Method for Providing a Substrate with an Antimicrobial Coating, and Coated Substrates Obtainable Thereby

Abstract

The invention relates to the field of antimicrobial materials, in particular to implantable and other medical devices exhibiting antimicrobial activity. Provided is a method for providing a substrate with an antimicrobial coating by immobilizing a quaternary ammonium compound onto the surface of said substrate, comprising the polycondensation of AB.sub.2 monomers comprising a secondary amine as A-group and blocked isocyanates as B-groups to obtain a low number average molecular weight polyurea of at least 2500 Da, contacting said polyurea with a surface grafted with coupling agent to covalently anchor the polyurea, and continuing polycondensation to obtain a hyperbranched polyurea coating; followed by immobilizing onto said coating a hydrophobic N-alkylated polyethylenimine (PEI) having antimicrobial properties.

Claims

1. A method for providing a substrate with an antimicrobial coating by immobilizing a quaternary ammonium compound onto the surface of said substrate, comprising the steps of: (i) providing a surface comprising reactive hydroxyl groups; (ii) covalently grafting onto said reactive hydroxyl groups a siloxane coupling agent comprising a blocked isocyanate group; (iii) polycondensation of AB.sub.2 monomers comprising a secondary amine as A-group and blocked isocyanates as B-groups to obtain a low number average molecular weight (M.sub.n) polyurea of at least 2500 Da; (iv) contacting said low molecular weight polyurea with the surface grafted with coupling agent to covalently anchor the polyurea, and continuing polycondensation by heating, optionally in the presence of AB.sub.2 monomers, to obtain a hyperbranched polyurea coating; followed by (v) immobilizing onto said hyperbranched polyurea coating a hydrophobic N-alkylated polyethylenimine (PEI) having antimicrobial properties.

2. Method according to claim 1, wherein said coupling agent is (2-oxo-N(3-triethoxysilyl)propyl)azepane-1-carboxamide.

3. Method according to claim 1 or 2, wherein said AB.sub.2 monomers are of the general formula ##STR00003## wherein R.sub.1 and R.sub.2 are aliphatic chains (CH.sub.2).sub.m and (CH.sub.2).sub.n wherein m and n are an integer in the range of 3 to 15, preferably 3 to 8, and L.sub.1 and L.sub.2 are blocking groups, preferably selected from caprolactam, phenol, oxime, triazole and malonic esters.

4. Method according to any one of the preceding claims, wherein step (iii) comprises polycondensation of AB.sub.2 monomers to a number average molecular weight in the range of 2500 to 5000 Da, preferably 3000-5000 Da.

5. Method according to any one of the preceding claims, wherein step (v) comprises preparing N-alkylated PEI by alkylating PEI in the presence of 1,8-Bis(dimethylamino)naphthalene, 1,6-di-t-butyl pyridine or 1,6-dimethyl pyridine.

6. Method according to any one of the preceding claims, wherein step (v) comprises immobilizing PEI to the hyperbranched polyurea coating followed by N-alkylation of PEI.

7. Method according to any one of claims 1-5, wherein step (v) comprises N-alkylation of PEI prior to immobilizing N-alkylated PEI to the hyperbranched polyurea coating.

8. Method according to any one of the preceding claims, wherein N-alkylation comprises alkylation with a linear or branched C.sub.5-C.sub.15 alkyl chain, preferably N-hexylation or N-dodecylation, and N-methylation.

9. Method according to any one of the preceding claims, wherein N-alkylation comprises a two-stage alkylation of PEI, preferably N-hexylation followed by N-methylation of N-hexyl-PEI.

10. Method according to any one of claims 1-8, wherein N-alkylation comprises a one-stage alkylation of PEI.

11. Method according to any one of the preceding claims, wherein said surface is a medical grade polymer, preferably selected from the group consisting of plasma-treated silicone rubber, such as plasma-treated medical grade polydimethylsiloxane elastomer (PDMS), polyurethane, and polyvinylchloride (PVC).

12. Method according to any one of claims 1 to 10, wherein said surface is a metal which is biocompatible with the mammalian body, preferably selected from the group consisting of titanium, titanium-alloy, tantalum and tantalum-alloy.

13. Method according to any one of the preceding claims wherein substrate is a medical device or implant.

14. Method according to claim 13, wherein the substrate is selected from the group consisting of a catheter, a prosthesis, an orthopedic implant and a cardiovascular implant.

15. A substrate coated with an antimicrobial coating, obtainable by a method according to any one of claims 1-14.

16. Coated substrate according to claim 15, comprising at least one therapeutic agent and/or a further antimicrobial agent.

17. Coated substrate according to claim 15 or 16, being a medical device or implant, preferably selected from the group consisting of a catheter, a prosthesis, an orthopedic implant and a cardiovascular implant.

18. A method for reducing microbial biofilm formation on the surface of a substrate, preferably a medical device or implant, comprising the step of providing at least part of the surface of said substrate with an antimicrobial coating obtainable by a method according to any one of claims 1-14.

19. A method for reducing or eliminating the incidence of an implant-associated infection, comprising implanting a coated medical device or implant according to claim 17 in a subject in need thereof.

Description

LEGEND TO THE FIGURES

[0049] FIG. 1: Microscopic images of glass coating with 5 wt % oligomer (20 zoom) according to the invention. Panels A and B show representative sections.

[0050] FIG. 2: Microscopic images of glass coating with 5 wt % monomer (20 zoom) according to the prior art. Panels A and B show representative sections.

EXPERIMENTAL SECTION

Materials

[0051] Glass slides were purchased from Waldemar Knittel (Braunschweig, Germany). Polydimethyl siloxane (PDMS) sheets were kindly provided by Atos Medical (Hrby, Sweden). Bishexamethylene triamine, (3-aminopropyl) triethoxysilane, polyethyleneimine (750 kDa, 50 wt % in water), iodomethane, 2-methyl-2-butanol, fluorescein disodium salt, 1-bromohexane and cetyltrimethylammonium chloride were all purchased from Sigma-Aldrich. Potassium hydroxide and dimethylformamide were purchased from Acros organic. Sulfuric acid, hydrogen peroxide and ethanol were obtained from Merck. Methanol and toluene were obtained from Lab-Scan. Carbonyl biscaprolactam (CBC; >99%) was kindly provided by DSM innovation center, ALLINCO (The Netherlands). All chemicals were used as received.

Example 1: Synthesis of 2-oxo-N(3-triethoxysilyl)propyl)azepane-1-carboxamide (Coupling Agent)

[0052] A three neck flask provided with a reflux condenser, a nitrogen inlet and a connector to a vacuum pump, was three times evacuated and flushed with nitrogen, to remove all oxygen. CBC (11.34 g, 45 mmol) and (3-aminopropyl) triethoxysilane (9.95 g, 45 mmol) were dissolved in 40 mL toluene. The reaction was carried out under nitrogen at 80 C. overnight.

[0053] After the solution was cooled down to room temperature toluene was removed under reduced pressure. The obtained coupling agent was stored under nitrogen. The liberated caprolactam was not removed. According to the NMR spectrum, the yield was more than 98%. .sup.1H-NMR (300 MHz, CDCl.sub.3) =0.60 (2H, m, SiCH.sub.2), 1.19 (9H, m, SiOCH.sub.2CH.sub.3), 1.65-1.74 (2H, m, SiCH.sub.2CH.sub.2 and 4H CONCH.sub.2(CH.sub.2).sub.3 (ring)), 2.67 (2H, m, NCOCH.sub.2 (ring)), 3.28 (2H, m, NHCH.sub.2), 3.79 (6H, m, SiOCH.sub.2), 3.97 (2H, m, CONCH.sub.2, ring), 9.27 (1H, br, NHCON).

Example 2: Application Coupling Agent

[0054] Glass slides (76 mm26 mm) were sonicated for 20 min at RT in dichloromethane. Subsequently, they were provided with silanol groups (SiOH) by using an oxygen plasma cleaner (Diener electronic, Femto) for 60 seconds under stable vacuum (2 mTorr).

[0055] PDMS sheets were cut into small pieces (2.01.5 cm) and immersed in toluene for 24 h at room temperature with stirring. Next, the pieces were transferred into hexane and stirred for 24 h at room temperature. The samples were dried overnight at 60 C. The PDMS samples were provided with silanol groups (SiOH) by using an oxygen plasma cleaner (Diener electronic, Femto) for 60 seconds under stable vacuum (2 mTorr).

[0056] The obtained hydrophilic slides were immersed in the 3 v/v % solution of coupling agent (see Example 1) in ethanol for 10 min at RT and then placed in vacuum oven and heated at 110 C. for 2 hours under vacuum. The unreacted coupling agent was removed by washing the slides in ethanol for 20 minutes in sonic bath at RT and then dried under nitrogen.

Example 3: Synthesis of AB.SUB.2 .Monomer

[0057] A three necked flask equipped with an inlet and outlet was flushed with nitrogen. Carbonyl biscaprolactam (23.22 g, 92 mmol) and bishexamethylene triamine (9.98 g, 46 mmol) were dissolved in 40 mL toluene and stirred at 80 C. for 20 h in a nitrogen atmosphere. The toluene solution was cooled down to room temperature (RT) and extracted five times with an aqueous solution containing 5 wt % CaCl.sub.2). After the aqueous layer had been removed, the organic layer was concentrated to half its volume using a rotary evaporator. Toluene was removed under reduced pressure and the product was dried in a vacuum oven at 40 C. overnight (Yield 80%). .sup.1H-NMR of AB.sub.2 monomer (400 MHz, CDCl.sub.3): 1.33-1.47 (m, 16H, CH.sub.2(CH.sub.2).sub.4CH.sub.2, 1.70 (m, 12H, CH.sub.2(CH.sub.2).sub.3CH.sub.2, ring), 2.56 (m, 4H, CH.sub.2NH), 2.67 (m, 4H, NCOCH.sub.2, ring), 3.25 (q, 4H, CONHCH.sub.2), 3.96 (t, 4H, CH.sub.2N, ring), 9.23 (br, 2H, CONH).

Example 4: Condensation of AB.SUB.2 .Monomers to a Low Average Number Molecular Weight Polyurea

[0058] A three-neck flask, provided with a reflux condenser, a nitrogen inlet and a connector to a vacuum pump, was three times evacuated and flushed with nitrogen, to remove all oxygen. To a solution of bishexamethylene triamine (BHTA) (8.81 g, 40.90 mmol) in dry DMF (50 mL) a solution of carbonyl biscaprolactam (20.64 g, 81.81 mmol) in DMF (60 mL) was added and the resulting mixture was stirred at 80 C. overnight under a nitrogen atmosphere. Subsequently, the temperature was raised to 145 C. after which the polymerization started. A number of oligomers with various molecular weights were prepared by heating the monomers during various polymerization times (table 1). The polymer solutions were precipitated in cold water (500 mL). The precipitates were isolated, washed with diethyl ether and dried in the vacuum oven at 40 C. for 48 h. .sup.1H-NMR (300 MHz, DMSO) =1.07 to 1.57 (8H, m, (CH.sub.2).sub.4), 1.61 (6H, m (CH.sub.2).sub.3, ring), 2.65 (2H, m, NCOCH.sub.2), 2.90 to 3.20 (2H, t, CON(CH.sub.2).sub.2, 2H, m, CONHCH.sub.2), 3.86 (2H, m, CH.sub.2NCO), 6.06 (1H, m, CH.sub.2NHCON(CH.sub.2).sub.2), 9.13 (1H, t, CONH).

TABLE-US-00001 TABLE 1 Molecular weights at various residence times Residence time (h) M.sub.n (Da) M.sub.w/M.sub.n BI end-groups 0.65 3,500 2.0 9 2 4,900 2.2 12 4 6,700 2.3 14 6 8,000 2.5 18 9 9,300 3.6 25 10 9,500 4.1 33 15 11,000 7.2 38

Example 5: Hyperbranched Coatings on Glass Slides

[0059] A. 100 L of a solution of low molecular weight polyurea (M.sub.n=4,900 Da; 5 wt % in ethanol) of Example 4 was dropped onto glass slides of Example 2 and spin-coated (2,000 rpm, 60 s). After evaporation of the ethanol the polymerization was continued at 145 C. for 2 h under nitrogen. Unreacted compounds were removed by sonication in ethanol for 20 min followed by extraction in DMF at 115 C. overnight and sonication again in 100 mL of ethanol for 20 min at RT. The coated glass slides were dried and stored under nitrogen. The microscopic images are shown in FIG. 1. The roughness of coated samples was 0.156-0.027 m, measured by the optical profilometer operating in non-contact mode using white light interferometry (STIL, France). A surface roughness below about 1 m is generally considered too rough and hence unsuitable for application as medical device. Not only causes a rough surface undesired friction e.g. when inserting it in the body, rougher surfaces also increase the rate of bacterial attachment.
B. As a comparative example, glass slides were coated with AB.sub.2 monomers as has been described in the prior art. Briefly, a solution of AB.sub.2 monomers (5 wt % concentrations in ethanol, 100 L) was dropped and spin-coated onto glass slides of example 2 (2,000 rpm, 60 s). After evaporation of the ethanol a bulk polymerization was carried out at 145 C. for 2 h under nitrogen. Unreacted compounds were removed by sonication in ethanol for 20 min followed by extraction in DMF at 115 C. overnight and sonication again in 100 mL of ethanol for 20 min at RT. The coated glass slides were dried and stored under nitrogen. The microscopic images of the coated samples is shown in FIG. 2. The roughness was 1.1410.35 m, measured by the optical profilometer operating in non-contact mode using white light interferometry (STIL, France).
These data show that pre-oligomerization approach of the present invention resulted in a much lower roughness (0.1560.027 m) than a coating obtained via the surface polymerization of monomers (1.140.35 m)

Example 6: Hyperbranched Coatings on PDMS Slides

[0060] A. 100 L of a solution of low molecular weight polyurea (M.sub.n=4,900 Da; 5 wt % in ethanol) was dropped onto PDMS slides of example 2 and spin-coated (2,000 rpm, 60 s). After evaporation of the ethanol the polymerization was continued at 145 C. for 2 h under nitrogen. Unreacted compounds were removed by sonication in ethanol for 20 min followed by extraction in DMF at 115 C. overnight and sonication again in 100 mL of ethanol for 20 min at RT. The coated glass slides were dried and stored under nitrogen. The surface topography (roughness) of coated samples was 0.790.15 m, measured by the optical profilometer operating in non-contact mode using white light interferometry (STIL, France).
B. As a comparative example, PDMS slides were also coated with AB.sub.2 monomers.
100 L of a solution of the AB.sub.2 monomer (5 wt % in ethanol) was dropped onto PDMS slides and spin-coated (2,000 rpm, 60 s). After evaporation of the ethanol the bulk polymerization was carried out at 145 C. for 2 h under nitrogen. Unreacted compounds were removed by sonication in ethanol for 20 min followed by extraction in DMF at 115 C. overnight and sonication again in 100 mL of ethanol for 20 min at RT. The coated glass slides were dried and stored under nitrogen. The surface topography (roughness) of coated samples was 2.350.32 m, measured by the optical profilometer operating in non-contact mode using white light interferometry (STIL, France).

Example 7: Modification of Hyperbranched Coating with Polyethyleneimines

[0061] A solution of poly(ethyleneimine) (PEI) in water (50 wt %) was freeze dried overnight (M.sub.w=750 kDa) and the residue was dissolved in methanol in 20 wt % concentration. 300 L of the PEI solution was dropped on coated glass slides of Example 6A and spin casted (2000 rpm, 60 s). The anchoring reactions were carried out at 125 C. for 24 h under nitrogen. Unreacted PEI was removed with methanol using ultrasonic bath for 45 min at RT and dried under nitrogen.

Alkylation of PEI Functionalized Hyperbranched Coating

[0062] In a round bottom flask provided with a reflux condenser, coated slides comprising tethered PEI were immersed in 75 mL 1-bromohexane and heated under nitrogen at 90 C. overnight. Next 1.07 g of 1,8-bis(dimethylamino)naphthalene (proton sponge) in 25 mL of tert-amyl alcohol was added. The reaction was continued for another 6 hours at 90 C. Afterwards, the coatings were three times sonicated in methanol for 20 min at RT and dried under nitrogen. A second alkylation step was done in a round bottom flask fitted with a reflux condenser. Samples were immersed in a solution of 10 mL iodomethane in 75 mL tert-amyl alcohol. The alkylation reaction was carried out at 42 C. overnight; the samples were subsequently sonicated in methanol for 20 min at RT and followed by extraction in methanol at 65 C. for 1 day and another sonication in methanol for 20 min at RT. The obtained coatings were dried and stored under nitrogen.

Example 8: Antibacterial Properties of the Coated Surfaces

Bacterial Culture Preparation

[0063] Bacteria were grown aerobically overnight at 37 C. on a blood agar plate from a frozen stock solution. Plates were kept at 4 C. and used no longer than for 2 weeks. A single bacterial colony from a blood agar plate was inoculated in 10 mL trypton soya broth (TSB, OXOID, England) in case of Gram-positive bacteria or 10 mL Todd Hewitt broth (THB, OXOID, England) in case of Gram-negative bacteria, and incubated at 37 C. for 24 h. This pre-culture was used to inoculate 200 mL of TSB or THB and incubated for 16 h. Bacteria were harvested by centrifugation at 5000g for 5 min and washed twice with phosphate buffered saline (PBS buffer, 10 mM potassium phosphate, 150 mM NaCl, pH 7). Bacteria were sonicated on ice for 310 s at 30 W (Vibra Cell model 375, Sonics and Materials, USA) to break up aggregates if needed. This established procedure did not cause cell lysis.

Petrifilm Assay

[0064] The Petrifilm assay (Petrifilm Aerobic Countplate, 3M, USA) was used to determine killing bacteria on contact under nutrient rich conditions. Bacterial concentrations of 10.sup.6, 10.sup.5 and 10.sup.4 per mL in PBS buffer were used. The bottom of the Petrifilm containing gelling agent was first swelled with 1 mL sterile demi water for 30 min. Next, 10 L of bacterial suspensions at the appropriate concentration was dropped on coated and un-coated samples that were placed on the bottom film of the Petrifilm. After the top-lid was placed on the coated samples of example 6. the Petrifilms were incubated at 37 C. for 48 h. Bacterial suspension without a sample in between the films (10 L) was used as a control. After the incubation time numbers of the CFU per cm.sup.2 were counted. The results with various bacterial strains is given in Table 2.

TABLE-US-00002 TABLE 2 Antibacterial properties of coated substrate against various bacterial strains and at various concentrations. Control* Bacterial strain 10.sup.2 10.sup.2 10.sup.3 10.sup.4 10.sup.5 Staphylococcus 74 5 0 0 0 0 epidermidis ATCC12228 Staphylococcus 58 9 0 0 3 1 epidermidis 3081 Escherichia coli 80 7 0 6 2 43 4 ATCC 1110 Escherichia coli Hu734 77 4 12 7 140 16 *In the control experiment a calculated number of 100 bacteria were suspended on the substrate to check the viability.

[0065] These data show that the antibacterial coating of example 6 kills >99% of the Staphylococcus epidermidis ATCC 12228, Staphylococcus epidermidis 3081, Escherichia coli ATCC 1110 and about 85% of Escherichia coli Hu734.

Example 9: Bactericidal Properties Against Gram Negative Bacteria in the Presence of EDTA

[0066] Coated PDMS slides described in example 7 (PDMS-PEI.sup.+) were evaluated according the procedure of example 8 for their bactericidal effect against Gram negative bacteria (strains ATCC 15597 and Hu 734), in the absence or presence of EDTA. The results (Table 3) show that a permeabilisor, such as EDTA, has a profound influence on the antibacterial properties of said coatings against Gram negative bacteria. EDTA as such has no antibacterial properties at all.

TABLE-US-00003 TABLE 3 Bacterial challenge per sample (5 cm.sup.2) Sample 10.sup.2 10.sup.3 10.sup.4 E. coli ATCC 15597 (triplicates) Control (viability check) 79 3 PDMS uncoated 65 6 TMTC TMTC PDMS-PEI.sup.+ 6 3 65 8 TMTC control + 0.1 mM EDTA 76 4 control + 1 mM EDTA 78 2.5 PDMS-PEI.sup.+ + 0.1 mM EDTA 0 3 1.5 41 4.5 PDMS-PEI.sup.+ + 1 mM EDTA 0 0 25 6.sup. E. coli Hu 734 (triplicates) Control (viability check) 78 4 PDMS uncoated 73 5 TMTC TMTC PDMS-PEI.sup.+ 12 7 140 16 TMTC control + 0.1 mM EDTA 76 8 control + 1 mM EDTA 75 6 PDMS-PEI.sup.+ + 0.1 mM EDTA 2 1.5 12 4 38 8.sup. PDMS-PEI.sup.+ + 1 mM EDTA 0 4 2.5 26 6.5