Method for grafting polymers on metallic substrates

Abstract

A method of conferring modified properties, e.g. modified physical and/or biochemical properties, to a metallic substrate surface, including at least two steps being (i) a first step including at least exposing the substrate surface to a hetero-bifunctional anchoring molecule carrying at least a silane group and at least a A.sub.1 group, the A.sub.1 group being optionally introduced within the anchoring molecule via a preliminary functionalizing step, and (ii) a second step of exposing the substrate surface to a polymer carrying at least three groups A.sub.2 capable of reacting with A.sub.1 in a thiol-ene reaction, the number average molecular weight of the polymer being greater than 1 000 g/mol.

Claims

1. A method of conferring modified properties to a metallic substrate surface, the surface being formed at least partly by metal oxides, comprising: (i) a first step comprising at least exposing said substrate surface to a hetero-bifunctional anchoring molecule carrying at least a silane group and at least a A.sub.1 group, said A.sub.1 group being optionally introduced within said anchoring molecule via a preliminary functionalizing step, and (ii) a second step of exposing the substrate surface to a polymer carrying at least three groups A.sub.2 capable of reacting with A.sub.1, the second step being carried out after the first step, the group A.sub.1 being an alkenyl group or SH and the group A.sub.2 being SH or a group SSR when A.sub.1 is an alkenyl group and A.sub.2 being an alkenyl group when A.sub.1 is SH, with R being a polymer, wherein said polymer has a number average molecular weight between 2,000 and 5,000,000 g/mol.

2. Method according to claim 1, wherein the anchoring molecule is covalently attached to the substrate surface as a result of the first step and wherein the polymer is covalently attached to the anchoring molecule as a result of the second step.

3. Method according to claim 1, wherein the modified properties conferred to the metal substrate surface are chosen from the group consisting of hydrophilic character; improved hydrophobic character; cytotoxic properties; antibiotic, bactericidal, viricidal and/or fungicidal properties; cell-adhesion property; improved biocompatibility; protein repellency; adhesion property; electric conductivity property and reactivity property rendering said surface able to immobilize biomolecules.

4. Method according to claim 1, wherein said polymer is chosen from the group consisting of polyethylenes, polyacrylamides, polyacrylates, polyvinyl polymers, polystyrenes, polyalcohols, polyvinylalcohols, polyallylalcohols, polyvinylbenzyl polymers, polyamines, polyethyleneimines, polyallylamines, polymethacrylates, polymethacrylamides, polyethers, polyethylene glycols, polyesters, poly(DL-lactides), polyamides, polyurethanes, poly(ethylene-alt-succinimides), polysaccharides, dextrans, celluloses, hydroxyethylcelluloses, methylcelluloses, polyureas, polyanilines, polypeptides, polypyrroles and polythiophenes, their mixtures, copolymers and derivatives.

5. Method according to claim 1, wherein said polymer is a polyether or a polysaccharide.

6. Method according to claim 1, wherein the anchoring molecule is of the following formula (I): ##STR00003## wherein: X represents a bivalent group chosen from the group consisting of a (C.sub.1-C.sub.18)alkylene group optionally interrupted by 1 to 3 (C.sub.1-C.sub.4)alkenylene groups and/or 1 to 3 (C.sub.5-C.sub.10)arylene groups and/or optionally substituted by 1 to 3 (C.sub.1-C.sub.4)alkenyl groups and/or 1 to 3 (C.sub.5-C.sub.10)aryl groups, a bifunctional statistical polymer having a molecular mass of about 1,000 to 10,000 g/mol, and a 1,m-phenylene group with m=2, 3 or 4, R1, R2 and R3 represent independently a substituent chosen from the group consisting of a hydrogen atom, halogen atoms, (C.sub.1-C.sub.6)alkyl groups, (C.sub.1-C.sub.6)alkoxy groups, A.sub.1 represents either SH or RaC=CRbRc and Ra, Rb and Rc represent independently a substituent chosen from the group consisting of a hydrogen atom and (C.sub.1-C.sub.6)alkyl groups.

7. Method according to claim 1, wherein the group A.sub.2 is an alkenyl group RaC=CRbRc and A.sub.1 is SH, with Ra, Rb and Rc representing independently a substituent chosen from the group consisting of a hydrogen atom and (C.sub.1-C.sub.6)alkyl groups.

8. Method according to claim 1, wherein the substrate is a titanium-based material, a silicon-based material comprising a silicium oxide surface, or an iron-based material comprising an iron oxide surface.

9. Method according to claim 1, wherein the second step (ii) is carried out in the presence of a photoinitator in a content of at most 10% by weight with respect to the total volume of the solution.

10. Method according to claim 1, wherein the second step (ii) is carried out with photoactivation, in the presence of a photoinitator in a content of 0 to 1% by weight with respect to the total volume of the solution.

11. Method according to claim 1, wherein the substrate is chosen from the group consisting of medical implants and research tools.

12. Method according to claim 1, wherein the method confers an anti-adhesive property to medical implants and to surgical instruments, or confers an adhesive and/or cell-growth promoting property to medical implants or for internal/external bone-fracture fixation, and implants used in maxillofacial and craniofacial treatments.

13. A method of conferring modified properties to a metallic substrate surface, the surface being formed at least partly by metal oxides, comprising: (i) a first step comprising at least exposing said substrate surface to a hetero-bifunctional anchoring molecule carrying at least a silane group and at least a A.sub.1 group, said A.sub.1 group being optionally introduced within said anchoring molecule via a preliminary functionalizing step, and (ii) a second step of exposing the substrate surface to a polymer carrying at least one group A.sub.2 capable of reacting with A.sub.1, the second step being carried out after the first step, the group A.sub.1 being an alkenyl group and the group A.sub.2 being SH or a group SSR with R being a polymer, the number average molecular weight of said polymer being greater than 1,000 g/mol.

Description

DESCRIPTION OF FIGURES

(1) The figures are intended for purposes of illustrating and are not meant to be limiting the scope of the present invention.

(2) FIG. 1 represents schematically and not true to scale a layer of anchoring molecules covalently attached to a metallic substrate surface.

(3) FIG. 2 represents schematically and not true to scale a polymer layer covalently attached to a layer of anchoring molecules on a metallic substrate surface.

(4) The following examples illustrate the present invention without limiting its scope.

EXAMPLES

Materials

(5) Solvents were purchased from SDS (Peypin, France).

(6) Titanium substrates were purchased from Goodfellow (Lille, France).

(7) Silicon (100) wafers covered by a native oxide layer were purchased from Neyco (Paris, France).

(8) MeO-PEG-SH (Mw=5000 g/mol) was purchased from Rapp polymere (Tiibingen, Germany).

(9) Allyl-modified methylcellulose was prepared according to Example 7 (copolymer 11) as described in WO 2008/041187.

(10) 10-Undecenyltrichlorosilane was purchased from ABCR (Karlsruhe, Germany).

(11) 2,2-Dimethoxy-2-phenylacetophenone (IRGACURE 651) and 2-Benzyl-2-(dimethylamino)-4-morpho linobutyrophenone (IRGACURE 369), O-(2-Mercaptoethyl)-O-methyl-hexa(ethylene glycol) as well as FITC labelled BSA and fibrinogen were purchased from Sigma-Aldrich (Lyon, France).

A. Surface Modification of Metal Substrates

Ex. 1: Vinyl-Comprising Anchoring Molecules/PEG-SH on Ti Substrate

1.1 Preparation of Substrate for Monolayer Coating

(12) The titanium plates to be used as substrate were subjected to deep UV treatment using a UV grid lamp from UVP (Cambridge, UK), providing 20 mW/cm.sup.2 (=185 nm), at room temperature during 10 min.

1.2 Deposition of the Vinyl-Comprising Anchoring Molecules

(13) The substrate plates prepared according to step 1.1 were exposed to a monolayer deposition solution prepared by mixing 100 L of 10-Undecenyltrichlorosilane with 100 mL of dry toluene solvent. The coating procedure was performed in Ar for 120 min at room temperature. Samples were withdrawn from the silane solutions and washed several times with CHCl.sub.3, ethanol and then dried under nitrogen stream.

1.3 Grafting with a Thiol-Group Comprising Oligo(Ethylene Glycol)

Comparative

(14) A mixture of 10 L 2,2-Dimethoxy-2-phenylacetophenone (20 mM in ethylene glycol) and 10 L of O-(2-Mercaptoethyl)-O-methyl-hexa(ethylene glycol) was pipetted onto the substrate obtained in step 1.2 and covered with a quartz cover slide. The assembly was exposed to UV light for 10 seconds at room temperature, using a BIO-LINK BLX (Vilber Lourmat, France), providing 7.2 mW/cm.sup.2 (=254 nm) at the surface. The slide was removed from the substrate and rinsed several times with ethanol and water.

1.4 Grafting with a Thiol-Group Comprising Poly(Ethylene Glycol)

Invention

(15) 5 mg of MeO-PEG-SH (Mw=5000 g/mol) were solubilized in 1 mL of a saturated water solution of photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure-2959, Ciba-Geigy, 0.1 wt-% in water), pipetted onto the substrate obtained in step 1.2 and covered with a quartz cover slide. The assembly was exposed to UV light, for 1 minute at room temperature, using a CL-1000L crosslinker (UVP, USA), providing 5.2 mW/cm.sup.2 (=365 nm), at the surface. The slide was removed from the substrate and rinsed several times with ethanol and water.

Ex. 2: Vinyl-Comprising Anchoring Molecules/PEG-SH on Si Substrate

2.1 Preparation of Substrate for Monolayer Coating

(16) The silicon substrates were cleaned in chloroform, acetone, and ethanol, then blown dry in a filtered nitrogen stream and cleaned by immersion in a piranha solution (H.sub.2SO.sub.4/H.sub.2O.sub.2, 70:30 v/v, 80 C.) for 20 min and via ozonolysis to remove organic contaminants. This treatment was followed by three rinsings with deionized water and by drying under a filtered nitrogen stream.

2.2 Deposition of the Vinyl-Comprising Anchoring Molecules

(17) The substrate plates prepared according to step 2.1 were exposed to a monolayer deposition solution prepared by mixing 100 L of 10-Undecenyltrichlorosilane with 100 mL of dry toluene solvent. The coating procedure was performed in Ar for 120 min at room temperature. Samples were withdrawn from the silane solutions and washed several times with CHCl.sub.3, ethanol and then dried under nitrogen stream.

2.3 Grafting with a Thiol-Group Comprising Poly(Ethylene Glycol)

Invention

(18) 5 mg of MeO-PEG-SH (Mw=5000 g/mol) were solubilized in 1 mL of a saturated water solution of photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure-2959, Ciba-Geigy, 0.1 wt-% in water), pipetted onto the substrate obtained in step 2.2 and covered with a quartz cover slide. The assembly was exposed to UV light, for 1 minute at room temperature, using a CL-1000L crosslinker (UVP, USA), providing 5.2 mW/cm.sup.2 (=365 nm), at the surface. The slide was removed from the substrate and rinsed several times with ethanol and water.

Ex. 3: Thiol-Comprising Anchoring Molecules/Allyl-Modified Methylcellulose on Si Substrate

3.1 Preparation of Substrate for Monolayer Coating

(19) The silicon substrates were modified according to the procedure described under 2.1.

3.2 Deposition of the Anchoring Molecules and Functionalization with a Thiol Group

(20) 10-Undecenyltrichlorosilane was deposited according to the procedure described under 2.2. Then the alkene terminated surface is reacted with a solution of dithiothreitol (20 mM) and 2,2-Dimethoxy-2-phenylacetophenone (20 mM) in DMF during 10 min at room temperature using a BIO-LINK BLX (Vilber Lourmat, France), providing 7.2 mW/cm.sup.2 (=254 nm) at the surface.

3.3 Grafting with an Allyl-Modified Methylcellulose

(21) 1 mg of allyl-modified methylcellulose were solubilized in 1 mL of a saturated water solution of photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure-2959, Ciba-Geigy, 0.1 wt-% in water), pipetted onto the substrate obtained in step 3.2 and covered with a quartz cover slide. The assembly was exposed to UV light, for 1 hour at room temperature, using a CL-1000L crosslinker (UVP, USA), providing 5.2 mW/cm.sup.2 (=365 nm), at the surface. The slide was removed from the substrate and rinsed several times with ethanol and water.

Ex. 4: Thiol-Comprising Anchoring Molecules/Allyl-Modified Methylcellulose on Ti Substrate

4.1: Preparation of Substrate for Monolayer Coating and Deposition of the Anchoring Molecules and Functionalization with a Thiol Group

(22) After cleaning, surfaces (titanium oxide TiO.sub.2) were treated with a mercaptosilane: (3-mercaptopropyl)trimethoxysilane (MPTS) according the protocol published in Langmuir, 18 (2002) 846-854.

4.2: Preparation of Allyl-Modified Methylcellulose

(23) In a round flask (250 mL) is dissolved, part by part, methylcellulose (1.5 g; 8.67.10-3 mol of units) in a cold solution of aqueous NaOH (400 mg in 200 mL of water at 0 C.). After complete dissolution (transparent, viscous, and slightly foamy solution) allylbromide (4 mL; 5 eq./unit) is added. The solution becomes milky white and is vigorously stirred at room temperature for 24 h. The polymer is then flocculated by rotating the solution in a warm bath (50-60 C.), several times until all polymer is aggregated (in a spongy gel). This gel is removed from the solution by filtration and dialyzed 2 days in water baths (3 L) for removal of reactants in excess (dialysis tube have to be rehydrated in water for 15 min before use). The polymer solution is then dried in an oven or lyophilized to obtain modified methycellulose as either a transparent film or a white porous material. Yield 60-70%. .sup.1H-NMR (300 MHz, D.sub.2O): 5.80 (m, 1H), 5.30 (m, 2H), 2.9-4.5 (m, 227H); ATR-FTIR (diamond): 3450 cm1, 2830-3000 cm1, 1615 cm1, 1054 cm1, 1300-1500 cm1.

4.3: Grafting with a Vinyl-Modified Methylcellulose

(24) The silanized titanium surface is incubated in an aqueous solution of methylcellulose bearing vinyl groups (1 mg/mL) and a photoinitiator (Irgacure 2939 (2-Hydroxy-4-(2-hydroxyethoxy)-2-methylpropiophenone)), 0.1% w/v) under light exposure (=254 or 365 nm, 7.2 mW/cm2 or 5.2 mW/cm2) during 60 min. Then the surface is washed several times in water, ethanol.

B. Investigation of Modified Surface Properties

B.1. Bacterial Adhesion

(25) The titanium substrates obtained in example 1 (steps 1.3, 1.4 and 4.3), as well as an unmodified titanium substrate were immersed in 20 mL of LB medium containing 100 L of a bacterial innoculum (GFP labeled E. coli MG1655). The bacteria were allowed to grow overnight at 37 C. Then incubated substrates were washed with phosphate buffered saline in order to remove non adhesive bacteria.

B.2. Optical Microscopy

(26) The substrates obtained in the preceding bacterial adhesion experiment were examined by optical microscopy using a Leica DMRX upright optical microscope. The images were recorded with a Retiga EXi CCD camera (QImaging, USA) and the bacteria counted on the image.

B.3. Contact Angle Measurements

(27) The silicon substrates obtained in example 2 (steps 2.2 and 2.3) and in example 3 (step 3.3) were measured with a CA goniometer (Digidrop, GBX). The CAs were determined using water at room temperature (25 C.). For dynamic (advancing (.sub.A) and receding (.sub.R) CA measurement, water droplets (about 4 L) were added and withdrawn from the surface, respectively. Measurements were taken at three different locations on each sample surface. The CA data reported was determined by averaging the averaged values of three samples (n=3) which were prepared in independent experiments.

B.4. Results

(28) The results of the bacterial adhesion experiment for example 1 are summarised in table 1 and show that an anti-adhesive property has been conferred to the substrate surface by the method according to the invention.

(29) TABLE-US-00002 TABLE 1 Bacterial density (E. coli MG1655, bacteria/cm.sup.2) Control (unmodified Ti substrate) 1.9 10.sup.5 Oligo(ethylene glycol)-modified Ti substrate 1.6 10.sup.4 (step 1.3, comparative) Poly(ethylene glycol)-modified Ti substrate 1.9 10.sup.3 (step 1.4, invention)

(30) The results of the bacterial adhesion experiment for example 4 are summarised in table 2 and show that an anti-adhesive property has been conferred to the substrate surface by the method according to the invention.

(31) TABLE-US-00003 TABLE 2 Bacterial density (E. coli MG1655, bacteria/cm.sup.2) Control (unmodified Ti substrate) 9637 Ti modified with MPTS 11523 Methylcellulose-modified Ti substrate 75 (step 4.3, invention)

(32) The results of the contact angle measurements are summarized in tables 3 and 4 and show that a hydrophilic property has been conferred to the substrate surfaces by the method according to the invention.

(33) TABLE-US-00004 TABLE 3 Water contact angles () advancing receding Alkene-modified Si substrate (step 2.2, 101 94 intermediate SAM of invention) Poly(ethylene glycol)-modified Si substrate 54 18 (step 2.3, invention)

(34) TABLE-US-00005 TABLE 4 Water contact angles () advancing receding Methylcellulose-modified Si 58 12 substrate (step 3.3, invention)