Means and methods for providing a substrate with a biocidal 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 comprising providing a substrate that is coated with a polyamine-functionalized polymer, and contacting said polyamine-functionalized polymer with an aqueous salt solution comprising at least one salt having a polarizability α.sub.37 at least 4 Å.sup.3 determined at 37° C. The salt solution may comprise one or more of NaI, KI, NaBr, KBr, NaClO.sub.4, KClO.sub.4, Na.sub.2SO.sub.4, K.sub.2SO.sub.4, Na.sub.3PO.sub.4, K.sub.3PO.sub.4, Mg(NO.sub.3).sub.2, Ca(NO.sub.3).sub.2, (NH.sub.4).sub.2SO.sub.4, NH.sub.4NO.sub.3, MgSO.sub.4, CaSO.sub.4, and Al(NO.sub.3).sub.3.

Claims

1. A method for providing a substrate with an antimicrobial coating, comprising providing a substrate that is covalently coated with a polyamine-functionalized polymer, wherein said polyamine is non-quaternized, and contacting said polyamine-functionalized polymer with an aqueous salt solution comprising at least one salt having a polarizability α.sub.37 of at least 4 Å.sup.3 determined at 37° C.

2. Method according to claim 1, wherein said aqueous salt solution comprises at least one salt having a polarizability α.sub.37 of at least 4.5 Å.sup.3.

3. Method according to claim 1 or 2, wherein said aqueous salt solution comprises an ammonium salt, an alkaline metal salt or earth alkaline metal salt of an anion selected from the group consisting of Br.sup.−, I.sup.−, ClO.sub.4.sup.−, SO.sub.4.sup.2−, NO.sub.3 and PO.sub.4.sup.3−.

4. Method according to any one of claims 1-3, wherein said shielding composition comprises one or more of NaI, KI, NaBr, KBr, NaClO.sub.4, KClO.sub.4, Na.sub.2SO.sub.4, K.sub.2SO.sub.4, Na.sub.3PO.sub.4, K.sub.3PO.sub.4, Mg(NO.sub.3).sub.2, Ca(NO.sub.3).sub.2, Zn(NO.sub.3).sub.2, NaNO.sub.3, (NH.sub.4).sub.2SO.sub.4, NH.sub.4NO.sub.3, MgSO.sub.4, NH.sub.4I, CaSO.sub.4, and Al(NO.sub.3).sub.3.

5. Method according to any one of claims 1-4, wherein said polyamine-functionalized polymer comprises non-alkylated polyethyleneimine (PEI).

6. Method according to any one of claims 1-5, followed by contacting said polyamine-functionalized polymer with one or more proteinaceous substances.

7. Method according to any one of claims 1-6, wherein the substrate is a medical grade material, preferably selected from the group consisting of medical grade polyethylene, polydimethylsiloxane elastomer (PDMS), polyurethane, and polyvinylchloride (PVC).

8. Method according to any one of claims 1-6, wherein the substrate is a material which is biocompatible with the mammalian body, preferably selected from the group consisting of ceramics, stainless steel alloys, titanium, titanium-alloy, tantalum and tantalum-alloy.

9. Method according to any one of the preceding claims, wherein the polymer coating is covalently associated with at least part of the outer surface of the substrate.

10. Method according to any one of the preceding claims, wherein the polymer coating comprises a polyurea coating, preferably a hyperbranched polyurea coating.

11. Method according to claim 10, wherein said hyperbranched polyurea coating is obtained by a) providing a surface, optionally comprising reactive hydroxyl groups, and covalently grafting onto said (hydroxylated) surface a coupling agent; b) polycondensation of AB.sub.2 monomers comprising a secondary amine as A-group and blocked isocyanates as B-groups to obtain a number average molecular weight polyurea of at least 1500 Da; and c) contacting said low molecular weight polyurea with the surface grafted with a 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.

12. Method according to claim 11, wherein said coupling agent is ##STR00005##

13. A coupling agent of the formula ##STR00006##

14. The use of the coupling agent according to claim 13 in the interphase region between an inorganic substrate and an organic substrate.

15. The use according to claim 14, in the interphase region between an inorganic substrate selected from a glass, a metal, and a mineral substrate, and an organic substrate selected from an organic polymer, a coating, and an adhesive.

16. The use of the coupling agent according to claim 13, in the interphase region between (i) a solid substrate, such as a glass, a metal, a polymer, or a mineral substrate, and (ii) an antimicrobial coating, preferably an antimicrobial coating comprising non-quaternized PEI.

17. A method for providing a coupling agent of claim 13, comprising reacting dopamine or a salt thereof with carbonyl biscaprolactam (CBC) in a suitable solvent in the presence of a base.

18. Method according to claim 17, comprising reacting stoichiometric amounts of CBC, dopamine hydrochloric acid salt and trimethylamine in a polar solvent having a boiling point of 80° C. or higher, preferably DMF, in a nitrogen atmosphere.

19. A method of providing a solid substrate with a coating material, preferably a polymer coating, more preferably an antimicrobial polymer coating, comprising contacting at least part of the surface of the substrate with a coupling agent according to claim 13 to form a chemical bond between the solid surface and the coating material.

20. Method according to claim 19, wherein the solid substrate is a medical device or implant, preferably selected from the group consisting of a catheter, a prosthesis, an orthopedic implant and a cardiovascular implant.

21. A coated substrate obtainable by a method according to any one of claims 1-12, 19 or 20.

Description

LEGEND TO THE FIGURES

[0089] FIG. 1: .sup.1H NMR spectrum of the dopamine-based coupling agent N-(3,4-dihydroxyphenethyl)-2-oxooazepane-1-carboxamide (CaBiDA).

EXPERIMENTAL SECTION

Example 1: Synthesis of Coupling Agent CaBiDA

[0090] Stoichiometric amounts of carbonyl biscaprolactam (CBC; 1.419 g, 5.631 mmol), dopamine hydrochloric acid salt (1.068 g, 5.631 mmol), trimethylamine (0.784 mL, 5.631 mmol) and 10 mL DMF were added to round bottom flask which was equipped with a reflux condenser, and stirred at 80° C. for 24 h in a nitrogen atmosphere. After cooling down the solution the coupling agent was precipitated by adding 50 mL of an aqueous CaCl.sub.2) solution (5 wt %) and then dissolved in 10 mL CHCl.sub.3. After removal of the aqueous layer, the organic layer was washed two times with 50 mL of an aqueous solution of CaCl.sub.2 (5 wt %) and once with 50 mL of a saturated brine solution. After removal of the aqueous layer, the organic layer was dried on anhydrous magnesium sulfate (MgSO.sub.4). After drying, MgSO.sub.4 was filtered off and the solvent was completely removed in a rotavapor under reduced pressure (yield 97%). The 1H NMR spectrum shown in FIG. 1 confirmed the structure of CaBiDA.

[0091] A reaction scheme is given in scheme 1.

##STR00003##

Example 2: Synthesis of AB.SUB.2 .Molecules and Hyperbranched Polyurea Polymer (HBP)

[0092] Carbonyl biscaprolactam (CBC, 5.805 g, 23 mmol), bishexamethylene triamine (2.495 g, 11.5 mmol) and 10 mL xylene were added into a round-bottom flask equipped with a reflux condenser and stirred heated at 80° C. for 8 h in a nitrogen atmosphere to obtain the AB.sub.2 monomer. Next, the temperature was raised to 145° C. for 1 h reaction to induce prepolymerization, yielding a number average molecular weight of 1,500 Da.

[0093] The solution was cooled down to room temperature, 5 mL of xylene was added and extracted three times with an aqueous CaCl.sub.2 solution (5 wt %), and once with a saturation brine solution. After removal of the aqueous layer, the organic layer was dried on anhydrous magnesium sulfate (MgSO.sub.4). After drying MgSO.sub.4 was filtered off and the solvent was completely removed in a rotavapor under reduced pressure, yielding a waxy solid. In scheme 2 an overview of the reactions is depicted.

##STR00004##

Example 3: Pre-Treatment of a Titanium Substrate

[0094] Titanium samples (Ti, 10 mm×10 mmxl mm) were cleaned by dispersing them in a sonication bath in n-hexane for 10 min, followed by 30 min ultrasonic immersion in 40 mL of 10 M HCl solution, subsequently immersed in 40 mL milli-Q water and sonicated for 30 min. Then, the Ti samples were rinsed with acetone and dried at room temperature (RT). Next, the Ti samples were oxidized in a mixture of 25% NH.sub.4OH, 30% H.sub.2O.sub.2 and H.sub.2O (1:1:5 v/v) at 80° C. for 20 min. After this treatment. Ti samples were washed with milli-Q water, followed by washing with acetone and dried at RT.

Example 4: Application of Coupling Agent

[0095] A) The pre-treated Ti substrates (Example 3) were immersed into a solution of the coupling agent (Example 1) in ethanol (3 wt %) for 10 min, and then heated at 110° C. for 2 h in a vacuum oven. The unreacted coupling agent was removed by sonicating the samples with ethanol for 20 min at RT and dried.

[0096] B) Alternatively, a pre-treated Ti substrate (Example 3) was immersed into a PBS solution comprising CaBiDA and NaIO.sub.4 (molar ratio 2:1) at a pH of 7.0 and incubated at 37° C. overnight, and then washed ultrasonically with ethanol for 10 min and dried.

Example 5: Immobilization of Hyperbranched Polymer

[0097] A solution of HBP (Example 2; 5 wt % in ethanol) was spin-coated (2000 rpm, 60 s) on the Ti pieces covered with the coupling agent (Example 4A). Then the samples were heated for 2 h at 145° C. in a nitrogen atmosphere. Non-anchored polymers were removed by an extraction in DMF for 2 h at 115° C. Next, the coated Ti pieces were sonicated in methanol for 20 min at RT and dried.

Example 6: Functionalization with Polyethyleneimine (PEI)

[0098] 100 μL of PEI solution (20 wt % in methanol) was dropped on the coated titanium samples (example 5) and spin coated (2000 rpm, 60 s). The anchoring reactions were carried out at 125° C. for 3 hours in a nitrogen atmosphere. Unreacted PEI was removed by sonication with methanol for 20 min at RT and dried.

Example 7: Enhancing the Charge Density of PEI

[0099] The PEI-coated samples (example 6) were immersed in several aqueous solutions comprising different types of salts for 2-20 h at room temperature (see Table 1), followed by 3 times washing with pure water and sonicating once for 10 min in pure water. The resulting charge density was measured according to the fluorescein method (example 8), and expressed as percentage increase relative to the charge density prior to immersion. Furthermore, the antibacterial properties of a number of samples was tested in a Petri film assay with S. epidermidis as bacterial strain (example 9).

TABLE-US-00001 TABLE 1 Charge densities and antibacterial properties of PEI-coated titanium substrates after exposure to aqueous solutions comprising different types and concentrations of salts. Charge Incubation Polarizability density Anti- Salt Time (h) According to * increase bacterial None — No NaCl (10 mM) 20 3.359  0% No NaI (1 mM) 20 7.443 39% Yes NaI (10 mM) 2 7.443 ND Yes NaI (10 mM) 8 7.443 ND Yes NaI (10 mM) 20 7.443 81% Yes NaI (25 mM) 20 7.443 105%  Yes NaI (40 mM) 20 7.443 134%  Yes NaI (100 mM) 20 7.443 135%  Yes Mg(NO.sub.3).sub.2 (10 mM) 20 7.475 26% Yes Zn(NO.sub.3).sub.2 (1 mM) 2 ND ND Yes NaNO.sub.3 (1M) 2 4.313 63% Yes # Li et al. (J. Phys. Chem. B, 2017, 121, 6416-6424) who determined salt polarizability values at 37° C. presented as α.sub.37 in the last column of Table 1. ND: not determined

Example 8: Two-Step N-Alkylation of PEI (Comparative Example)

[0100] In a round bottom flask provided with a reflux condenser, coatings comprising tethered PEI sample (example 6) were N-alkylated (quaternized) by immersion in 20 mL C.sub.6H.sub.13Br at 90° C. for 4 h. Next, 1.07 g of a proton sponge (1,8-bis(dimethylamino)naphthalene) in 25 mL of tert-amyl alcohol was added. The reaction was continued for 3 h. The coated samples were rinsed with methanol and subsequently sonicated for 20 min at RT. The obtained coatings were dried and placed into round bottom flask in a nitrogen atmosphere for a second alkylation step. In a round bottom flask was provided with a reflux condenser and the samples were immersed in 20 mL CH.sub.3I at 42° C. for 24 h. The coated samples were rinsed with methanol and subsequently sonicated for 20 min at RT, then dried and stored in bottle in a nitrogen atmosphere. The increase of the charge density due to both alkylation steps was 109%. The antibacterial properties were determined as described in example, with S. epidermidis bacteria and all were killed.

Example 9: Charge Density Measurements

[0101] Samples were immersed at RT in 15 mL of a 1 wt % fluorescein (disodium salt) solution in demineralized water for 10 min, washed four times with 50 mL water, followed by sonication in 50 mL water for 5 min at RT to remove any dye not complexed with cationic species, and dried with an air flow to remove residue water. Next, the samples were placed in 10 mL of a 0.1 wt % cetyltrimethylammonium chloride solution in demineralized water and sonicated for 10 min at RT to desorb complexed fluorescein dye. Subsequently, 10 v/v % of 100 mM phosphate buffer, pH 8, was added to a total volume of 11 mL and UV/VIS measurements (Spectra max M2 UV/VIS spectrophotometer) carried out at 501 nm. The charge density was calculated according to the method as described by Steven Roest, Henny C. van der Mei, Ton J. A. Loontjens, Henk J. Busscher, in Applied Surface Science 356 (2015) 325-332. The results are indicated in Table 1.

Example 10: Antibacterial Evaluation by the Petrifilm Test

[0102] S. epidermidis ATCC 12228 was first streaked on a blood agar plate from a frozen stock solution (7 v/v % DMSO) and grown overnight at 37° C. on blood agar. One colony was inoculated in 10 mL tryptone soya broth (TSB, Oxoid, Basingstoke, UK) and incubated at 37° C. for 24 h. This culture was used to inoculate a main culture of 200 mL TSB, which was incubated for 16 h at 37° C. Bacteria were harvested by centrifugation for 5 min at 5000 g and 10° C. and subsequently washed two times with 10 mM potassium-phosphate buffer, pH 7.0.

[0103] The ability of the coatings to kill adhering staphylococci was evaluated by the Petrifilm assay. The Petrifilm assay employed is based on culturing of organisms that survive contact with the coatings under nutrient-rich conditions. The Petrifilm Aerobic Count plate (3M Microbiology, St. Paul, Minn., USA) consists of two films: a bottom film containing standard nutrients, a cold-water gelling agent and an indicator dye that facilitates colony counting and a top film enclosing the sample within the system. The bottom film containing the gelling-agent was first swelled with 1 mL sterile demineralized water for 40 min and transferred to the transparent top film before usage. Next, 10 μL bacterial suspensions with the different concentrations were placed on coated slides (1 cm×1 cm). After closure of the Petrifilm system with a slide in between, the staphylococcal suspension spread over the entire surface area of the samples, enabling calculation of the bacterial challenge per cm2 from the dimensions of the samples and the bacterial concentration in suspension. Petrifilms were incubated at 37° C. for 48 h after which the numbers of CFU were counted. As a control, 10 μL of the bacterial suspension was inoculated on Petrifilm without a sample in between.

Example 11: Covalently Attached Antibacterial Coating on PDMS

[0104] PDMS pieces (2×2.5 cm.sup.2) were placed in an air plasma equipment (Femto system from Diener-electronic, Germany) at 100 W, for 1-2 min (at 1.7×10.sup.−1 mbar air). The obtained hydrophilic PDMS pieces were immersed in the 3 v/v % solution of CaBiDA in absolute ethanol for 10 min at RT and then placed in vacuum oven and heated at 110° C. for 2 h under vacuum. The unreacted coupling agent was removed by washing PDMS pieces in ethanol for 20 min in sonic bath at RT and dried under vacuum and stored under nitrogen.

[0105] PDMS slides were submerged in a solution of hyperbranched polymer (5 wt % in ethanol) and subsequently spin coated (2000 rpm, 60 s). On heating at 145° C. for 2 h fixation on the coupling agent and a continued polymerization of the hyperbranched polymer on the surface was carried out under a nitrogen flow. Non-anchored polymers were removed by an extraction in 200 mL DMF at 115° C. for 2 h. Next PDMS pieces were sonicated in absolute ethanol for 20 min at RT and dried and stored under nitrogen.

[0106] A solution of polyethyleneimine (PEI) in water (50 wt %) was freeze dried overnight (M.sub.w=750 kDa) and the residue was dissolved in methanol in 20 wt % concentrations. 200 μL of the PEI solution was dropped on the sample covered with the hyperbranched polymer and spin coated (2000 rpm, 60 s). The anchoring reactions were carried out on aluminium plate at 125° C. for 3 under nitrogen. Unreacted PEI was removed with methanol using ultrasonic bath for 45 min at RT and dried under nitrogen.

[0107] The PEI-coated PDMS piece were immersed at RT in 10 mmol/L NaI solution for 2, 8 and 20 h respectively, followed by 3 times washing with pure water and sonicating once for 10 min in pure water. The resulting charge density was measured according to the fluorescein method and expressed as percentage increase relative to the charge density prior to immersion.

[0108] The charge density increase of the sample after 20 h immersion was 30%. The antibacterial properties of the samples after 2 and 8 h immersion were tested with Staphylococcus Epidermidis as described in Example 10. No surviving bacteria were observed.

Example 12: CaBiDA Shows Improved Adhesion

[0109] The CaBiDA coupling agent was applied on a titanium substrate using the NaIO.sub.4 route as described in example 4B. The siloxane coupling agent 2-oxo-N(3-triethoxysilyl)propyl)azepane-1-carboxamide (CaBiTES) previously disclosed in WO2016/043584 was used as comparative example. The samples were immersed in water for 4 days at room temperature. The contact angles measured before and after immersion are presented in Table 2.

TABLE-US-00002 TABLE 2 Contact angles of titanium samples covered with CaBiDA or CaBiTES. Coupling agent Before immersion After 4 days immersion CaBiDA 38.8 37.8 CaBiTES 71.0 46.9

[0110] The contact angle of CaBiDA remained the same, while the contact angle of CaBiTES went down from 71.0 to 46.9 degrees, indicating a reduced stability of CaBiTES under wet conditions.

REFERENCES

[0111] .sup.1 Bactericidal Properties of Flat Surfaces and Nanoparticles Derivatized with Alkylated Polyethylenimines, Jian Lin, Shuyi Qiu, Kim Lewis, and Alexander M. Klibanov, Biotechnol. Prog., 2002, Vol. 18, No. 5, 1082-1086. [0112] .sup.2 PLOS, Unusual Salt and pH Induced Changes in Polyethylenimine Solutions, Kimberly A. Curtis, Danielle Miller, Paul Millard, Saswati Basu, Ferenc Horkay, Preethi L Chandran, Sep. 29, 2016, 1-20. [0113] .sup.3 A Shape-Adaptive, Antibacterial-Coating of Immobilized Quaternary-Ammonium Compounds Tethered on Hyperbranched Polyurea and its Mechanism of Action Lia A. T. W. Asri, Mihaela Crismaru, Steven Roest, Yun Chen, Oleksii Ivashenko, Petra Rudolf, Joerg C. Tiller, Henny C. van der Mei, Ton J. A. Loontjens, and Henk J. Busscher, Adv. Funct. Mater. 2014, 24, 346-355. [0114] .sup.4 Woongmo Sung, Zaure Avazbaeva, and Doseok Kim, Salt Promotes Protonation of Amine Groups at Air/Water Interface, J. Phys. Chem. Lett. 2017, 8, 3601-3606. [0115] .sup.5 Antimicrobial Surfaces, Joerg C. Tiller, Advances in Polymer Science 2010, 240(1):193-217. [0116] .sup.6 Permanent, non-leaching antibacterial surfaces-2: How high density cationic surfaces kill bacterial cells, Hironobu Murata, Richard R. Koepsel, Krzysztof Matyjaszewskic, Alan J. Russell, Biomaterials 28 (2007) 4870-4879. [0117] .sup.7 Macromol Biosci. 2012; 12(9): 1279-1289, Poly(ethylene imine)s as antimicrobial agents with selective activity, Katherine Gibney, Iva Sovadinova, Analette I. Lopez, Michael Urban, Zachary, Ridgway, Gregory A. Caputo, and Kenichi Kuroda. [0118] .sup.8 Charge properties and bacterial contact-killing of hyperbranched polyurea-polyethyleneimine coatings with various degrees of alkylation Steven Roest, Henny C. van der Mei, Ton J. A. Loontjens, Henk J. Busscher, Applied Surface Science 356 (2015) 325-332.