WO2022203596A1 - Matériaux antimicrobiens à large spectre - Google Patents

Matériaux antimicrobiens à large spectre Download PDF

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WO2022203596A1
WO2022203596A1 PCT/SG2022/050147 SG2022050147W WO2022203596A1 WO 2022203596 A1 WO2022203596 A1 WO 2022203596A1 SG 2022050147 W SG2022050147 W SG 2022050147W WO 2022203596 A1 WO2022203596 A1 WO 2022203596A1
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antimicrobial
pdms
alkyl
aryl
alkenyl
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PCT/SG2022/050147
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English (en)
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Yugen Zhang
Arunmozhiarasi Armugam
Siew Ping TEONG
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Agency For Science, Technology And Research
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures 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
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/48—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with two nitrogen atoms as the only ring hetero atoms
    • A01N43/50—1,3-Diazoles; Hydrogenated 1,3-diazoles
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P3/00—Fungicides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/785—Polymers containing nitrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G73/00—Macromolecular compounds obtained by reactions forming a linkage containing nitrogen with or without oxygen or carbon in the main chain of the macromolecule, not provided for in groups C08G12/00 - C08G71/00
    • C08G73/06—Polycondensates having nitrogen-containing heterocyclic rings in the main chain of the macromolecule
    • C08G73/0605—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms
    • C08G73/0616—Polycondensates containing five-membered rings, not condensed with other rings, with nitrogen atoms as the only ring hetero atoms with only two nitrogen atoms in the ring
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/48—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule in which at least two but not all the silicon atoms are connected by linkages other than oxygen atoms
    • C08G77/54—Nitrogen-containing linkages
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J3/00—Processes of treating or compounding macromolecular substances
    • C08J3/24—Crosslinking, e.g. vulcanising, of macromolecules
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L79/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing nitrogen with or without oxygen or carbon only, not provided for in groups C08L61/00 - C08L77/00
    • C08L79/04—Polycondensates having nitrogen-containing heterocyclic rings in the main chain; Polyhydrazides; Polyamide acids or similar polyimide precursors
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L83/00—Compositions of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon only; Compositions of derivatives of such polymers
    • C08L83/04—Polysiloxanes
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/04—Polysiloxanes
    • C08G77/12—Polysiloxanes containing silicon bound to hydrogen
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G77/00—Macromolecular compounds obtained by reactions forming a linkage containing silicon with or without sulfur, nitrogen, oxygen or carbon in the main chain of the macromolecule
    • C08G77/04—Polysiloxanes
    • C08G77/20—Polysiloxanes containing silicon bound to unsaturated aliphatic groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J2383/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
    • C08J2383/04—Polysiloxanes

Definitions

  • the present invention relates to an antimicrobial polymer, its method of preparation and its use.
  • Peritoneal silicone catheters impregnated with rifampicin, triclosan and trimethoprim using the swelling to evaporation method has been shown to inhibit colonization of methicillin-resistant Staphylococcus aureus (MRSA) for 90 days in a flow model
  • foley catheters impregnated with a combination of rifampicin, triclosan, and sparfloxacin has been shown to delay the colonization of catheter associated urinary tract infection (CAUTI) pathogens for 7 to 12 weeks in laboratory settings.
  • CAUTI catheter associated urinary tract infection
  • Casting or blending of biomaterials with antimicrobial agents to produce composite biomaterials has also been demonstrated. However, such materials still face similar issues as surfaces that are coated with antimicrobial compounds.
  • PDMS polydimethylsiloxane
  • Various polymers have been used in fabrication of a wide range of medical devices. Of these, polydimethylsiloxane (PDMS) elastomers have emerged as a promising lead for catheter development.
  • PDMS is innately biocompatible, chemically stable, transparent and mechanically elastic. Nevertheless, being chemically inert means that PDMS lacks reactive functional groups on its surface, and pre-treatment steps such as high-energy treatments or chemical etching are usually required to introduce useful functional groups for surface modification. Impregnation of useful antimicrobials or biocides into commercially available silicon catheters heavily depends on the solubility of the antimicrobial compounds in organic solvents.
  • an antimicrobial polymer comprising a polysiloxane polymer crosslinked with an antimicrobial crosslinker, wherein the antimicrobial crosslinker comprises an antimicrobial agent terminated on both ends with a linker that covalently bonds with the polysiloxane, wherein the antimicrobial crosslinker comprises no more than 10% by weight of the total weight of the antimicrobial polymer.
  • the antimicrobial agent may be embedded and crosslinked into the network of the polysiloxane polymer, and may exhibit a slow, prolonged and sustained release of the antimicrobial agent and thus long-term antimicrobial efficacy.
  • the polymer as defined above may possess the same physical properties as an unmodified polysiloxane polymer while exhibiting antimicrobial activity.
  • complete bacterial ( Staphylococcus aureus ) and fungi ( Candida albicans ) eradication may be observed for up to 60 days despite continuous microbial challenge.
  • the antimicrobial polymer as defined above may have remarkable biocidal effect (contact killing) for several multi drug resistant (MDR) clinical isolates such as Escherichia coli, Serratia marcescens, VR-Enterococcus faecium, MR-Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, ESBL Enterobacter aerogenes, Candida parapsilosis, Candida tropicalis and Candida auris, which may include the ESKAPE family of pathogens, suggesting that the antimicrobial polymer as defined above may have versatile applications against a wide range of microbes.
  • MDR multi drug resistant
  • the antimicrobial polymer as defined above may withstand prolonged incubation with continuous challenge with microbes for up to 60 days against single culture bacterial or fungi or their co-culture. Further advantageously, the antimicrobial polymer as defined above may effect complete elimination of microbes within 30 seconds of exposure, indicating that the polymer is capable of fast killing kinetics. More advantageously, the antimicrobial polymer as defined above may be recycled over multiple uses, stored at room temperature for long-term usage and may be biocompatible.
  • a method of preparing the antimicrobial polymer as defined above comprising the step of mixing: a) an antimicrobial crosslinker, wherein the antimicrobial crosslinker comprises an antimicrobial agent terminated on both ends with a linker; b) a polysiloxane precursor; and c) curing agent, to form a mixture, wherein the antimicrobial crosslinker comprises no more than 10% by weight of the total weight of the antimicrobial polymer.
  • the method as defined above may be a facile and simple method for fabricating antimicrobial polysiloxane-based materials, whereby the method may involve modifying the terminals of a polymeric antimicrobial agent with vinyl groups which may directly react with a polysiloxane precursor such as polydimethylsiloxane (PDMS) precursors.
  • a polysiloxane precursor such as polydimethylsiloxane (PDMS) precursors.
  • PDMS polydimethylsiloxane
  • the polysiloxane polymer may be crosslinked with the antimicrobial agent in a one -pot synthesis.
  • the method may be beneficial for fabricating antimicrobial biomaterials for manufacturing devices used in medical and healthcare applications.
  • the method may result in the successful preparation of the antimicrobial polymer despite the polysiloxane polymer being highly hydrophobic and the antimicrobial agent being highly hydrophilic. That is, the method may be able to achieve a typically difficult chemical reaction between a hydrophobic moiety and a hydrophilic moiety.
  • only minor changes may be observed to the surface hydrophobicity of PDMS materials.
  • the method as defined above may be useful in preparing medical devices. Compared to conventional products that may rely on surface grafting/modification, coating or impregnation to introduce antimicrobial properties to the device, the method as defined above may facilitate easier and facile manufacture of devices that may have inherent antimicrobial properties. No additional steps to coat or impregnate the device with antimicrobial agents may be needed. In addition, the amount of antimicrobial agents to be incorporated during fabrication may be easily regulated during the one-time casting process using the method as defined above. In another aspect, there is provided an antimicrobial polymer obtainable by the method as defined above.
  • a medical device comprising the antimicrobial polymer as defined above.
  • the antimicrobial polymer as defined above may have a wide range of applications, especially in biomedical devices such as catheters.
  • the antimicrobial polymer as defined above may enable any polysiloxane- based device or product to be made with inherent antimicrobial properties.
  • a device or product may have natural disinfection and antifouling properties.
  • the antimicrobial polymer as defined above may be used directly as the bulk material, rather than a coating, of a medical device such as a catheter, as the antimicrobial polymer may retain the bulk properties of the unmodified polysiloxane polymer while having the inherent antimicrobial properties . This may advantageously avoid requiring additional antimicrobial coatings or surface modification to be performed on the device.
  • the antimicrobial polymer as defined above may have the potential to be used in antifouling/antibiofilm applications in catheters that may require to be kept in place during a long-term under continuous use.
  • Alkyl as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C12 alkyl, more preferably a C1-C10 alkyl, most preferably C1-C6 unless otherwise noted.
  • suitable straight and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t-butyl, hexyl, and the like.
  • the group may be a terminal group or a bridging group.
  • Alkenyl as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain.
  • the group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z.
  • Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl.
  • the group may be a terminal group or a bridging group.
  • Alkynyl as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2 - 12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain.
  • Exemplary structures include, but are not limited to, ethynyl and propynyl.
  • the group may be a terminal group or a bridging group.
  • Aryl as a group or part of a group denotes (i) an optionally substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring.
  • aryl groups include phenyl, naphthyl, and the like; (ii) an optionally substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a C5-7 cycloalkyl or C5-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl.
  • the group may be a terminal group or a bridging group.
  • an aryl group is a CVC 1 x aryl group.
  • Arylalkyl means an aryl-alkyl- group in which the aryl and alkyl moieties are as defined herein.
  • Preferred arylalkyl groups contain a Ci-12 alkyl moiety.
  • Exemplary arylalkyl groups include benzyl, phenethyl, 1 -naphthalenemethyl and 2-naphthalenemethyl.
  • the group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.
  • Alkylaryl means an alkyl-aryl— group in which the aryl and alkyl moieties are as defined herein. Preferred alkylaryl groups contain a Ci-12 alkyl moiety. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the aryl group.
  • Alkylarylalkyl means an alkyl-aryl-alkyl group in which the aryl and alkyl moieties are as defined herein. Preferred alkylarylalkyl groups contain a Ci-12 alkyl moiety. The group may be a terminal group or a bridging group. If the group is a ter inal group it is bonded to the remainder of the molecule through the aryl group.
  • Cycloalkyl refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 12 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane.
  • a cycloalkyl group typically is a C3-C12 alkyl group. The group may be a terminal group or a bridging group.
  • Heteroaryl either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur.
  • heteroaryl examples include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, lH-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phen
  • a heteroaryl group is typically a Ci-Cis heteroaryl group.
  • a heteroaryl group may comprise 3 to 8 ring atoms.
  • a heteroaryl group may comprise 1 to 3 heteroatoms independently selected from the group consisting of N, O and S.
  • the group may be a terminal group or a bridging group.
  • Heterocyclyl refers to saturated, partially unsaturated or fully unsaturated monocyclic, bicyclic or polycyclic ring system containing at least one heteroatom selected from the group consisting of nitrogen, sulfur and oxygen as a ring atom.
  • heterocyclic moieties include heterocycloalkyl, heterocycloalkenyl and heteroaryl.
  • the term "about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • an antimicrobial polymer comprising a polysiloxane polymer crosslinked with an antimicrobial crosslinker, wherein the antimicrobial crosslinker comprises an antimicrobial agent terminated on both ends with a linker that covalently bonds with the polysiloxane, wherein the antimicrobial crosslinker comprises no more than 10% by weight of the total weight of the antimicrobial polymer.
  • the antimicrobial agent may be a linear polymer.
  • the antimicrobial agent may comprise multiple imidazolium groups.
  • the antimicrobial agent may have the following formula (I): wherein:
  • R 1 and R 2 are each independently selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl;
  • R 3 is each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;
  • X 1 is each independently a halogen; and p is an integer selected from 2 to 50.
  • the compound of formula (I) may have a molecular weight in the range of 1000 to 3000.
  • the antimicrobial crosslinker may have the following formula (II): wherein:
  • R 1 , R 2 and R 4 are each independently selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl;
  • R 3 is each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;
  • L 1 is each independently the linker
  • X 1 and X 2 are each independently a halogen; and p is an integer selected from 2 to 50.
  • the antimicrobial crosslinker when cross-linked with the polysiloxane polymer, may have the following formula (III): wherein: R 1 , R 2 and R 4 are each independently selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl;
  • R 3 is each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;
  • X 1 and X 2 are each independently a halogen; and p is an integer selected from 2 to 50 and
  • the linker L 1 of the antimicrobial crosslinker when crosslinked with the polysiloxane polymer, may be chemically transformed to form a covalent bond with the polysiloxane polymer.
  • R 1 , R 2 and R 4 may each independently be selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl.
  • the aryl may be ortho-benzene, meta-benzene or para-benzene.
  • R 1 and R 4 may each independently have a structure selected from the group consisting of the following formulae (IV) to (VI): and any combination thereof, wherein q may be independently 0 or an integer selected from 1 to 12, and * may indicate where the structure is attached to the rest of Formula (I), (II) or (III). q may be 0 or q may be an integer from 1 to 12, 1 to 2, 1 to 5, 1 to 10, 2 to 5, 2 to 10, 2 to 12, 5 to 10, 5 to 12 or 10 to 12. q may each independently be 0 or 1.
  • R 1 may be alkylarylalkyl.
  • the aryl of the alkylarylalkyl may be ortho-benzene, meta-benzene or para-benzene.
  • the aryl of the alkylarylalkyl of R 1 may be ortho-benzene or may have a structure of formula (IV), wherein both instances of q is 1.
  • R 4 may be alkylaryl.
  • the aryl of the alkylarylalkyl may be ortho-benzene, meta-benzene or para- benzene.
  • the aryl of the alkylarylalkyl of R 4 may be para-benzene or may have a structure of formula (VI), wherein q is 0 or 1.
  • R 4 may have a structure of formula (VI), wherein one q is 0 and the other q is 1.
  • R 2 may be alkenyl.
  • R 2 may be a C to Cn alkenyl.
  • R 2 may be a C4 alkenyl, or butenyl.
  • R 2 may be n-butenyl, 2-butenyl or isobutenyl.
  • R 2 may be 2-butenyl.
  • R 3 may each independently be selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl.
  • R 3 may be hydrogen.
  • X 1 and X 2 may each independently be a halogen.
  • the halogen may be fluorine, chlorine, bromine or iodine.
  • X 1 may be bromine and X 2 may be chlorine.
  • p may be an integer selected from 2 to 50, 2 to 5, 2 to 10, 2 to 20, 5 to 10, 5 to 20, 5 to 50, 10 to 20, 10 to 50 or 20 to 50. p may be an integer from 5 to 10.
  • crosslinker is covalently bonded to the polysiloxane.
  • L 1 may each independently be the linker.
  • L 1 may be selected from the group consisting of vinyl, ester, amide, urethane, hydroxy and any combination thereof.
  • L 1 may be vinyl.
  • the vinyl group may advantageously be involved in the curing reaction of the polysiloxane, thereby facilitating crosslinking of the antimicrobial agent with the polysiloxane.
  • the antimicrobial agent may have the following structure:
  • the antimicrobial agent having the above structure may be the imidazolium polymer PIM45.
  • PIM45 may have excellent antimicrobial efficacy and the polymer chains may be typically capped with imidazole groups, facilitating further functionalization.
  • the antimicrobial crosslinker may have the following structure:
  • the antimicrobial crosslinker when cross-linked with the polysiloxane, may have the following structure: wherein * indicates where the crosslinker is covalently bonded to the polysiloxane.
  • the polysiloxane polymer may be a poly(alkyl)siloxane polymer or polydi(alkyl)siloxane polymer.
  • the alkyl of the poly(alkyl)siloxane or polydi(alkyl)siloxane polymer may be methyl, ethyl, propyl, butyl, pentyl or hexyl.
  • the polysiloxane polymer may be poly dime thylsiloxane (PDMS).
  • PDMS may be innately biocompatible, chemically stable, transparent and mechanically elastic. PDMS may have a wide range of applications, especially in medical devices such as catheters. PDMS may advantageously retain its mechanical properties provided that the amount of additional component (in this case antimicrobial agent) is limited to be below the critical amount of 10 wt% based on the total weight of the antimicrobial polymer.
  • additional component in this case antimicrobial agent
  • the antimicrobial crosslinker may comprise no more than 10% by weight of the total weight of the antimicrobial polymer.
  • the antimicrobial crosslinker may comprise about 0.1% to about 10%, about 0.1% to about 0.2%, about 0.1% to about 0.5%, about 0.1% to about 1%, about 0.1% to about 2%, about 0.1% to about 5 %, about 0.2% to about 0.5%, about 0.2% to about 1%, about 0.2% to about 2%, about 0.2% to about 5%, about 0.2% to about 10%, about 0.5% to about 1%, about 0.5% to about 2%, about 0.5% to about 5%, about 0.5% to about 10%, about 1% to about 2%, about 1% to about 5%, about 1% to about 10%, about 2% to about 5%, about 2% to about 10% or about 5% to about 10% by weight of the total weight of the antimicrobial polymer.
  • the antimicrobial polymer may prevent the growth of microbes such as bacteria and fungi on its surface.
  • the bacteria may be selected from the group consisting of Escherichia coli, Staphylococcus aureus, Klebsiella pneumonia, Serratia marcescens, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter aerogenes ESBL- sensitive, Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecium and Proteus mirabilis.
  • the fungi may be selected from the group consisting of Candida albicans, Candida tropicalis, Candida parapsilosis, Candida auris, Aspergillus fumigatus, Aspergillus flavus, Aspergillus clavatus, Cryptococcus neoformans, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii, Fusarium solani Fusarium oxysporum, Fusarium verticillioides, Fusarium proliferatum Stachybotrys chartarum), and Aspergillus niger.
  • a method of preparing the antimicrobial polymer as defined above comprising the step of mixing: a) an antimicrobial crosslinker, wherein the antimicrobial crosslinker comprises an antimicrobial agent terminated on both ends with a linker; b) a polysiloxane precursor; and c) curing agent, to form a mixture, wherein the antimicrobial crosslinker comprises no more than 10% by weight of the total weight of the antimicrobial polymer.
  • the total weight of the antimicrobial polymer may be the combined weight of the antimicrobial crosslinker, the polysiloxane precursor and the curing agent.
  • the antimicrobial crosslinker may be prepared by reacting the antimicrobial agent having the following formula (I): wherein:
  • R 1 and R 2 are each independently selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl;
  • R 3 is each independently selected from hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heteroaryl, or heterocyclyl;
  • X 1 is each independently a halogen; and p is an integer selected from 2 to 50, with a linker precursor having the following formula (VII):
  • R 4 is selected from aryl, arylalkyl, alkylarylalkyl, alkylaryl, alkyl, cycloalkyl, alkenyl or alkynyl, wherein the aryl, arylalkyl, alkylarylalkyl or alkylaryl is optionally substituted with alkyl, alkenyl or alkynyl; and
  • L 1 is the linker
  • X 2 may be chlorine
  • R 4 may be alkylaryl, wherein the aryl of the alkylarylalkyl may be para-benzene or may have a structure of formula (VI), wherein q is 1 or 0, and L 1 may be vinyl.
  • R 4 may have a structure of formula (VI), wherein one q is 0 and the other q is 1.
  • the polysiloxane precursor may have the following formula (VIII): (VIII), wherein R 5 may be each independently hydrogen or alkyl; a may be an integer in the range of 10 to 1000; and L 2 may each independently be a reactive group. a may be an integer in the range of 10 to 1000, 10 to 100 or 100 to 1000.
  • the reactive group L 2 may be vinyl, alkylene or any mixture thereof.
  • the curing agent may have the following formula (IX):
  • R 5 may be each independently hydrogen or alkyl; and b and c may independently be an integer in the range of 10 to 1000.
  • R 5 may be methyl.
  • b and c may independently be an integer in the range of 10 to 1000, 10 to 100 or 100 to 1000.
  • the polysiloxane precursor and curing agent may be contacted at a ratio of in the range of about 50:1 to about 5:1 by weight, about 50:1 to about 20:1, about 50:1 to about 10:1, about 50:1 to about 5:1, about 20:1 to about 10:1, about 20:1 to about 5:1, or about 10:1 to about 5:1 by weight.
  • the polysiloxane precursor and curing agent may be contacted at a ratio of about 10: 1 by weight.
  • the mixing step may be performed in a solvent.
  • the solvent may be tetrahydrofuran, ethanol, toluene or any mixture thereof.
  • the solvent may be tetrahydrofuran.
  • the method may further comprise the step of adding a platinum catalyst to the mixture.
  • Platinum may be a good catalyst for the hydrosilylation or curing of polysiloxane and its derivatives.
  • the method may further comprise the step of degassing the mixture.
  • the degassing step may be performed under vacuum.
  • the method may further comprise the step of curing the mixture.
  • the curing step may be performed at a temperature in the range of about 60 °C to about 80 °C, 60 °C to about 70 °C, about 60 °C to about 70 °C or about 70 °C to about 80 °C for a duration in the range of about 6 hours to about 24 hours, about 6 hours to about 12 hours, about 6 hours to about 18 hours, about 12 hours to about 18 hours, about 12 hours to about 24 hours or about 18 hours to about 24 hours.
  • antimicrobial polymer as defined above in or as at least part of an antimicrobial surface.
  • antimicrobial polymer as defined above in or as at least part of a medical device.
  • the medical device may be selected from the group consisting of catheter, intravenous line, endotracheal tube, nephrostomy tube, gastronomy tube, traction pin, defibrillator, shunt, endoscopic catheter and joint implant.
  • Any polysiloxane-based device or product material may be made using the microbial polymer as defined above.
  • FIG. 1 is a scheme showing the preparation of the antimicrobial PDMS-PIM composite biomaterial.
  • Fig. 1A shows the synthesis of PIM45 -vinyl and
  • Fig. IB shows the synthesis of PDMS-PIM.
  • Fig. 2 is a scheme showing the preparation of the antimicrobial PDMS-PIM composite biomaterial.
  • Fig. 1A shows the synthesis of PIM45 -vinyl
  • Fig. IB shows the synthesis of PDMS-PIM.
  • Fig. 2 is a scheme showing the preparation of the antimicrobial PDMS-PIM composite biomaterial.
  • Fig. 1A shows the synthesis of PIM45 -vinyl
  • Fig. IB shows the synthesis of PDMS-PIM.
  • Fig. 2 shows the synthesis of PDMS-PIM.
  • FIG. 2 shows the ⁇ NMR spectra of PIM45-vinyl and solution released from PDMS-PIM5, compounds proposed to be PIM-Si and PIM45-vinyl.
  • FIG. 3 refers to calibration curves for determination of PIM45-vinyl and PIM45 concentrations in PBS. UV spectrum scan was carried out on the elution product to determine the l, m ax for the antimicrobials in PBS and subsequently calibration curves that follows Beer-Lambert Law were constructed for Fig. 3A: 0-100 ⁇ g/mL and Fig. 3B: 63-1000 ⁇ g/mL PIM45-vinyl; and Fig. 3C: 0- 100 pg/mL and Fig. 3D: 3-50 pg/mL parent PIM45 compounds in PBS, respectively.
  • FIG. 4 refers to a 1 H-NMR spectrum of the PIM antimicrobial compound released from PDMS- PIM.
  • the biomaterial 100 mg in 1 mL of D O was incubated at room temperature on a shaking incubator at 300 rpm.
  • the D O solution containing the released active compounds were subjected to 1 H NMR spectroscopy.
  • a minute amount of PIM component released from PDMS-PIM5 showed the presence of the vinyl group (expected chemical shifts 6.75 ppm (doublet of doublets) and 5.82 ppm (doublet)).
  • Dimethyl sulfone was used as an internal control.
  • FIG. 5 refers to graphs showing the evaluation of the controlled release of antimicrobial from composite biomaterial. Quantitation of imidazolium PIM45 and PIM45 -vinyl derivatives that was released/eluted in PBS over Fig. 5 A: 5 x 1 hour cycles and Fig. 5B: 5 x 1 day cycles. Quantitation was carried out using spectrophotometric assay based on calibration curves that were generated from the parent PIM45 -vinyl compound.
  • FIG. 6 refers to a scheme showing the hydrosilylation reaction of PIM45 -vinyl and the hydrolysis reaction of PDMS-PIM.
  • FIG. 7 refers to a graph showing the antimicrobial potency of the PIM45 eluted in a solution of unmodified PDMS and PDMS-PIM1 after 1, 2, 4 and 5 cycles. * in the graph denotes zero (0) CFU on the agar plate for undiluted samples.
  • Fig. 8 refers to a graph showing the antimicrobial potency of the PIM45 eluted in a solution of unmodified PDMS and PDMS-PIM1 after 1, 2, 4 and 5 cycles. * in the graph denotes zero (0) CFU on the agar plate for undiluted samples.
  • Fig. 8 refers to a graph showing the antimicrobial potency of the PIM45 eluted in a solution of unmodified PDMS and PDMS-PIM1 after 1, 2, 4 and 5 cycles. * in the graph denotes zero (0) CFU on the agar plate for undiluted samples.
  • Fig. 8 refers to a graph showing the antimicrobial potency of the PIM
  • FIG 8 refers to a graph showing the antimicrobial potency of the PDMS-PIM biomaterial after the elution and release of PIM45.
  • the eluted composite biomaterial was subjected to antimicrobial assay against S. aureus.
  • * denotes zero (0) CFU on the agar plate for undiluted samples.
  • FIG. 9 refers to images showing the surface topology of PDMS and PDMS-PIM materials taken by Field Emission Scanning Electron Microscopy (FESEM) and Atomic Force Microscopy (AFM). Scale bar represents 1.0 pm.
  • FESEM Field Emission Scanning Electron Microscopy
  • AFM Atomic Force Microscopy
  • FIG. 10 refers to a graph showing AFM analysis on surface roughness of the biomaterials. Results are depicted as root mean square (RMS) values (Rq, nm) for respective unmodified PDMS and PDMS-PIM biomaterial.
  • RMS root mean square
  • FIG. 11 refers to a graph showing the surface wettability analysis of pristine PDMS and the antimicrobial PDMS-PIM composite biomaterials (with 0.5 wt%, 1 wt%, 2 wt% and 5 wt% PIM45) by contact angle determination. The results are expressed as mean and standard deviation of 3 x 3 replicates.
  • FIG. 12 refers to a graph showing the outcome of the surface antimicrobial assay for PDMS and PDMS-PIM materials (PDMS-PIM0.5, PDMS-PIM1 and PDMS-PIM5).
  • the surface antimicrobial assay was carried out using representative of Gram negative bacteria (E. coli #15036); Gram positive bacteria (S. aureus #6538) and Fungi (C. albicans #10231) microbes following the JIS Z 2801-2010 standard protocol at 24 hour time point using neat/pure media.
  • * denotes zero (0) CFU on the agar plate for undiluted samples. The results are expressed as mean and standard deviation of three replicates.
  • FIG. 13 is a graph showing the rapid killing efficacy of PDMS-PIM material carried out based on the JIS Z 2801 standard protocol with 30 second exposure of microbes. * denotes zero (0) CFU on the agar plate for undiluted samples. The results are expressed as mean and standard deviation of three replicates.
  • Fig. 14 is a graph showing the rapid killing efficacy of PDMS-PIM material carried out based on the JIS Z 2801 standard protocol with 30 second exposure of microbes. * denotes zero (0) CFU on the agar plate for undiluted samples. The results are expressed as mean and standard deviation of three replicates.
  • Fig. 14 is a graph showing the rapid killing efficacy of PDMS-PIM material carried out based on the JIS Z 2801 standard protocol with 30 second exposure of microbes. * denotes zero (0) CFU on the agar plate for undiluted samples. The results are expressed as mean and standard deviation of three replicates.
  • Fig. 14 is a graph showing the rapid killing
  • FIG. 14 refers to a graph showing the durability of the PDMS-PIM biomaterial in microbial co culture.
  • the biomaterials were challenged with microbes (5. aureus and C. albicans ) at 48 to 72 hour intervals.
  • the antimicrobial potency evaluation was carried out at 15 days, 30 days and 60 days endpoint.
  • FIG. 15 refers to a graph showing the potential to recycle/reuse the recovered PDMS-PIM disk. Each cycle indicates a 15 day period that the biomaterials were periodically challenged with the microbes at 48 to 72 hour intervals, before being assessed for the antimicrobial potency. The results are expressed as mean and standard deviation of three replicates. * indicates zero (0) CFU on the agar plate for undiluted samples.
  • FIG. 16 refers to a graph showing the efficacy of antimicrobial activity of PDMS-PIM1 on clinical pathogens. Potency of PDMS-PIM1 biomaterial against clinical pathogens was evaluated following JIS Z 2801-2010 protocol. The results are expressed as mean and standard deviation of three replicates. * denotes zero (0) CFU on the agar plate for undiluted samples
  • FIG. 17 refers to a graph showing the eradication of biofilm by PDMS-PIM1.
  • the biofilms were formed on glass cover slips.
  • PDMS and PDMS-PIM1 material were overlaid on the biofilm and incubated at 35 °C for respective time points.
  • the biofilm bacteria inoculate was harvested in 10 ml, PBS and plated on LB agar plates following appropriate dilution. The results are expressed as mean and standard deviation of three replicates. * denotes zero (0) CFU on the agar plate for undiluted samples.
  • FIG. 18 refers to a graph showing eradication of biofilm by PDMS-PIM1 in solution.
  • PDMS material and PDMS-PIM1 material were immersed in 10 ml PBS. Tubes (2 cm x 3) with preformed biofilm in lumen were then incubated in the presence of PDMS-PIM material of varying weights, for 1 hour at 100 rpm/35 °C.
  • the viable colonies were determined by plate assay in both experiments. The results are expressed as mean and standard deviation of three replicates. * denotes zero (0) CFU on the agar plate for undiluted samples.
  • FIG. 19 refers to a graph showing the hemocompatibility assessment carried out using red blood cell hemolysis assay.
  • PDMS-PIM biomaterial (5 mg and 10 mg) in 100 pL of red blood cells was incubated at 35 °C at 150 rpm for 1 hour.
  • the lysis of RBC was determined spectrophotometrically at 576 nm. The results are expressed as mean and standard deviation of three replicates.
  • FIG. 20 refers to a graph showing the cytocompatibility evaluation carried out using L929 fibroblast cell viability assay.
  • PDMS-PIM biomaterial (5 mg and 10 mg) was added to the L929 cells (in 96 well plate) in 100 pL of DMEM complete media. The plate was incubated at 35 °C with 5% CO2 for 24 hours. Cell viability was determined using the Alamar Blue reagent. The results are expressed as mean and standard deviation of three replicates.
  • Microbes Escherichia coli (E. coli, ATCC 15036), Staphylococcus aureus (S. aureus, ATCC 6538), Candida albicans (C. albicans, ATCC 10231) and murine fibroblast cell line (NCTC clone 929; L929, ATCC® CCL1TM) were purchased from American Type Culture Collection (ATCC.org).
  • Klebsiella pneumonia (CI0027), Serratia marcescens (CI0183A1), Pseudomonas aeruginosa (CI0183A2), Acinetobacter baumannii (RI0139A), Enterobacter aerogenes ESBL- sensitive (RI0006A2), Methicillin resistant Staphylococcus aureus (R10309N), Vancomycin resistant Enterococcus faecium (RI0252N1), Candida tropicalis (RI0243A), Candida parapsilosis (ATCC 22019), and Candida auris (NCPF 8977) were obtained from National Centre for Infectious Disease, Singapore.
  • PDMS based silicone materials are naturally hydrophobic.
  • the static contact angle of PIM45- vinyl and pristine polydimethylsiloxane (PDMS) surfaces were measured by OCA15 contact angle analyzer (Future Digital Scientific Corp., Westbury, New York, USA). Deionized (DI) water (2 pL) at 3 pL/sec, was used for all measurements. All samples were analyzed in triplicates, and the static contact angle data were presented as mean ⁇ SD.
  • the surfaces of the PDMS and PDMS-PIM material was characterized by Field Emission Scanning Electron Microscopy (FESEM, JEOL JSM-7400E). Briefly, the material was washed with water followed by 70% ethanol and air dried. The dried disk was then coated with thin platinum film using high resolution sputter coater (JEOL, JFC-1600 Auto Fine Coater; coating conditions: 20 mA, 30 s) and viewed under SEM.
  • FESEM Field Emission Scanning Electron Microscopy
  • the stability of the antimicrobial compound in the casted composite PDMS material was evaluated by both quantitative and qualitative assessments. All experiments were carried out in at least three replicates.
  • the concentration of released PIM45 -vinyl was determined using a spectrophotometric method. UV spectrum scan was carried out for PIM45 -vinyl in solution.
  • the antimicrobial property of the fabricated biomaterial was determined using the Japanese Industrial Standard (JIS Z 2801 : 2010; Protocol (2010)). Briefly, 100 pL microbial in a suspension of 10 6 CFU/mL in pure media (tryptic soy broth (TSB) or yeast mannitol broth (YMB); unless otherwise specified), was added onto the surface of a composite PDMS biomaterial (2.5 cm x 2.5 cm) disk. A plastic film (2 cm x 2 cm) was then overlayed on the suspension and incubated at appropriate temperature for 24 hours. The biomaterial disk was rinsed with 9.9 mL of TSB/YMB following incubation and the cell suspension was plated on Luria-Bertani (LB) agar plates.
  • LB Luria-Bertani
  • CFU/mL colony forming units
  • the biomaterial disk was rinsed with 9.9 mL of TSB/YMB following incubation, and diluted further at lOx serial dilution with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a cell suspension 40 pL was then plated on Luria-Bertani (LB) agar plates. The plates were incubated at 36 °C for 16 to 18 hours and the number of viable microbes indicative of colony forming units (CFU) was recorded. The number of CFUs was multiplied by volume and the respective dilution factor to compute for CFU/mL.
  • Representative gram negative bacteria (E. coli), gram positive bacteria (S. aureus) and fungi (C. albicans) were used for the assessments. All experiments were carried out in three replicates. Biofilm formation and anti-biofilm assay (time-kill study)
  • Microbes 100 ⁇ L at 106 CFU/mL in media were applied on sterile glass cover slips in six well plates.
  • the microbial inoculate was allowed to grow and attach itself onto the cover slip for 72 hours to form a mature biofilm.
  • the cover slips were then rinsed with PBS to remove unattached, planktonic bacterium, following the growth of biofilm. Formation of the biofilm was confirmed following crystal violet staining.
  • the PDMS-PIM material (in triplicate) were then placed over the biofilm (on cover slips). Following incubation, the bacteria samples were harvested at intervals of 1 hours, 2 hours, 4 hours and 24 hours.
  • a plate assay was carried out to determine colony forming units of viable bacteria.
  • the harvesting was performed by rinsing the PDMS-PIM material and the coverslip with 10 mL PBS to recover the microbes.
  • Serial dilution (10x) was carried out using phosphate buffered saline (PBS).
  • a cell suspension 40 pL was plated on Luria-Bertani (LB) agar plates. The plates were incubated at 36 °C for 16 to 18 hours and the number of viable microbes indicative of colony forming units (CFU) was recorded. The number of CFUs was multiplied by volume and respective dilution factors to compute for CFU/mL. All experiments were carried out in three replicates.
  • the continuous microbial challenge protocol was similar to the contact killing assay (JIS Z 2801) with an extended incubation period.
  • the biomaterial disk 2.5 cm x 2.5 cm
  • Microbial suspension at 10 6 CFU/mL in the respective media (TSB for S. aureus or YMB for C. albicans or both) was applied on the disk surface and covered with thin plastic films (2 cm x 2 cm).
  • the samples were incubated at 37 °C for stipulated time points. Bacterial suspension was periodically inoculated/refreshed every 48 hours to 72 hours incubation and continued until the endpoint.
  • the respective PDMS and PDMS-PIM materials were rinsed with 10 mL PBS and diluted further at lOx serial dilution with phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • a cell suspension 40 pL was plated on Luria-Bertani (LB) agar plates. The plates were incubated at 36 °C for 16 to 18 hours and the number of viable microbes indicative of colony forming units (CFU) was recorded. The number of CFUs was multiplied by volume and respective dilution factors to compute for CFU/mL.
  • Representative gram negative bacteria ( E . coli), gram positive bacteria (S. aureus ) and fungi (C. albicans ) were used for the assessments. All experiments were carried out in three replicates.
  • Both a red blood cell hemolysis assay and a alian cell viability assay was used to evaluate the biocompatibility of the composite biomaterial.
  • fresh blood mice
  • saline (0.9% NaCl)
  • RBC red blood cells
  • PBS phosphate buffered saline
  • the biomaterial, PDMS or composite PDMS-PIM (5 mg or 10 mg) was added to the wells and incubated for 1 hour at 35°C for RBS lysis reaction to occur.
  • the plates were centrifuged at 2200 rpm/5 minutes following the incubation period. An aliquot (100 pL) of the supernatant was transferred to a new 96 well plate.
  • the hemoglobin released upon RBC lysis was determined spectrophotometrically at 576 nm using a spectrophotometer. The percentage of lysis was calculated using TritonXIOO treated positive control and untreated control RBC. The data was analyzed and expressed as mean and standard deviation of three replicates for quantification.
  • L929 murine fibroblast cells were cultured in DMEM complete media (supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 pg/mL streptomycin) at 37 °C in 100% humidity and 5% CO2.
  • Fibroblast cells were seeded at 1.0 x 10 4 cells/well (100 pL) in 96 well plate for overnight growth. The overnight culture of L929 in 96 well plate were incubated with 5 mg and 10 mg PDMS and PDMS-PIM material.
  • Cell viability assay was carried out using the Alamar Blue reagent according to manufacturer’s protocol (Thermofisher Inc). Briefly 10 pL of Alamar Blue Reagent was added to 90 pi of complete DMEM media (DMEM containing 10% FBS and 1% Penicillin/Streptomycin) in the wells and incubated for 1 to 4 hours at room temperature. Fluorescent intensity of viable cells were read at 570 nm/590 nm. The cell viability was calculated as the ratio of the absorbance of treated cells to the absorbance of the control groups (untreated). All experiments were performed in triplicate in three independent experiments.
  • PDMS polydimethylsiloxane
  • the modified PDMS material was prepared with less than 5% of PIM45-vinyl and the product was denoted as PDMS-PIMx, where x represents weight% of PIM45 in PDMS.
  • PDMS Sylgard®R 184 (Dow Corning Corporation, Midland, Michigan, USA) is a heat curable polydimethylsiloxane (PDMS) supplied as a two-part kit consisting of pre -polymer (base) and cross-linker (curing agent) components.
  • the pre -polymer and cross-linker were mixed at 10:1 weight ratio and casted in a Petri dish (in accordance with the manufacturer’s recommendation).
  • the composite material was degassed under vacuum to remove air bubbles and cured overnight at 70 °C.
  • the cured PDMS sheet was cut into square pieces of 2.5 cm x 2.5 cm, with a thickness around 1 mm.
  • the PDMS samples were then washed with ethanol and dried at 60 °C before use.
  • PIM45-vinyl imidazolium antimicrobial polymer (“PIM45-vinyl”)
  • PIM45 -vinyl was carried out using PIM45 having an imidazole terminal and 4- vinylbenzyl chloride.
  • the imidazolium polymer material, PIM45 was synthesized by adding 4- vinylbenzyl chloride (56 mg, 3.0 eq. of PIM45) to a 1.25 mL EtOH solution containing PIM45 (250 mg, 0.122 mmol).
  • the mixture in a sealed vial, was constantly mixed at 65 °C for 16 hours.
  • the suspension was transferred to 15 mL centrifuge tube and purified by repetitive precipitation with THF.
  • the compound was then dried under reduced pressure at 90 °C. A faint yellow powder of PIM45-vinyl was obtained.
  • the product was verified by proton nuclear magnetic resonance ( ⁇ NMR) spectrum (Fig. 2)
  • PIM45-vinyl (0.011 g to 1.11 g, 0.1 to 10 wt%) in 1 to 10 ml THF was mixed thoroughly with the PDMS base and curing agent at a 10:1 ratio by weight. The solvent and any air bubbles were then removed from the quasi-solid substance by degassing under vacuum and curing at 70 °C overnight.
  • PDMS biomaterials (PDMS-PIM0.5, 1, 2, 5) with different PIM loadings (0.5 wt%, 1 wt%, 2 wt% and 5 wt%) were synthesized.
  • the PDMS sheet cured in a square petri dish was cut into square pieces of 2.5 cm x 2.5 cm, with a thickness around 1 mm. The PDMS samples were rinsed with ethanol and dried at 60 °C before use.
  • Example 5 Contact killing assay The surface antimicrobial properties of the fabricated biomaterials was evaluated using the Japanese Industrial Standard (JIS Z 2801: 2010) protocol. The microbes (in neat media) were exposed to composite PDMS-PIM for 24 hours. The data revealed that PDMS-PIM0.5 and PDMS-PIM1 have potent biocidal activity against E coli, S aureus and C albicans (Fig. 12).
  • a time killing assay was performed with PDMS-PIM1 and PDMS-PIM5 to assess the shortest exposure time needed to eliminate microbes on the surface of PDMS material. It was found that the bactericidal property of PDMS-PIM 1 was effective within 30 seconds of microbial exposure (Fig. 13), exhibiting complete eradication of the microbes in a single inoculation.
  • the surface antimicrobial assay was repeated over longer durations to study the stability and durability of the PDMS-PIM biomaterial.
  • the antimicrobial PDMS-PIM disks were challenged with respective microbes every 48 to 72 hours. The study was terminated at 45 days for single culture while three time points at 15 days, 30 days and 60 days were recorded for co-culture assays. In a single culture assay, complete bactericidal effect was observed for up to 45 days for PDMS- PIM biomaterials against both S. aureus and C. albicans. In contrast, the unmodified PDMS biomaterial exhibited colonization at 10 n CFU/mL and 10 10 CFU/mL for S. aureus and C. albicans, respectively (Table 4).
  • a S. aureus and C. albicans co-culture was initiated in the laboratory and tested on the composite PDMS-PIM materials (Fig. 14). Microbes in this co-culture was identified by the phenotypic characteristics of the colonies. S. aureus forms golden colored colonies that can be distinguished from the white colonies formed by C. albicans. PDMS-PIM1, PDMS-PIM2 and PDMS-PIM5 materials were shown to fully eradicate co-cultured bacterial and fungal colonization after 15 days in culture. At the 30 day time point, reduction in antimicrobial potency was observed for PDMS- PIM1 against both S. aureus and C. albicans.
  • PDMS-PIM2 While PDMS-PIM2 exhibited moderate efficacy against C. albicans, it fully eradicated S. aureus (Fig. 14), in a dose dependent manner (Table 5). It is noteworthy that PDMS-PIM5 eliminated both S. aureus and C. albicans even after 60 days in co-culture, demonstrating excellent durability. Such qualities make PDMS-PIM5 suited for practical medical applications, especially in long-term indwelling urinary catheters which typically could remain in place for more than 30 days.
  • the PDMS-PIM materials recovered on day 15 of co-culture incubation were subjected to a further 2 cycles (15 days / cycle) of co-culture incubation (Fig. 15). It was found that PDMS-PIM5 could last for 3 cycles of 15 days each (45 days in total) with washing and rinsing carried out every 15 days. Thus, PDMS-PIM5 exhibited stable antimicrobial properties after 3 cycles of periodical rinsing or washing. Reuse of the recovered PDMS-PIM material also confirmed that PDMS-PIM5 could effectively eradicate colonization of bacteria in cocultures.
  • Example 6 Efficacy against clinical isolates
  • PDMS-PIM 1 In order to assess the potential value of PDMS-PIM in medical device fabrication, the composite biomaterial was tested for its antimicrobial efficacy on 11 clinical isolates. These microbes represented various drug/antibiotic resistant (MDR) species often related to nosocomial infections in healthcare settings, especially in catheter associated urinary tract infection (CAUTI). It was found that PDMS-PIM 1 inhibited the growth of all the clinical isolates including Enterococcus faecium-VR Enterococcus; Staphylococcus aureus-MRSA; Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter species-ESBL Enterobacter, S. marcescens, E. coli, C. parapsilosis, C. tropicalis and C. auris (Fig. 16).
  • MDR drug/antibiotic resistant
  • ESKAPE refers to six nosocomial pathogens that exhibit multidrug resistance (MDR) and virulence, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
  • MDR multidrug resistance
  • virulence namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.
  • MDR bacteria are the most notorious group of bacteria that impose serious threats to the global health, including death by rendering even the most effective drugs ineffective.
  • PDMS-PIM biomaterial could effectively and efficiently eliminate the colonization by all the members of ESKAPE group of pathogens upon a 24 hour incubation, as well as MRS A and Vancomycinresistant Enterococcus (VRE) pathogens (Fig. 16) and respective Candida sp of clinical isolates.
  • VRE Vancomycinresistant Enterococcus
  • Biofilms were formed on polystyrene, PDMS, low density polyethylene (tube) and glass cover slips and were subsequently exposed to the PDMS-PIM1 material. It was found that PDMS-PIM1 could effectively eradicate biofilm colonies on all the surfaces. A time dependent assay showed that biofilm colonies on glass cover slips were eradicated within 1 hour of contact with PDMS-PIM composite material (Fig. 17). Biofilm formed in a tube could be eliminated using an eluate from 100 mg of PDMS-PIM1 material in 10 mL PBS solution (Fig. 18).
  • Biocompatibility assays also showed that the PDMS- PIM biomaterial is biocompatible and safe. 10 mg PDMS-PIM5 exhibited 1.44 ⁇ 0.56 % hemolysis, while 10 mg PDMS-PIM1 showed 0.3 ⁇ 0.26% hemolysis (Fig. 19).
  • Cell viability assays using murine derived fibroblast L929 cells also proved that the PDMS-PIM composite biomaterial is non-toxic to mammalian cells in culture, and as such, are biocompatible.
  • Catheter associated infections pose a serious health threat and remains a challenge in healthcare settings worldwide. Patients requiring indwelling catheters, especially urinary catheters, often get infections within days of catheter use. Though antimicrobial-coated catheters are currently available, their efficacy in clinics are still far from satisfactory.
  • PDMS-PIM antimicrobial imidazolium polymer
  • PDMS-PIM biomaterial exhibited potent antimicrobial activity and fast killing property with 30 seconds of exposure to the microbes resulting in complete eradication of the microbes.
  • the material demonstrated long lasting activity that could inhibit the colonization of both S. aureus and C. albicans for at least 60 days in culture. Eradication of microbial colonization on a subsequent inoculation of the recovered and reused material further confirmed that the PDMS-PIM material could be recycled for usage.
  • the PDMS-PIM material does not cause any change or adverse effects on the mechanical performance of the fabricated materials, either pristine or used products. The tensile property of the fabricated materials were also not altered with extended use, thus confirming their durability.
  • the ESKAPE family of MDR bacteria are the most notorious group of bacteria that pose a serious global health threat and account for up to 87% of all hospital acquired infections. This family of bacteria has been listed as “high priority” under the WHO priority listing. It is noteworthy that PDMS-PIM biomaterials could efficiently and effectively inhibit the colonization of all the members of ESKAPE group of pathogens as well as the WHO priority pathogens such MRSA and VRE. The susceptibility of these drug resistant bacteria towards PDMS-PIM as shown in Example 6 further emphasises that the composite biomaterials in this disclosure in the form of catheters could prove beneficial in preventing catheter-associated infections especially in CAUTI.
  • the composite PDMS-PIM biomaterial is biocompatible as shown in Example 8 and exhibits stable antimicrobial activity up to at least 6 months storage at room temperature, as indicated in Example 9.
  • the antimicrobial polymer as defined above may be suitable for use in any kind of surface or product that requires antimicrobial properties.
  • the antimicrobial polymer may be useful in medical devices such as catheters, as the antimicrobial polymer itself may be used as the bulk material to form the medical device, without relying on additional antimicrobial treatment to effect the antimicrobial property.
  • the antimicrobial polymer as defined above may be useful in other medical devices such as intravenous lines, endotracheal tubes, nephrostomy tubes, gastronomy tubes, traction pins, defibrillators joint implants, shunts, and endoscopic catheters.
  • the method of preparing the antimicrobial polymer may be useful in easily preparing medical devices having inherent antimicrobial properties.

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Abstract

La présente invention concerne un polymère antimicrobien comprenant un polymère de polysiloxane réticulé avec un agent de réticulation antimicrobien, l'agent de réticulation antimicrobien comprenant un agent antimicrobien terminé aux deux extrémités par un lieur qui se lie de manière covalente au polysiloxane, l'agent de réticulation antimicrobien comprenant pas plus de 10 % en poids du poids total du polymère antimicrobien. La présente invention concerne également un procédé de préparation du polymère antimicrobien et les utilisations du polymère antimicrobien.
PCT/SG2022/050147 2021-03-24 2022-03-17 Matériaux antimicrobiens à large spectre WO2022203596A1 (fr)

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