AN ANTIMICROBIAL SUBSTRATE, A METHOD AND A COMPOSITION FOR PRODUCING IT Technical field The present invention generally relates to the field of antimicrobials. More specifically, the present inven- tion relates to an antimicrobial substrate having adhered to at least a part of its surface an organosilicon qua- ternary ammonium salt compound, a method for producing said substrate and a composition for use in the produc- tion of said substrate.

Technical background An antimicrobial is an agent that prevents microbi- ological contamination by destroying (killing), inhibit- ing the growth or reproduction of, and/or removing micro- organisms, such as bacteria, fungi, yeasts, algae, and virus.

One of the most common classes of antimicrobials is quaternary ammonium salts (QAS), such as dodecyltrimethyl ammonium bromide (DTAB).

A sub-class of QAS is organosilicon quaternary ammo- nium salt compounds (also referred to as quaternized or- ganosilanes). These compounds and different uses thereof are well known within the field. Examples of some prior art references describing antimicrobial uses of quater- nized organosilanes are US Pat. Nos. 3,560, 385; 3,730, 701 ; 3, 794,736 ; 3, 814, 739; US 3,730, 701; 3,794, 736; 3,817, 739; 3,860, 709 ; 3,865, 728; 4,282, 366; 4,504, 541; 4,564, 456; 4,615, 937; 4,692, 374,4, 408,996 ; 4,414, 268; 4,425, 372 ; 4, 395,454 ; 4,411, 928 ; 4, 822,667 ; and 4,835, 019.

Organosilicon quaternary ammonium salt compounds are bacteriostatic/bactericidal, fungistatic/fungicidal, spo- rostatic, algistatic/algicidal, and viricidal.

Most cells have a net negative charge and are thus attracted to positively charged organosilicon quaternary ammonium salt compounds.

3- (trimethoxysilyl) propyl-dimethyloctadecyl ammonium chloride (Formula I), also called TMS (sold by Aegis En- viromental Management Inc under the trademark AEM 5772/5, previously Dow Corning 5700, CAS No 27668-52-6) is an ex- ample of a commercially commonly used quaternized or- ganosilane. TMS may be prepared by quaternization of di- methyloctadecylamine with 3-chloropropyltrimethoxysilane.

Formula I In aqueous media, it is believed that TMS is con- verted to 3- (trihydroxysilyl) propyl-dimethyloctadecyl am- monium chloride. This compound is capable of binding to a wide variety of natural and synthetic substrate surfaces, such as wood ; metal ; glass ; leather; plastics, e. g. poly- ethylene and polypropylene ; rubber ; ceramics; paper and fabrics, e. g. cellulose, cotton, polyamides, and polyes- ters. Therefore, a common method of adhering it to a sub- strate surface is to add a dilute solution of TMS in methanol to water and then treat the substrate with the thus obtained solution to provide an antimicrobial coat- ing.

3- (trihydroxysilyl) propyl-dimethyloctadecyl ammonium chloride binds to the surface either through (i) ionic bonds between 0-on a negatively charged surface possess- ing acidic hydroxyl groups and the positively charged am- monium ion, through (ii) covalent bonds between OH on a surface possessing non-acidic hydroxyl groups and the

- Si-OH group, or through (iii) electrostatic attraction between the negative charge that exist on most non- hydroxylated surfaces and the positively charged ammonium ion. It is also believed that intermolecular siloxane po- lymerisation (-Si-O-Si-bonds) occurs on the surface be- tween the surface-associated molecules.

The Cis hydrocarbon chain quaternary ammonium portion of TMS is believed to provide the antimicrobial proper- ties of the compound.

The killing of microorganisms by the action of a quaternized organosilane, such as TMS, is however be- lieved to be a rather slow process. If a lot of microor- ganisms are adsorbed by a substrate surface, such as a cleansing cloth, modified with an antimicrobial quater- nized organosilane, the microorganisms are believed to become rather loosely attached to the substrate surface and as a consequence alive, i. e. not yet killed, microor- ganisms may leak from the substrate surface.

It would be a great advantage if this problem could be avoided.

Furthermore, the surface of a material coated with a quaternized organosilane is rather hydrophobic thus giv- ing the material a rather poor absorption capacity of hy- drophilic liquids, such as blood and water. A poor ab- sorption capacity of hydrophilic liquids may be a disadvantage in certain applications, such as cleansing mops and cloths. High hydrophobicity is also a disadvantage in those applications where the an- timicrobial material is to be washed and re-used, such as clothing and articles of beddings. Another application wherein a high hydrophobicity is a disadvantage is a water filter. However, when increasing the hydrophilicity of an antimicrobial material, it is also of great importance not to impair the antimicrobial activity of the material.

Summary of invention An object of the invention is to provide an antim- icrobial substrate having adhered to at least a part of its surface an organosilicon quaternary ammonium salt compound, said substrate having an improved adhesion of microorganisms, preferably an increased surface charge density and thus the ability to more strongly adsorb mi- croorganisms, or even the ability to adsorb more microor- ganisms per surface area of the substrate.

It is also preferred that said surface charge den- sity is preserved during washing of the substrate.

Another object of the invention is to provide an an- timicrobial substrate having adhered to at least a part of its surface an organosilicon quaternary ammonium salt compound, said substrate having an increased hydrophilic- ity but preserved antimicrobial activity.

A substrate having a surface modified according to the present invention may be used in several different applications.

According to a first aspect of the invention, there is provided an antimicrobial substrate having adhered to at least a part of its surface an organosilicon quater- nary ammonium salt compound and a cationic polymer.

Said cationic polymer is preferably hydrophilic.

The cationic polymer is preferably a polyethylene imine or polyhexamethylene biguanide hydrochloride (PHMB). Both these polymers comprise-NH-in the poly- meric backbone.

Said organosilicon quaternary ammonium salt compound is preferably 3- (trimethoxysilyl) propyl-dimethyloctadecyl ammonium chloride.

According to a second aspect of the invention, there is provided a method for producing an antimicrobial sub- strate, as described above, comprising adhering an or- ganosilicon quaternary ammonium salt compound to at least a part of the substrate surface, and adhering a cationic polymer to at least a part of the substrate surface.

Said cationic polymer is preferably hydrophilic.

The cationic polymer is preferably a polyethylene imine or polyhexamethylene biguanide hydrochloride (PHMB).

Said organosilicon quaternary ammonium salt compound is preferably 3- (trimethoxysilyl) propyl-dimethyloctadecyl ammonium chloride.

According to a third aspect of the invention, there is provided a composition for use in the production of an antimicrobial substrate, as described above, said compo- sition comprising an organosilicon quaternary ammonium salt compound and a cationic polymer.

Other features and advantages of the present inven- tion will become apparent from the following description of the invention.

Detailed description of the invention The invention relates to an antimicrobial substrate having adhered to at least a part of its surface an or- ganosilicon quaternary ammonium salt compound and a cati- onic polymer, preferably hydrophilic.

Most microbial cells are negatively charged and hy- drophobic. Thus, most microorganisms are attracted to positively charged, hydrophobic surfaces. Without being bound by any theory, it is believed that the cationic polymer adhered to the substrate surface according to the invention provides an increased (compared to a substrate surface having a quaternized organosilane but no cationic polymer adhered thereto) positive surface charge density on the substrate surface. The increased positive surface charge density in turn provides an increased electric field strength in a specific medium, such as air.

The increased positive surface charge density re- sults in an increased microorganism adhesion, thus pre- venting leakage of alive microorganisms from the sub- strate surface. In some applications, even more microor- ganisms per surface area may be adhered to the substrate.

The cationic polymer adhered to at least a part of the substrate surface is preferably hydrophilic, thus providing an increased (compared to a substrate surface having a quaternized organosilane but no cationic, hydro- philic polymer adhered thereto) hydrophilicity but pre- served antimicrobial activity.

Examples of cationic polymers for use according to the invention are polyethylene imines, polyhexamethylene biguanide hydrochloride (PHMB), cationic starch, polyDAD- MAC (polydimethyldiallyl ammonium chloride), polyalumin- ium chloride, cationic polyamides, cationic polyamines, such as polyamine-epichlorohydrin resins, and cationic derivatives of polyacrylamides. Other cationic polymers, which may be used according to the invention, are also known to persons skilled in the art.

It shall be noted that PHMB itself has been classi- fied as an antibacterial compound.

PHMB comprises an average of 12 biguanides per mole- cule.

Polymers with a small number of repeating units, such as 2-20 units, are often referred to as oligomers.

Thus, PHMB may be referred to as an oligomer or a short polymer.

The cationic polymer may be a branched or linear polymer, but is preferably a branched polymer. A branched polymer may be physically and/or mechanically attached to the substrate surface by entanglement of its polymer chain in the organosilane network on the substrate sur- face, formed as described in the Technical Background.

It shall be noted that the cationic polymer also may be chemically attached, such as by covalent bonds, to the substrate surface.

The cationic polymer is preferably polyethylene imine or PHMB. Both these polymers comprise-NH-in the polymeric backbone.

Said polyethylene imine is preferably a branched polyethylene imine.

Moreover, said polyethylene imine preferably has an average molecular weight within the range of 800 to 750 000.

Suitable antimicrobial organosilicon quaternary am- monium salt compounds for use according to the invention are represented by Formula II: Formula II wherein R1 is an C1-30 alkyl group, preferably C8-30 alkyl group, R2 and R3, R4 and R5 each independently are an Cl-30 alkyl group or hydrogen, and X is a counter ion, such as Cl@, Br-, I-or CH3COO-.

Examples of organosilicon quaternary ammonium salt compounds for use according to the invention are 3- (triethoxysilyl) -propyl-dimethyloctadecyl ammonium chlo- ride, 3- (tri-methoxysilyl) propyl-methyl-dioctyl ammonium chloride, 3- (trimethoxysilyl) propyl-dimethyldecyl ammo- nium chloride, 3- (trimethoxysilyl)-propyl-methyldidecyl ammonium chloride, 3- (trimethoxy-silyl) propyl- dimethyldodecyl ammonium chloride, 3- (tri-methoxysilyl)- propyl-methyldidodecyl ammonium chloride, 3- (trimethoxy- silyl) propyl-dimethyltetradecyl ammonium chloride, 3- (trimethoxy-silyl) propyl-methyldihexadecyl ammonium chlo- ride, and 3- (trimethoxysilyl) propyl-dimethyloctadecyl am- monium chloride.

More preferably, the organosilicon quaternary ammo- nium salt compound is a 3- (trimethoxysilyl) propyl- dimethyloctadecyl ammonium halide, most preferably 3- (trimethoxy-silyl) propyl-dimethyloctadecyl ammonium chlo- ride (Rl=-Cl8H37, R2 =-CHs, R3 =-CH3, R4 =-C3H6-, R5 = - CH3, and X = Cl).

The substrate according to the invention may com- prise 3-8% (w/w) TMS, on dry weight basis.

An exemplary embodiment of the substrate according to the invention comprises TMS and a polyethylene imine in a weight ratio of 1: 1.

Another exemplary embodiment of the substrate ac- cording to the invention comprises TMS and a polyethylene imine in a weight ratio of 40: 1.

Thus, the ratio between TMS and PEI may be within the range of from 1: 1 to 40: 1, but the desired effect ac- cording to the invention may also be obtained outside this range.

Still another exemplary embodiment of the substrate according to the invention comprises TMS and PHMB in a weight ratio of 1: 1.

The substrate according to the invention adsorbs, and/or absorbs, and retains microbes, such as bacteria, fungi and/or virus.

The substrate according to the invention can thus be used for removing microorganisms from surfaces, such as biological surfaces, including skin and wounds, construc- tion surfaces, including building surfaces, furniture surfaces and automative surfaces, air and water.

Examples of microbes being adsorbed/absorbed and re- tained by the substrate according to the invention are Staphylococcus strains, such as Staphylococcus aureus, Streptococcus strains, Fusarium strains, Salmonela strains, Shigella strains, Yersinia strains, Eschera coli, Bacillus cereus, calivirus, Norwalk virus and simi- lar virus, Campylobacteria, Clostridium botulinum, C. perfringes, Listeria monocytogenes, Penicillium, and As- pergillus.

The surface of the substrate (or the entire sub- strate), according to the invention, to which the or- ganosilicon quaternary ammonium salt compound and the cationic polymer are adhered may be of wood ; metal; glass; leather ; plastics, such as polyethylene and poly-

propylene; rubber; ceramics ; paper; non-woven or woven fabrics of inorganic or organic fibers, such as naturally occurring fibers, polymeric fibers, composite fibers, etc. For instance, fibers of cellulose, cotton, wool, glass, stone, calcium sulphate, carbon, polyamides, poly- olefins, and polyesters may be used for preparing non- woven or woven fabrics.

For material surfaces lacking hydroxyl functionality (i. e. lacking-OH groups), such as polyethylene and poly- propylene, it may be advantageous to hydrolyze the sur- face before applying the organosilicon quaternary ammo- nium compound. Hydrolyzing may, for instance, be per- formed by contacting the surface with an aqueous acid, such as sulphuric acid, an alkaline hydroxide, such as sodium hydroxide or hydrogen peroxide.

Examples of applications (substrate) include sani- tary equipment, such as a surface cleansing cloth for hard or semi-hard surfaces (e. g. furniture, walls, floors, etc) or a mop textile ; water and air filters, such as for use in breathing masks and in venting systems in prenatal incubators, buildings or vehicles; liquid ab- sorbing material in food packages ; clothing, including training clothes, such as intimate apparel, stockings and socks; protective clothing, including different working clothes, such as cooking, laboratory and medical/surgical clothes; shoes, including shoe soles; sanitary articles, such as sanitary napkins, panty liners, diapers, and in- continence guards; refreshers/wet wipes ; napkins; hand- kerchiefs; paper and textile towels ; wound com- presses/cloths (for instance, for treatment of eczema and burn injuries) ; adhesive dressings; plaster ; medi- cal/surgical cloths/clothing, gloves, face masks and cov- erings, including pre-surgery coverings and paper and plastic film coverings for medical examination tables ; plastic film for use in a laboratory; agricultural plas- tic film for storage of hay; coverings in general ; arti- cles of beddings, such as sheets, quilts, blankets, quilt

covers, mattress covers, pillows, and pillow cases; fi- brous, plastic and rubber gloves, including disposable gloves, such as latex and PVC ; containers, wrappings, and bags/sacks, including food packaging (e. g. plastic film) and storage of other objects, such as sand and dirty washing.

An embodiment of the substrate according to the in- vention is a substrate, such as a refresher, in the form of a layered structure comprising a first layer having adhered to at least part of its surface an organosilicon quaternary ammonium salt compound and a cationic, pref- erably hydrophilic, polymer, and a second layer having a surface comprising one or more skin treating agent.

The surface of the second layer is preferably ar- ranged on a side of the substrate being opposite to the side of the substrate on which the surface of the first layer is arranged.

Examples of skin treating agents are wound-cleansing preparations, such as a saline solution ; wound-healing preparations, such as an ointment, a viscous liquid or gel, possibly containing nourishing substances and/or growth factors ; and other skin lotions, creams and oint- ments, such as a moisture lotion, a sunscreen lotion, or a suntan lotion.

The invention also relates to a method for producing the above described antimicrobial substrate, said method comprising adhering an organosilicon quaternary ammonium salt compound to at least a part of the substrate sur- face, and adhering a cationic, preferably hydrophilic, polymer to at least a part of the substrate surface.

The organosilicon quaternary ammonium salt compound may be applied before, after or simultaneously with the cationic polymer.

The organosilicon quaternary ammonium salt compound and the cationic polymer may be comprised in the same aqueous composition, and thus applied on the substrate surface in a single step.

Preferably, the organosilicon quaternary ammonium salt compound and the cationic polymer are applied simul- taneously on the substrate surface using a single aqueous composition comprising both compounds.

Hence, the invention also relates to a composition, preferably an aqueous composition, comprising an or- ganosilicon quaternary ammonium salt compound and a cati- onic polymer.

The organosilicon quaternary ammonium salt compound, is preferably a compound according to Formula II and may be any one of the compounds exemplified above.

The organosilicon quaternary ammonium salt compound in the composition according to the invention is prefera- bly TMS.

The cationic polymer in the composition according to the invention may be any one of the polymers exemplified above, but is preferably polyethylene imine or PHMB. Both these polymers comprise-NH-in the polymeric backbone..

Said polyethylene imine is preferably a branched polyethylene imine.

Moreover, said polyethylene imine preferably has an average molecular weight within the range of 800 to 750 000.

An exemplary embodiment of the composition according to the invention comprises TMS and a polyethylene imine in a weight ratio of 1: 1.

Another exemplary embodiment of the composition ac- cording invention comprises TMS and PHMB in a weight ra- tio of 1: 1.

Another exemplary embodiment of the composition ac- cording to the invention comprises TMS and a polyethylene imine in a weight ratio of 40: 1.

Thus, the ratio between TMS and PEI in the composi- tion according to the invention may be within the range of from 1: 1 to 40: 1, but the desired effect according to the invention may also be obtained outside this range.

The invention will now be illustrated by means of the following non-limiting examples.

Example 1: Production of modified cloths 0.5 1 of an aqueous solution of 3- (trimethoxy- silyl) propyl-dimethyloctadecyl ammonium chloride (TMS), 0. 5% by weight, and a branched polyethylene imine (PEI) (CAS No 25987-06-8), 0.5% by weight was prepared by add- ing TMS and PEI to water and stirring for 15 minutes at room temperature.

An alkali washed cloth of polyester (80%) and poly- amide (20%), 10 g, was immersed during stirring for 30 min in the TMS: PEI solution, squeezed and heated at 150°C for 15 minutes.

Example 2: Evaluation of bacteria adsorption Alkali washed cloths of polyester (80%) and polyam- ide (20%) were treated according to the procedure de- scribed in Example 1 using polyethylene imines of differ- ent molecular weights (800,2 000,25 000,50 000, and 750 000). The polyethylene imine of molecular weight 50 000 was ethoxylated.

In addition, a similar cloth was treated according to the procedure described in Example 1 except that poly- ethylene imine was replaced by polyhexamethylene bigua- nide hydrochloride (PHMB).

An untreated similar cloth, a similar cloth treated with an aqueous solution containing merely TMS (0. 5%), and a similar cloth treated with an aqueous solution con- taining merely polyethylene imine having a molecular weight of 800 (0. 5%) were used as comparative samples.

Bacteria adsorption to the above cloths (except the cloth treated with merely polyethylene imine) were evalu- ated using strains of Staphylococcus aureus (Gram posi- tive) and Escherichia coli (Gram negative). Each bacteria strain were suspended in a saline solution (0.9% NaCl) and the optical density (OD) of the suspensions at 420 nm

were measured. The suspensions were found to contain about 2.3 x 109 S. aureus/ml and about 1.1 x 10'E. coli/ml, respectively.

A small piece (1 g) of each cloth was incubated in 30 ml of each bacteria suspension for 30 minutes, where- upon the cloths were removed from the suspensions and the optical density of each suspension measured. The results are presented in Table 1 and Table 2.

Table 1 Escherichia coli Treatment Initial OD OD after 30 A minutes TMS + PEI 0.184 0.029 0.155 (800) TMS + PEI 0.179 0.058 0.121 (2 000) TMS + PEI 0.178 0.033 0.145 (25 000) TMS + PEI 0.177 0.039 0. 138 (50 000) TMS + PEI 0.184 0.020 0.164 (750 000) TMS + PHMB 0.183 0.051 0.132 Comparative samples untreated 0.178 0.176 0.002 TMS 0. 178 0.107 0.071 Table 2 Staphylococcus aureus Treatment Initial OD OD after 30 A minutes TMS + PEI 0.179 0.029 0.150 (800) TMS + PEI 0.178 0.037 0.141 (2 000) TMS + PEI 0.176 0.054 0.122 (25 000) TMS + PEI 0.177 0.037 0.140 (50 000) TMS + PEI 0.181 0.026 0.155 (750 000) TMS + PHMB 0.181 0.024 0.157 Comparative samples untreated 0.175 0.133 0.042 TMS 0. 178 0.048 0.130

As shown by the results presented in Table 1 and 2, more bacteria adhere to the cloths according to the in- vention than to the cloths used as comparative samples.

Example 3: Evaluation of wettability The relative wettability (~hydrophilicity) of the cloths used in Example 2 was estimated by applying a drop (100 p. l) of an aqueous solution of CUS04 (1 M) on each cloth and measuring the time period until the drop was absorbed by the cloth.

The applied water drop was instantaneously absorbed by the untreated cloth and the cloth treated with only PEI (800), respectively.

No absorption was observed for the cloth treated with only TMS.

For the cloths treated with TMS and PEI of different molecular weights, the absorption times were found to be between 10 and 80 minutes.

The cloth treated with TMS and ethoxylated PEI (50 000) showed the shortest absorption time, i. e. the high- est wettability.

The other cloths showed wettabilty according to the following (highest wettability to the left):

TMS+PEI (750,000) > TMS+PEI (25,000) > TMS+PEI (800) > TMS+PEI (2,000) Example 4: Determination of surface charge and wetting properties An alkali washed cloth of polyester (80%) and poly- amide (20%) was treated according to the procedure de- scribed in Example 1 using a polyethylene imine having a molecular weight of 750 000, with the exception that the concentration of TMS was 1% by weight and the concentra- tion of PEI was 250 ppm.

A similar cloth treated with an aqueous solution containing merely TMS (0. 5%) was used as comparative sam- ple.

Surface charge The surface charge on each cloth was measured using a static sensor, 3M Static Sensor, model 709. All cali- bration measurements complied with MIL-STD-45662A.

A mat, connected to ground, was placed on a table.

The sensor was kept approximately 10 mm above the mat and put to zero. Each cloth was then placed on the mat and the sensor was placed 10 mm above the cloth surface. The charge was measured at three different locations on each cloth and the measurements were repeated three times. The sensor was re-zeroed against the grounded mat between each set of measurement.

The results from the charge measurements are pre- sented in Table 3.

Table 3 Surface charge Surface charge (arbitrary units) average (arbitrary units) Cloth treated with TMS 1st location 780, 805, 820 802 2nd location 927, 825, 900 884 3rd location 100, 195, 175 157 Cloth treated with TMS and PET 1St location 1480, 1685, 1638 1601 2nd location 1870, 1930, 1821 1874 3rd location 1225 t 1269 r 1311 1268

As shown in Table 3, the cloth treated with both TMS and PEI has a significantly higher positive surface charge, a surface charge average of 1581, than the cloth treated with merely TMS, a surface charge average of 614.

It was also shown that the charge varies over the cloth surface. The surface charge was, for instance, found to be highest in location 2, i. e. in the centre of the cloth.

Moreover, the surface charge on the cloth treated with merely TMS appears to be more heterogeneous than the cloth treated with both TMS and PEI.

Wetting properties The wetting properties of the cloths were determined by measurements in a Dynamic Absorption Tester (DAT), manufactured by Fibro Systems AB. The spreading and pene- tration of liquid droplets, in this example water, can be followed with a time resolution of about 20 ms. A droplet of the liquid is pumped from a syringe attached to a screw pump and the drop is automatically applied to the surface by a short stroke of an electromagnet. A CCD cam- era connected to a PC follows the spreading and absorp- tion of the droplet. During the first second, 50 images are captured and stored for later analysis. After the first second, the images are analysed on-line and less images are then captured, 5-10 images/second.

The images were analysed with respect to contact an- gle.

The results from the water contact angle measure- ments (average values of 3-5 measurements) are presented in Table 4.

Table 4 Water contact angle (degrees) Cloth treated with TMS 105 Cloth treated with TMS and PEI 95

The results show that the cloth treated with merely TMS are more hydrophobic than the cloth treated with both TMS and PEI.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent for one skilled in the art that various changes and modifications can be made therein without de- parting from the spirit and scope thereof.