-associates N-brominated PHMG have very high antimicrobial activity. The bromine concentration released from the particles is very high and probably is the cause for the high antimicrobial activity. The PHMG particles (the control media, without Br) also possess some antimicrobial activity as reported in the literature.

In order to evaluate the efficiency of the particles, the particle-associates N- brominated PHMG was washed with 8L of the water and the activity and bromine released was determined. The results are given in Table 11. The results show that the inactivation efficiency of the washed particle-associates N-brominated PHMG decreased compared to the unwashed particles, but still remain highly active.

Table 11 : Log reduction of MS2 bacteriophages in different conditions

Example 13: mixtures of PHMG and PEG

Polyethylene glycol (PEG) was admixed with PHMG is order to reduce its solubility and control its release rate into water.

PEG/PHMG cylinder units were prepared by injection molding; the two water soluble polymers PEG MW=20,000 g/mole or PEG MW=35,000 g/mole and PHMG, MW=3,200 g/mole were dissolved in water at PHMG content of 5% 10% 15% and 20% per PEG carrier and dried by lyophilization to obtain a white powder. The homogenous powder mixture of PEG/PHMG was mixed with 15% CaCC>3 dry powder as filler and 0.5% HPN-68L as nucleating agent and injection molded into cylinder units. The use of PEG was to control PHMG dissolution rate. The addition of CaCC>3 as filler and HPN- 68L as nucleating agent stabilizing the injection presses by allowing a better heat transfer and more consolidation centers in the bulk, the stabilizer needed due to low viscosity of melted PEG which makes it difficult for the injection process.

PHMG concentration in water was determined by UV at 254nm. The release of PHMG to water was tested as the cylinder unit mounted upright on top of a water purification filter where water added to the filter chamber must contact the PEG-PHMG cylinder. Fig. 14 presents the cylinder dissolution rate. Antimicrobial activity:

To evaluate the bactericidal efficiency of PHMG mixed with PEG, bactericidal efficiency was assessed in 3 separate water purifiers comprising the 10% PHMG in PEG mixture, according to developing countries standard (minimum 3 log reduction of E. coli and minimum 3 log reduction of MS2). Technical details of the materials used are given in Table 12. The microorganisms used to assess biological activity are given in Table 8 above.

Table 12: technical data of materials

The flush between challenges was performed on reverse osmosis and tap water basis (2/3 RO+ 1/3 tap). The water used for the challenge was RO water with sea salt addition (in order to achieve TDS of 200-300 ppm).

Before each challenge 5L of influent wash water was passed through each purifier. In each challenge point 2.8L of contaminated water was passed through each purifier.

After passing 250ml of the water, the samples were collected in 2 different ways: (i) collecting the outlet water into 250ml sterile bottle with SDS (Sodium dodecyl sulfate, 0.05%- PHMG neutralization solution). The PHMG released from the purifiers was neutralized immediately. Another 250ml was collected into a sterile bottle containing 0.05% SDS. One sample was tested in Bactochem laboratory.; (ii) collecting the outlet water into 2L sterile box without SDS. After collecting 2L of the outlet water, 250 ml was transferred into a sterile 250ml bottle with 0.05% SDS for precluding of PHMG action. Another 250ml was transferred into a sterile bottle containing 0.05% SDS. One sample was tested in Bactochem laboratory. The total contact time between PHMG and microorganisms was 30-40 minutes.

E. coli concentration was checked by filtration method (incubation in 44.5°C, 24 hr). Filtration of 1 ml and 100ml of the sample. MS2 bacteriophages concentration was checked by double layer method, incubation in 35°C, for 24 hr.

HPC bacteria were tested only in samples collected after stagnation. 0.1 of each sample from each purifier was spread evenly on TSA plates in order to determine HPC concentration. In addition, HPC bacteria were tested in the inlet water used for the influent wash water. Results of the antibacterial activity tests are presented in Table 13.

The inactivation results for MS2 bacteriophages show that the purifiers exceed the requirements of the developing countries standard at all of the challenge points tested, for both samples tested ('on-line' and 2L sample). Regarding E. coli bacteria, the results received for the 'on-line' sample exceed the requirements of the developing countries standard for all of the challenge points tested.

Sampling after stagnation was also carried out to verify no subsequent growth of bacteria. 2.5L of influent flash water was poured to the upper tank and filtrated through the purifier. The sampling was done as in the challenges but the difference is that this time the collection was at the first void volume. The first outlet water was collected into 250ml sterile bottle with 0.05% SDS and tio-sulfate and then collecting 2L of the outlet water into a box without SDS and transferring 250ml into a sterile bottle with 0.05% SDS and tio-sulfate for neutralization. Results are provided in Table 14. After stagnation periods no growth of E. coli, MS2 or HPC was seen in none of the purifiers.

Contact MS2 log reduction E. coli log reduction

Test

Collecting time (log PFU/lml) (log CFU/lml) point

(min) Inlet 1 2 3 Inlet 1 2 3

On contact 3-4 >3.2 >3.2 >3.2 3.6 3.2 3.3

15L 3.2 5.3

2L 31-40 >3.2 >3.2 >3.2 >5.3 3.9 >5.3

On contact 2 >3.6 >3.6 >3.6 3.0 2.7 4.4

62L 3.6 4.7

2L 28-33 >3.6 2.7 >3.6 2.5 1.4 3.4

On contact 2-3 >3.4 >3.4 >3.4 >4.5 4.5 4.5

125L 3.4 4.5

2L 31-44 >3.4 >3.4 >3.4 >4.5 >4.5 >4.5

On contact 2-3 >3.4 >3.4 >3.4 4.0 >4.5 >4.5

190L 3.4 4.5

2L 30-45 >3.4 >3.4 >3.4 2.3 0.9 >4.5

On contact 2-3 >3.5 >3.5 >3.5 5.4 >5.4 >5.4

225L 3.5 5.4

2L 31-44 >3.5 >3.5 >3.5 1.6 1.3 1.8

On contact 2 >3.4 >3.4 >3.4 4.8 5.1 3.4

250L 3.4 5.1

2L 31-44 >3.4 >3.4 >3.4 4.3 0.9 >5.1 Table 13: E. coli bacteria and MS2 phages log reduction in the tested purifiers according to developing countries standard (criterion: at least 3 log reduction)

Table 14: E. coli, MS2 and HPC bacteria in effluent in the tested purifiers after stagnation periods.

Claims

CLAIMS:

1. An antimicrobial agent comprising modified polyhexamethylene guanidine (PHMG), said modified PHMG being selected from a cross-linked PHMG, a complexed PHMG, a grafted PHMG, a surface-associated PHMG, an N-halogenated PHMG, a mixture of PHMG and polyethylene glycol (PEG), and combinations thereof.

2. The antimicrobial agent of claim 1, wherein said modified PHMG has a molecular weight of between about 1 ,000 and 20,000 g/mole.

3. The antimicrobial agent of claim 2, wherein said modified PHMG has a molecular weight of between about 2,000 and 5,000 g/mole.

4. The antimicrobial agent of any one of claims 1 to 3, wherein said modified PHMG is substantially water insoluble.

5. The antimicrobial agent of claim 1, wherein said modified PHMG being a cross- linked PHMG comprising PHMG and a cross-linking agent.

6. The antimicrobial agent of claim 1, wherein the cross-linked PHMG is at least 0.1% cross-linked.

7. The antimicrobial agent of claim 1 , wherein the cross-linked PHMG is between 1 % and 30% cross-linked.

8. The antimicrobial agent of any one of claims 5 to 7, wherein the molar ratio between said PHMG and said cross-linking agent is between 99.9: 0.1 to 70:30.

9. The antimicrobial agent of any one of claims 5 to 8, wherein said cross-linking agent is selected from a triamine, a polyetheramine (Jeffamine), poly(vinyl amine), polyallylamine, a diisocyanate or polyisocyanate, a di or polyacyl halide, a dialdehyde or polyaldehyde, a diepoxide or polyepoxide, and poly(aminomethyl methacrylate).

10. The antimicrobial agent of claim 9, wherein said cross-linking agent is a triamine.

11. The antimicrobial agent of claim 9, wherein said cross-linking agent is a polyetheramine.

12. The antimicrobial agent of claim 9, wherein said cross-linking agent is a polyallylamine.

13. The antimicrobial agent of claim 9, wherein said cross-linking agent is a diisocyanate or polyisocyanate being selected from phenylene di and tri-isocyanates, triphenylmethane trisocyanate and hexamethylene diisocyanate.

14. The antimicrobial agent of claim 9, wherein said cross-linking agent is an acyl- halide being selected from citroyl chloride, phenylene tricarboxyloyl chloride and poly(acryloyl chloride).

15. The antimicrobial agent of claim 9, wherein said cross-linking agent is a di or polyaldehydes being selected from glutar aldehyde, polyaldehydes based on oxidized oligo and polysaccharides.

16. The antimicrobial agent of claim 9, wherein said cross-linking agent is diepoxide selected from 1,3-butadienediepoxide and 4-vinylcycl-l-cohexene diepoxide.

17. The antimicrobial agent of claim 1, wherein said modified PHMG is a complexed PHMG comprising PHMG and a complexing agent.

18. The antimicrobial agent of claim 17, wherein said complexing agent is at least one polyanion.

19. The antimicrobial agent of claim 18, wherein the molar ratio between said PHMG and said polyanion is between about 10:90 to 90: 10.

20. The antimicrobial agent of claim 17 or 18, wherein said polyanion is selected from an from an acrylic acid polymer, a sulfate containing polymer, alginate, a polymer comprising hyaluronic acid moieties, homopolymers or copolymers of methacylic or acrylic acid, and carboxy methyl cellulose.

21. The antimicrobial agent of claim 1, wherein said modified PHMG is a grafted PHMG, having side chain moieties grafted onto the PHMG backbone.

22. The antimicrobial agent of claim 21, wherein said side chain moieties are selected from primary amines, aldehydes, isocyanates and guanidine groups.

23. The antimicrobial agent of claim 21 or 22, wherein the distance between two adjacent side chains is between 1 and 9 monomers/PHMG carbons.

24. The antimicrobial agent of any one of claims 21 to 23, wherein the side chain moieties have a molecular weight of between 300 to 3,000 g/mole.

25. The antimicrobial agent of claim 1, wherein said modified PHMG is a surface- associated PHMG, the PHMG being associated to at least a portion of an external surface of a particle.

26. The antimicrobial agent of claim 25, wherein said association is substantially not labile.

27. The antimicrobial agent of claim 26, wherein said association is selected from ionic association, hydrophobic association, amphiphilic association, covalent association, hydrogen bonding and combinations thereof.

28. The antimicrobial agent of any one of claims 25 to 27, wherein said portion is at least 10% of the external surface of the particle.

29. The antimicrobial agent of claim 28, wherein said particle is substantially coated with said PHMG.

30. The antimicrobial agent of any one of claims 25 to 29, wherein said particle is selected from a substantially spherical particle, a flake, a pellet, a substantially cylindrical particle, and a fiber.

31. The antimicrobial agent of any one of claims 25 to 30, wherein said particle has an average size of between 100 and 10,000 nm.

32. The antimicrobial agent of any one of claims 25 to 31, wherein said particle is selected from a silica particle, a polystyrene particle, and a particle having amine groups on its external surface.

33. The antimicrobial agent of claim 1, wherein said modified PHMG is an N- halogenated PHMG.

34. The antimicrobial agent of claim 33, wherein said N-halogenated PHMG is between 5 to 60% halogenated.

35. The antimicrobial agent of claim 33 or 34, wherein said N-halogenated PHMG is halogenated with bromine or chlorine.

36. The antimicrobial agent of claim 35, wherein upon contact with water, active bromine or active chlorine is released from said N-halogenated PHMG.

37. The antimicrobial agent of claim 1, wherein said modified PHMG is a mixture of PHMG and polyethylene glycol (PEG).

38. The antimicrobial agent of claim 37, wherein said PEG has a molecular weight of between 10,000 and 50,000 g/mole.

39. The antimicrobial agent of claim 38, wherein said PEG has a molecular weight of between 20,000 and 40,000 g/mole.

40. The antimicrobial agent of any one of claims 37 to 39, wherein the weight ratio between PHMG and PEG is between 1 :99 and 30:70.

41. The antimicrobial agent of claim 40, wherein the weight ratio between PHMG and PEG is between 5:95 and 25:75.

42. A self-sterilizing product comprising the antimicrobial agent of any one of claims 1 to 41.

43. The self-sterilizing product of claim 42, being selected from a water filtering medium, a water filter, a water purifier, a water container, and a water conduction tube.

44. Use of the antimicrobial agent of any one of claims 1 to 41 in the manufacturing of a product having a self-sterilizing surface.

45. A method for reducing bacterial concentration in water, the method comprising contacting the water with the antimicrobial agent of any one of claims 1 to 41.

46. A method for preventing growth of bacteria on a surface being in contact with water for at least a period of time, the method comprising incorporating the antimicrobial agent of any one of claims 1 to 41 onto said surface.

47. The method of claim 46, wherein said surface is coated with the antimicrobial agent of any one of claims 1 to 41.

48. The method of claim 47, wherein said surface consists of the antimicrobial agent of any one of claims 1 to 41.

49. A coating disposed on a substrate surface, the coating comprising an antimicrobial agent according to any one of claims 1 to 41.

50. The coating according to claim 49, wherein the substrate is designed for water applications.

51. The coating according to claim 49, wherein the substrate to be coated is designed for biological uses or devices used in medical applications.

52. The coating according to claim 49, wherein the substrate being any region of a storage container or a delivery system for use in food packaging, food and beverage containers, food and beverage preparation or disposing equipment.

53. The coating according to claim 49, wherein the substrate is of a product selected from a filtration device, a water line, a reusable water container, a hydration system, a water bottle and a container.

54. A product implementing an antimicrobial agent according to any one of claims 1 to 42, the product being selected from personal products and industrial products.

55. The product according to claim 54, being selected from products for sporting activities, orthodontic devices, face or breathing masks, pacifiers, contact lenses, food preparation surfaces, food packaging, reusable water containers, hydration systems, water bottles, computer keyboards, telephones, rental car steering wheels, health club equipment, whirlpool spas and humidifiers.

56. The product according to claim 55, being selected from filtration devices and water lines, reusable water containers, hydration systems, water bottles and containers.