WO2025003679A1

WO2025003679A1 - Antimicrobial coated substrate

Info

Publication number: WO2025003679A1
Application number: PCT/GB2024/051654
Authority: WO
Inventors: Tim NICHOL; Tom Smith
Original assignee: Sheffield Hallam University
Current assignee: Sheffield Hallam University
Priority date: 2023-06-29
Filing date: 2024-06-27
Publication date: 2025-01-02
Anticipated expiration: 2025-12-29
Also published as: GB202309937D0

Abstract

The invention relates to a substrate having a hybrid coating chemically bonded thereto, wherein the coating optionally comprises an antimicrobial component releasably captured within a polysiloxane based network of silicon-carbon bonds and silicon- oxygen bonds. The invention extends to a biocompatible implant comprising such a coated substrate, and a method of preparing such a coated substrate.

Description

Antimicrobial Coated Substrate

Field of the Invention

The invention relates to an antimicrobial coated substrate. More specifically, the present invention relates to a substrate having a hybrid coating chemically bonded thereto to provide a physical barrier against microbial contamination, wherein the coating comprises a polysiloxane-based network of silicon-carbon bonds and siliconoxygen bonds, and wherein the coating optionally comprises an antimicrobial component releasably captured within said polysiloxane-based network. Also provided are methods for preparing such coated substrates, and biocompatible implants comprising such coated substrates.

Background of the Invention

Many skeletal defects, which may result from traumatic injuries, congenital defects, aging or neoplastic disorders, require correction through challenging reconstructive surgery, many of which will require implantation of a prosthetic, i.e. an artificial device configured for implantation into the body to replace a missing or damaged body part. A typical, non-limiting, example is total hip arthroplasty involving replacement of both the acetabulum and the head and neck of the femur. As the general population ages, the number of arthroplasty procedures undertaken have increased significantly over the last 20 years.

Following surgery for prosthetic implantation, including arthroplasties, a proportion of patients will develop serious infections, which require post-surgical exchange or loss of the prosthetic, alongside continued treatment with anti-infectives over prolonged periods of time. The pathogenesis of these prosthetic joint infections results from the formation of a bacterial biofilm, resultant from bacteria adhering to the surface of the prosthetic joint. There is, therefore, a continuing demand for anti-infective materials for use in such reconstructive processes. The demand for such materials is not limited to orthopaedic and craniomaxillofacial surgery, but also for use in repairing endodontic defects and in dental implants. Arthroplasty procedures, of the type indicated above, may be divided into two general types namely, cemented and cementless procedures, with selection between the two being determined primarily by bone density. In a cemented procedure, a bone cement such as poly(methyl 2-methylpropenoate) is used to attach the prosthetic components to the bone. Cement based arthroplasty procedures significantly reduce the risk of surgical infection. In particular, the use of antimicrobial (typically gentamicin, clindamycin, vancomycin or combinations thereof) loaded bone cement has been shown, in isolation, to significantly reduce infection rate. The antibiotic loaded bone cement provides a direct, continuous prophylactic dose to the infected area. However, the antibiotic release into the bloodstream must be controlled so as to avoid toxic and allergic responses, and to prevent a low, prolonged release that would increase selection pressures leading to antimicrobial resistance.

However, cement-based arthroplasty is disadvantageous, as joint manipulation over time causes wear of the cement, which leads to the formation of stress cracks in the cement and ultimately cement erosion and an unstable joint. Biological fixation, without cement, is therefore advantageous, albeit with a slower recovery rate due to the time required for secondary/biological fixation (bone in growth into the prosthetic).

There is, therefore, a need for a substrate, and in particular a biocompatible implantable substrate, which is particularly suitable for use with implantable prosthetics, where cement is not utilised, that provides a barrier against infection, and optionally exhibits antibacterial characteristics to significantly reduce, and preferably eliminate, post operative infections.

WO 2006/115805 discloses a biocompatible composite for use in contact with body fluids for orthopaedic implantation. The composites are formed as a sol-gel coating layered onto a substrate with a pharmaceutically active compound incorporated in the coating. In particular, the sol-gel layer may comprise an antibiotic that is configured to be released following implantation of the composite so as to reduce the risk of post surgical infection.

However, a number of disadvantages exist with conventional antibacterial sol-gel coatings including primarily, the integrity of such coatings and the rate and duration of antibiotic release in vivo. WO 2010/023483 discloses a biocompatible implant comprising a room temperature curable, sol-gel derived hybrid organic-inorganic silica-based coating that is configured to encapsulate an antimicrobial component, only releasing said antimicrobial component in a controllable manner upon introduction of a fluid, e.g., a biological fluid, to the organic-inorganic network.

However, whilst the coatings disclosed in WO 2010/023483 addressed many of the abovementioned disadvantages, in particular, those of coating integrity and rapid I uncontrolled antimicrobial release, associated with conventional antibacterial sol-gel coatings, a number of disadvantages still exist. In particular, and whilst the coatings can be formed by curing at room temperature (and so permitting the incorporation of heat-sensitive antimicrobial agents), the curing process occurs over several hours. Therefore, whilst such a slow curing process may be tolerated for a coating that applied to a medical implant at the manufacturing stage, it would not be suitable for application at the point of use, e.g., in the operating theatre.

Accordingly, it is an aim of the present invention to develop a room temperature curable coating for use in biocompatible implants that, like those of WO 2010/023483, that comprises a sol-gel derived, strongly adhered coating configured for the controlled release of an antimicrobial component, but which can also be rapidly cured at ambient temperature, thereby opening the possibility of such coatings be applied to medical implants at the point of use.

Statements of Invention

The present invention, in its various aspects, is as set out in the accompanying claims.

The inventors provide a substrate comprising a sol-gel derived coating that provides a physical barrier against microbial contamination and, optionally, is configured for the controlled release of an antimicrobial component. In particular, the coating is configured such that, when present, the antimicrobial component is captured within an organic-inorganic oxide network and is released only at the appropriate time (e.g., during and post surgery), allowing the substrate to be stored prior to use, without losing its antimicrobial functionality. As indicated above, controlled release is of considerable importance given the risk of toxic and allergic responses to the antimicrobial and/or the risk of antimicrobial resistant strains. The present sol-gel derived coating is configured specifically provide a physical barrier against microbial contamination and, optionally, to encapsulate an antimicrobial component and to release it only in response to the introduction of a fluid, in particular a biological fluid, on and into coating network.

The inventors provide both a method of preparing the barrier, and optionally an antimicrobial, coating and a barrier I antimicrobial coated substrate. Turning firstly to the method, the formulation of the sol prior to application on the substrate, has been optimised to not only to increase the available storage time of the sol prior to application and to ensure the effectiveness of the antimicrobial-substrate coating is not reduced, at least below required levels, but also to rapidly cure at ambient temperature. Accordingly, there may be provided a two-stage preparatory method in which any antimicrobial to be added is independently suspended in a solution optimised for storage of the antimicrobial, which can then be mixed with the separately prepared sol a short time prior to coating. Such a process is particularly suitable the incorporation of unstable antimicrobials.

The organic-inorganic oxide sol is optimised such that the resultant solid organic- inorganic oxide network provides a barrier against microbial contamination and, when present, does not impede a continuous or otherwise controlled release of the antimicrobial during and after surgical implantation (where the substrate is a prosthetic, for example). Importantly, when present, the antimicrobial is immobilised within the dry, cured coating allowing the implant to be physically handled and manipulated by a surgeon during arthroplasty procedures.

However, when a fluid, in particular a bodily fluid, is introduced into the porous network, the antimicrobial, if present, is mobilised to provide controlled release into the patient. As will be appreciated, the encapsulation and indeed the dispersion of the antimicrobial within the sol-suspension mixture prior to coating is important to optimise so as to achieve the desired concentration distribution of antimicrobial through the thickness of the coating.

The inventors provide a sol-gel derived biocompatible coating that is optimised specifically to cure rapidly (i.e., within 10 minutes, and preferably within 5 minutes or less) to touch at ambient room temperatures (i.e., 25°C or less). Further, once cured, a thick coating is provided that adheres firmly to the substrate. The coating comprises comparative, if not enhanced, durability over known systems, advantageously exhibiting minimal wear resistance whilst providing a physical barrier against microbial contamination and, optionally, a controlled, sustained release of the biologically active compound from the porous network.

The inventors have realised that by forming the coating via a sol-gel process using a siloxane monomer such as hexamethyldisiloxane and optionally further precursors such as a silane and/or a silicate for the sol component, a porous network is created is ideally suited as a coating for biological implants such as prostheses and fixation devices, including screws, pins, bone plugs and the like. Utilising a siloxane monomer, in contrast to a polysiloxane precursor as disclosed in WO 2010/023483, is advantageous for a number of reasons, in particular, such coatings have been found to rapidly cure at low temperature whilst surprisingly retaining desirable coating properties conventionally associated with the use of polysiloxane precursor (e.g. increased bonding strength to the substrate; improved flexibility of the coating; controlled porosity of the network; and a crack free, non-brittle coating.

The coating thickness can be controlled as the siloxane monomer precursor may be readily cross linked with other particles during polymerisation and so forming part of the resultant network. Cross linking agents and curing agents may be incorporated at the sol-gel stage so as to facilitate network formation during gelation.

Hydrophobicity has been found to be particularly important for the controlled release the antimicrobial component. This may be tailored by variation of any one or a combination of i) the concentration and/or chain length of polysiloxane formed via polymerisation of the precursors in the sol-gel (and the resulting coating network); and ii) the extent and nature of functional groups extending from the polysiloxane.

The polysiloxane may comprise functional groups/or functionalised side chains extending from the main Si-0 backbone. These functional groups and side chains may comprise any oxygen or nitrogen based groups with functionalised side chains comprising for example, acrylic, epoxy or other functionalised groups including organosilanes and/or hybrid organic-inorganic silicate, siloxane and silane compounds.

The synergistic combination of the Si-0 and Si-C bonds provides for the possibility of creating porous coatings having a thickness as high as around 100 pm, with coatings of the order of 5 - 10 m being routinely prepared. This is in contrast to many conventional sol-gel films in this field where maximum coating thickness of not greater than 200 nm are possible without undesirable cracking. Importantly, the coating may be configured to remain in contact with the substrate for significant time periods (of the order of months or years) prior to degradation. Accordingly, the antimicrobial component, incorporated within the porous network, may be released into a biological environment for a much greater time period over many existing systems. The longevity of the coating is also advantageous so as to provide a physical batter against microbial contamination, and to prevent corrosion and degradation of the coated implant after the pharmaceutically active compound has been completely released from the network.

It is also expected that, consistent with previous observations disclosed in Bone Joint J 2021 ; 103-B(3):522-529 using coatings formed from a polysiloxane precursor as disclosed in WO 2010/023483, the coated substrates of the present invention will not impair bone healing rates.

The present coating may be formed as a single or multiple layer system on the substrate. Importantly, when present, the antimicrobial is released from the network whilst the coating is maintained at the substrate. By repeating coating and curing steps during formation, it is possible to create a multilayer sol-gel derived coating in which, for example, a layer positioned towards the substrate- coating interface comprises different chemical and/or physical/mechanical properties to a layer at the outermost region of the coating. Importantly, the hydrophobic property of the coating may be tailored by adjustment of the relative concentrations of the silane, silicate and/or siloxane precursors so as to optimise the release rate of the antimicrobial from the network and the stability of the coating at the substrate.

Significant reductions in curing times to achieve the desired cross- linking/condensation of the network are also possible with the coatings of the present invention compared to those of WO 2010/023483. In particular, cure times of around 1 hour may be achieved at temperatures between 55° - 75° for the coatings of WO 2010/023483, whereas ambient temperature cure times of less than 10 mins, and preferably less than 5 minutes, may be achieved for the coatings of the present invention. According to a first aspect of the invention there is provided a substrate having a hybrid coating chemically bonded thereto, the coating obtainable by a sol-gel process using a siloxane monomer precursor of General Formula (I):

(R¹)3-Si-O-Si-(R¹)3 General Formula (I); wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6, preferably a C1-3 alkyl, group; and wherein said coating comprises a polysiloxane based network of silicon-carbon bonds and silicon oxygen bonds.

In preferred embodiments, said coating comprises an antimicrobial component releasably captured within said polysiloxane based network of silicon-carbon bonds and silicon oxygen bonds.

In particular, the antimicrobial component is releasably captured within said network in response to the introduction of a fluid into the coating. Preferably, the polysiloxane- based network is formed as a porous network, allowing fluid to flow into and out of the network. Porosity of the network may be controlled at the sol- gel stage of the process so as to achieve the desired release rate of antimicrobial component.

In preferred embodiments, the siloxane monomer precursor of General Formula (I) is hexamethyldisiloxane (HMDS), i.e. a compound of Formula (la):

(CH₃)3-Si-O-Si-(CH₃)3 Formula (la)

Optionally, the substrate comprises or consists of a metal, preferably a metal selected from iron, tantalum, tungsten, gold, silver, copper, nickel, cobalt, chromium, magnesium and/or zinc, or any alloy thereof. Preferred metal alloys are stainless steel, a titanium based alloy, a cobalt based alloy, a chromium based alloy and/or a magnesium based alloy.

Alternatively the substrate may be non-metallic. Suitable non-metallic substrates include ceramics or glass ceramic hybrids; pyrolytic carbon; fiberglass; textiles; plastics, preferably a plastic selected from a polyethylene, polycarbonate, polypropylene, polystyrene, polyvinyl chloride, polyurethane, polyamide, polytetrafluoroethylene, polymethyl methacrylate and polyetheretherketone; mineral based materials, preferably hydroxyapatite or zirconia; silica, silicate or a silicon containing polymer; glass; latex; or rubber. The substrate may comprise a medical tool or medical apparatus associated with patient care and/or surgical and/or dental procedures, including orthopaedic, craniomaxillofacial and/or dental surgery, or the correction of endodontic defects. In addition, the substrate may comprise a structure associated with food preparation including by way of example, food preparation surfaces. Moreover, the substrate may comprise structures designed to be frequently contacted by liquid, in particular water, such as wash basins, toilets, baths, showers and tiles etc.

Optionally, the coating may comprise a dopant species captured or chemically bonded to the polysiloxane based hybrid organic-inorganic oxide network. In particular, the present coating is preferably formed by incorporating particles, optionally nano particles (i.e. particles of any shape with at least two dimensions in the range from about 1x10“⁹ to 1x10“⁷ m), at the sol-gel stage of coating formation. The particles may comprise a silane, a silicate and/or other dopant particles such as Y-AI2O3 and hydroxyapatite.

The siloxane monomer derived polysiloxanes within the network may be chemically bonded to one another by cross linking agents. The cross linking agents may comprise non- functionalised organic hydrocarbons or functionalised hydrocarbons or other organic, inorganic and/or organic-inorganic cross linking agents. The particles incorporated in the sol-gel phase chemically bond to the polysiloxane during the condensation process. The resulting network comprises substantially linear polysiloxane with Si-0 repeating units and organic side chains extending from the main Si-0 backbone. The organic side chains may comprise any alkyl, aryl and/or mixed alkyl-aryl groups. These alkyl or aryl groups may be substituted with additional functionalised groups along the Si-0 backbone, where the functionalised groups comprises any elements selected from period table groups 5 to 7 including in particular nitrogen, phosphorus, oxygen, sulphur and chlorine.

Where the organic side group, directly bonded to the Si-0 backbone is alkyl, the alkyl group may comprise between 1 to 20 carbon atoms. Optionally, the alkyl, aryl and/or mixed alkyl-aryl groups that are attached directly to the Si-0 backbone may be functionalised by comprising nitrogen, phosphorous, oxygen, sulphur and/or chlorine atoms. Optionally, the Si-0 backbone may comprise at least one functional side chain bonded directly to either the Si-0 backbone or at least one of the alkyl, aryl or alkyl- aryl side groups. When present, the antimicrobial component may comprise a uniform concentration distribution through the coating thickness from an external facing region to the substrate-coating interface. Alternatively, the coating may comprise a substantially non-uniform concentration distribution of the antimicrobial component through the coating thickness from the external facing region to the substrate-coating interface.

The coating may comprise a multilayer structure, wherein each layer optionally has a different concentration of the antimicrobial component. In such embodiments, one or more layer may be free of any antimicrobial components. This would allow different concentrations of antimicrobial to be released over time. For example, a concentration rich layer of antimicrobial may be provided towards an outermost region of the coating so as to release large concentrations during and immediately after surgery whilst an inner coating layer may have a relatively lower antimicrobial concentration or even be free of said antimicrobial. The multilayer structure may be formed by multiple sol-gel coating and curing steps.

Optionally, for multi-layer systems a region of the coating positioned towards the substrate-coating interface may be more hydrophobic than a region positioned towards the outermost surface of the coating. Also, the coating at the substrate-coating interface may comprise a greater hardness than the outmost region of the coating. Optionally, the coating or outermost layer of the coating at the substrate-coating interface may comprise hydroxyapatite so as to improve bone-in-growth.

The present coating may also further comprise one or more biologically active components configured to promote in vivo bone regrowth at the region of the coating, where for example the coating is applied to a prosthesis. Such functionalised components may also be configured to prevent destruction of the bone by osteoclasts around the prosthesis, to generally promote desired cell proliferation and to improve the performance of the prosthesis by molecular interaction and/or chemical reaction with the host's biological system in vivo. Such additional biologically active components may include: osteogenic proteins (including but not restricted to one or more recombinant human bone morphogenic proteins); carboxymethyl chitosan; other biologically active proteins, DNA, extracellular matrix components and analogues thereof; calcium based compounds; phosphorus based compounds; biologically active agents derived from vitamins; multi-molecular complexes and assemblies; and nanoparticles. Dopant nanoparticles considered to be advantageous for bone regrowth include active species containing calcium and/or phosphorous and agents derived from vitamins.

Alternatively or additionally, the present coating may further comprise one or more components selected from anti-inflammatory, analgesic, antineoplastic and anti- angiogenic agents. Such pharmaceutically active compounds are well known to those of skill in the art, and examples of which can be readily chosen and incorporated into the present coating without undue burden.

The coating of the present invention may utilise any silicate based precursor including, but not limited to, an organosilicate and/or a silane based precursor including in particular an organosilane.

As used herein, the term 'hybrid coating' refers to a sol-gel derived coating formed from at least two different silicon based precursors. Accordingly the hybrid coating of the subject invention comprises at least a first silicon centre, derived from a first precursor bonded to carbon and a second silicon centre, derived from a second precursor bonded to oxygen. That is, at least two silicon centres differ throughout the network by the number of respective carbon and/or oxygen bonds at each silicon centre.

In particular, the sol-gel derived polysiloxane based coating may be derived from any one or a combination of the following additional precursors incorporated within the coating network during the sol-gel phase: any organically modified silane selected from the group consisting of alkylsilanes, methyltrimethoxysilane, methyltriethoxysilane, dimethyldiethoxysilane; trimethylethoxysilane; vinyltrimethoxysilane; vinyltriethoxysilane; ethyltriethoxysilane; isopropyltriethoxysilane; butyltriethoxysilane; octyltriethoxysilane; dodecyltriethoxysilane; octadecyltriethoxysilane; aryl-functional silanes; phenyltriethoxysilane; aminosilanes; aminopropyltriethoxysilane; aminophenyltrimethoxysilane; ammopropyltrimethoxysilane; acrylate functional silanes; methacrylate-functional silanes; acryloxypropyltrimethoxysilane; carboxylate; phosphonate; ester; sulfonate; isocyanate; epoxy functional silanes; chlorosilanes; chlorotrimethylsilane; chlorotriethylsilane; chlorotrihexylsilane; dichlorodimethylsilane; trichloromethylsilane; N,O-Bis (trimethylsilyl)-acetamide (BSA); N,O-Bis (trimethylsilyl) trifluoroacetamide (BSTFA); hexamethyldisilazane (HMDS); N- methyltrimethylsilyltrifluoroacetamide (MSTFA); N-methyl-N-(t- butyldimethylsilyl)trifluoroacetamide (MTBSTFA); trimethylchlorosilane (TMCS); trimethylsilyimidazole (TMSI); and combinations thereof.

In particularly preferred embodiments, the present coating may be derived from any one or a combination of the following additional precursors: tetraethoxy orthosilicate; tetraethyl orthosilicate (TEOS); methyltriethoxy orthosilicate (MTEOS); phenyltriethoxy orthosilicate (PTEOS); octyltriethoxy orthosilicate (OTEOS); dimethyldiethoxy orthosilicate (DMDEOS); methyltrimethoxy orthosilicate (MTMOS); trimethoxymethyl silane (MTMS); phenyltrimethoxy orthosilicate (PTMOS); tetramethoxy orthosilicate; and tetramethyl orthosilicate (TMOS). An exemplary coating is derived from the following additional precursors: TEOS, TMOS and MTMS.

The term 'alkyl' refers to a linear, branched, cyclic, or any combination thereof saturated hydrocarbon. The term 'substituted’, when used in the context of a hydrocarbon (e.g. an alkyl) group, refers to one or more of the hydrogens on the hydrocarbon group being replaced by another substituent, such as hydroxyl, cyano, nitro, mercapto, alkylthio, halo, alkylamino, dialkylamino, alkoxy, and tri alkoxysilyl.

The antimicrobial component of the coating is not particularly limited, and may comprise any one or a combination of compounds having antimicrobial, and preferably broad spectrum antimicrobial activity. As used herein, an antimicrobial component includes any component that provides antibacterial, antiviral and/or antifungal activity. Preferably the antimicrobial component is an antibacterial component.

However, preferred antimicrobial components for use in the coating of the invention are selected from one or more of the following: a protease; a furanone; an aminoglycoside; a beta-lactam; an antimicrobial peptide; a lipopeptide such as daptomycin or caspofungin; a glycopeptide; a macrolide; a lincosamide such as clindamycin; a ketolide such as telithromycin; an antifungal agent; a tetracycline; a quinolone; rifampicin; fusidic acid; colistin; a nitroimidazole antibiotic such as metronidazole; and fosfomycin.

A bacteriophage may also be provided as the antimicrobial component for use in the coating of the invention, either alone or in combination with one or more antimicrobial components as identified above. Preferred aminoglycosides are selected from: gentamicin; amikacin; arbekacin; kanamycin; neomycin; netilmicin; paromomycin; rhodostreptomycin; streptomycin; tobramycin; and/or apramycin.

Preferred beta-lactams are selected from: ampicillin; amoxicillin; benzyl penicillin; oxacillin; cloxacillin; flucloxacillin; ampicillin/flucloxacillin; temocillin; azlocillin; mezlocillin; piperacillin; piperacillin/tazobactam; carbenicillin; ticarcillin; ticarcillin/clavulanate; dicloxacillin; flucloxacacillin; nafcillin; procaine benzylpenicillin; avibactam; phenoxymethylpenicillin; bezathine benzylpencillin; meropenem/vabrobactam; ampicillin/sulbactam; ticarcillin/clavulanic acid; ticarcillin; clavulanic acid; co-amoxiclav; piperacillin/tazobactam; aztreonam; Cephalosporins such as cefazolin, cefalexin, cefaclor, cefotaxime, cefoxitin, ceftazidime, cefuroxime, ceftaroline, cefotetan, cefixime, cephalosporin C, ceftaroline, ceftobiprole, ceftriaxone and cefepime; and Carbapenems such as dorapenem, meropenem, imipenem and ertapenem.

Preferred antimicrobial peptides are selected from: polymyxins; and/or nisin.

Preferred glycopeptides are selected from: vancomycin; and/or teicoplanin.

Preferred macrolides are selected from: erythromycin; clarithromycin; azithromycin; dirithromycin; roxithromycin; and/or telithromycin.

Preferred tetracyclines are selected from: tetracycline; doxycycline; and/or tigecycline.

Preferred quinolones are selected from nalidixic acid; ciprofloxacin; levofloxacin; gatifloxacin; moxifloxacin; and/or chloroquin.

Preferred antifungal agents are selected from azoles, polyenes, allyam ines, echinocandins, flucytosine, grisofulvin and risofulvin.

Preferred azoles are selected from: fluconazole, itraconazole, clotrimazole, ketoconazole, voriconazole, posaconazole, isavuconazonium, oteseconazole, miconazole.

Preferred echinocandins are selected from: caspofungin, micafungin, anidulafungin Preferred polyenes are selected from: nystatin, natamycin, amphotericin B.

Preferred allylamines are selected from: terbinafine, naftifine. In particularly preferred embodiments, the antimicrobial component is an aminoglycoside, and is most preferably gentamicin.

According to a second aspect of the invention there is provided a biocompatible implant comprising the coated substrate according to the first aspect of the invention. In some embodiments the biocompatible implant is for permanent implantation in the body. In other embodiments, the biocompatible implant is for transient implantation in the body.

As used herein, the term ‘biocompatible implant’ refers to any foreign material configured for implantation into a mammalian subject, which is formed partially or preferably entirely from one or more coated substrates according to the first aspect of the invention.

Preferably, the biocompatible implant is a prosthetic or fixation device, including by way of example, bone screws, pins, rivets, support structures, cadges, nails, plates, wire or plugs. As used herein, the term “prosthetic” refers to an artificial device configured for implantation into the body to replace a missing or damaged body part, non-limiting examples of which include; artificial limbs, artificial joints or joint surfaces, dental implants, heart valves, stents, cosmetic implants, implantable contraceptives, and/or synthetic implantable such as grafts, cartilage repair devices.

According to a third aspect of the invention there is provided a method of preparing a coated substrate, preferably a coated substrate according to the first aspect of the invention, the method comprising: (i) preparing a sol using a siloxane monomer of General Formula (I):

(R¹)3-Si-O-Si-(R¹)3 General Formula (I), wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6, preferably a C1-3 alkyl, group; (ii) coating the surface of a substrate with said sol; and (iii) curing said sol on the surface of said substrate to form a sol-gel derived hybrid coating chemically bonded to the substrate, wherein said coating comprises a polysiloxane based network of silicon-carbon bonds and silicon oxygen bonds.

In preferred embodiments, the coating contains an antimicrobial component. In such embodiments, the method comprises: (i) preparing a sol using a siloxane monomer of General Formula (I): (R¹)3-Si-O-Si-(R¹)3 General Formula (I), wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6, preferably a C1-3 alkyl, group; (ii) providing a preparation comprising an antimicrobial component; (iii) combining said sol and said antimicrobial component to form a mixture; (iv) coating the surface of a substrate with said mixture; and (v) curing said mixture on the surface of said substrate to form a sol-gel derived hybrid coating chemically bonded to the substrate, wherein said antimicrobial component is releasably captured within a polysiloxane based network of silicon-carbon bonds and silicon-oxygen bonds.

In preferred embodiments, the curing step comprises curing said sol or said mixture on the surface of said substrate at a temperature of from about 1 °C to about 30°C, and more preferably at ambient (i.e from about 15 to about 25°C) temperature. Preferably, said sol or said mixture is cured on the surface of said substrate for a time period of from about 2 minutes to about 20 mins, and more preferably about 5 to 10 mins. Most preferably, said sol or said mixture is cured for a period of about 5 minutes.

Preferably, the preparation further comprises sterilising the coating, for example prior to implantation. Sterilisation may comprise exposure of the coating to gamma radiation, or by any other suitable physical and/or chemical means. Alternatively, the method may entail the combination of pre-sterilized components.

The method is particularly useful for providing an antimicrobial releasing coating to a medical tool or biocompatible implant such as a prosthetic or fixation device at the point of use.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprise” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics or compounds described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

Embodiments of the invention will now be described by way of example only with reference to the following figures where:

Figure 1 illustrates a cross sectional view of a prosthetic comprising a coating of the according to the present invention;

Figure 2 shows sol gel coated test substrates for gentamicin elution testing: A) Titanium B) Ti coated Titanium C) HA coated Titanium

Figure 3 shows sol gel coated, 3D printed titanium test substrates for gentamicin elution testing.

Figure 4 shows the elution of gentamicin from sol gel coated test substrates at specific time points over a 168 hour period. Results are shown as mean +/- SD. A) Ti-coated Titanium (Ti-Ti); Polyetheretherketone (PEEK); HA-coated Titanium (HA-Ti); and 3D printed titanium (3D). B) Al-coated Titanium (TiAl); Cobalt-Chromium (CoCr); Al- coated Cobalt Chromium (CoCrAI); and Titanium (Ti).

Figure 5 shows the cumulative total elution of gentamicin from sol gel coated test substrates over a 168 hour test period. Results are shown as mean +/- SD (n=5). The minimum inhibitory concentration (MIC) of gentamicin for Staphylococci is shown as a red dotted line. Ti-coated Titanium (TiTi); Polyetheretherketone (PEEK); HA-coated Titanium (HA-Ti); 3D printed titanium (3D printed); Al-coated Titanium (TiAl); Cobalt- Chromium (CoCr); Al-coated Cobalt Chromium (CoCrAI); and Titanium (Ti).

Figure 6 shows BS EN ISO 2409:2020 standard for measuring coating delamination.

Figure 7 shows examples of cross scratch test samples showing A) PEEK, B) Ti and C) CoCr

Figure 8 shows Barrier testing of sol gel coating. Cell culture filter devices were coated with 50pl, 100pl or brushed on sol gel. After curing the filters were inoculated and incubated for 72h. Growth of plates can be seen above and growth every 24h was recorded as - for no growth or +, ++ and +++ for growth

Figure 9 shows SEM image and corresponding silicon EDX image analysis following post interference fit coating integrity analysis. The top image shows the SEM analysis and below the EDX analysis of Si. The presence of Si is indicated as a yellow colour.

The present invention finds particular application within the medical field and in particular for coating devices to be implanted in the human or animal body. Referring to Figure 1 , a prosthetic 100 is coated with the present antimicrobial encapsulating network 101. In particular, Figure 1 illustrates a prosthetic 100 coated over its entire surface area with coating 101. However, as will be appreciated, the coating may be applied over specific regions of the outer surface of the substrate 100 as desired.

The silane and silicate based sol, which forms the bulk of coating 101 , is prepared independently of the aqueous-based antimicrobial suspension. This allows the sol- gel component to be optimised to create the desired porous network whilst optimising the antimicrobial suspension to ensure the biologically active species maintains its viability in successfully inhibiting bacterial colonisation.

The silane and silicate based sol and the microbial suspension are then mixed prior to coating on substrate 100. Coating 101 is applied by any conventional technique including dip, spray or spin techniques. Coating 101 is then cured so as to allow the sol- gel to bond chemically with the outmost surface of substrate 100 so as to provide a secure coating resistant to the various torsional, sheer and impact loading forces imparted to prosthetic 100 as the joint is manipulated throughout the lifetime of the implantation. The hybrid organic- inorganic coating can be configured specifically to chemically bind to a plurality of different substrate materials including for example, glass, metal, ceramic and mineral surfaces.

Coating 101 is positioned in direct contact with the outer surface of substrate 100 to form a substrate-coating interface 103. An external facing surface 102 of coating 101 is therefore presented and configured to be in direct contact with either bone or soft biological tissue. The antimicrobial, freely suspended in the sol-suspension mixture, is encapsulated in the solid network of the coating extending between substrate-coating interface 103 to the external facing surface 102 of coating 101 . Multiple coating layers 101 may be applied one on top of another over substrate 100 to form a multilayer structure. This may involve repeated application of the solsuspension mixture followed by sequential curing of each wet layer. Accordingly, it is possible to produce a multilayered coating having a variable or uniform antimicrobial concentration gradient from outermost surface 102 to substrate-coating interface 103.

An experimental investigation was undertaken utilising the antimicrobial gentamicin encapsulated within a sol-gel coating for potential use as an antibacterial coating/film on a cementless prosthetic.

One aim of the experimental investigation was to determine if the sol-gel derived coat could be rapidly, e.g., within 5 minutes, cured at ambient temperature without loss of integrity of the bond between coating 101 and substrate 100.

A further aim was to determine if gentamicin is released from the coating whilst maintaining its viability as an antibiotic.

Example 1 : Sol gel coating preparation

The base hybrid sol was prepared using an acid catalysed process. The precursors; tetraethylorthosilicate (TEOS), tetramethylorthosilicate (TMOS), trimethoxymethylsilane (MTMS), and hexamethyldisiloxane (HMDS) were stirred with isopropanol and nitric acid [0.07 M] in the volumetric ratio of 0.5:1 :1 :0.2:4.4:4.9. The reaction was catalysed by the addition of isopropanol and nitric acid to the remaining precursors whilst stirring vigorously.

Where required, gentamicin was added to the base hybrid sol to a final concentration of 1 .25 wt% w/v (i.e. 12.5 mg gentamicin per ml of sol) prior to coating application.

A thin layer of coating was applied to each test sample using a small modelling brush, and allowed to cure at room temperature (around 25°C) for 5 minutes. All subsequent experiments were carried out immediately after the 5 min curing time had elapsed.

Example 2: Elution testing

For elution testing, the gentamicin containing sol gel coating of Example 1 was applied to eight different substrates (Fig. 2): Titanium (Ti); Ti-coated Titanium (Ti-Ti); hydroxyapatite-coated Titanium (HA-Ti); Al-coated Titanium (TiAl); Cobalt-Chromium (CoCr); Al-coated Cobalt Chromium (CoCrAI); and polyetheretherketone (PEEK).

All samples (n=5) were submerged into 8 ml 0.1 M ammonium acetate buffer solution (pH 7.2) and incubated at room temperature with gentle agitation. At regular time points (t=0, 1 , 2, 4, 8, 24, 50, 72, 168 hours) the elution buffer was collected and replaced with fresh ammonium acetate buffer. The first time point (t=0) was taken less than 5 mins from initial submersion of the sample.

In addition, 3D printed titanium samples (3D), shown in Fig. 3, were also coated (n=3) and elution carried out over the same time points up to 168 hours.

Quantification of eluted gentamicin was carried out by LC-MS-MS using a Phenomenex Luna C18 column (150 mm x 1 mm) coupled to an Agilent 6420 triple quadrupole LC/MS system. The isocratic mobile phase was 50% methanol, 0.2% formic acid (v/v) and the flow rate was set at 0.05 mL min’¹. The mass spectrometer was operated with an electrospray ionisation (ESI) source in positive ion mode using Selected Ion Monitoring to measure an ion fragment of 478 m/z, corresponding to protonated gentamicin C1 (478 m/z). Elution of gentamicin was quantified by comparison against a gentamicin standard curve.

Results

All tested samples showed an initial release of gentamicin following submersion, the rate of which slowed down over the 168 h period. Notably, release of gentamicin is still apparent after 168 hours. The initial concentration of gentamicin released exceeded the minimum inhibitory concentration (MIC) of Staphylococci by 3-fold (Ti) to 51 -fold (3D printed) (Figs 4 and 5).

Example 3: Cross hatch scratch testing

Cross hatch scratch testing was carried out using and Elcometer 107 Cross hatch cutting device. Test samples were coated with gentamicin containing sol gel according to Example 1 , and a 6 mm x 6 mm grid scratched onto the surface. Adhesive tape was applied to the surface of the coated sample and removed after 30 s. The level of delamination of coating was scored based upon BSI EN ISO 2409:2020 (Fig 6). Results

During the elution experiments of Example 2, there was some evidence of flaking of the cured sol gel coating from the Ti ) sample, but no evidence from any of the other samples was found. Therefore, subsequent adhesion testing analysis was carried out on the smoothest samples, i.e. , Ti , CoCr and PEEK, with the expectation that these samples would provide the less robust coatings in terms of adhesion compared to less smooth coated samples.

As shown in Figure 7 and Table 1 , all samples showed a similar performance with an overall score of 2 for the adhesion test which equates to 5-15% removal from the grid surface.

Table 1 : Average adhesion score following cross hatch scratch testing (n=5)

Example 5: Microbial barrier assay

The ability of the sol gel coating per se (absent any antimicrobial component), to prevent penetration of bacteria was tested using the barrier method as set out in Salisbury and Percival (2018). Cultures of S. aureus (SH1000), and E. coli (clinical isolate) were grown on Mueller Hinton broth and Mueller Hinton Agar plates. Gentamicin-free sol gel coatings were brushed on and added to cell culture inserts at different volumes and allowed to cure as set out in Example 1 and placed on top of the agar. Following inoculation with a 1 x 10⁸ CFU bacterial suspension, the test inserts were placed on agar plates and incubated at 37°C for up to 72 hours. Results:

As shown in Figure 8, cell culture inserts containing no sol gel showed growth after 24h for E. coli (clinical isolate) and S. aureus (SH1000) inoculated samples. No growth was seen from S. aureus with sol gel coated inserts. No growth was seen with brushed on sol gel and 100 pl volume sol gel coatings on the E. coli inoculated filter.

Example 6: Coating Integrity analysis (interference fit)

In order to determine the performance of sol gel under interference fit conditions rod samples of titanium, PEEK, 3D printed titanium and hydroxyapatite-coated titanium were coated with a gentamicin containing sol gel coating according to the process set out in Example 1 and hammered into artificial bone block by interference fit. The samples were imaged by scanning electron microscopy (SEM) analysis and the presence of silicon was determined by Energy Dispersive X-ray (EDX) analysis. SEM with EDX analysis was carried out at the Henry Royce Institute, University of Manchester.

Results:

Of all the tested samples, the PEEK rods were the only samples that showed significant variability, as the initial test showed little or no silicon following interference fit into bone block. However, all subsequent repeats all clearly indicated an intact coating. All other test samples showed intact coatings following interference fit in all replicates analysed (Fig 9).

Discussion

The results above demonstrate the feasibility of using an antimicrobial sol gel for coating medical devices with preparation and application during the surgical procedure. The coatings of the invention were mixed for less than 1 minute, simply applied using a small brush and allowed to cure for 5 minutes at ambient temperature before immediately carrying out the testing procedures. Such a coating application and curing protocol was chosen to mimic the conditions which would likely be needed for perioperative application of the coating by the surgical team. All antimicrobial containing coatings provided an initial release exceeding the MIC for Staphylococci (the most common causative organisms for surgical site infections) and elution rates continued to exceed the MIC for 8 - 168 hours within this test. It should be noted that the current coating used a gentamicin concentration of 1 .25% as this is the concentration used in bone cement, however the coatings can incorporate much higher concentrations of antibiotic within the coating itself to provide higher elution concentrations.

In the elution tests detailed here, the antibiotic is eluted into 0.1 M ammonium acetate buffer, which is used to maintain a physiological pH whilst allowing ease of analysis using the LC-MS system. Previous work has eluted gentamicin from sol gel into a complex solution containing cell culture medium and foetal calf serum, and has shown that there is no significant difference in elution rates when compared to ammonium acetate.

In the samples shown here it was clear that samples with smooth flat surfaces showed the least amount of gentamicin elution and those with coated surfaces and greater surface area showed increased gentamicin elution. This is consistent with the increased surface area having more coating and therefore greater concentrations of gentamicin. The presence of a coated surface and the increased porosity of 3D printed samples also aided adhesion of coating to sample with the greatest elution rate of gentamicin within the first hour at 51 fold greater than the MIC for Staphylococci. Slight delamination was seen on the smooth Ti and so for the adhesion test all smooth surfaced samples were chosen as ‘worst case scenario’ candidates. All samples showed similar performance with an average of <15% delamination from sample surface.

The performance of the coating during interference fit implantation was assessed, where samples were coated with sol gel containing gentamicin and hammered into Sawbone blocks. The EDX analysis of the samples detected the silicon sol gel coating intact on all the samples with the exception of PEEK, where some variation was detected. The difference between the first analysis and subsequent testing was attributed to simple experimental error during sample preparation. The other materials all show clear evidence of an intact coating after interference fit indicating that the coating has bonded strongly to the surface of these materials. To address the regulatory requirements for a medical device, it was decided that the property of the coating perse as a barrier to bacterial penetration should be assessed. The coating of cell culture filters with sol gel prevented the penetration and growth of E. coli (clinical isolate) and S. aureus (SH1000) cultures onto agar plates even in the absence of any antimicrobial component. Over 72 hours, sol gel coatings were able to sufficiently prevent growth on Mueller Hinton agar plates demonstrating that this would prevent bacteria penetrating onto the implants surface.

In summary, the sol-gel gentamicin coating of the present invention is suitable for use a fast-cure coating for orthopaedic devices not least due to the following advantageous properties:

• cure time of < 5 mins;

• strong adhesion on a variety of surfaces and materials;

• release of antibiotic beginning <5 mins and continuing over a 1 week period;

• intact following interference fit testing; and

• provides a barrier to bacterial penetration.

Claims

1. A substrate having a hybrid coating chemically bonded thereto, the coating obtainable by a sol-gel process using a siloxane monomer precursor of General Formula (I):

(R¹)3-Si-O-Si-(R¹)3 General Formula (I); wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6 group; and wherein said coating comprises a polysiloxane based network of siliconcarbon bonds and silicon oxygen bonds.

2. The substrate according to claim 1 , wherein said coating comprises an antimicrobial component releasably captured within said polysiloxane based network of silicon-carbon bonds and silicon oxygen bonds.

3. The substrate according to claim 2, wherein said antimicrobial component releasably captured within said network in response to the introduction of a fluid into the coating.

4. The substrate according to any of the preceding claims, wherein said polysiloxane-based network is formed as a porous network.

5. The substrate according to any of the preceding claims, wherein the compound of General Formula (I) is a compound of Formula (la):

(CH₃)3-Si-O-Si-(CH₃)3 Formula (la)

6. The substrate according to any of the preceding claims, wherein said substrate comprises or consists of a metal; metal alloy; ceramic; glass ceramic hybrid; pyrolytic carbon; fiberglass; textile; plastic; mineral based material; silica, silicate or silicon containing polymer; glass; latex; or rubber.

7. The substrate according to any of the preceding claims, wherein said coating comprises one or more additional dopant species captured or chemically bonded to the polysiloxane based hybrid organic-inorganic oxide network, wherein the additional species are optionally selected from a silane, a silicate, particles, nano particles, Y-AI2O3 and/or hydroxyapatite.

8. The substrate according to any of claims 2 to 7, wherein said coating comprises a multilayer structure, and optionally wherein each layer comprises a different concentration of said antimicrobial component.

9. The substrate according to any of claims 2 to 7, wherein said coating comprises a uniform concentration distribution of said antimicrobial component through the coating thickness from an external facing region to the substrate-coating interface.

10. The substrate according to any of claims 2 to 7, wherein said coating comprises a substantially non-uniform concentration distribution of the antimicrobial component through the coating thickness from an external facing region to the substrate-coating interface.

11. The substrate according to any of the preceding claims, wherein the coating further comprises one or more biologically active components configured to encourage in vivo bone regrowth at the region of the coating, wherein said biologically active component(s) are optionally selected from: osteogenic proteins; recombinant human bone morphogenic proteins; carboxymethyl chitosan; biologically active proteins; DNA; extra cellular matrix components and analogues thereof; calcium based compounds; phosphorus based compounds; biologically active agents derived from vitamins, multi-molecular complexes and assemblies; and nanoparticles.

12. The substrate according to any of the preceding claims, wherein the coating further comprises one or more components selected from anti-inflammatory, analgesic, antineoplastic and anti-angiogenic agents.

13. The substrate according to any of the preceding claims, wherein the antimicrobial component is selected from one or more of: a protease, a furanone, an aminoglycoside, a beta-lactam, an antimicrobial peptide, a lipopeptide, a glycopeptide, a macrolide, a lincosamide, a ketolide, an antifungal agent, a tetracycline, a quinolone, rifampicin, fusidic acid, colistin, a nitroimidazole antibiotic, and fosfomycin.

14. A biocompatible implant, preferably a prosthetic or fixation device, comprising the coated substrate according to any of the preceding claims.

15. A method of preparing a coated substrate, the method comprising:

(i) preparing a sol using a siloxane monomer of General Formula (I):

(R¹)3-Si-O-Si-(R¹)3 General Formula (I), wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6 alkyl group;

(ii) coating the surface of a substrate with said sol; and

(iii) curing said sol on the surface of said substrate to form a sol-gel derived hybrid coating chemically bonded to the substrate, wherein said coating comprises a polysiloxane based network of silicon-carbon bonds and silicon-oxygen bonds.

16. A method of preparing a coated substrate, the method comprising:

(i) preparing a sol using a siloxane monomer of General Formula (I):

(R¹)3-Si-O-Si-(R¹)₃ General Formula (I), wherein each R¹ independently represents hydrogen, hydroxy or an optionally substituted C1-6 alkyl group;

(ii) providing a preparation comprising an antimicrobial component;

(iii) combining said sol and said antimicrobial component to form a mixture;

(iv) coating the surface of a substrate with said mixture;

(v) curing said mixture on the surface of said substrate to form a solgel derived hybrid coating chemically bonded to the substrate, wherein said antimicrobial component is releasably captured within a polysiloxane based network of silicon-carbon bonds and silicon-oxygen bonds.

17. The method according to claim 15 or claim 16, wherein the step of curing said sol or said mixture is carried out on the surface of said substrate at a temperature of from about 1 °C to about 30°C, and/or a time period of from about 2 minutes to about 20 minutes.

18. The method according to any of claims 15 to 17, further comprising the step of sterilizing the coating, wherein the said sterilizing optionally comprises exposing said coating to gamma radiation.