The present invention relates to binding of quaternary ammonium to a substrate, for example a fabric, in order to render the substrate antimicrobial.

Background of the Invention

The biocidal effect of quaternary ammonium polymers (quats) has been know for mo- re than half a century, for example as disclosed in 1941 in US patent No 2,261,002 by Ritter or in 1945 in US 2,388,614 by Kirby and Lonz, both assigned to DuPont. From the same period is US 2,271,378 by Searle describing quats for cellulose products to hold them germ-free. The combination of methyl and dodecyl and tetradecyl alkyl groups in polymeric quaternary ammonium has been disclosed in US 5,352,833 by Merianos.

The use of quats on cloth has been described as far back as the 70'ies. For example, US 3,898,336 by Rembaum et al. or US 3,670,048 by Magat et al. from 1972 discloses a polyester textile with quats grafted to the surface. The use of a cellulose non- woven quat-web for cleaning of skin, for example in dry mode, has been described in US 4,615,937 by Bouchette and, further, in US 4,929,498 by Suskind and Miller for primarily wet mode, where Latex is used as a binder for the fibres on which or- ganosilicon quats are bound. Polyehtyleneimine (PEI) is mentioned as one possible starting polyamine. WO 2006 071191 by Schonemyr et al assigned to Appeartex dis- closes PEI derivatives of various kinds on fibres, for example cotton fibres.

The binding of quats on substrate has been an issue of ongoing study, because the quats, on the one hand, should be antimicrobially efficient and, on the other hand, stable bound to the substrate in order to achieve a long lasting effect. Covalent bind- ing to substrates has been proposed in US 5,783,502 by Swanson assigned to BSI Corporation disclosing a virucidal fabric with quats covalently bound to the surface of the fibres, for example cotton. For the covalent binding, photochemical coupling is proposed, for example, by azidonitrophenyl groups. Covalent binding of quats on glass surfaces is disclosed in International Patent Application Wo2002/085542 by Tiller et al and assigned to MIT. Patent application US2010/03212 by Kis et al. and assigned to Novartis discloses an antimicrobial article, for example a coated textile, which comprises a carrier, a linking element, a spacer, for example a PEI, and a quaternary ammonium group.

International Patent Application Wo2000/033778 by Batich et al. assigned to Quick- Med discloses a superabsorbent polymer matrix with an enhanced surface area and an antimicrobial compounds coupled by non-siloxane bonds, for example covalent bonds, to the polymer matrix, the antimicrobial compounds being quaternary ammo- nium, for example. International Patent Application Wo2003/039602 by Batich and assigned to Quick-Med discloses a substrate, for example a wound dressing, having a quat coating covalently bound to the substrate. International Patent Application Wo2007/24972 by Liesenfeld et al. assigned to Quick-Med discloses a wound dressing with a non-leachable antimicrobial, for example quaternary ammonium, and a releasable agent, for example doxycycline.

International Patent Application Wo2008/127416 by Haldar et al. assigned to MIT discloses PEI with quaternary ammonium, for example having methyl groups and do- decyl groups, bound non-covalently but stably to a substrate, where the polymeric quat coating can be applied in the same manner as paint to the substrate. The preparation of the coating includes preparation of a PEI by acid hydrolysis which is quater- nised by dodecylation, followed by methylation before application to the surface of the substrate. The conclusion of advantages of non-covalent binding of quats to surfaces is also followed in the article "One-Step, Painting-Like Coating Procedures To Make Surfaces Highly and Permanently Bactericidal" by Park, Wang, and Klibanov published in February 2006 in Biotechnol. Prog. 2006, 22, 584-589. In this article, it is pointed out that surface derivatization methods involve several synthetic steps as well as harsh reagents at elevated temperatures, why covalent attachment of polymers is probably not a viable option for a general, single- step procedure for making solid surfaces bactericidal.

As it appears from the prior art, there are ongoing efforts to improve fabrics with quat coating in order to bind the quat properly to the fabric. However, no apparent consensus has yet been found, and there is still need for improvements in the art. When delineating from the field of antimicrobial quat binding on surfaces and turning to other non-related fields, especially microbiology, a special covalent photoreactive binder is used for binding biological entities to various surfaces, namely SANPAH, Succinimidyl-6(4'-azido-2'-nitrophenylamino)hexanoate. US 4,679,220 discloses use of SANPAH for binding ATPase to a Dextran surface of a colloidal gold particle. Co- valent binding of biological entities on a polymer surface is disclosed in US 4,716,122 by Schaefers assigned to Organogen, where aryl-azide groups are used to covalently bind reaction components of immunological reactions to a carrier material, for example consisting of a synthetic resin, especially surface-treated polyethylene. The preferred provider for such aryl-azide groups is SANPAH. US 2009/117166 by Myung et al discloses antibody binding to a polymer surface through a SANPAH-Collagen- SANPAH intermediate layer. US 2009/081797 by Fadeev discloses a polymer surface which has been plasma-treated to form functional groups, which form covalent bonds with a conjugate to which a biologically active compound is attached. An option for the conjugate is SANPAH. In addition, potentially, fluorescin (quaternary ammonium compound) can be used as a cell marker.

However, SANPAH has not yet been proposed for covalent binding of antimicrobial polymeric quaternary ammonium compounds to substrates, and no advice has been given, how SANPAH can, actually, be used to achieve such a proper binding. Object of the Invention

It is an object of the invention to provide an improvement in the art for binding of antimicrobial polymeric quaternary ammonium compounds to substrates, especially with a covalent binding.

Description of the Invention

This object is achieved by the following method for providing a coating on a sub- strate, where the coating comprising a PEI-based polymer having antimicrobial quaternary ammonium. The method comprises

- providing polyethyleneimine, PEI,

- quaternising the PEI, and

- using methylation of the quaternised PEI before photochemically coupling the PEI to the substrate with a photoreactive coupling agent.

In addition, the method comprises reacting the photoreactive coupling agent with the PEI before the methylation process. Optionally, the photoreactive coupling is a covalent coupling.

In experiments, it has surprisingly turned out that the inclusion of the photo-reactive coupling agent in the polymer should be made before the methylation process, because the methylation can have detrimental effect on the photoreaction with the coupling agent.

For example, the method comprises dodecylation of the quaternised PEI before methylation. Optionally, the method comprises binding a protecting agent to some of the quaternary ammonium sites of the PEI before dodecylation and removing the protecting agent after dodecylation. A potential protecting agent is Trityl, although other agents are possible. Advantageously, the antimicrobial polymeric quaternary ammonium has antimicrobi- ally active quaternary ammonium with three dodecyl groups. As an alternative, the antimicrobially active quaternary ammonium has two dodecyl groups and one methyl group. As even further alternative, other combinations of alkyl groups may be part of the antimicrobial quaternary ammonium in the polymer.

For example, ANPAH is used in a photochemical reaction as a coupling agent between the substrate and the antimicrobial polymeric quaternary ammonium. ANPAH is 6-[4'-azido-2'-nitrophenylamino]hexanoyl and illustrated below.

For the photochemical coupling, the substrate is provided with the antimicrobial polymeric quaternary ammonium including the ANPAH and exposed to light. Although visible light would induce photochemical reaction, the binding is, typically, performed with UV irradiation of the coating on the substrate.

As it has turned out, ANPAH is suited as a photochemical coupling agent, especially, when using a polyethyleneimine (PEI) based polymer. A PEI is illustrated below.

The PEI can, optionally, be a branched PEI, for example implying a ratio of primary to secondary to tertiary amines of 1:2: 1. An ANPAH-PEI polymer can be created, which may then be bound to the substrate with covalent attachment of the ANPAH on the substrate. Advantageously, the ANPAH-PEI polymer is an N-alkyl-N-ANPAH-PEI polymer, especially, an N-alkyl-N-methyl-ANPAH-PEI polymer. Such a polymer can be created by providing PEI, quaternising PEI, and using methylation of the quaternised PEI before photochemical coupling of the PEI to the substrate. When taking offset in the International Patent application WO2008/127416, the method includes formation of PEI, for example by acid hydrolysis, quaternisation by dodecylation followed by methylation. Adding ANPAH to this scheme, a polymer is created of the form N-dodecyl-N-methyl- ANPAH-PEI. Also in this case, the inclusion of the photo-reactive ANPAH in the polymer should be made before the methylation process, for example after the dodecylation process, because the methylation can have detrimental effect on the photoreactive process.

From such reaction scheme, it is possible to create an antimicrobial polymeric quaternary ammonium that has antimicrobially active quaternary ammonium with two dode- cyl groups and one methyl group. An example is illustrated below.

Alternatively, the active quaternary ammonium may have two methyl groups and one dodecyl group, which is an embodiment for an antimicrobial quat as described for a non-covalent binding in International Patent Application Wo2008/127416 by Haldar et al. assigned to MIT.

As a further alternative, it is possible to obtain a coating with antimicrobial quaternary ammonium groups in the polymer having three dodecyl groups, which is believed to have a higher antimicrobial efficiency that one or two dodecyl groups.

When using ANPAH, the photochemical reaction, advantageously, involves a reaction between the ANPAH and hydroxyl groups of a substrate. In a concrete embodiment, ANPAH is photochemical coupled to hydroxyl groups of the substrate.

For example, in the method, ANPAH is provided by reacting SANPAH with PEI for creation of an ANPAH-PEI polymer, where the succinimidyl entity is split off during the reaction.

In some experiments, the PEI based polymer was branched, hydrophopbic, and poly- cationic.

For substrates, a large variety of materials and forms are possible. Materials include cellulose materials, for example cotton or regenerated cellulose, as well as synthetic polymer, for example polyester, polyamide, acetate polymers, or polyolefins.

Examples of possible substrates include non-exclusively hard polymer surfaces, for example from medical devices, and soft surfaces including fabrics. Special application is found in antimicrobial clothing, towels, wipers, sheets, gauze, wound dressings, bandages, and air masks. Examples of microbes against which such antimicrobial coatings can be used include bacteria, virus, and fungi, especially human pathogenic bacteria and viruses. The types of counteracted bacteria include both Gram-positive as well as Gram-negative bacteria, such as Escherichia coli and Staphylococcus aureus. The counteracted viruses include both enveloped virus, for example influenza viruses, as well as non- enveloped viruses, for example polio viruses.

Thus, the invention also comprises a method of cleaning skin and killing microbes, the method comprising providing a fabric coated with an antimicrobial polymeric quaternary ammonium according to the above, and contacting human skin with the fabric, for example under dry conditions, that is without wetting the fabric first. During contact, pathogenic microbes are picked up from the skin with the fabric and eliminated by contact with the antimicrobial polymeric quaternary ammonium in the fabric. For example, the invention comprises a method of cleaning skin and killing microbes, the method comprising providing a fabric coated with an antimicrobial polymeric quaternary ammonium covalently to a substrate with ANPAH as a coupling agent between the substrate and the antimicrobial polymeric quaternary ammonium, and contacting human skin with the fabric, during contact picking up pathogenic microbes from the skin with the fabric, and eliminating the microbes by contact with the antimicrobial polymeric quaternary ammonium.

The advantage of the fabric being capable of cleaning skin under dry conditions makes the fabric suitable for use in rural areas and under emergency situations, where the access to water is scarce. The fabric could be used as a long-lasting cleaning wiper that reduces risks for spread of diseases.

The above purpose is also achieved with the following method in which antimicrobial polymeric quaternary ammonium as part of a coating is bound covalently to a sub- strate by photochemical reaction with a coupling agent between the substrate and the antimicrobial polymeric quaternary ammonium. In this case, the special aspect is Aspect 1 in that the coupling agent is ANPAH. An example of a substrate is a fabric, the term fabric including microfibre textiles. This method of Aspect 1 may be combined with features selected from the above description and/or combined with following aspects, optionally in selected groups:

Aspect 2: optionally, the antimicrobial polymeric quaternary ammonium is a polymer based on polyethyleneimine, PEI;

Aspect 3: optionally, reacting ANPAH with PEI and as a result creating an ANPAH- PEI polymer;

Aspect 4: optionally, the ANPAH-PEI polymer is an N-alkyl-N-methyl-ANPAH-PEI polymer;

Aspect 5: optionally, the method comprises providing PEI, quaternising PEI, and us- ing methylation of the quaternised PEI before photochemical coupling of the PEI to the substrate, for example, also reacting ANPAH with PEI before the methylation process;

Aspect 6: optionally, the ANPAH-PEI polymer is an N-dodecyl-N-methyl- ANPAH- PEI polymer, for example, the method comprises dodecylation of the quaternised PEI before methylation.

Aspect 7: optionally, the method comprises reacting ANPAH with PEI after dodecylation;

Aspect 8: optionally, the antimicrobial polymeric quaternary ammonium has antim- icrobially active quaternary ammonium groups with two dodecyl groups and one methyl group; alternatively, it may have two methyl groups and one dodecyl group;

Aspect 9: optionally, the reacting of ANPAH with PEI comprises dissolving SANPAH and dissolving PEI and reacting the dissolved SANPAH with the dissolved PEI to form an ANPAH-PEI polymer;

Aspect 10: optionally, the PEI based polymer is branched, for example hydrophopbic and/or polycationic, in addition;

Aspect 11: optionally, the method comprises providing a substrate with hydroxyl groups and photochemical coupling the ANPAH to the hydroxyl groups of the substrate. The initial purpose is also achieved with a substrate, for example a fabric, including microfibre textiles, the substrate having an antimicrobial coating, preferably produced by a method according to any of the above aspects, wherein the coating comprises antimicrobial polymeric quaternary ammonium bound covalently to the substrate with ANPAH as a coupling agent between the substrate and the antimicrobial polymeric quaternary ammonium. Optionally, the coating comprises an N-alkyl-N-methyl- ANPAH-PEI polymer, for example an N-dodecyl-N-methyl-ANPAH-PEI polymer. As a further option, the antimicrobial polymeric quaternary ammonium has antimicro- bially active quaternary ammonium groups with two dodecyl groups and one methyl group.

Detailed Description of the Invention In the following, the invention is described in further detail with reference to a specific embodiment of creating a coating material.

As will be explained in the following, the synthesis procedure for the coating with the antimicrobial polymeric quaternary ammonium implies an initial protection step.

Protection

In this rotection step, branched polyethyleneimine, BPEI, as illustrated below,

is protected by binding Trityl to some of the quaternary ammonium sites of the PEI, which can be illustrated by the following. Tr

HM

0.5s 0.45K 0.05ί·;

The ratios given in the above illustrations are only exemplary and not exclusively binding for the invention.

In experiments, a branched polyethyleneimine, BPEI, was used with a weight of 750 kDa, although the weight could also be different and is not limited to this specific number. The following parameters for weight, amount, temperatures and durations of actions described in connection with a concrete experimental embodiment are exemplary but can be slightly modified.

In this concrete exemplary experimental embodiment, BPEI was provided by 50 wt in water, why, as an initial step, it was dehydrated in a lyophilizer to provide dry BPEI. An amount of 5.2g of this dry BPEI was dissolved in 25 mL anhydrous di- methylformamide under Ar. An amount of 6 mL anhydrous dichloromethane was added. An amount of 6 mL of 0.5M trityl chloride solution in anhydrous dichloromethane was drop- wise added at -10°C in a NaCl/ice water bath with a flushing of the generated HC1 gas with Ar. The mixture was stirred overnight for about 12 hrs, allowing the ice bath to melt and return to room temperature. The polymer precipitated overnight. Volatiles were removed under vacuum.

The final polymer was redissolved in methanol and subject of dialysis in 3.5kDa molecular-weight cutoff (MWCO) regenerated cellulose dialysis membrane (Spectrum Labs) against 2L methanol for at least 8hrs, repeated once more with fresh 2L methanol. The methanol was subsequently removed from the dialyzed polymer under vacuum to yield 4.5g of N-Tr-BPEI. By NMR, it was found that 1.9% of amines were tritylated.

Dodecylation

The N-Tr-BPEI of the previous step was subsequently subject to dodecylation. An amount of l.Og of N-Tr-BPEI, 3.8g K2C03, 14 mL tert-amyl alcohol (dried over molecular sieves), and 17 mL 1-bromododecane were combined. Using a connection to a condenser, it was flushed and left with an Ar balloon, stirred at 95°C for 96 hrs, and then centrifuged at 6000 rpm for 30 min at room temperature to remove carbonate salts. The supernatant was dialyzed against 2L of hexane for at least 8hrs, repeated once with fresh 2L hexane. The final yield was 4.0g N,N-dodecyl,Tr-BPEI

Deprotection

In order to make the trityl protected sites accessible for the ANPAH, the following deprotection step was performed. An amount of l.Og of N,N-dodecyl,Tr-BPEI was dissolved in 20 mL dichloromethane to which 20 mL trifluoroacetic acid were added at room temperature with a stirring for 1 hr. Further, 20 mL methanol was added with continued stirring for further 15 min. After that, volatiles were removed under vacuum. To this polymer solution, 50 mL of dialysis solution (5% pyridine and 0.5% tetraethylammonium bromide in chloroform) were added to neutralize acids. The neutralized solution was dialyzed against 1L of dialysis solution for at least 8hrs, repeated once with fresh 1L of dialysis solution and then twice with 1L of fresh chloroform. From the dialyzed polymer, volatiles were removed under vacuum to yield 0.78g N-dodecyl-BPEI.

Derivitization of the coupling agent

After the dodecylation step, the coupling agent was introduced. For this purpose, 0.75g of N-dodecyl-BPEI were dissolved in 5 mL of anhydrous dichloromethane to which 8.8 micro-liter of diisopropylethylamine was added as well as an amount of 50mg SANPAH dissolved in 5mL anhydrous dichloromethane. The mixture was stirred in dark at room temperature for 1 hr followed by dialysis against 1L of dichloromethane overnight in the dark. The solvent was removed under vacuum from the dialyzed polymer to yield is 0.63g of N,N-ANPAH,dodecyl-BPEI

Methylation

As already outlined above, a successful coating was obtained, if the methylation step was performed after the introOduction of the ANPAH to the PEL For the methylation, 0.5g of N,N-ANPAH,dodecyl-BPEI was dissolved in 10 mL anhydrous dichloromethane, after which 1 mL iodomethane was added and the solution stirred at 37 °C for 24hrs in sealed condenser in the dark. Volatiles were removed under vacuum to yield 0.5g of N,N,N-ANPAH,dodecyl,methyl-BPEI. Photoreactive coupling to the surface

The final N,N,N-ANPAH,dodecyl,methyl-BPEI, or in another notation, N-dodecyl-N- methyl-N-ANPAH-BPEI was dissolved in dichloromethane and coated to a substrate with subsequent illumination by UV radiation for 10 minutes.

Experiments showed substantial antimicrobial effect against E coli Comments

By the method above, a coating was obtained with antimicrobial quaternary ammonium groups in the polymer having three dodecyl groups.

However, the method may also be used for a coating with antimicrobial quaternary ammonium in the polymer having two or only one dodecyl group. Alternatively, other combinations of alkyl groups may be part of the antimicrobial quaternary ammonium in the polymer.