WO2012049250A2 - Procédé pour immobiliser des principes actifs cationiques sur des surfaces - Google Patents

Procédé pour immobiliser des principes actifs cationiques sur des surfaces Download PDF

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WO2012049250A2
WO2012049250A2 PCT/EP2011/067893 EP2011067893W WO2012049250A2 WO 2012049250 A2 WO2012049250 A2 WO 2012049250A2 EP 2011067893 W EP2011067893 W EP 2011067893W WO 2012049250 A2 WO2012049250 A2 WO 2012049250A2
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Prior art keywords
hydrophobin
antimicrobial agent
weight
antimicrobial
cationic
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PCT/EP2011/067893
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German (de)
English (en)
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WO2012049250A3 (fr
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Marcus Fehr
Ulf Baus
Thomas Subkowski
Catharina Hippius
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Basf Se
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Publication of WO2012049250A3 publication Critical patent/WO2012049250A3/fr

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N47/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid
    • A01N47/40—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides
    • A01N47/42—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom not being member of a ring and having no bond to a carbon or hydrogen atom, e.g. derivatives of carbonic acid the carbon atom having a double or triple bond to nitrogen, e.g. cyanates, cyanamides containing —N=CX2 groups, e.g. isothiourea
    • A01N47/44—Guanidine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/08—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing solids as carriers or diluents
    • A01N25/10—Macromolecular compounds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/24—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing ingredients to enhance the sticking of the active ingredients
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D5/00—Coating compositions, e.g. paints, varnishes or lacquers, characterised by their physical nature or the effects produced; Filling pastes
    • C09D5/14—Paints containing biocides, e.g. fungicides, insecticides or pesticides

Definitions

  • One goal in the application of antimicrobial agents is to immobilize one or more drugs that reduce the proliferation and / or infectivity of microorganisms on the surface of substrates such that the agents are not uncontrolled during use washed down the surface and thus the protection is lost. Since different surfaces are treated very differently, for example, washed or exposed to the weather, various immobilization techniques are used. These methods often also cause inactivation of the antimicrobial agents or they do not firmly bond the antimicrobial agents to the surfaces such that the contaminants are washed out too quickly and the surfaces lose their antimicrobial properties.
  • An object of the present invention is to develop the simplest possible method for immobilizing one or more antimicrobial active substances that can be used for different active substances and different surfaces.
  • the method using the cysteine-rich protein hydrophobin should also not lead to inactivation of the antimicrobial effect.
  • cationic antimicrobial agents eg the antiseptic polyhexamethylene biguanide
  • a method for immobilization on surfaces is therefore of particular interest for the class of cationic antimicrobial agents.
  • the adsorption of the antimicrobial agent polyhexamethylene biguanide on cellulose substrates was described. The active substance is applied to hydrophilic, anionic surfaces (see R.
  • antimicrobial peptides presuppose model surfaces having functional groups or introducing functional groups on inert polymer surfaces (such as silicone and PVC) (see S. Haynie et al., Antimicrobial Agents and Chemotherapy, 02-1995, 301-307; Humblot et al., Biomaterials 30, 2009, 3503-3512).
  • the functional groups or the antimicrobial active substances to be immobilized have to be activated in order to form covalent bonds.
  • introducing functional groups and enabling them are additional steps that involve both technical effort and cost.
  • covalent immobilization significantly reduces the effects of antimicrobial agents such that the so-treated surfaces are not able to effectively prevent bacterial colonization (see V. Humblot 2009, Bagheri et al., Antimicrobial Agents and Chemotherapy , 03-2009, 1 132-1141).
  • a technical alternative to the covalent immobilization of cationic drugs is the adsorption by means of polyelectrolyte layers.
  • the cationic active substance can itself be a polyelectrolyte (see US2007 / 0243237) or the cationic active substance can be embedded in a polyelectrolyte layer (see US2009 / 0258045).
  • US2007 / 0243237 describes a process for applying an anti-microbial coating in which a negatively charged polyelectrolyte component and a positively charged polyelectrolyte component are applied as a film to a substrate, wherein at least one the components have anti-microbial activity.
  • a known biocidal component can be covalently bound to a charged polymer.
  • a coated structure is described, which is coated on the one hand with a charged, anti-microbial peptide and on the other hand with a poly-electrolyte component.
  • Hydrophobins are known as small, cysteine-rich proteins, which, for. B. occur in filamentous fungi such as Schizophyllum commune. Naturally occurring hydrophobins often have about 100 to 150 amino acids. They usually have eight cysteine units in the molecule. Hydrophobins can be isolated from natural sources, but they can also be obtained by genetic engineering, as disclosed, for example, in WO 2006/082251 or WO 2006/131564.
  • hydrophobins as emulsifiers, thickeners, surface-active substances, for hydrophilicizing hydrophobic surfaces, for improving the water resistance of hydrophilic substrates, for producing oil-in-water emulsions or for water-in-oil emulsions.
  • pharmaceutical applications such as the production of ointments or creams and cosmetic applications such as skin protection or the production of hair shampoos or hair rinses are proposed.
  • WO 2006/082253 discloses formulations for coating surfaces, eg. B. of finely divided inorganic or organic particles, with hydrophobins. For this purpose, the aqueous hydrophobin solutions are applied to the surface to be coated.
  • WO 2006/103215 discloses the use of hydrophobins for stain-resistant treatment of hard surfaces, such as floors.
  • WO 2006/103230 discloses the use of aqueous formulations of hydrophobins for the surface treatment of hardened mineral building materials.
  • WO 2004/000880 describes the noncovalent binding of antibodies, enzymes, peptides, lipids, nucleic acids and carbohydrates to hydrophobin-treated surfaces.
  • US 7393448 describes the non-covalent inclusion of substances, themselves smaller than the protein hydrophobin, in a hydrophobin coating on a sensor surface.
  • hydrophobins in the context of the present invention is to be understood below as meaning, in particular, polypeptides of the general structural formula (I)
  • each X is an amino acid sequence consisting of amino acids independently selected from the 20 naturally occurring amino acids (Phe, Leu, Ser, Tyr, Cys, Trp, Pro, His, Gin, Arg, Met, Thr, Asn, Lys, Val, Ala, Asp, Glu, Gly).
  • the radicals X may be the same or different.
  • the numerical indices standing at X each represent the number of amino acids in the respective amino acid sequence (partial sequence) X, and each amino acid residue in each X may independently be the same or different from the adjacent residues.
  • C represents cysteine, alanine, serine, glycine, methionine or threonine, at least four of the radicals named C being cysteine, and the indices n and m independently of one another represent natural numbers between 0 and 500, preferably between 15 and 300 and indicate the number of amino acid residues containing in the respective amino acid sequence X.
  • the polypeptides according to the formula (I) are further characterized by the property that at room temperature after coating a glass surface, they increase the contact angle of a water droplet of at least 20 °, preferably at least 25 ° and particularly preferably 30 °, in each case compared with the contact angle of a water droplet of the same size with the uncoated glass surface.
  • the amino acids designated C 1 to C 8 are preferably cysteines. However, they can also be replaced by other amino acids of similar space filling, preferably by alanine, serine, threonine, methionine or glycine. However, at least four, preferably at least 5, more preferably at least 6 and in particular at least 7, of the positions C 1 to C 8 should consist of cysteines.
  • Cysteines can either be reduced in the proteins according to the invention or form disulfide bridges with one another. Particularly preferred is the intramolecular formation of CC bridges, in particular those having at least one, preferably 2, more preferably 3 and most preferably 4 intramolecular disulfide bridges.
  • cysteines, serines, alanines, glycines, methionines or threonines are also used in the positions indicated by X, the numbering of the individual C positions in the general formulas may change accordingly.
  • the radicals X n and X m may be peptide sequences that are naturally also linked to a hydrophobin.
  • residues may be peptide sequences that are not naturally linked to a hydrophobin.
  • Including such radicals X N and / or X m are to be understood, in which a naturally occurring in a hydrophobin peptide sequence is extended by a non-naturally occurring in a hydrophobin peptide sequence.
  • X n and / or X m are naturally non-hydrophobin-linked peptide sequences, such sequences are generally at least 20, preferably at least 35 amino acids long. These may be, for example, sequences from 20 to 500, preferably 30 to 400 and particularly preferably 35 to 100 Act amino acids. Such a residue, which is not naturally linked to a hydrophobin, will also be referred to below as a fusion partner.
  • the proteins may consist of at least one hydrophobin part and one fusion partner part which in nature do not coexist in this form.
  • Fusion hydrophobins from fusion partner and hydrophobin part are described, for example, in WO 2006/082251, WO 2006/082253 and WO 2006/131564.
  • the fusion partner portion can be selected from a variety of proteins. Only a single fusion partner can be linked to the hydrophobin moiety, or several fusion partners can also be linked to a hydrophobin moiety, for example at the amino terminus (X n ) and at the carboxy terminus (X m ) of the hydrophobin moiety. However, it is also possible, for example, to link two fusion partners with a position (X n or X m ) of the protein according to the invention.
  • fusion partners are proteins which occur naturally in microorganisms, in particular in Escherischia coli or Bacillus subtilis.
  • fusion partners are the sequences yaad (SEQ ID NO: 16 in WO 2006/082251), yaae (SEQ ID NO: 18 in WO 2006/082251), ubiquitin and thioredoxin.
  • fragments or derivatives of said sequences which comprise only a part, for example 70 to 99%, preferably 5 to 50%, and particularly preferably 10 to 40% of said sequences, or in which individual amino acids or nucleotides are opposite the said sequence are changed, wherein the percentages in each case refers to the number of amino acids.
  • the assignment of the sequence names to the DNA and polypeptide sequence and the corresponding sequence protocols can be found in the application WO 2006/103225 (page 13 of the description and sequence listing) and in the present application.
  • the fusion hydrophobin in addition to the fusion partner mentioned as one of the groups X n or X m or as a terminal component of such a group on a so-called affinity domain (affinity tag / affinity tail) on.
  • affinity domains include (His) k , (Arg) k , (Asp) k , (Phe) k , or (Cys) k groups, where k is generally a natural number from 1 to 10. It may preferably be a (His) k group, where k is 4 to 6.
  • the group X n and / or m X may consist exclusively of such an affinity domain or a naturally or non-naturally to a hydrophobin comparable knüpfter radical X n and X m is extended by a terminal affinity domain.
  • the hydrophobins used in the invention can also in their Polypeptide be modified, for example by glycosylation, acetylation or by chemical crosslinking, for example with glutaraldehyde.
  • One property of the hydrophobins or their derivatives used according to the invention is the change of surface properties when the surfaces are coated with the proteins.
  • the change in the surface properties can be determined experimentally, for example, by measuring the contact angle of a water drop before and after coating the surface with the specific protein and determining the difference between the two measurements.
  • the implementation of contact angle measurements is known in principle to the person skilled in the art. The measurements refer to room temperature and water drops of 5 ⁇ and the use of glass slides as substrate. The exact experimental conditions for an exemplary method for measuring the contact angle are shown in the experimental part.
  • the fusion proteins used according to the invention have the property of increasing the contact angle by at least 20 °, preferably at least 25 °, particularly preferably at least 30 °, in each case compared with the contact angle of a water droplet of the same size with the uncoated glass surface.
  • Particularly preferred hydrophobins for practicing the present invention are the dewA, rodA, hypA, hypB, sc3, basfl, basf2 hydrophobins. These hydrophobins including their sequences are disclosed, for example, in WO 2006/082251 and in the sequence listing below. Unless stated otherwise, the sequences given below refer to the sequences disclosed in WO 2006/082251.
  • the fusion proteins yaad-Xa-dewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf1-his (SEQ ID NO: 24) with the polypeptide sequences given in parentheses and the nucleic acid sequences coding for them (SEQ ID NO 19, SEQ ID NO 21, SEQ ID NO 23), in particular Sequences according to SEQ ID NO: 19, 21, 23.
  • preference is given to using the hydrophobin yaad-Xa-dewA-his (SEQ ID NO: 19 / SEQ ID NO: 20).
  • proteins which, starting from the amino acid sequences shown in SEQ ID NO. 20, 22 or 24 shown polypeptide sequences by exchange, insertion or deletion of at least one, up to 10, preferably e, more preferably 5% of all amino acids, and still possess the biological property of the source proteins to at least 50% particularly preferred embodiments.
  • the biological property of the proteins is hereby understood as the change in the contact angle already described by at least 20 °.
  • Particularly suitable derivatives for carrying out the present invention are from yaad-Xa-dewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-Xa-basf1-his (SEQ ID NO: 24) derivatives derived from truncation of the yaad fusion partner.
  • yaad-Xa-dewA-his SEQ ID NO: 20
  • yaad-Xa-rodA-his SEQ ID NO: 22
  • yaad-Xa-basf1-his SEQ ID NO: 24
  • the truncated residue should comprise at least 20, preferably at least 35, amino acids.
  • a truncated radical having 20 to 293, preferably 25 to 250, more preferably 35 to 150 and for example 35 to 100 amino acids can be used.
  • a cleavage site between the hydrophobin and the fusion partner or the fusion partners can be used to cleave off the fusion partner and release the pure hydrophobin in underivatized form (for example, by BrCN cleavage of methionine, factor Xa, enterokinase, thrombin, TEV cleavage Etc.).
  • the protein yaad40-Xa-dewA-his (SEQ ID NO: 26 in WO 2007/014897) is preferably used which has a yaad radical which is shortened to 40 amino acids.
  • the hydrophobins used in the process according to the invention for the purification of hydrophobic surfaces can be prepared chemically by known methods of peptide synthesis, such as, for example, Merrifield solid-phase synthesis.
  • Naturally occurring hydrophobins can be isolated from natural sources by suitable methods. As an example, let Wösten et. al., Eur. J. Cell. Bio. 63, 122-129 (1994) or WO 1996/41882.
  • a genetic engineering preparation for hydrophobins without fusion partner from Talaromyces thermophilus is described in US 2006/0040349.
  • the production of fusion proteins can preferably be carried out by genetic engineering methods in which a nucleic acid sequence coding for the fusion partner and a hydrophobin part, in particular DNA sequence, are combined in such a way that the desired protein is produced in a host organism by gene expression of the combined nucleic acid sequence.
  • a production method for example, is disclosed by WO 2006/082251 or WO 2006/082253.
  • the fusion partners greatly facilitate the production of hydrophobins. Fusion hydrophobins are produced in genetically engineered processes with significantly better yields than hydrophobins without fusion partners.
  • the fusion hydrophobins produced by the Wrtsorganismen according to the genetic engineering process can be worked up in a manner known in principle and purified by means of known chromatographic methods.
  • the simplified processing and purification process disclosed in WO 2006/082253, pages 1 1/12 can be used.
  • the fermented Cells are first separated from the Fermetationsbrühe, digested and the cell debris of the inclusion bodies (inclusion bodies) separately.
  • inclusion bodies for example by acids, bases and / or detergents can be digested in a manner known in principle in order to liberate the fusion hydrophobins.
  • the inclusion bodies with the fusion hydrophobins used according to the invention can generally be completely dissolved within about 1 h already using 0.11M NaOH.
  • the solutions obtained can - if necessary after setting the desired pH - are used without further purification for carrying out this invention.
  • the fusion hydrophobins can also be isolated from the solutions as a solid.
  • the isolation can preferably be effected by means of spray granulation or spray drying, as described in WO 2006/082253, page 12.
  • the products obtained by the simplified work-up and purification process comprise, in addition to residues of cell debris, usually about 80 to 90% by weight of proteins.
  • the amount of fusion hydrophobins is generally from 30 to 80% by weight with respect to the amount of all proteins.
  • the isolated products containing fusion-hydrophobins can be stored as solids and dissolved for use in the respective desired media.
  • the fusion hydrophobins can be used as "pure" hydrophobins for carrying out this invention, and cleavage is advantageously carried out after isolation of the inclusion bodies and their dissolution in the hydrophobin used, at least one fusion hydrophobin having a polypeptide sequence selected from the group of yaad-Xa-dewA-his (SEQ ID NO: 20), yaad-Xa-rodA-his (SEQ ID NO: 22) or yaad-xa-basf1-his (SEQ ID NO: 24) and yaad40-Xa-dewA-his (SEQ ID NO: 26 in WO 2007/014897).
  • a fusion hydrophobin with a truncated fusion partner such as Protein yaad40-Xa-dewA-his (SEQ ID NO: 26 in WO 2007/014897) which has a yaad residue truncated to 40 amino acids
  • a fusion hydrophobin with a truncated fusion partner such as Protein yaad40-Xa-dewA-his (SEQ ID NO: 26 in WO 2007/014897) which has a yaad residue truncated to 40 amino acids
  • Cationic antimicrobial agents have also long been used in disinfectants.
  • the present invention relates to a process for the immobilization of one or more antimicrobial agents on the surface of a substrate comprising the surface treatment with at least one hydrophobin (H) and at least one cationic antimicrobial agent (W).
  • H hydrophobin
  • W cationic antimicrobial agent
  • the present invention relates to a method for immobilizing one or more antimicrobial active substances on the surface of a substrate in which a cationic antimicrobial agent (W) is a higher molecular weight, cationic compound, for example a poly-cationic substance.
  • W cationic antimicrobial agent
  • This can be z. B. may be a compound, e.g. contains more than 3, in particular more than 5 and often more than 10 cationic groups.
  • the present invention also relates to a method for immobilizing an antimicrobial agent on the surface of a substrate in which a low molecular weight, cationic compound is used as the cationic antimicrobial agent (W).
  • the present invention also relates to a method for immobilizing an antimicrobial agent on the surface of a substrate in which a higher molecular weight quaternary ammonium compound, a higher molecular weight polyiminocarbonyl compound or a higher molecular weight polyethyleneimine-peptide conjugate is used as the cationic antimicrobial agent (W) becomes.
  • the present invention also relates to a method for immobilizing antimicrobial agents on the surface of a substrate in which a higher molecular weight cationic compound and a low molecular weight cationic compound are used together.
  • the present invention further relates to a method for immobilizing an antimicrobial agent on the surface of a substrate in which a fusion protein is used as the hydrophobin (H).
  • the present invention further relates to a method of immobilization as described above comprising treating the surface with at least one composition comprising at least one hydrophobin (H) and / or at least one cationic antimicrobial agent (W).
  • at least one composition comprising at least one hydrophobin (H) and / or at least one cationic antimicrobial agent (W).
  • the present invention relates, on the one hand, to a method for immobilizing an antimicrobial agent on the surface of a substrate, the method comprising the following steps: a) wetting the surface of the substrate with a composition (Z1) comprising the following components: (i) at least one solvent (L1),
  • solvent (L1) contains at least 60% by weight of water
  • the present invention relates to a method for immobilizing an antimicrobial active substance on the surface of a substrate, wherein the method comprises, as a step, wetting the surface of the substrate with a composition (Z3) comprising the following components:
  • the solvent contains at least 60% by weight of water
  • the present invention also relates to a method for immobilizing an antimicrobial active substance on the surface of a substrate, in which the concentration of the hydrophobin component (H) in the composition (in particular in one of the compositions (Z1) and / or (Z3)) 0, 05 to 5,000 ppm.
  • the present invention further relates to a method of immobilizing an antimicrobial active substance on the surface of a substrate, wherein the concentration of the cationic antimicrobial agent (W) in the composition (in particular in the compositions (Z2) and / or (Z3)) is 0.05 Wt .-% to 20 wt .-% is.
  • the concentration of the cationic antimicrobial agent (W) in the composition depends, inter alia, on the type of active substance (or combination of active substances) and the type of surface to be treated. Frequently, from 0.05% to 10% by weight of the cationic antimicrobial active ingredient is sufficient
  • the present invention also relates to a method for immobilizing an antimicrobial active substance on the surface of a substrate, wherein the concentration the additive (A) in the composition (in particular in one of the compositions (Z1) and / or (Z3)) is 0.01% by weight to 3% by weight and the concentration of the auxiliary components (HK) in the composition is 0 , 01 wt .-% to 3 wt .-% is.
  • concentration of the auxiliary component (HK) in the composition depends, inter alia, on the type of active ingredient (or the combination of active ingredients), the nature of the auxiliary component and the nature of the surface to be treated.
  • the present invention further relates to a method for immobilizing an antimicrobial agent on the surface of a substrate, wherein the surface is a hydrophobic surface made of silicone or a polymeric or copolymeric plastic from the group polyethylene PE, polypropylene PP, polyvinyl chloride PVC, polyethylene terephthalate PET, polyurethane PUR, linoleum and rubber.
  • the invention also provides a composition for immobilizing an antimicrobial agent on the surface of a substrate, comprising (or consisting of) the following components:
  • auxiliary components optionally 0.01 to 3 wt .-% of one or more auxiliary components (HK). the sum of all components being just 100% by weight.
  • the invention also relates to a composition for immobilizing an antimicrobial active substance on the surface of a substrate, comprising (or consisting of) the following components: 80 to 99.95% by weight of solvent (L), where the solvent
  • auxiliary components optionally 0.01 to 3% by weight of one or more auxiliary components (HK), the sum of all components being just 100% by weight,
  • the invention further relates to the use of a composition comprising at least one hydrophobin (H) and at least one cationic antimicrobial agent (W) for immobilizing the antimicrobial active substance on the surface of the substrate.
  • a composition comprising at least one hydrophobin (H) and at least one cationic antimicrobial agent (W) for immobilizing the antimicrobial active substance on the surface of the substrate.
  • the use of a composition is also of interest, the composition containing a fusion hydrophobin (H) and at least one polycationic antimicrobial agent (W) for immobilizing the polycationic antimicrobial agent on the surface of the plastic substrate.
  • Typical examples of cationic antimicrobial active substances (W) in the context of the present invention are:
  • Cationic surfactants quaternary ammonium compounds, ampho-surfactants, alkylamines and amine derivatives, cationic polymers, antimicrobial peptides and proteins, peptido-mimetics and quaternary phosphonium compounds.
  • the cationic antimicrobial active substances (W) may be of low molecular weight (molecular weight less than 1000 g / mol) (such as, for example, benzalkonium chloride (284 g / mol) or cetyltrimethylammonium bromide (364 g / mol)).
  • Tallow alkyl, coco alkyl and soya alkyl chlorides, bromides or hydroxides
  • Cocoalkyl and soya alkyl chlorides, bromides or methyl sulphates
  • Alkyltrimethyl alkyl of C8-C18, saturated and unsaturated, and tallow alkyl, cocoalkyl and soya alkyl chlorides, bromides or methyl sulphates
  • Benzalkonium Chloride Alkyl-dimethyl-benzyl-ammonium chloride
  • Alkyl-dimethyl compounds alkylbenzyl-ammonium chloride
  • Cocos-dimethyl benzyl ammonium chloride Cocos-dimethyl benzyl ammonium chloride
  • the cationic antimicrobial agents (W) are often higher molecular weight (molecular weight greater than 1000 g / mol) or high molecular weight, which can improve the long-term immobilization on the surface.
  • cationic antimicrobial active substances are in particular quaternary ammonium compounds (so-called "quats"), which are described, for example, for the antibacterial finishing of textiles in the literature often cover a wide germ spectrum with excellent effects.
  • quats quaternary ammonium compounds
  • Karl Heinz Walljunusser Sterilization Disinfection - Preservation, 5th Edition, Georg Thieme Verlag Stuttgart, New York 1995, page 586 ff. This substance class is described in detail. It is known that quaternary ammonium compounds have a bactericidal effect, in particular, if at least one of the four substituents on the quaternary nitrogen has a chain length of 8 to 18 C atoms, preferably of 12 to 16 C atoms.
  • the other substituents may, for. B. straight or branched alkyl radicals or radicals with heteroatoms or radicals with aromatics th. Frequently one or more benzyl radicals are bonded to the quaternary nitrogen in the molecule. Also quaternary ammonium compounds having two methyl groups, an n-alkyl group having between 10 to 18 carbon atoms and a 3-trimethoxysilylpropyl group can be used.
  • quaternary ammonium compounds often have the property of being well soluble in water. This property is contrary to the aqueous application in the industrial finishing process. At the same time, however, this property leads to such compounds being washed off the substrates quickly, since adhesion to the surface is possible primarily by means of van der Waals forces and possibly ion pair bonds.
  • the precursors of quats namely tertiary amines, can also be quaternized with, for example, 3-chloropropyltrimethoxysilane. There are z. B.
  • the cationic antimicrobial active compounds (W) are preferably polycationic compounds which carry a plurality of (eg more than 5, often more than 10) positively charged groups.
  • the cationic antimicrobial agents (W) may, for. B. also be conjugates of polyethyleneimines with peptides.
  • the cationic antimicrobial agents (W) kill or prevent their multiplication by interacting with the negatively charged membrane and / or the negatively charged cell wall of the bacteria, disrupting vital processes (such as preserving the membrane gradient). For the interaction with the negatively charged cell components, the cationic charge of the antimicrobial agents is essential.
  • cationic antimicrobial active substances can also bind via electrostatic interactions and / or hydrogen bonds to hydrophilic, anionic surfaces such as cellulose or anionic polyelectrolytes.
  • hydrophilic, anionic surfaces such as cellulose or anionic polyelectrolytes.
  • the binding via electrostatic interaction causes inactivation of the antimicrobial effect, since the cationic charges of the antimicrobial agents are no longer available for interaction with the anionic cell components of the bacteria.
  • cationic antimicrobial active substances can also be adsorbed on hydrophobic polymer surfaces without polar, hydrophilic groups and without anionic charges in such a way that an antimicrobial effect remains even after intensive washing of the polymer surfaces. Such surfaces can z.
  • Example of silicone, linoleum, rubber or plastics in particular from the group polyethylene PE, polypropylene PP, polyvinyl chloride PVC, polyethylene terephthalate PET, polyurethane PUR exist.
  • the surfaces can be planar but also arbitrarily shaped, for example, surfaces of medical devices.
  • hydrophobin coating of surfaces despite the anionic charges of the hydrophobin is suitable for improving the binding of the cationic antimicrobial active substances on completely different surfaces without inactivating them.
  • the binding of the active substances with the help of hydrophobin leads thereby to an extension of the spectrum of action.
  • a synergistic effect on the antimicrobial properties could be observed by the combination of hydrophobin and cationic antimicrobial agent.
  • the binding of the cationic antimicrobial active substances (W) to the hydrophobin coating can be carried out as a simple adsorption via noncovalent interactions (a) or through a combination of noncovalent and covalent interactions (b).
  • the known hydrohobins bind to a variety of different surfaces. After binding of the hydrophobins, the surface properties can be dominated by the properties of the hydrophobins.
  • the hydrophobin properties generally correspond to the sum of the typical protein properties of the hydrophobins (i) and the properties of individual amino acids or groups of similar amino acids (ii) on the surface of the hydrophobins.
  • the typical protein properties of hydrophobins (i) also result from the three-dimensional arrangement of their amino acids. In addition to binding to any surfaces, the associated change in surface polarity can be of importance above all else.
  • a hydrophobin coating in particular hydrophobic surfaces of hydrophilic surfaces, but in principle also hydrophilic surfaces, can be made more hydrophobic.
  • the properties of the amino acids (ii) are determined essentially by their functional groups. Thus, different amino acids have different functional groups, e.g.
  • a hydrophobin coating typically also brings functional groups and charges to the surfaces. This is especially true for inert surfaces of e.g. Silicon, polypropylene, polyethylene, PVC, glass, ceramics, titanium oxide and metals and their alloys are of great benefit.
  • (a) cationic antimicrobial agents usually involves all types of noncovalent interactions (hydrophobic interactions, Van der Waals forces, hydrogen bonds, and ionic interactions).
  • the ionic interactions may be of particular interest.
  • the cationic antimicrobial active substances (W) can also be bound to the surfaces by the negative charges of the aspartic acid and glutamic acid residues of the hydrophobins without this resulting in inactivation.
  • the coating of surfaces by hydrophobins (H) and the binding of cationic antimicrobial agents can be carried out in one step (i) or in two steps (ii). Accordingly, the present invention comprises a method for immobilization with application of only an active substance-containing composition but also a method for immobilization with application of several active substance-containing compositions.
  • the first step is the coating of the surface by at least one hydrophobin (H).
  • the surface is e.g. treated with a composition (usually aqueous solution) of at least one hydrophobin (H).
  • the cationic antimicrobial active substance (W) z. B. applied with a composition (usually aqueous solution) and z. B. non-covalently adsorbed to the hydrophobin coating.
  • the surface is often brought into contact with a solution of at least one cationic antimicrobial active substance (which may also contain other components).
  • a composition (often an aqueous solution) of at least one hydrophobin (H) and the antimicrobial cationic active substances (W) is prepared and the surface to be treated is treated with this solution.
  • this solution may contain further substances which are necessary for the specific application and for the stabilization of the solution.
  • the treatment of the surfaces can be done by overlaying, dipping, spin-coating or spraying. The simple adsorption is particularly advantageous for polymeric and for applications in which a covalent bond is not possible, advantageous.
  • the simple adsorption of the cationic antimicrobial active substances (W) can be enhanced by covalent bonds (b). This may be advantageous, especially in the case of low molecular weight active substances.
  • the antimicrobial cationic active ingredients can be coupled to the hydrophobins. In the coupling, in particular, the functional groups of the hydrophobins (H) or the functional groups of the cationic antimicrobial agents are activated and subsequently reacted with the respective functional groups of the opposite side.
  • the coupling can be carried out on the hydrophobin coating on the surface (ii) or with the free hydrophobins (i).
  • the hydrophobin is first bound to the surface. Then the hydrophobin coating or the cationic antimicrobial active substance (W) are activated and subsequently both components are added together. Alternatively, the cationic, antimicrobial active substance (W) can initially be adsorbed only on the coating and the covalent bond subsequently be attached.
  • hydrophobin-drug conjugates are prepared in solution which can then be adsorbed to the surface. The synthesis of the hydrophobin-active agent conjugates also takes place by activation of the functional groups of the hydrophobins or of the cationic antimicrobial active ingredients.
  • the activation of the functional groups can be carried out by cross-linking substances.
  • the choice of cross-linker depends on the type of functional groups to be coupled. For the coupling of amines, for example:
  • amines for the coupling of amines to thiols are, for example: bifunctional N-hydroxy-succinimide haloacetyl crosslinkers,
  • Carbodiimides such as 1-ethyl-3- [3-dimethylaminopropyl] carbodiimides, hydrochlorides or ⁇ , ⁇ '-dicyclohexylcarbodiimides in combination with N-hydroxysuccinimides or N-hydroxysulfosuccinimides.
  • N-hydroxysuccinimides or N-hydroxysulfosuccinimides.
  • hetero-bifunctional N- [p-maleimidophenyl] isocyanates hydroxyl functions and thiols can be conjugated.
  • bifunctional crosslinkers with maleinimide or pyridyldithiol functionalities are suitable.
  • the invention is illustrated by the accompanying figures Fig.1 to Fig.8 and by the following examples.
  • the graph in Fig. 1 shows schematically the effect of the antimicrobial equipment on the colonization of the surface of a substrate by microorganisms.
  • On the left side (A) one can see schematically that the surface is heavily colonized by microorganisms without antimicrobial equipment (black dots).
  • the microorganisms form a biofilm that is firmly anchored to the surface.
  • On the right side (B) it can be seen that after antimicrobial treatment of the surface, the microorganisms that attempt to colonize the surface have been killed.
  • the number of microorganisms on the surface equipped with the composition according to the invention (cfu / cm A 2) is significantly lower than in the case of the untreated surface. Ideally, the antimicrobial finish will completely prevent colonization. In the illustrated schematic case, a few living microorganisms are still present on the treated surface.
  • the graph in Fig. 2 shows the anti-biofilm activity of silicone surfaces after adsorption of the drug polyhexamethylene biguanide (Reputex 20).
  • Fig. 2A shows the number of germs (cfu / cm 2 ) of Staphylococcus epidermidis (DSM 1798); Fig. 2B shows E. coli; Fig. 2C shows C P. mirabilis. Shown without washing, with 1x washing and 3x washing, and 1 hour and 24 hours in PBS (phosphate based saline solution).
  • DSM 1798 Staphylococcus epidermidis
  • Fig. 2B shows E. coli
  • Fig. 2C shows C P. mirabilis. Shown without washing, with 1x washing and 3x washing, and 1 hour and 24 hours in PBS (phosphate based saline solution).
  • the graph in Fig. 3 shows the anti-biofilm activity of silicone surfaces after adsorption of Reputex 20 using hydrophobin A.
  • Fig. 3A shows S. epidermidis
  • Fig. 3B shows E. coli
  • Fig. 3C shows P. mirabilis.
  • the graph in Fig. 4 shows the anti-biofilm activity of silicone surfaces after adsorption of the drug component PEI-P18 conjugate.
  • Fig. 4A shows S. epidermidis
  • Fig. 4B shows E. coli.
  • the graph in Fig. 5 shows the anti-biofilm activity of silicone surfaces after adsorption of the active ingredient component PEI-P18 conjugate with the aid of hydrophobin.
  • Fig. 5A shows S. epidermidis
  • Fig. 5B shows E. coli.
  • the graph in Fig. 6 shows the anti-biofilm activity of silicone surfaces against E. coli after adsorption and covalent attachment of the antimicrobial peptide P18 by means of hydrophobin A.
  • Fig. 6A shows the number of germs after 3x washing with PBS before Biofilmassay
  • Fig. 6B shows after 10x washing with PBS before Biofilmassay.
  • Control is a silicone surface without antimicrobial finish for normal biofilm development. Further control is adsorbed P18 without covalent binding.
  • the graph in FIG. 7 shows the anti-biofilm activity of silicone surfaces against S. epidermidis after adsorption of polyhexamethylene biguanide (Reputex 20) by means of hydrophobin B in a one-step process.
  • the silicone surfaces were incubated with an aqueous solution containing 500 ppm Hydrophobin B and 2% Reputex 20 at pH 4.
  • the graph in FIG. 8 shows the anti-biofilm activity of silicone surfaces against S. epidermidis after adsorption of Reputex 20 with the aid of hydrophobin B in the one-step process.
  • the silicone surfaces were incubated with an aqueous solution containing 100 ppm Hydrophobin B, 1% Reputex 20 and 0.3% Luviquat hold at pH 6.
  • the effect of the antimicrobial finish of the surfaces tested was determined by a static biofilm assay.
  • the surfaces to be tested were cut into circular slices which fit into the holes of 24-well microtiter plates.
  • the surfaces were immobilized on the bottom of the microtiter plates using grind-grease.
  • biofilms were carried out from a preculture.
  • 20 ml of the commercially available TSBY medium (Difco Laboratories, MI, USA) was transfected using a stationary overnight culture of Staphylococcus epidermidis DSM 1798, Escherichia coli DSM 5698, Proteus mirabilis DSM 4479 or Escherichia coli BL21 (DE3) with an optical density of 0 , 1 inoculated.
  • the preculture was incubated for 2 to 4 hours with agitation (at 200 rpm) and 37 ° C until an optical density of 2 to 3 was reached.
  • the preculture was diluted to an optical density of 0.0004 in 5% TSBY in saline (0.9% NaCl) (approximately 10E5 cfu / ml). Of these, 1 ml was added to the surfaces in the 24-well plate and then incubated at 37 ° C with gentle swirling at 50 rpm (revolutions per minute) so that a biofilm could be formed on the surface.
  • the biofilm on the surfaces was analyzed after one hour (1 h) and after 24 hours (24 h) (or 5 hours optional).
  • the planktonic cells were removed for this purpose.
  • the surfaces were then removed from the microtiter plate and washed briefly in saline, saline (0.9% NaCl). Then the adherent cells in the biofilm were detached from the surface by sonication.
  • the surfaces were placed in a Falcon tube (50 ml volume) with 2 ml Saline transferred and sonicated for five minutes in an ultrasonic water bath.
  • the resulting bacterial suspension was diluted and plated on TSBY agar plates. After 18 hours at 37, the resulting colonies were counted and recalculated to the number of cells on the surface.
  • the silicone surfaces Prior to adsorption, the silicone surfaces (15 mm diameter discs suitable for 24-well microtiter plates) were washed twice with an SDS solution (10 mg / ml in ultrapure water). Subsequently, rinsing was done twice with ultrapure water and the surfaces were degreased by immersion in ethanol (70% in ultrapure water), sterilized and subsequently dried.
  • the silicone surfaces to be coated were incubated for 1 hour at 400 rpm on the Eppendorf shaker with the Reputex 20 solution. Afterwards the surfaces were dried.
  • the surfaces were washed before the biofilm assay according to different protocols.
  • the surfaces were washed once, three times and ten times with 1 ml PBS.
  • the surfaces were shaken for one minute in the Eppendorf shaker at 400 rpm (revolutions per minute) during each washing step.
  • the wash solution was then removed, discarded and replaced with new ones.
  • the antimicrobial finished surfaces were incubated for one hour and 24 hours in 500 ml PBS.
  • FIG. 2 graphically shows the anti-biofilm activity of silicone surfaces (number of germs in cfu / cm 2 after adsorption of polyhexamethylene biguanide for three organisms (in each case in comparison to the control (without active ingredient):
  • the immobilization of the antimicrobial active substance is not sufficient.
  • the antimicrobial finish of the silicone surfaces was done in two steps.
  • the hydrophobin A was bound to silicone as the hydrophobin component (H).
  • the surfaces were incubated for at least 3 hours with 0.5 mg / ml of hydrophobin A in binding buffer (50 mM tri-HCl, 1 mM CaCl 2 at pH 8.0).
  • binding buffer 50 mM tri-HCl, 1 mM CaCl 2 at pH 8.0.
  • Preference is given to using the hydrophobin yaad-Xa-dewA-his (SEQ ID NO: 20) (see WO 2006/082251). Following was washed twice with ultrapure water and dried the surface.
  • polyhexamethylene biguanide (Reputex 20) was adsorbed to the hydrophobin coating.
  • the surfaces were overlaid with 1 ml each of 50 mg / ml Reputex 20 in PBS and incubated for one hour at room temperature. Following was washed once with 1 ml of ultrapure water.
  • the surfaces were washed according to various protocols before the biofilm assay.
  • the surfaces were washed once, three times and ten times with 1 ml PBS.
  • the surfaces were shaken for one minute in the Eppendorf shaker at 400 rpm (revolutions per minute) for each washing step.
  • the wash solution was then removed, discarded and replaced with new ones.
  • the antimicrobial finished surfaces were incubated for one hour and 24 hours in 500 ml PBS.
  • Example 1 The effect of the antimicrobial finished surfaces was determined as described in Example 1.
  • the antimicrobial treatment by adsorption of the antimicrobial active ingredient with the aid of hydrophobin A also acts after intensive washing against the biofilm formation by S. epidermidis.
  • polyhexamethylene biguanide By the hydrophobin coating polyhexamethylene biguanide is better retained on the surface, so that even after intensive washing enough drug is present to prevent biofilm formation by S. epidermidis.
  • Figure 3 shows the anti-biofilm activity of silicone surfaces after adsorption of polyhexamethylene biguanide with the aid of hydrophobin A for various microorganisms, wherein an improved fixation of the active ingredient could be observed.
  • P18cys corresponds to the amino acid sequence of P18 with an additional C-terminal cysteine residue (KWKLFKKIPKFLHLAKKFC).
  • P18cys was prepared by synthesis (e.g., from Bachem AG, Bubendorf, Switzerland).
  • SMCC is a heterobifunctional crosslinker with an amino-reactive and a thiol-reactive component.
  • the amino functions of PEI were activated with SMCC.
  • the PEI was diluted to 100 mg / ml in PBS (Phosphate Buffered Saline: 10 mM potassium phosphate, 137 mM NaCl, pH 7.5), the pH was adjusted to 7 to 8 with 4 M HCl, and this solution was then subjected to overnight PBS dialysed.
  • PBS Phosphate Buffered Saline: 10 mM potassium phosphate, 137 mM NaCl, pH 7.5
  • PBS Phosphate Buffered Saline: 10 mM potassium phosphate, 137 mM NaCl, pH 7.5
  • this solution was then subjected to overnight PBS dialysed.
  • the PEI thus treated was then at a concentration of
  • Uncoupled P18cys and low molecular weight substances were removed by dialysis against PBS.
  • the PEI-P18 conjugates were analyzed by SDS-Page methods.
  • Example 5 Antimicrobial finishing of silicone surfaces by adsorption of PEI-P18 conjugates
  • the PEI-P18 conjugates synthesized as in Example 4 were adsorbed as an active ingredient component on silicone surfaces. Prior to adsorption, the silicone surfaces (15 mm diameter discs suitable for 24-well microtiter plates) were washed twice with an SDS solution (10 mg / ml in ultrapure water).
  • the surfaces were washed before the biofilm assay according to different protocols.
  • the surfaces were washed ten times with 1 ml PBS.
  • the surfaces were shaken for one minute in the Eppendorf shaker at 400 rpm (revolutions per minute) for each washing step.
  • the wash solution was then removed, discarded and replaced with new ones.
  • the antimicrobial finished surfaces were incubated for one hour and 24 hours in 500 ml PBS.
  • the effect of the antimicrobial finished surfaces was determined as described in Example 1.
  • silicone surfaces can be antimicrobially treated.
  • the antimicrobial finish is capable of preventing biofilm formation by S.
  • the active ingredient that remains on the surface after washing is sufficient to prevent biofilm formation.
  • the PEI-P18 conjugates are not retained on the surface so strongly that after intensive washing, enough active ingredient is present to prevent biofilm formation by E. coli.
  • FIG. 4 shows the anti-biofilm activity of silicone surfaces after adsorption of antimicrobial PEI-P18 conjugates to:
  • the antimicrobial finish of the silicone surfaces was done in two steps.
  • hydrophobin A was bound to silicone.
  • the surfaces were incubated for at least 3 hours with 0.5 mg / ml of hydrophobin A in binding buffer (50 mM tri-HCl, 1 mM CaCl 2 at pH 8.0). Following was washed twice with ultrapure water and dried the surface.
  • the adsorption of the conjugates was carried out in the second step from a solution of 10 mg / ml (P18 equivalents) in PBS.
  • the silicone surfaces to be coated were incubated with the solution for one hour at 400 rpm on the Eppendorf shaker. Afterwards the surfaces were dried.
  • the surfaces were washed before the biofilm assay according to different protocols.
  • the surfaces were washed ten times with 1 ml PBS.
  • the surfaces were shaken for one minute in the Eppendorf shaker at 400 rpm for each wash step.
  • the wash solution was then removed, discarded and replaced with new ones.
  • the antimicrobial finished surfaces were incubated for one hour and 24 hours in 500 ml PBS.
  • Example 1 The effect of the antimicrobial finished surfaces was determined as described in Example 1.
  • the antimicrobial finish by adsorption of the PEI-P18 conjugates with the aid of hydrophobin A also works after intensive washing against the biofilm formation by E. coli. Due to the hydrophobin coating, the PEI-P18 conjugates are permanently retained on the surface, so that even after intensive washing, enough active ingredient is present to prevent biofilm formation by E. coli.
  • FIG. 5 shows the anti-biofilm activity of silicone surfaces after adsorption of PEI-P18 conjugates with the aid of hydrophobin in:
  • Example 7 Antimicrobial finish of silicone by adsorption and covalent linkage with the antimicrobial peptide P18
  • the peptide P18 was covalently bound to an existing hydrophobin coating on silicon surfaces (15 mm diameter discs suitable for 24-well microtiter plates).
  • the amino functions of the hydrophobin on the surface were activated by EDC in the presence of NHS.
  • Activation was carried out by incubating the hydrophobin coating on the silicone surfaces in 24-well plates for 30 minutes at room temperature in a solution of 760 ⁇ ultrapure water, 40 ⁇ MES buffer (20 mM, pH 6), 100 ⁇ EDC (250 mM, pH 6 to 6.8) and 100 ⁇ M NHS (250 mM, pH 7.0 to 8.0).
  • 100 ⁇ P18 solution (10 mM in 100 mM P18 NaC0 3, pH 8.5
  • incubated for a further 60 minutes at room temperature. After the reaction was washed with ultrapure water.
  • FIG. 6 shows the anti-biofilm activity of silicone surfaces against E. coli after adsorption and covalent binding of the antimicrobial peptide P18 with the aid of hydrophobin A:
  • the surfaces were washed three times before biofilm and ten times with 1 ml of PBS each time. For this purpose, the surfaces were shaken for one minute in the Eppendorf shaker at 400 rpm for each wash step. The wash solution was then removed, discarded and replaced with new ones.
  • Example 1 The effect of the antimicrobial finished surfaces was determined as described in Example 1 ( Figure 6). In addition, biofilm formation was measured even after 5 hours. While the samples to which the antimicrobial peptide was immobilized without covalent binding no longer show any inhibition after washing three times and ten times, a marked reduction in the biofilm formation of E. coli on the silicone surfaces with covalently immobilized P18 can be seen.
  • Example 8 Antimicrobial finishing of silicone surfaces in one-step process by simultaneous treatment with polyhexamethylene biguanide and hydrophobin B
  • the antimicrobial finish of the silicone surfaces was carried out in the one-step process.
  • Two aqueous compositions were prepared: a) which contains, as hydrophobin component (H), 500 ppm of hydrophobin B (see WO 2006/082251) and 2.0% by weight of polyhexamethylene biguanide (Reputex 20),
  • hydrophobin component (H) 100 ppm of hydrophobin B and 1, 0 wt .-% Polyhexamethylenbiguanid (Reputex 20) and 0.3% by weight of an additive (Ludiquat Hold).
  • the antimicrobial finish of the silicone surfaces is done in one step.
  • the surfaces were incubated for 1 h with 1 ml of a solution of 500 ppm Hydrophobin B and 2% Reputex 20 at pH 4. Afterwards the surfaces were dried.
  • FIG. 7 shows the anti-biofilm activity of silicone surfaces against S. epidermidis after adsorption of Reputex 20 with the aid of hydrophobin B in the one-step process.
  • the silicone surfaces were incubated with an aqueous solution containing 500 ppm Hydrophobin B and 2% Reputex 20 at pH 4.
  • the antimicrobial finish of the silicone surfaces by adsorption of Reputex 20 by means of hydrophobin B in the one-step process shows a similarly good effect as the adsorption described in Example 3 using hydrophobin A in the two-step process.
  • an active substance such as polyhexamethylene biguanide can be adsorbed so well that, after intensive washing, sufficient active substance is still present to completely prevent biofilm formation by S. epidermidis.
  • Example 9 Antimicrobial finishing of silicone surfaces in one-step process by simultaneous treatment with polyhexamethylene biguanide, hydrophobin B and an auxiliary component (HK)
  • the antimicrobial finish of the silicone surfaces was done in a one-step process.
  • auxiliaries for example cationic, zwitterionic or nonionic surfactants or polymers.
  • FIG. 8 shows the anti-biofilm activity of silicone surfaces against S. epidermidis after adsorption of Reputex 20 using single-step hydrophobin B.
  • the silicone surfaces were incubated with a solution of 100 ppm Hydrophobin B, 1% Reputex 20 and 0.3% Luviquat hold at pH 6.

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Abstract

L'invention concerne un procédé pour immobiliser un principe actif antimicrobien sur la surface d'un substrat, selon lequel la surface du substrat est traitée avec une composition contenant les constituants suivants: (i) un solvant, (ii) une hydrophobine (H), (iii) un principe actif (W) antimicrobien cationique, (iv) éventuellement des additifs (A) ou des constituants auxiliaires (HK), ledit procédé permettant d'obtenir un apprêt antimicrobien durable de substrats.
PCT/EP2011/067893 2010-10-13 2011-10-13 Procédé pour immobiliser des principes actifs cationiques sur des surfaces WO2012049250A2 (fr)

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