ANTIM1CROB1ANOS MATERIALS NON-LIXIVANTS OF ANNIHILATION BY CONTACTFIELD OF THE INVENTIONThe present invention relates to non-leaching antimicrobial materials, specifically, to the provision of antimicrobial materials capable of contact-killing microorganisms, as well as methods for the manufacture and use of said materials.
BACKGROUND OF THE INVENTIONThe constant threat of bacterial contamination and the associated health repercussions have made conservative ubiquitous portions of drugs and packaged foods. However, conservatives sometimes have undesirable side effects, especially in pharmaceutical products. The growing concern of the consumer regarding the harmful effect of the preservatives in recent years has required their reduction or preferably, the total elimination, without the risk of bacterial contamination, thus promoting the need to develop new methods of packaging and cost storage. effective to avoid bacterial contamination. The problem is acute in the pharmaceutical area, especially in the ophthalmic industry, which is currently driven by the need to direct the issuance of patient sensitivity towards conservatives in eye solutions. Burnstein, N. L. et al., Trans. Qphthalmol. Soc, 104: H02 (1985); Collins, H.B. et al., Am, J. Optom & Phvsiolog. Optics, 51: 215 (A89). A similar problem exists in the areas of food, medical devices, health care and water purification. The mode of action of all surfaces resistant to the currently known infection is through one of the following mechanisms: (i) dissolution of an antimicrobial component to the contact solution, or (ii) chemically bound antimicrobials. The first is achieved by mixing an antimicrobial compound with a plastic material. The composite material is then either molded to a device or applied as a coating. The bacterial action of said coatings depends on the diffusion of the biotoxic agent to the solution. In the literature, many examples of this type have been reported. Another variant of this type involves the hydrolysis or dissolution of the matrix containing an antimicrobial compound, thus effecting its release towards the solution. However, high levels of preservatives are released to contact solutions in long-term applications. In the latter mechanism, a bioactive compound is covalently bound either directly to the surface of the substrate or to a polymeric material that forms a non-dissolving surface coating. The antimicrobial compounds in said coatings exhibit a greatly reduced activity, unless it is aided by the hydrolytic cleavage of either the bound antimicrobial or the coating itself. In any case, high levels of preservative have to be released to the solution in order to produce an antimicrobial action. Several products for external or internal use in humans or animals can serve to introduce undesirable bacterial, viral, fungal and other infections. These products include medical devices, surgical gloves and implements, catheters, implants and other medical devices. To avoid such contamination, said devices can be treated with an antimicrobial agent. In the patents of E.U.A. Nos. 3,566,874 3,674,901; 3,695,921; 3,705,938; 3,987,797; 4,024,871; 4,318,947 4,381,380; 4,539,234; 4,612,337; 3,699,956; 4,054,139; 4,592,920 4,603,152; 4,667,143 and 5,019,096 methods have been proposed for preparing a medical device resistant to infection. However, such methods are complicated and unsatisfactory. The above known antimicrobial coatings usually leach the material into the surrounding environment. Many are specifically designed to release antimicrobial agents (see, U.S. Patent No. 5,019,096). There is a need for medical devices and other products, which are capable of resisting microbial infection when used in the area of the body to which they are applied, which provide this resistance over time, and which do not leach antimicrobial materials into the body. ambient.
COMPENDIUM OF THE INVENTIONIt is an object of the invention to provide antimicrobial non-lixiviation materials for contact annihilation, which are capable of contact killing microorganisms, but which do not leach significant amounts of antimicrobial materials into the surrounding environment. The antimicrobial materials can be deposited on the surface of a substrate to form an antimicrobial coating of contact annihilation on the surface, can be mixed with a polymer and sneaked onto an antimicrobial, freestanding object or film, or can be incorporated into a vehicle to provide a global antimicrobial, which can be applied as desired to form an antimicrobial layer of contact annihilation. The antimicrobial materials of the present invention are molecularly designed to allow a complex or matrix bound biocide to retain high activity without elution of any biocide towards contact solutions, vehicles or other materials. The antimicrobial activity comes from the sustained biocidal action, cooperative of its components. The selective transfer of a component from the interior of the matrix directly to the microorganism after contact is achieved through a mechanism of "loose controls" after coupling and penetration of the microorganism in the membrane of the cell. The antimicrobial material, therefore, maintains a long-term efficacy without releasing toxic eluate products into the surrounding environment. The antimicrobial material of the present invention comprises a combination of an organic material, which is capable of forming a matrix, and a broad spectrum biocide complexed with or associated with the organic material. The biocide interacts sufficiently and strongly with the organic material that the biocide does not leach or easily dissociate from the organic material. The organic material must possess two important properties: it must be able to bind or complex, reversibly, with the biocide, and must be capable of introducing the biocide into the cell membrane of a microorganism with which it makes contact. The organic material is preferably substantially insoluble in water, and is capable of dissolving in or adhering to the cell membrane surrounding the microorganism. Preferred organic materials are those that can be immobilized on a surface or incorporated into a vehicle and which bind the biocide in such a way that they preferentially release the biocide towards a microorganism, which makes contact with the material, but not towards the surrounding environment . The biocide is preferably a low molecular weight metallic material that is toxic to microorganisms and is capable of complexing with or reversibly binding to the organic matrix material, but whichpreferentially binds to cellular proteins of microorganisms. When a microorganism makes contact with the antimicrobial material, the organic material is coupled or penetrates at least the outer portion of the lipid bilayer of the cell membrane of the microorganism sufficiently to allow the introduction of the biocide to the microorganism, wherein all the Cell proteins or proteins in the lipid bilayer compete effectively for the biocide due to favorable binding constants. The result is a contact annihilation delivery system that selectively transfers the biocide through or to the cell membrane of the microorganism after contact without elution of the biocide in the solution, thus maintaining long-term efficacy. The only mode of action of the antimicrobial material currently described offers a high activity coupled with the substantially low leachable products. The organic materials useful in the present invention comprise materials that are capable of: 1) reversibly binding or complex form with a biocide, and 2) introduce the biocide to the membrane of the microorganism cell after contact. A preferred class of materials are those having the aforementioned properties, which are capable of complexing and / or joining an antimicrobial metal material. A highly preferred class is organic materials, which can be dissolved in, or adhere to, and penetrate at least the outer portion of the lipid bilayer membrane of a microorganism. For this purpose, surfactants, such as cationic compounds, polycationic compounds, anionic compounds, polyanionic compounds, non-ionic compounds, poly-ionic compounds or zwitterionic compounds are useful. Organic materials that are currently most preferred for use in the invention include cationic or polycationic materials, such as biguanide compounds. In a preferred embodiment of the present invention, the organic material is a polymer capable of forming a matrix. It is understood that the term "polymer", as used herein, includes any organic material comprising three or more units, and includes oligomers, polymers, copolymers, terpolymers, etc. In one aspect, the organic material can be an adduct formed through the reaction of the organic material with an entanglement agent or a chain extension agent. The crosslinking agents that can be used in the present invention are those multifunctional organic compounds, which react with the organic material to form an adduct to which it can be entangled to form an interlaced network or matrix. Suitable entangling agents include, for example, multifunctional compounds containing organic groups such as isocyanates, epoxides, carboxylic acids, acid chlorides, acid anhydrides, succimidyl ether aldehydes or ketones, and may further include multifunctional compounds such as alkyl methanesulfones. , alkyl trifluoromethanesulfonates, alkyl paratoluen methansulfones, alkyl halides and multifunctional epoxides. As used herein, the term "multifunctional" refers to compounds having at least three functional groups. The chain extension agents that can be used in the present invention are monofunctional or difunctional organic compounds, which react with the organic material to form an adduct, but which are not necessarily capable of being interlaced, and which with hydrophobic, that is, substantially insoluble. Suitable chain extension agents include, for example, monofunctional or difunctional aliphatic hydrocarbons, heteroaliphatic hydrocarbons, aromatic and heteroaromatic hydrocarbons, organosilanes and perfluoro compounds. Examples of chain extension agents include bis-glycidyl ethers of bisphenol A, bisepoxides such as glycol a, β-bisglycidyl polyethylene, poly [bisphenol A-copechlorohydrin glycidyl end-blocked and N, N-diglycidyl-4-glycid i loxian ilin. In a preferred embodiment, the organic material comprises a biguanide compound. The biguanide compound can be a polymer comprising repeating biguanide units, or it can be a co-polymer containing biguanide units in one or more organic materials. For example, the biguanide polymer can be a copolymer formed by reacting a polyepoxy compound and a biguanide compound. In a presently preferred embodiment, the organic material comprises an adduct formed by reacting the polyhexamethylene biguanide with an epoxide, such as N, N-bismethylene diglycidylaniline. The resulting adduct can then be applied to a substrate and then dried to form a non-interlaced matrix or can be cured to form an interlaced network or matrix. The biocide can be any antimicrobial material, which is capable of binding or forming a complex, not lixíviablemente, with the organic material, but which, when put in contact with a microorganism, preferentially transfers proteins to the microorganism. For this purpose, metallic materials are preferred, which bind to cellular proteins of microorganisms and are toxic to microorganisms. The metallic material can be a metal, metal oxide, metal salt, metal complex, metal alloy or mixtures thereof. Examples of such materials include, for example, silver, zinc, cadmium, lead, mercury, antimony, gold, aluminum, copper, platinum and palladium, their salts, oxides, complexes and alloys, and mixtures thereof. The appropriate metallic material is selected based on the use that will be given to the device. The currently preferred metallic materials are the silver salts. In a presently preferred embodiment, a silver halide, most preferably, silver iodide, is used. In one aspect, the antimicrobial material comprises a complex of a polycationic ligand compound and a metallic material. The polycationic compound and the metallic material form a coordination complex, stable, which can be isolated, having antimicrobial properties. In a preferred embodiment, the polycationic compound is a polymer. In another preferred aspect, the same polycationic compound has antimicrobial activity. In a presently preferred embodiment, the polycationic compound is polyhexamethylene biguanide and the metal is silver, most preferably, silver iodide. The complex is preferably in dry form, such as a powder, comprising fine particles of the complex. The invention further comprises liquid compositions for forming an antimicrobial, non-leaching, annihilation layer by contact, on a surface. In one embodiment, the composition is a two-part composition comprising a first solution, dispersion or suspension of an organic material, and a second solution, dispersion or suspension of a biocide. If an interlaced coating or film is desired, the first solution, dispersion or suspension will also contain the interlacing agent. As a first step, the entanglement agent and the organic material can be reacted to form a non-interlaced adduct. To form a coating or layer of non-leaching, contact annihilation on a substrate, the first composition is applied to the substrate under conditions sufficient to immobilize the organic material on the substrate, forming a matrix. If an entanglement agent is present, the matrix can be cured to induce entanglement. The matrix is then exposed to the solution of the biocide material under conditions sufficient to induce the biocide to be non-leachable, complexed with or associated with the matrix. In another embodiment, the liquid composition is a one part composition comprising a solution, dispersion or suspension of the organic material, the biocide, and optionally, the crosslinker. To form the contact annihilation coating on a substrate, this composition is applied to the substrate under conditions sufficient to immobilize the organic material on the substrate, forming a matrix, wherein the biocide is not leachably bound to or associated with the matrix. The dry powder, and the liquid compositions of two parts or a part can also be used to make films, microbeads or other solid, self-stable antimicrobial forms, as described in detail below. As used herein, the term "freestanding" means not joining a substrate. The invention further provides methods for making the compositions of the present invention, and applying them to various substrates to form the antimicrobial coatings or layers on the substrates, or by mixing them with a carrier. Generally, the compositions are made by combining the organic polycationic material with the metal biocide under appropriate conditions to form the complex. Conditions may vary depending on the polycationic materials and the selected metal biocide. In a modality, the complex is formed by contacting a liquid solution of the polycationic material with a liquid solution of the biocidal metal material, resulting in the formation of the complex as a precipitate from the solution. The precipitate can then be dried and milled to form a powder. To make the dry or powdered compositions of the invention, the organic polycationic material and the metal biocide can first be combined in a liquid vehicle to form a solution, dispersion or suspension of the complex, which can then be dried to evaporate the liquid. The drying step can be carried out in any suitable way to obtain the desired product, including spray drying, air drying, heating, etc. In one embodiment, a powder form of the complex can be prepared by combining a liquid solution of the organic material and the biocide to form a solution, dispersion or suspension of the complex of organic material: biocide. The solution, dispersion or suspension is then cast as a film on a non-stick substrate and dried to form a film. The film is then separated from the non-stick substrate and ground to a powder. The term "non-sticky substrate" means a substrate to which the coating or film formed from the complex will not be bonded, and from which it is removed intact. In another embodiment, a complex is formed between an interlaced form of the organic material and the biocide metal material. In this embodiment, the organic material is reacted with an entanglement agent to form an adduct. The adduct is then cured to induce entanglement. The resulting interlaced material is then contacted with the biocide metal material under conditions sufficient to form the complex. In a preferred embodiment, the organic material is polyhexamethylene biguanide (PHMB) or an adduct formed through the reaction of PHMB with an epoxy functional compound, preferably N, N-methylenebisdiglidylaniline (MBDGA). The adduct is formed by reacting PHMB and MBDGA by heating a mixture of the two components at a temperature of about 90 to about 95 ° C for about 15 minutes. The PHMB or PHMB adduct is then combined with the metal biocide, preferably a silver salt, thereby forming a precipitate containing a PHMB complex: MBDGA: Agl. The silver salt currently preferred is silver iodide. The resulting precipitate is then dried and can be milled to form a fine powder of the complex. Techniques for making dry powders of complexes based on organic materials and / or biocides other than PHMB and silver can be achieved using reaction conditions and drying protocols known and available to those skilled in the art. To make the liquid compositions of the invention, a solution, dispersion or suspension of an organic material can be made, or, if appropriate, as available from the manufacturer. For example, polycationic polymers may be available in the form of a resin (ie, a liquid carrier), which may be suitable for use as such, or with a slight adjustment, for example, in the solid content. The polymer resins can also be mixed with other resins or compounds, or the polymers can be reacted with or derivatized from other polymers or compounds to form copolymers, functionalized polymers or adducts. The binding and / or reaction conditions will depend on the selected materials. In one embodiment, a liquid solution of a polycationic organic material, such as PHMB, can be used as the organic material. The PHMB can be used as such, or it can be reacted with another organic polymer or compound to form a copolymer, adduct or functionalized derivative. The protocols for the formation of copolymers, adducts and derivatives are well known in the chemical field. In a currently preferred , described above in relation to the process for forming the dry composition, the PHMB is first reacted with an epoxy functional compound to form an adduct. The resulting liquid solution containing the adduct can be applied as such and subsequently impregnated with the biocide as described below, or it can be combined with the biocide to form a solution, dispersion or suspension of an adduct complex of PHMB: biocide. In the currently preferred embodiment, the PHMB is reacted with MBDGA to form the adduct, and the adduct is combined with a silver salt, preferably silver iodide, to form a PHMB complex: MBDGA: Agl. As will be readily apparent to those skilled in the art, these techniques can be used to make complexes of organic materials and biocides based on materials other than PHMB, MBDGA and Agi provided the materials have the required functional characteristics, ie, the ability of forming a complex, which has antimicrobial properties and will not leach or release the biocide to a liquid or other substance in contact with the complex, but which preferably will transfer the biocide to a microorganism in contact with the complex. The method for applying the liquid compositions to form an antimicrobial coating generally comprises providing a solution, dispersion or suspension to the organic material, and, if a non-interlaced material is desired, coating the solution, dispersion or suspension of the organic material on the substrate. , and, apply the coating, forming a matrix. If an interlaced coating is desired, the organic material is first combined with an interlacing agent. Typically, both the organic material and the interlayer will be in liquid form (e.g., in a solution, dispersion or suspension), and the two solutions are combined, forming a liquid mixture. The liquid can be an organic solvent, an aqueous liquid or a mixture of an organic solvent and an aqueous liquid. The organic material and the entanglement agent are then reacted to form an adduct. The resulting adduct can be stored for later use, or it can be immediately applied to a substrate. Liquid compositions containing the organic material (with or without the added interlayer) can be applied to the substrate of choice through any suitable means for applying a liquid coating, for example, spraying, brushing, dripping, calendering, bar coating or curtain. The method selected for applying the composition to the substrate will depend on several factors, including the thickness of the desired coating and the nature and configuration of the substrate. If necessary, the surface to be coated can be cleaned or treated before applying the polymer solution. The resulting coating is dried to form the matrix, and, if interlacing is desired, it is subjected to interlacing conditions, forming an interlaced network. The entanglement conditions may include thermal cure, ultraviolet cure, chemical cure or other curing methods. The matrix is then contacted with a solution of the biocide under conditions sufficient to deposit the biocide material in the matrix, so that the biocide material is not leachably associated with or bound to the matrix. Another embodiment of the method for making the coatings of the present invention comprises first combining the organic material and the biocide, then applying the mixture to the substrate to form the matrix as described. If an interlaced coating is desired, the organic material and the crosslinking agent are reacted to form an adduct as described above, then the adduct is combined with the biocide. The resulting adduct / biocide mixture can be stored for later use, or can be applied immediately to a substrate and cured as described above to induce entanglement, thus forming the polymer network having the biocide not leachably associated therewith or bound thereto. In the methods of the invention described above, the amounts and / or concentrations of the materials used will depend on the nature and stoichiometry of the materials used, and the desired final product. In the presently preferred embodiments, the concentration of the solution, dispersion or suspension of the organic material, or the resin of the organic adduct formed by the reaction of the polymer and the interlayer, is typically in the range of about 0.5 to about 20% by weight. Typically, a polymer interlacing ratio in the range of about 1: 1 to about 3: 1 (percent by weight) will form interlaced networks, which will non-leach the biocide and will preferentially transfer the biocide to the microorganism after contact, as described at the moment. Solutions of the biocide material typically ranging from about 0.005 to about 0.5% by weight can be used to impregnate the matrix with the biocide. In another embodiment of the method herein, a self-supporting antimicrobial material can be formed using the antimicrobial material herein. In this embodiment, using the two-part compositions described above, a solution, suspension or dispersion of the organic material is cast onto a non-sticky substrate and dried to form a film. If an interlaced material is desired, the organic material and the interlayer are first combined and reacted to form an adduct, as described above, and a solution, suspension or dispersion of the adduct is cast to form the film. The film is cured to induce entanglement, as described. The film is then contacted with a solution, dispersion or suspension of biocide material to deposit the material within the matrix of the organic material. Then, the film is separated from the substrate and used as desired. Alternatively, the interlaced or non-interlaced freestanding films can be cast using the liquid compositions of a part described above. The self-stable antimicrobial materials can also be prepared using the antimicrobial materials of the present invention in other physical forms in addition to films, including microbeads or solid forms, for example, which can be prepared by mixing the antimicrobial powder with a suitable vehicle, then casting or by molding the object using well-known techniques. In another embodiment, an antimicrobial powder can be formed by casting a free-standing film, as described above, then grinding the film to a powder. The powder can also be formed through precipitation from the solution, the complex between the organic material and the biocide metal material, drying the precipitate and grinding it to form the powder. The powder has antimicrobial contact killing properties similar to the films and coating described above. The antimicrobial powder can be incorporated into a vehicle, such as a gel, cream or liquid, and applied to a surface to form an antimicrobial layer. For example, a formulation comprising the antimicrobial powder dispersed in a pharmaceutically acceptable carrier can be used as a topical antiseptic and applied to a wound. In a preferred embodiment, the antimicrobial materials of the present invention are used to form an annihilation surface by contact on a substrate. To provide the annihilation surface by contact on the substrate, the organic compound can be attached to and / or immobilized on the substrate through any suitable method, including covalent binding, ionic interaction, coulombian interaction, hydrogen bonding, entanglement (for example, as interlaced (cured) networks) or as interpenetration networks, for example. In a presently preferred embodiment, the organic matrix is formed by first reacting polyhexamethylene biguanide with N, N-bismethylene diglycidylaniline to form an adduct. The stable coating solutions of the resulting adduct have been obtained in both absolute ethanol and aqueous ethanol. The adduct can be applied to the surface of a substrate either by drip coating, brushing or spraying. Once applied to the substrate, the coating is dried to form a matrix. The coating can be cured (e.g., through heating) to induce entanglement, thereby forming an interlaced polymer network on the substrate. The resulting optical coating is clear, resistant to most solvents and temperature changes, and does not delaminate, flake or crack. The coating typically has a thickness of about 10 microns, although the thickness of the coating can be varied by well-known techniques, such as increasing the solids content of the resin. A broad spectrum metallic antimicrobial, preferably a silver compound, is then introduced into the polymer network so that it is trapped as submicron particles, and complexed with the functional groups in the polymer. Alternatively, the metallic material is combined with the liquid containing the polymer before applying it to the substrate. In the currently preferred embodiment, the broad spectrum antimicrobial is a silver halide, preferably silver iodide. The antimicrobial materials of the present invention are unique in the following aspects: i) The unique nature of the antimicrobial material uses a cooperative effect of its components. This results in a high biocide activity, while maintaining non-significant leachable products towards the solutions where it is in contact, ii) The mechanism of action is essentially a mediated surface, so organisms succumb only by contact with the material due to the non-leaching property associated with it. iii) The ability of said surfaces to remain completely inert in solution in the absence of contamination by microorganisms. iv) The ability of these surfaces to remain viable over multiple organism attacks without reducing their bioactivity. v) the use of said biocide material on an interior or exterior surface of a device, thus eliminating the possibility of microbial colonization on the surface, vi) user friendliness and cost effectiveness of the coating for all types of applications. vii) Adaptability to exist in manufacturing technology, thus enabling a large-scale production with minimal cost. The above objects, aspects and advantages and others will be better understood from the following specification when read together with the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGSFigure 1A is a schematic graphic illustration of the matrix / biocide complex of the present invention, prior to contacting the coating with the microorganisms. Figure 1B is a schematic graphic illustration of the contact annihilation ability of the matrix / biocide complex of the present invention during contact of the coating with the microorganisms. Figures 2A-D are a schematic graphic illustration of a preferred method for applying the matrix / biocide complex of the present invention to a substrate. Figure 2A shows the matrix immobilized on the substrate, with chains of organic material forming arms or tentacles that exit to the surrounding environment. Figure 2B shows the immobilized matrix impregnated with a biocide compound, with biocide deposits deposited within the matrix and molecules of the biocidal compound attached to the tentacles. Figure 2C shows a microorganism in contact with the matrix / biocide complex, wherein the polymer chains are coupled and dissolved in the cell membrane of the microorganism. Figure 2D shows the penetration of the cell membrane and the transfer of the biocide from the network to the microorganism, causing the death of the cell. Figure 3 is a graph illustrating the bioactivity of a preferred coating of the present invention, a matrix formed from PHMB entangled in complex with silver salts, treated as a function of surface area to volume against the microorganism Pseudomonas aeruginosa in saline regulated in its pH with phosphate at 30 ° C.
DETAILED DESCRIPTIONThe antimicrobial materials of the present invention can be combined with a variety of vehicles to form bulk antimicrobial compositions, or they can be coated on a variety of substrates to form an antimicrobial coating. Both bulk antimicrobials and coatings are non-leaching and contact killing. That is, they do not leach significant amounts of antimicrobial components into the surrounding environment, and will kill most of the microorganisms that come into contact with them. The term "microorganism", as used herein, includes bacteria, blue-green algae, fungi, yeast, mycoplasms, protozoa and algae. The term "biocide", as used herein, means the annihilation of microorganisms, or inhibition of the growth of microorganisms, which may be reversible under certain conditions.
As used herein, the terms "non-leachable" or "substantially non-leachable" represent that none or very small amounts (e.g., below a certain threshold) of organic and / or biocide material dissolve in a liquid environment . Preferably, this threshold is not higher than 1 part per million (ppm), and most preferably is less than 100 parts per billion (ppb). Organic materials useful in the present invention comprise materials that are capable of: 1) reversibly binding or complexing with the biocide, and 2) introducing the biocide to the membrane of the microorganism cell. A preferred class of materials is one that has the aforementioned properties, which are capable of being immobilized on a surface and which preferentially join a biocidal metal material in such a way that they allow the release of the metal biocide towards the microorganism, but not towards the contact environment. Very preferred is the class of organic materials, which can dissolve in, adhere to, break or penetrate the lipid bilayer membrane of a microorganism. For this purpose, surfactants may be used, such as cationic compounds, polycationic compounds, anionic compounds, polyanionic compounds, non-ionic compounds, polyanionic compounds or zwitterionic compounds. Organic materials that are currently highly preferred for use in the invention include cationic or polycationic compounds, such as biguanide compounds. Preferred cationic materials include benzalkonium chloride derivatives, a 4- [1-tris (2-hydroxyethyl) ammonium-2-butenyl] poly [1-dimethylammonium-2-butenyl] -α-tris (2-hydroxyethyl) chloride. ammonium, and biguanides of the formula:Y ^ f-NH-C-NH-C-NH-X-Jn-Yz NH NHor their water soluble salts, wherein X is any aliphatic, cycloaliphatic, aromatic, substituted aliphatic, substituted aromatic, heteroaliphatic, heterocyclic or heteroaromatic compound, or a mixture of any of these, and Yi and Y2 are any aliphatic, cycloaliphatic compound, aromatic, substituted aliphatic, substituted aromatic, heteroaliphatic, heterocyclic or heteroaromatic, or a mixture of any of these, and wherein n is an integer equal to or greater than 1. Preferred compounds include, for example, chlorhexidine (available from Aldrich Chemical Co. ., Milwaukee, Wl) or a polyhexylmethylene biguanide (available from Zeneca Biocides, Inc. of Wilmington, DE). The aforementioned organic materials can be modified to include a thiol group in their structure so as to allow binding of the compound to a metal substrate, or they can be derivatized with other functional groups to allow direct immobilization on a non-metallic substrate. For example, the aforementioned organic materials can be conveniently functionalized to incorporate groups such as hydroxy, amine, epoxy, alkyl or alkoxy functionalities to allow direct immobilization to a surface. In a preferred embodiment of the present invention, the organic material comprises a polycationic material, which is* interlaced to form the matrix. The crosslinking agents that can be used in the present invention are those that react with the polycationic material to form an adduct, which can then be reacted to form an interlaced network or matrix. Suitable entangling agents include, for example, compounds containing organic multifunctional groups, such as isocyanates, epoxides, carboxylic acids, acid chlorides, acid anhydrides, succinimethyl ether aldehydes and ketones, and organic compounds such as alkyl methanesulfones, alkyl trifluoromethanesulfonates, alkyl paratoluen methanesulfones, alkyl halides and organic epoxides. In a presently preferred embodiment, a polyhexamethylene biguanide polymer is reacted with an epoxide, such as N, N-methylene bis diglycidylaniline, which is then cured to form an interlaced network. The biocidal material may be any antimicrobial material, which is capable of non-leachable bonding or complexing with the organic matrix, but which, when contacted with the microorganism, is preferentially transferred to the microorganism. For this purpose, metallic materials that are toxic to microorganisms are preferred. The metallic material can be a metal, metal oxide, metal salt, metal complex, metal alloy or mixture thereof. Examples of such metals include, for example, silver, zinc, cadmium, lead, mercury, antimony, gold, aluminum, copper, platinum, and palladium, their oxides, salts, complexes, and alloys, and mixtures thereof. The appropriate metallic material is selected based on the use that will be given to the device. The presently preferred metallic materials are the silver compounds. The biocide material can be introduced into the matrix either contemporaneously with or after application of the organic material to a surface. The invention also provides a substrate, wherein the surface is partially coated with additional organic materials, and / or biocide materials, or both. Examples of organic and biocide materials that can be used are discussed below. The use of a combination of at least two different organic and biocide materials can improve the antimicrobial properties of the coating. Different types of microorganisms can exhibit different degrees of sensitivity to different organic and biocide materials. In addition, the use of two or more different organic and biocide materials can significantly reduce the problem of selecting microorganisms that have resistance to organic and biocide materials in the coating that can occur only when they are used. The quantity and / or type of the antimicrobial coating, which is used in a particular application will vary depending on several factors, including the type and amount of contamination, which is likely to occur, and the size of the antimicrobial surface. The amount of antimicrobial used will be a minimum amount necessary to maintain the sterility of the liquid. As stated above, this amount will vary depending on several considerations. In a preferred embodiment, the organic material, whether or not it is interlaced, forms an insoluble, non-leachable matrix having a unique configuration: some of the organic material exits into the surrounding environment, i.e., the "arms" or "tentacles" of the material organic are projected from the matrix and into the surrounding environment. This phenomenon can be understood by referring to Figures 1 and 2, which are schematic graphic illustrations of a preferred coating of the present invention wherein the organic material is an entangled biguanide polymer and the biocidal material is a silver halide salt , preferably silver iodide. Figures 1A and 1B and Figures 2A-D show the polymer matrix having tentacles projecting into the surrounding environment, and the silver salt is deposited in the tanks and on the tentacles. Without wishing that it is bound by theory. It is believed that when a microorganism makes contact with the coating, the tentacles of the biguanide polymer dissolve in the surrounding lipid bilayer of the microorganism, thereby introducing silver molecules into the interior of the microorganism or into proteins within the membrane of the cell. The silver salt has a higher affinity for certain proteins in the microorganism than for the polymer, and, therefore, forms complex with the cellular proteins and is transferred to the microorganism, thus annihilating. Specifically, it is believed that silver forms complexes with the sulfhydryl and amino groups of cellular proteins. In this embodiment, the silver salt binds or impregnates the matrix and the polymer's tentacles so that the silver is substantially non-leachable. Again, without wishing that it is bound by theory, it is believed that the silver salt forms complexes with functional groups in the polymer, and that the complexed silver resists the leaching into the environment of liquids and other materials (e.g. gels) in contact with the coated surface. However, when the coating is exposed to cellular proteins, silver preferentially forms complex with the proteins. In a presently preferred embodiment, the polymeric material is polyhexamethylene biguanide (PHMB), the entanglement agent is N, N-methylenebisdiglycidylaniline (MBDGA), and the silver salt is a silver halide, most preferably, silver iodide. In this embodiment, the liquid composition is made by combining a solution of polyhexamethylene biguanide with a solution of the entanglement agent, and reacting the mixture under conditions sufficient to form a non-interlaced PHMB-MBDGA adduct. The ratio of PHMB to MBDGA is preferably in the range of about 1: 1 to 3: 1 by weight. The PHMB-MBDGA mixture is heated to about 95 ° C for about 2 hours in a closed reactor to form the adduct. The concentration of the resulting adduct resin is preferably in the range of about 0.5 to about 20% by weight. To form an antimicrobial contact annihilation coating, the adduct resin solution is placed as a coating on the desired substrate, and heated to a temperature sufficient to induce entanglement between the adducts, thereby forming an interlaced network or matrix. Sufficient temperatures for entanglement are typically in the range of about 70 ° C to about 200 ° C. The resulting interlaced network is then saturated with silver by immersing the coating for about two minutes in a silver iodide / potassium iodide solution. Silver solutions having a concentration of about 0.005 to about 0.5% can be used for this step. Silver iodide forms deposits in the matrix, and joins the tentacles. Silver iodide has sufficient affinity for the PHMB polymer to form an insoluble complex that will not leach into environmental solutions or other materials in contact with the material, even at elevated temperatures. However, when a microorganism makes contact with the coating, the tentacles break the lipid bilayer membrane of the microorganism, thus introducing the silver iodide into the microorganism. Silver iodide has a higher affinity for certain proteins within the microorganism than for the PHMB-MBDGA matrix, and forms complexes with these proteins, that is, silver is preferentially transferred from the coating to the microorganism. Silver accumulates to toxic levels in the microorganism and annihilates it. Deposits of silver iodide within the matrix fill the silver iodide in the tentacles spent on the microorganism by restoring the equilibrium for complex formation (Agí + PHMB ^ z - i [PHMBAgl]). This invention also includes coated substrates, freestanding films, powders and articles made in accordance with the above methods. The present invention provides stable, adherent coatings or layers using the coating formulations herein on a wide variety of materials, including those commonly used in membranes and in the manufacture of medical devices. The antimicrobial coatings according to the present invention can be applied, for example, to woods, metals, paper, synthetic polymers (plastics), natural and synthetic fibers, natural and synthetic rubbers, cloth, glass and ceramics. Examples of synthetic polymers include elastically deformable polymers, which may be thermoset or thermoplastic, such as, for example, polymers and copolymers of polypropylene, polyethylene, polyvinyl chloride, polyethylene terephthalate, polyurethane, polyesters, rubbers such as polyisoprene or polybutadiene, polytetrafluoroethylene, polysulfone and polyester sulfone. The substrate may be a deformable plastic or metal medicament container, such as a tube of toothpaste, wherein the container may remain deformed after the assortment of each dose. Other polymeric materials, including polymeric materials, which are used for the preparation of membranes or filter paper, also serve as substrates. Examples of organic polymeric materials include a polyamide (e.g., nylon), polycarbonate, polyacrylate, polyvinylidene fluoride, cellulosics (e.g., cellulose) and Teflon®. The substrate can be either hydrophilic or hydrophobic. With the exception of silicone and Teflon®, which may be required prior to activation of the surface with techniques such as plasma, chemical oxidation or metal sensitization, eg, a primer, no surface activation is necessary. The inorganic materials to which the coatings of the present may be applied include glass fiber materials, ceramics such as alumina or silica and metals. Concrete substrates and concrete ceramics can also be used. The coating or layer can be applied directly to many surfaces without an anterior surface modification.
Studies simulating one year of contact between the coating and aqueous solutions at room temperature resulted in less than 100 ppb of any active ingredient in the solution. The extract solutions by themselves (solutions that have been in contact with the coating) did not show any antimicrobial or mammalian cell toxicity. The coated surface remains totally inert and bio-active after being exposed to various physical and chemical stresses including: low temperature, ethanol, boiling water, prolonged exposure to solutions of variable pH and solutions of high ionic strength, as well as sterilization through conventional methods (eg wet autoclave, ethylene oxide, irradiation?, ethanol). Surface coatings, freestanding films and formulations containing the antimicrobial powder according to the present invention exhibit antimicrobial activity against gram positive and gram negative bacteria and yeast, and are resistant to the development of fungi. The treated surfaces completely annihilate organisms at attack levels of 106-108 CFU / ml in 8 to 20 hours at 30 ° C, depending on the type of organism. Tables 1 and 2 (in Example 4) list the bioactivity of the coated surfaces towards different attack organisms. The coating makes the surfaces resistant to the bio-film, which is coupled with its chemical inertness, particularly making it suitable for many applications in devices. The antimicrobial materials of the present invention have been successfully applied to the surface of microporous membranes, including within the pores as shown by SEM-EDX. Stable, uniform coatings have been obtained in a variety of membrane materials with almost no reduction in their flow property. The coated microporous membranes annihilate microorganisms after contact and are resistant to the phenomenon of "past bacterial development", which occurs even in membranes with a pore size of 0.2 μM sterilization in long-term contact applications. Said membranes are, therefore, well suited for incorporation into devices used in long-term filtration applications such as multiple dose dispensers for conservative free formulations, water purification systems and in any application where it is desirable to utilize properties of membrane barrier for more than a day. The mechanism of action is one in which the antimicrobial materials are activated only after contact with the microorganism. Once the microorganism accumulates a toxic amount of silver, it succumbs and separates from the surface. The coating or other treated surface, therefore, remains active only if the viable organism makes contact with it, and is inverted to be inert in its absence. This unique property so that the biological activity is triggered by the contact of the bacterial cell allows the coating to work "intelligently". For such contact mechanism to be effective, the annihilation rate is expected to vary as a function of the total ratio of surface area of coated substrate to the volume of the bacterial suspension in contact with it (S / V ratio) at constant temperature . As shown in the Examples, time experiments were performed for annihilation in variable diameter coated polyethylene tubes that were inoculated with predetermined volumes of a suspension containing 10 6 CFU / ml of Pseudomonas aeruginosa in pH regulated saline with phosphate (PBS). . The reduction in organism concentration was measured as a function of time at constant temperature for 20 hours. The experimental results are summarized in Figure 3. There is no substantial difference in the rate of annihilation for S / V ratios ranging from 2.5 to 5 cm "1; Similar results were obtained for a 1.5 ratio. For the larger diameter tubes tested (S / V = 0.5), however, viable organisms were detected at low levels, which can be attributed to a reduction in the probability of organisms in contact with the surface with increasing levels. No toxic components were found in free solutions of organism in contact with the coated tubes under identical conditions when they were tested both chemically and biologically, which supports the proposed contact mechanism for cell death. Such a distinction could not be evident if sterilization occurred through controlled dissolution or diffuse elution of the coating components towards the solution; in any case, high levels of active components could be present in solution. The antimicrobial materials of the present invention can be used to form contact annihilation coatings or layers on a variety of substrates. As shown in the Examples, the material forms a contact, non-leaching annihilation surface in materials that are used in medical devices, which are implanted, inserted or in intimate contact with a patient, such as catheters, urological devices , blood collection and transfer devices, tracheostomy devices, valves, stents, intraocular lenses and in personal care and health products, which are in contact with the patient or another user, such as toothbrushes, cases for contact lenses and dental equipment. The antimicrobial materials of the present invention in powder form can be dispersed or dissolved in a vehicle and used as a topical antiseptic, wound dressing or topical disinfectant. Such vehicles may include creams, gels, lotions, soaps or other topically applicable materials. The materials can be used in medical devices and devices and health care products, consumer products, baby care products, personal hygiene products, bathroom accessories including showers, toilet seats, collectors and backstops , kitchen surfaces, surfaces for food preparation and packaging, water storage, treatment and supply systems, biosensing systems and laboratory and scientific equipment. For example, contact lens cases are a proven contributor to the spread of eye pathogens and disease. A lens case coated with a coating of the present invention has been shown to kill in vitro all clinically relevant pathological strains of microorganism without leaching toxic chemicals into the contact lens solution (see Example 2 and 4). Once a bio-film has been formed on an untreated contact lens case, it resists virtually all types of disinfection products currently available for the care of contact lenses. In this way, the bio-film serves as a reservoir for bacteria that re-contaminate the lenses each time they are stored in the case. The treated lenses are compatible with all the disinfectant solutions tested to date. The use of the coating allows the sterilization of the lenses using ordinary saline as the soaking solution. The antimicrobial materials herein can be used to coat toothbrushes with ordinary nylon bristles (see Example 2). The treated toothbrush annihilates the pathogens commonly found in the mouth of the human being and on the surfaces of the bathroom, while untreated toothbrushes encourage their development. It is believed that toothbrushes are partly responsible for the spread of oral and dental disease. In vitro and in vivo testing programs examined the types, number of annihilation rates for organisms commonly found in the mouth. The tests indicated that the treated toothbrush virtually eliminated all of these pathogens in a 12-hour period. The inert coating is not eluted from the brush and, therefore, has no flavor and poses no risk to the consumer. The materials herein may also be used to provide an antimicrobial layer or to kill microbes in dental instruments, dental floss, and other devices for use in the mouth. Bio-film formation is a major problem in many applications of water containment, water filtration and water supply. Once a bio-film is formed, it typically resists additional treatment and acts as a constant source of microbial contamination. The prevention of the formation of the bio-film is the key to the maintenance of high quality water systems. The materials herein can be used to prevent the formation of bio-film in many water treatment products. For example, water containers and water purification systems used in camping, residential, commercial and military applications, which need to be periodically emptied, disinfected and rinsed. The treatment with the antimicrobial materials herein could eliminate the costs and hazards with this procedure, as well as the risks associated with improper maintenance of these water storage systems. The antimicrobial materials herein are also useful in point-of-use water purification filters, which trap bacteria and nutrients commonly found in all water systems. The bio-films formed in these filters usually spread the bacteria into the water stream in amounts that exceed the standard safety limits. Treated filters could offer a longer service life and significantly reduce the bio-hazard potential. Surfaces in medical offices, such as treatment tables, or consoles in a typical dental office have proven to be a major source of bacterial contamination, possessing potential risks to the health of the patient and the equipment. Although water supplies are treated routinely to reduce bio-contamination, the water that remains in the lines in the dental console can promote the formation of bio-films. The coating or treatment of these surfaces with the antimicrobial materials of the present invention can prevent the formation of bio-film on these substrates. The antimicrobial materials herein have been tested against the bacteria most commonly found in water. The treated pipe withstood repeated attempts to induce the formation of bio-film at several very high attack levels, while the untreated control pipe developed an extensive bio-film (see Examples 5 and 11). The treated pipe showed no trace of chemical elution in the water. The materials herein can also be applied to woven and non-woven fabrics used in hospitals and in health care supplies ranging from face masks to bed sheets. The materials can be applied in a form of spray or rub, which can be applied to surfaces in order to make them antimicrobial. Long-term resident catheters have a high risk of infections (2% -9%), which increases the patient's discomfort, the risk of systemic infections and the patient's stay in the hospital. Catheters treated with the antimicrobial materials herein can reduce the presence of infection caused by bacteria. The materials can also be used in urinary catheters, implants and inserts designed to work with the incontinence suffered by the increased risk of infection. Coatings made with the materials herein have been shown to kill microorganisms in human urine. The antimicrobial materials of the present invention can be used to treat standard biological plastic laboratory equipment for applications requiring low microbiological contamination, for example, laboratory tools for cell culture.
EXAMPLES EXAMPLE 1 Preparation of the PHMB-BMDGA SolutionsPolyhexamethylene biguanide (PHMB) (available as a 20% aqueous solution of Zeneca Biocides, Wilmington, DE) was distilled to remove the water, and the PHMB was redissolved in absolute ethanol to give 20% by weight of solution. This solution was used to prepare the resins presented below. (a) Further 312 ml of the 20% PHMB solution in ethanol were diluted with 600 ml of ethanol. This solution was added to a solution of N, N-methylene bis diglycidylaniline (MBDGA) (Aldrich Chemical Company, Milwaukee, Wl) containing 37.60 g of MBDGA dissolved in 119.9 ml of acetonitrile and 280.1 mole of ethanol. The resulting mixture was heated to 95 ° C in a closed reactor for two hours, forming an adduct of PHMB-MBDGA. The adduct solution was cooled and filtered (Scientific Grade 417 filter). The resulting adduct solution contained 10% by weight of the PHMB adduct: MBDGA having a PHMB: MBDGA ratio of 1.5: 1. (b) 330 ml of the 20% PHMB. This solution was combined with100 ml of a solution of sodium hydroxide (NaOH) containing 66 grams of NaOH, 66 ml of water and 34 ml of ethanol. The mixture was added to a solution containing 40 g of MBDGA, 120 ml of acetonitrile and 280 ml of ethanol. The resulting solution was heated at 95 ° C for 2 hours to form the PHMB adduct: MBDGA. The solution was cooled and filtered as described above. The resulting adduct solution contained 10% by weight of the PHMB adduct: MBDGA having a PHMB: MBDGA ratio of 1.5: 1. The resins were characterized according to the following procedures: 1. The film formation was tested through an immersion test with PE / PP (polyethylene / polypropylene) where the PE / PP samples were immersed in the resin solutions made in (a) and (b) above, and dried by blowing hot air, and film formation was observed; 2. The ratio of polymer to interlayer (PHMB-MBDGA) in the resin solution was tested through UV / visible spectroscopy; 3. The gelation time of the resin mixture was tested. The resins were diluted with ethanol at a concentration of1%. The film formation of the diluted resins was tested through the immersion test with PE / PP as described above. Both resins formed a coherent film. The resins were stored in closed containers at room temperature.
EXAMPLE 2 Coating of Plastic ArticlesSeveral plastic articles were coated using the coating solutions described in Example 1. 1. Cases for contact lenses: the polyethylene and polypropylene cases for contact lenses were coated according to the following procedure: The contact lens cases they were cleaned by immersing them in absolute ethanol for 5 minutes and dried. The clean kits were immersed in the antimicrobial coating solution (Example 1a or 1b) for 1 to 2 minutes. The sample cases were dried by blowing hot air. The entanglement was induced by heating the cases to 120 ° C for the polyethylene cases and to 200 ° C for the polypropylene cases for 2 hours. The cassettes were allowed to cool, rinsed with water at 60 ° C to remove any unbound polymer, then dried at 60 ° C for 1-3 hours. The coated cassettes were immersed in a 0.05% solution of silver iodide / potassium iodide in alcohol for 2 minutes. The kits were rinsed with aqueous alcohol to remove any silver without binding. The cases were then rinsed with water and dried. 2. Bristles of Toothbrushes: Toothbrushes were coated with nylon bristles according to the procedure described for the contact lens cases, except that the crosslinking reaction was performed at 120-140 ° C. Polyurethane and Polyvinyl ride Catheters: polyurethane and polyvinyl ride catheters were coated according to the procedure described for contact lens cases, except that the entanglement reaction was performed at 80-120 ° C for polyurethane and at 120 ° C for polyvinyl chloride. Dental Water Line Unit and Filters Tubes: polyurethane and polyether sulfone membrane tubing and housing were coated according to the procedure described for the contact lens cases, except that the interlacing reaction was performed at 80-120 ° C for polyurethane and at 120-140 ° C for polyethersulfone. Interlacing Procedure for Silicone Parts: parts were pre-cleaned in 100% ethyl alcohol (reagent grade) to remove dust, grease and other contaminants. They were then subjected to an alkaline chemical attack by immersing them in 0.1M NaOH in a 90% ethanol solution (10% water) at room temperature and ultrasound was applied for 2 minutes. They were then coated in a manner identical to that of the contact lens cases. Coating Procedure for Teflon Parts: the parts were subjected to a surface pre-treatment through oxygen plasma for 5 minutes in a plasma reactor. After they were coated in an identical way to that of the contact lens cases. Coating Procedure for Nylon Sheets: nylon sheets were pre-cleaned with 100% ethyl alcohol (reagent grade) to remove dust, grease and other contaminants. The formulation of a coating resin part was diluted with 100% ethyl alcohol to the desired concentration of 1% by weight. The clean nylon sheet was immersed in the coating resin for a period of 1-2 minutes. Then, the sheet was carefully removed from the coating resin bath and the excess adhesion resin was allowed to drain. The coating on the nylon sheet was dried by placing it in an oven at 70 ° C for 3-4 minutes. Then, the dried resin coating was entangled through thermal curing at 120 ° C for a period of 2 hours. The cured samples were removed from the oven and allowed to cool to room temperature. This procedure was used to coat bristles of non-woven brushes, non-woven nylon and cellulose fibers.
EXAMPLE 3 Membrane Coating ProcedurePolyethersulfone and nylon membranes were cleaned as described in Example 2 above. The membranes were coated with the antimicrobial resin solution described in Example 1 (1a or 1b) and dried. The coatings were then entangled by heating at 120 ° C for about 2 hours. The resulting interlaced coatings were rinsed with water to remove any unbound polymer, rinsed with acidic water or pH regulator [pH 2-2.5], fwed by another rinse with water, then dried. The silver was deposited in the interlaced polymer matrix by immersing the coated membrane in a 0.05% solution of a complex of silver iodide / potassium iodide in aqueous alcohol. The unbound silver iodide was removed by washing with ethanol. The membrane was rinsed with water, then dried at 70 ° C for 30 minutes.
EXAMPLE 4 Ability to Annihilate by ContactThe coated articles described in Example 2 and the membranes described in Example 3 were exposed to a variety of bacteria of the fwing genera: Pseudomonas, Staphylococcus, Serratia, Klebsiella, Bacillus, Enterococcus and Aspergillus, and a fungus of the genus Candida. The species of microorganisms used are listed in Tables 1 and 2. The articles and membranes were incubated with the microorganisms at 37-30 ° C for at least 20 hours, and for more than 504 hours (21 days). The results are shown in Tables 1 and 2.
TABLE 1Organism Attack Time for Annihilation Complete annihilation at 30 ° C Pseudomonas dimunata 10b CFU / ml 20 hours Pseudomonas cepacia 106 CFU / ml 20 hours Staphylococcus aureus 106 CFU / ml 20 hours Serratia marcescens 106 CFU / ml 20 hours Escherichia coli 106 CFU / ml 20 hours Klebsiella pnuemoniae 106 CFU / ml 20 hours Bacillus subtilis 106 CFU / ml 20 hours Bacillus cerius 106 CFU / ml 20 hours Staphylococcus epidermidis 105 CFU / ml 72 hours Enterococcus faecalis 106 CFU / ml 20 hours Candida albicans 106 CFU / ml 168 hours Aspergillus niger 105 CFU / ml without development * 21 days at 25 ° CTABLE 2 Passed Bacterial Development Development of Tested Membranes with 106 CFU / mL Pseudomonas aeruginosa in PBS at 30 ° CAMS = antimicrobial surfaceEXAMPLE 5 Kinetics of the Antimicrobial ActionThe coating acts after contact with the microorganism, intercalating first in the membrane of the cell and secondly transferring the bio-toxic agent directly to the contact organism. The subsequent time for the annihilation experiment was carried out in polyethylene tubes with various diameters, coated with the PHMB-MBDGA-silver coating described in Example 1. The coatings were applied as described in Example 2 for the lens cases contact. The tubes were inoculated with predetermined volumes of initial concentrations of up to 109 CFU / ml of Pseudomonas aeruginosa (ATCC # 9027) in PBS and incubated at 30 ° C for 20 hours. At several time points, the tubes were sampled and the microorganisms were plated and counted. The treated tubes demonstrated significant antimicrobial activity even when the volume to surface (S / V) ratios exceeded 4: 1. The results are given in Figure 3. Additional evidence for the mechanism of contact annihilation was provided through the fwing experiment. Polypropylene tubes were coated as described in example 2 for the contact lens cases. The coated tubes and untreated controls were attacked with 106 CFU / ml of Pseudomonas aeruginosa in PBS at 30 ° C for 20 hours. An organism account through standard plate placement techniques did not show any viable microorganism, that is, a complete elimination (6 log reduction) compared to untreated tubes. The solution containing dead bacteria from the coated tubes was digested in 0.1M nitric acid and analyzed for the presence of silver. It was found that the silver concentration was approximately 600 ppb. A coated tube containing PBS in template (without bacteria) incubated during the same time did not show any detectable silver in the solution (less than 10 ppb).
EXAMPLE 6 No Leachability of coatingsTo stimulate an aging of about 1 year at room temperature, the membranes with a very large surface area were coated as described in Example 3. The coated membranes were immersed in water, saline and saline solutions regulated at their pH with phosphate at 70 ° C for 5 days. The test solutions were analyzed through spectroscopy methods with sensitivities of less than 10 parts per billion (ppb) of active ingredients, ie, PHMB, MBDGA, silver and iodide. The following levels were found. Silver: less than 10 ppb (below the detection limit)PHMB: less than 100 ppb (below the detection limit) MBDGA: less than 300 ppb (below the limit of quantification) Iodide: less than 50 ppb (below the limit of quantification). These analytical results were further confirmed by testing the contact solutions to demonstrate that they did not present any antimicrobial activity by attacking them with silver-sensitive Escherichia coli (ATCC # 8739) at a concentration of 106 CFU / ml. No increase in numbers of microorganisms was detected after 20 hours.
EXAMPLE 7 ToxicityTo determine mammalian cell toxicity, coated polypropylene tubes as described in Example 2 for the contact lens cases were aged in saline regulated at their pH at 50 ° C for 48 hours. The test solutions were evaluated for toxicity with mouse fibroblast cells and showed no toxicity to the cells.
EXAMPLE 8 Mechanical ResistanceThe coated surfaces as described in Example 2 were subjected to the Sutherland rub test with 0.2812 kg / cm2 for 50 shocks and remained viable while the rubbed surface showed no antimicrobial activity.
EXAMPLE 9 InertiaThe coating remained completely inert and bio-active after being exposed to a variety of physical and chemical stresses: • Low temperature (-15 ° C), 24 hours • Ethanol and boiling water, 1 hour • Prolonged exposure to acidic and basic solutions variable pH (4-10), 12 hours • Highly ionic solution (2% sodium chloride), 24 hours• Autoclave (121 ° C for 15 minutes) • Long-term exposure to urine (35 ° C for 7 days) • Attacked with 0.7% human serum albumin in pH regulated saline with phosphate in accelerated aging tests (observed a small increase in non-bioavailable silver eluble products due to the complex formation of the protein) • Exposure to blood products • Used by human volunteers for 3 days. No reaction was observed on the skin.
EXAMPLE 10 Bio-Surface ActivityThe coating annihilates microorganisms by contact, but is non-toxic to mammalian cells. In laboratory tests, the treated surfaces (polypropylene, polyethylene, nylon and polyethersulfone) effectively eliminated all tested human pathogens, including bacteria, yeast and fungi. • Bacillus cereus (ATCC # 11778) -106 CFU / ML in 20 hours • Escherichia coli (ATCC # 8739) -106 CFU / ML in 20 hours Pseudomonas aeruginosa (ATCC # 9027) -106 CFU / ML in 20 hours Pseudomonas cepacia ( ATCC # 25416) -106 CFU / ML in 20 hours Pseudomonas diminuta (ATCC # 19146) -106 CFU / ML in 20 hours Klebsiella pneumoniae (ATCC # 13883) -106 CFU / ML in 20 hours Staphylococcus aureus (ATCC # 6538) - 106 CFU / ML in 20 hours Serratia marcescens (ATCC # 8100) -106 CFU / ML in 20 hours Enterococcus faecalis (ATCC # 19433) -106 CFU / ML in 20 hours Staphylococcus epidermidis (ATCC # 12228) -105 CFU / ML in 72 hours • Candida albicans (ATCC # 10231) -105 CFU / ML in 168 hours. Coated surfaces as described in Example 2 were attacked with three microorganisms in the indicated initial concentrations. The microorganisms were suspended in pH-regulated saline with phosphate and allowed to remain in contact with the treated surfaces for extended periods at 30 ° C. The solutions were then analyzed using standard plate placement methods. Although organism growth was documented on untreated surfaces, the microorganisms were completely eliminated in the samples treated in the specified period. These results were confirmed in thousands of tests conducted over three years. In addition, the treated surfaces were tested against Aspergillus niger. No fungal growth was detected during the 28-day trial period.
EXAMPLE 11 Prevention of Bio-Film FormationTo determine the efficacy against bio-film formation, the coated polyurethane tubes as described in Example 2 and the untreated tubes were attacked with a mixture of the following microorganisms, incubated in a 1% synthetic growth medium at room temperature: Pseudomonas diminuta (ATCC # 19146), Pseudomonas aeruginosa (ATCC # 9027), Klebsiella pneumoniae (ATCC # 13883), Bacillus cereus (ATCC # 11778), Escherichia coli (ATCC # 8739), Staphylococcus aureus (ATCC # 6538) . Within 24 hours, the microorganisms in the untreated tubes were developed from an initial concentration of 104 CFU / ml to an average of 3x105 CFU / ml. The untreated tubes did not present viable microorganisms. The tubes were then washed and filled with water. Eight days later, the untreated tubes still produced 105 CFU / ml (resulting from the bio-film established during the first day of incubation), while the treated tubes did not produce microorganisms.
EXAMPLE 12 Bacteria Resistant to AntibioticThe untreated and treated surfaces (as described in Example 2) were attacked with 106 CFU / ml of a methicillin-resistant strain and neomycin of Staphylococcus aureus (ATCC # 33592). The microorganism was suspended in saline regulated at its pH with phosphate and allowed to remain in contact with the surfaces. In 20 hours, the JTO treated surfaces. they had viable organisms, while the number of viable organisms on untreated surfaces remained unchanged.
EXAMPLE 13 Preparation of the Silver Iodide Complex with Poly (hexamethylenebiguanide)g of Cosmosil CQ (Zeneca, Biocides, Wilmington, DE), 10 ml of ethanol (EtOH) and 1.2 g of potassium iodide (Kl) were mixed together. The resulting solution was added dropwise to 400 ml of an aqueous ethanol solution (1: 1) containing 0.5% (w / v) silver iodide and 6% (w / v) potassium iodide. The precipitated white rubber type product was separated from the solution, rinsed with 50 ml of 50% (v / v) aqueous ethanol, and dried in a vacuum oven for 18 hours at 50 ° C. The silver-containing product obtained after drying was a semisolid clear yellow colored resin with a silver content of 10.7%.
EXAMPLE 14 Preparation of the Polyhexamethylene Biguanide Base (PHMB) from a Polyhexamethylene Hydrochloride Solution200 ml of Cosmosil CQ (Zeneca Biocides, Wilmington, DE) was neutralized by the addition of 200 ml of aqueous NaOH (40% by weight) with slow stirring, forming a precipitate. After filtering the supernatant liquid, the precipitate was suspended in 400 ml of alcohol. The PHMB suspension was diluted to 500 ml with additional alcohol and filtered. Ten ml of the aliquot (filtered) gave 0.7 g of the dried product (100 ° C, 15 minutes). The calculated yield of PHMB in the total solution was 35 g.
EXAMPLE 15 Preparation of PHMB-Epoxy Resin (PHMB-MBDGA)17.5 g of 4,4'-methylenebisdiglycidylaniline (MBDGA) (Aldrich Chemical Company, Milwaukee) was dissolved in 70 ml of acetonitrile. The resulting MBDGA solution was added dropwise to the PHMB solution described in Example 14 which had been preheated in a water bath at 80-90 ° C with stirring. The reaction mixture became cloudy during the addition. The reaction was allowed to proceed for approximately 30 minutes, at which time the solution became clear. The reaction vessel was removed from the bath and cooled. The pH of the cooled solution was adjusted to 3.65 through the slow addition of 2N alcoholic HCl in alcohol.
EXAMPLE 16 Preparation of the Silver Iodide Complex with the PHMB-MBDGA resinml of 10% (w / v) of an ethanol solution of PHMB-MBDGA resin (2: 1 w / w) (prepared as described in example 15 above) was mixed with 30 ml of anhydrous ethanol and 3.6 g of potassium iodide. The resulting solution was added dropwise to 400 ml of an aqueous solution of ethanol (1: 1 v / v) containing 0.5% (w / v) of silver iodide and 6% (w / v) of potassium iodide. The product of precipitated white rubber type was separated from the solution, rinsed with 50 ml of 50% (v / v) aqueous ethanol, and dried in a vacuum oven for 18 hours at room temperature. The silver containing the obtained product was a light yellow, transparent solid resin, with a softening point of 40-45 ° C and a silver content of 3.8%.
EXAMPLE 17 Preparation of the Silver Iodide Complex with PHMB-MBDGA Interlaced Resin500 ml of 10% (w / v) of an ethanol solution of the PHMB-MBDGA resin (2: 1 w / w) was prepared as in Examples 15-17 above. The volume of solution obtained was reduced to approximately 100 ml by evaporation of the solvent under vacuum in a rotary evaporator at a water bath temperature of 70 ° C. To achieve solvent removal, the resulting viscous resin solution was transferred to a glass beaker and placed in a vacuum oven at room temperature. After 30 minutes of drying, the oven temperature was increased to 75 ° C and the sample was left under vacuum for a further 16 hours. The solid product obtained was cured at 130 ° C for 2 hours in a regular oven. The cured resin was milled to make resin powder with a particle size of about 50 microns. A quantitative yield of the entangled PHMB-MBDGA resin (> 90%) was obtained at the end of the powder preparation process. 10 g of silver iodide and 55 g of potassium iodide were dissolved in a mixture of 50 ml of water and 150 ml of ethanol. The crosslinked resin powder (15 g) prepared as described above was immersed in the silver solution and left under stirring for 30 minutes. Then the solid matter was separated from the supernatant and resuspended in 100 ml of anhydrous ethanol. After 10 minutes of washing, the resin powder was recovered from the mixture, rinsed with a fresh portion of alcohol (50 ml) and dried under vacuum at room temperature for 16 hours. 22 g of the resin powder of entangled PHMB-MBDGA charged with silver with a silver content of 5.9% after a complete evacuation of the solvent was obtained. Before use, the resin powder was milled and sieved through a standard test sieve to obtain the particle size below 50 microns.
EXAMPLE 18The broad spectrum antimicrobial activity of the resin powder, done as described in Examples 13-17, was evaluated as follows. The powder was suspended in saline regulated at its pH with phosphate (PBS) at resin concentrations ranging from 0.05 mg / ml to 100 mg / ml. The suspensions were inoculated with the following attack microorganisms: Pseudomonas aeruginosa ATCC 9027 Escherichia coli ATCC 8739 Staphylococcus aureus ATCC 6538 Serratia marcescens ATCC 8100 Staphylococcus epidermidis ATCC 12228 Candida albicans ATCC 10231 All microorganisms were inoculated as a suspension in PBS to the resin suspensions in PBS. PBS solutions without resin powder were used as controls. A bacterial attack level of approximately 106 CFU / ml was maintained for all resin concentrations. After inoculation, the solutions were incubated at 30 ° C for 20 hours, then the number of viable microorganisms was determined through the standard extension plate method. A step criterion was established for the antimicrobial efficacy of the resin suspensions, which required the complete elimination of each type of microorganism during a period of 20 hours.
Test Results TABLE 1 Antimicrobial Efficacy of Resin Dust against BacteriaSample Organism Final Conc. * Shows Organism Conc.(mg / ml) (CFU / ml) (mg / ml) Final * (CFU / ml)Control P. aeruginosa 4.5x10 Control S. marcescens 3.1x105100 P. aeruginosa 0 1 S. marcescens 0P. aeruginosa 0 10 S. marcescens 01 P. aeruginosa 0 100 S. marcescens 0Control E. coli 1.75X 10b Control S. epidermidis 3.75x10 '100 E. coli 0 1 S. epidermidis 0E. coli 0 10 S. epidermidis 01 E. coli 0 100 S. epidermidis 0Control S. aureus 2.4x10 100 S. aureus 0 10 S. aureus 0 1 S. aureus 0 * Average value TABLE 2 Antimicrobial Efficacy of Resin Powder against Bacteria and YeastSample Organism Final Conc. * Shows Organism Conc.(mg / ml) (CFU / ml) (mg / ml) Final * (CFU / ml)Control P. aeruginosa 9, .3x104 Control S. marcescens 1.75x1061. 00 P,. aeruginosa 0 1.00 S. marcescens 00. 75 P,. aeruginosa 0 0.75 S. marcescens TCF / O *0. 50 P. aeruginosa 0 0.50 S. marcescens 00. 25 P. aeruginosa 0 0.25 S. marcescens TFC / 0 *0. 10 P. aeruginosa 0 0.10 S. marcescens 00. 05 P. aeruginosa 0 0.05 S. marcescens 0Control E. coli 8 .2x104 Control C. albicans 2.6x1051. 00 E. coli 0 1.00 C. albicans 00. 75 E. coli 0 0.75 C. albicans 00. 50 E. coli 0 0.50 C. albicans 00. 25 E. coli 0 0.25 C. albicans TCF / 0 **0. 10 E. coli 0 0.10 C. albicans 00. 05 E. coli * * * 0.05 C. albicans 0Control S. aureus 1. 3x105 1.00 S. aureus 0 0.75 S. aureus 0.50 S. aureus 0 0.25 S. aureus 0.10 S. aureus 0. 0.05 S. aureus 0 * Average value ** TCF / O - Number of colonies very below statistical significance *** Accounts not available Resin powder completely eliminated all microorganisms used in the study on the scale of varying concentrations in a period of 20 hours over the entire scale of resin concentrations.
EXAMPLE 19 Preparation of Extended Chain PHMB through Reaction with Hydrophobic Epoxide32.5 g of blocked end polyglycide (bisphenol A-co-hydrochlorohydrin) glycidyl (molecular weight 1075, Aldrich) were dissolved in 77 ml of N, N-dimethylformamide (DMF) with stirring. 250 ml of a 13% (by weight) solution of PHMB (base) in 250 ml of absolute ethanol was rapidly added. The cloudy solution was heated in a water bath at 90 ° C for 1 hour with stirring. A clear viscous solution was obtained, which was allowed to cool to room temperature and which is immiscible with water.
EXAMPLE 20 Preparation of Extended Chain PHMB through the Reaction of Hydrophobic Epoxide17.3 g of 4-glycidyloxyaniline of N, N-diglycidyl ether (Aldrich) were dissolved in 25 ml of N, N-dimethylformamide (DMF) with stirring. 130 ml of a 20% solution of PHMB was rapidly added. HCL (Cosmosil CQ, Zeneca Biocides, Delaware) followed by 70 ml of distilled water. The cloudy solution was refluxed in a water bath for two hours with stirring. A clear viscous solution was obtained, which was allowed to cool to room temperature. The resulting extended chain compound was immiscible with water or pure ethanol, but was miscible in a 50% aqueous ethanol solution.