Biomolecule attachment

Abstract: A method for making a medical device having a biomolecule immobilized on a substrate surface is provided. The method comprises the steps of: (a) oxidizing 2-aminoalcohol moieties with a periodate to form an aldehyde-functional material; (b) combining the aldehyde-functional material with an amino-functional material to bond the two materials together through an imine moiety; and (c) reacting the imine moiety with a reducing agent to form a secondary amine.

Description: The present invention relates to an oxidative process for linking a protein or a peptide biomolecule comprising a 2-aminoalcohol moiety to a substrate surface comprising a primary amine moiety, e.g. to the surface of an appropriate medical device, for example so as to enhance the biological and physical properties of a substrate surface. For many years, a number of medical devices, for example pacemakers, vascular grafts, stents, heart valves, etc., which contact bodily tissue or fluids of living persons or animals have been developed, manufactured, and used clinically. However, a major problem with such devices is that their surfaces tend to adsorb a layer of proteins from tissues and fluids such as tears, urine, lymph fluid, blood, blood products, and other fluids and solids derived from blood. The composition and organization of this adsorbed protein layer is thought to influence, if not control, further biological reactions. Adverse biological reactions such as thrombosis and inflammation can diminish the useful lifetime of many devices. Implantable medical devices also tend to serve as foci for infection of the body by a number of bacterial species. These device-associated infections are promoted by the tendency of these organisms to adhere to and colonize the surface of the device. Consequently, it has been of great interest to physicians and the medical industry to develop surfaces of materials used in medical devices that are less prone to promoting the adverse biological reactions that typically accompany the implantation of a device. One approach for minimizing undesirable biological reactions associated with medical devices is to attach various biomolecules to their surfaces for the attachment and growth of a cell layer which the body will accept. Biomolecules such as growth factors, cell attachment proteins, and cell attachment peptides have been used for this purpose. In addition, biomolecules such as antithrombogenics, antiplatelets, antiinflammatories/ antimicrobials, and the like have also been used to minimize adverse biomaterial-associated reactions. A number of approaches have been suggested for the attachment of such biomolecules. US-A-4,642,242, for example, discloses a method for attaching heparin to a polyurethane polymer by partially depolymerising the heparin to yield aldehyde functional groups which are then reacted with amine groups in the polyurethane polymer material. Other approaches typically require the use of coupling molecules such as glutaraldehyde, cyanogen bromide, p-benzoquinone, succinic anhydrides, carbodiimides, diisocyanates, ethyl chloroformate, dipyridyl disulphide, epichlorohydrin and azides which serve as attachment vehicles for coupling of biomolecules to substrate surfaces. For example, covalent attachment of biomolecules using water soluble carbodiimides is described by Hoffman et al., "Covalent Binding of Biomolecules to Radiation-Grafted Hydrogels on Inert Polymer Surfaces", Trans. Am. Soc. Artif. Intern. Organs 18 : 10-18 (1972); and Ito et al., "Materials for Enhancing Cell Adhesion by Immobilization of Cell-Adhesive Peptide", J. Biomed. Mat. Res. 25 : 1325-1337 (1991). The addition of coupling molecules to the substrate surfaces of medical devices, however, can add instability to the surfaces and increase the prospect for burial of the attached biomolecule in the coupling layer. Coupling molecules can also create undesirable cross-links between the biomolecules or create cross-links between surface functional sites, thereby inhibiting attachment or destroying the biological properties of the biomolecules. The use of coupling molecules also tends to decrease the specificity for attachment of the biomolecule to the substrate surface, thereby losing conformational control over the attachment process. Thus, what is needed is an alternative method for attaching biomolecules to a substrate surface, e.g. to the surface of a medical device, particularly a method which does not require the use of coupling molecules. We have now developed a method for directly linking a protein or a peptide biomolecule comprising a 2-aminoalcohol moiety to a substrate surface comprising a primary amine moiety. Thus viewed from one aspect the invention provides a method for linking a protein or a peptide biomolecule comprising a 2-aminoalcohol moiety to a substrate surface comprising a primary amine moiety, said method comprising the steps of: (a) treating said protein or peptide with a periodate whereby to form an aldehyde-functional protein or peptide; (b) reacting said aldehyde-functional protein or peptide with said substrate surface whereby to link the substrate surface thereto via an imine moiety; and (c) treating the resulting material with a reducing agent whereby to form a secondary amine linkage. Preferably, the method of the invention involves the steps of: combining a periodate with a protein or a peptide biomolecule having a 2-aminoalcohol moiety to form an aldehyde-functional biomolecule in an aqueous solution having a pH of about 4-9 and a temperature of about 0-50°C; combining the aldehyde-functional biomolecule with a substrate surface comprising a primary amine moiety to immobilize the biomolecule on the substrate surface through an imine moiety; and reacting the imine moiety with a reducing agent to form an immobilized biomolecule on the substrate surface through a secondary amine linkage. A "biomaterial" may be defined as a material that is substantially insoluble in body fluids and that is designed and constructed to be placed in or onto the body or to contact fluid of the body. Ideally, a biomaterial will not induce undesirable reactions in the body such as blood clotting, tissue death, tumor formation, allergic reaction, foreign body reaction (rejection) or inflammatory reaction; will have the physical properties such as strength, elasticity, permeability and flexibility required to function for the intended purpose; can be purified, fabricated and sterilized easily; and will substantially maintain its physical properties and function during the time that it remains implanted in or in contact with the body. Biomaterials suitable for use in the method of the present invention include those comprising a 2-aminoalcohol and/or an amine moiety. A "medical device" may be defined as a device that has surfaces that contact tissue, blood, or other bodily fluids in the course of their operation, which fluids are subsequently used in patients. This can include, for example, extracorporeal devices for use in surgery, such as blood oxygenators, blood pumps, blood sensors, tubing used to carry blood and the like, which contact blood which is then returned to the patient. This can also include endoprostheses implanted in blood contact in a human or animal body, such as vascular grafts, stents, pacemaker leads, heart valves, and the like, which are implanted in blood vessels or in the heart. This can also include devices for temporary intravascular use, such as catheters, guide wires, and the like, which are placed into the blood vessels or the heart for purposes of monitoring or repair. Protein or peptide biomolecules which possess carbon-carbon bonds bearing an amine moiety adjacent to a hydroxyl moiety (i.e. a 2-aminoalcohol moiety) are oxidizable with periodate (see, for example, US-A-5,362,852), which can be provided as periodic acid or salts thereof, such as sodium periodate, potassium periodate, or other alkali metal periodates. Typically, a stoichiometric amount of periodate is used to oxidize the 2-aminoalcohol moiety, although an excess could be used. Oxidation of such materials forms reactive aldehyde moieties. Oxidation is conveniently carried out in an aqueous solution, preferably an aqueous buffered solution, at a temperature which does not destroy the biological properties of the biomolecule. Generally, buffers having a pH in a range of about 4-9 can be used, with a pH of about 6-8 being desired for certain pH sensitive biomolecules. Generally, the oxidation is carried out a temperature of about 0-50°C, and preferably at a temperature of about 4-37°C. Depending on the biomolecule, oxidation reactions can be carried out for as little as a few minutes to as long as many days. Commonly, oxidation is complete within 24 hours. Long-term oxidation reactions are preferably performed in the dark to prevent "over-oxidation". Treatment times and temperatures for the oxidation process are in general inversely related. That is, higher treatment temperatures require relatively shorter treatment times. The limitations of time and temperature are governed by the effect of the treatment on the biological stability of the biomolecule. There is a wide latitude in determining the proper conditions for a particular protein or peptide system and these conditions can be readily determined by one skilled in the art by routine experimentation upon a reading of the information presented herein. Subsequent to oxidation, the reaction solution may be stored prior to attachment to a substrate surface at a temperature of about 4°C. Typically, the storage stability of the reaction solution at a neutral pH or slightly acidic pH can extend for 1-14 days and sometimes even months when stored in the dark. Typically, the oxidation of the 2-aminoalcohol moiety can result in the formation of two different aldehyde-functional materials. If desired, these aldehyde-functional materials can be separated by a variety of known techniques based on affinity, molecular weight, etc. The resultant aldehyde moieties may interact with sites on a substrate surface for covalent attachment of the protein or peptide biomolecules. Such substrate surface attachment sites comprise amine moieties, which react with aldehyde moieties forming imines. The substrate surface to which the biomolecule is to be coupled should contain an adequate density of amine moieties for attachment of the desired number of biomolecules. Substrates which do not contain amines on their surface can easily be aminated by a number of methods well known to those skilled in the art. For example, amines can be provided by plasma treating materials with ammonia gas as described by Holmes and Schwartz, "Amination of Ultra-high Strength Polyethylene using Ammonia Plasma" Composites Science and Technology 38 : 1-21 (1990). Alternatively, amines can be provided by grafting acrylamide to the substrate followed by chemical modification to introduce amine moieties by methods well known to those skilled in the art. Polyvinyl amines or polyalkylimines can also be covalently attached to polyurethane surfaces according to the method described in US-A-4,521,564 (Solomone et al.). Alternatively, for example, aminosilane can be attached to the surface as set forth in US-A-5,053,048 (Pinchuk), a grafted acrylamide-containing polymer can be attached by radiation grafting as set forth in US-A-3,826,678 (Hoffman et al.), or a grafted N-(3-aminopropyl) methacrylamide-containing polymer can be attached by ceric ion grafting as set forth in US-A-5,344,455 (Keogh et al.). Typically, when an aldehyde moiety (RCH(O)) reacts with a primary amine moiety (-NH 2 ), an equilibrium is set up with the reaction product, which is a relatively unstable imine moiety (-N=CHR). This coupling reaction can be carried out under the same conditions described above for the oxidation, which are designed to protect the protein or peptide biomolecule from damage. To stabilize the linkage between the biomolecule and the substrate surface, subsequent reductive alkylation of the imine moiety is carried out using a reducing agent (i.e. a stabilizing agent) such as, for example, sodium borohydride, sodium cyanoborohydride, and amine boranes, to form a secondary amine (-NH-CH 2 -R) . This reaction can also be carried out under the same conditions described above for the oxidation. Typically, however, the coupling and stabilizing reactions are carried out in a neutral or slightly basic solution and at a temperature of about 0-50°C. Preferably, the pH is about 6-10 and the temperature is about 4-37°C, for the coupling and stabilizing reactions. These reactions (coupling and stabilizing) can be allowed to proceed for just a few minutes or for many hours. Commonly the reactions are complete (i.e. coupled and stabilized) within 24 hours. Protein or peptide biomolecules useful in accordance with the method of the invention can be synthetically derived or naturally occurring. As long as the protein or peptide biomolecules include carbon-carbon bonds bearing an amine moiety adjacent to a hydroxyl moiety, they can be attached to an aminated substrate surface by the method of the present invention. Specific examples include synthetic or natural proteins or peptides, which contain N-terminal serine, N-terminal threonine, or 5-hydroxylysine (5-hydroxylysine is only known to occur naturally in collagen, but in principal can be placed anywhere in a synthetic peptide or protein). Some protein or peptide biomolecules are susceptible to conformational changes when brought into contact with a hydrophobic substrate surface. These conformational changes can lead to the exposure of internalized non-polar groups which can lead to hydrophobic interactions between the biomolecule and the surface. These hydrophobic interactions can cause the exclusion of water molecules that normally surround the biomolecule in solution. This exclusion of water molecules between the biomolecule and the surface strengthens the hydrophobic interaction and can cause further conformational change of the biomolecule. The degree of conformational change a biomolecule experiences may or may not destroy its biological properties. Therefore, one must take into account the hydrophobic nature of the substrate surface when attaching biomolecules which are prone to hydrophobic interactions. In these cases, it is preferred to create a hydrophilic environment on the substrate surface, thereby preventing any unwanted hydrophobic interactions between the biomolecule and the surface which might destroy the biological properties of the biomolecule. There are a number of surface-derivatization techniques, e.g. grafting techniques, well known to those skilled in the art for creating hydrophilic substrate surfaces. For example, techniques based on ceric ion initiation, ozone exposure, corona discharge, UV irradiation and ionizing radiation ( 60 Co, X-rays, high energy electrons, plasma gas discharge) are known. Substrates which may be modified by the method of the present invention include metals such as titanium/titanium alloys, TiNi (shape memory/super elastic), aluminum oxide, platinum/platinum alloys, stainless steels, MP35N, elgiloy, haynes 25, stellite, pyrolytic carbon, silver or glassy carbon; polymers such as polyamides, polycarbonates, polyethers, polyesters, polyolefins including polyethylenes or polypropylenes, polystyrenes, polyurethanes, polyvinyl chlorides, polyvinylpyrrolidones, silicone elastomers, fluoropolymers, polyacrylates, polyisoprenes, polytetrafluoroethylenes, and rubber; minerals or ceramics such as hydroxapatite; human or animal protein or tissue such as bone, skin, teeth, collagen, laminin, elastin or fibrin; organic materials such as wood, cellulose, or compressed carbon; and other materials such as glass, or the like. Substrates comprising these materials can be coated or uncoated, and derivatized or underivatized. The method of the invention can be used to modify substrates of any shape or form including tubular, sheet, rod and articles of proper shape. Preferably, the substrate is a biomaterial for use in a number of medical devices such as vascular grafts, aortic grafts, arterial, venous, or vascular tubing, vascular stents, dialysis membranes, tubing, or connectors, blood oxygenator tubing or membranes, ultrafiltration membranes, intra-aortic balloons, blood bags, catheters, sutures, soft or hard tissue prostheses, synthetic prostheses, prosthetic heart valves, tissue adhesives, cardiac pacemaker leads, artificial organs, endotracheal tubes, lenses for the eye such as contact or intraocular lenses, blood handling equipment, apheresis equipment, diagnostic and monitoring catheters and sensors, biosensors, dental devices, drug delivery systems, or bodily implants of any kind. An example of a biomolecule that can be used in the present invention is collagen. Collagen, which is found in connective tissue, has special amino acids, one of which is 5-hydroxylysine, which can be oxidized with a source of periodate to form a pendant aldehyde moiety. The aldehyde moieties formed by oxidation of collagen can be used to covalently attach the aldehyde-functionalised collagen to amine containing substrate surfaces. Such collagen-coated substrate surfaces can be used, for example, as cell seeding surfaces, cell binding surfaces, cell separating surfaces, tissue fixation, collagen-coated stems or collagen-coated vascular grafts. Thus, the present invention overcomes a number of problems associated with the use of medical devices. The invention provides an oxidative process for covalently attaching protein or peptide biomolecules comprising a 2-aminoalcohol moiety to substrate surfaces comprising a primary amine moiety, such as the surface of a biomaterial, e.g. for use in medical devices. Viewed from a further aspect the invention provides a product, i.e. a protein or peptide biomolecule-carrying substrate preparable by the method of the invention. Example 1 Periodate Oxidation of a Peptide Containing an N-terminal Serine A tripeptide made of three serine amino acids and a dipeptide made of two lysine amino acids, both obtained from Sigma Chemical Co. (St. Louis, MO), were incubated in sodium metaperiodate (NaIO 4 ) also obtained from Sigma Chemical Co. (St. Louis, MO). The tripeptide (0.90 mmoles) was incubated in the dark while shaking at ambient temperature for 3 hours in 10 ml deionized water containing 1.2 mmoles NaIO 4 . The resultant solution (2.5 µl) was added to 2 ml of a solution containing 0.8 g NaOH, 0.2 g 4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (available as PURPALD from Sigma Chemical Co., St. Louis, MO), in 20 ml deionized water, and shaken vigorously for 15 minutes at ambient temperature. The dipeptide (0.72 mmoles) was incubated in the dark while shaking at ambient temperature for 3 hours in 10 ml deionized water containing 1.2 mmoles NaIO 4 . The resultant solution (10 µl) (note that this amount is four times the amount used for the tripeptide) was then added to 2 ml PURPALD solution and shaken vigorously for 15 minutes at ambient temperature. The resultant solutions were then analyzed spectrophotometrically at 550 nm. Dickinson and Jacobsen (Chem. Commun., 1719 (1970)) describe the specific and sensitive reaction of aldehydes with PURPALD to yield purple-to-magenta-colored 6-mercapto-5-triazolo-(4,3-b)-s-tetrazines which can be measured spectrophotometrically at 550 nm. Sample absorbances obtained at 550 nm were 0.04 for the dipeptide and 1.81 for the tripeptide, which indicates that only the tripeptide which contained an N-terminal serine was successfully oxidized using periodate. The dipeptide of the two lysine amino acids lacks a moiety with carbon-carbon bonds bearing an amine moiety adjacent to a hydroxyl moiety. Example 2 Periodate Oxidation of a Peptide Containing an N-terminal Threonine A dipeptide made of N-terminal threonine and leucine amino acids obtained from Sigma Chemical Co. (St. Louis, MO) was incubated in sodium metaperiodate (NaIO 4 ) also obtained from Sigma Chemical Co. (St. Louis, MO). The dipeptide (4.3 mmoles) was incubated in the dark while shaking at ambient temperature for 3 hours in 10 ml deionized water containing 1.2 mmoles NaIO 4 . The resultant solution (10 µl) was added to 2 ml of the PURPALD solution described in Example 1 and shaken vigorously for 15 minutes at ambient temperature. After the 15 minutes of shaking at ambient temperature, the resultant solution was analyzed spectrophotometrically at 550 nm. Sample absorbance obtained at 550 nm was 0.62 indicating the periodate had successfully oxidized the N-terminal threonine amino acid present in the dipeptide. Example 3 Periodate Oxidation of a Peptide Containing an N-terminal Serine A pentapeptide made of N-terminal serine, aspartic acid, glycine, arginine, and glycine amino acids obtained from Sigma Chemical Co. (St. Louis, MO) was incubated in sodium metaperiodate (NaIO 4 ) also obtained from Sigma Chemical Co. (St. Louis, MO). The pentapeptide (0.01 mmoles) was incubated in the dark while shaking at ambient temperature for 3 hours in 2 ml deionized water containing 0.23 mmoles NaIO 4 . The resultant solution (10 µl) was added to 2 ml of the PURPALD solution described in Example 1 and shaken vigorously for 15 minutes at ambient temperature. After the 15 minutes of shaking at ambient temperature, the resultant solution was analyzed spectrophotometrically at 550 nm. Sample absorbance obtained at 550 nm was 0.74, which again indicates periodate had successfully oxidized the N-terminal serine amino acid present in the pentapeptide. Example 4 Oxidation of Collagen Mouse collagen, type IV, obtained from Sigma Chemical Co. (St. Louis, MO) was oxidized with sodium metaperiodate (NaIO 4 ). Collagen type IV is known to mediate the attachment of epithelial, endothelial, myoblasts and nerve cells in vivo and in vitro. Two collagen solutions were prepared by (i) mixing half a vial of collagen with 56 mg NaIO 4 in 5 ml deionized water and (ii) mixing half a vial of collagen in 5 ml deionized water. Both solutions were incubated in the dark for 2 hours while shaking at room temperature. The resultant solutions (each 100 µl) were added to 2 ml of the PURPALD solution described in Example 1 and shaken vigorously for 30 minutes at ambient temperature. After the 30 minutes of shaking at ambient temperature, the resultant solutions were analyzed spectrophotometrically at 550 nm. The PURPALD solution was used as the blank. Sample absorbances obtained at 550 nm were 0.03 for non-oxidized collagen and 0.25 for oxidized collagen, indicating that the collagen was oxidized to form aldehyde moieties. Example 5 Attachment of Periodate Oxidized Biomolecules to Aminated Substrates One technique for creating amines on substrate surfaces entails grafting substrate surfaces with acrylamide (AAm) and N-(3-aminopropyl)methacrylamide (APMA) monomers using ceric (Ce IV ) ions. The Ce IV ions create free radicals on ozone treated silicone and polystyrene surfaces and untreated polyurethane surfaces which initiate the graft copolymerization of the acrylamides. The amount of surface amination (the graft copolymerization of APMA and AAm) that takes place on the substrate surface can be measured via staining with ponceau S dye, a negatively charged dye molecule. This dye ionically associates with the primary amines on the aminated surface. Following grafting, a periodate oxidized biomolecule can be coupled to the amine containing derivatized substrate surface. The 2-aminoalcohol-containing biomolecule is first oxidized with sodium metaperiodate (NaIO 4 ) forming reactive aldehyde moieties. These aldehyde moieties are then used to covalently attach fibronectin to the primary amino moieties present on the substrate surface. Sodium cyanoborohydride (NaCNBH 3 ) is then used to stabilize the imine linkages. Specific procedures required for each of these steps are described below. Polystyrene 24 well tissue culture plates were ozone treated by placing the culture plates in an ozone reaction vessel for 30 minutes while oxygen, which contained ozone, was flowing at a rate of 1.3 cm 3 /min. The oxygen containing ozone was created by flowing the oxygen through a corona discharge apparatus, which exposed the flowing oxygen to an 8000V electrical potential. Following ozone treatment, the plates were soaked in nitrogen purged deionized water for 30 minutes at ambient temperature. Following the 30 minute soak in nitrogen purged deionized water, the plates were grafted with acrylamide (AAm) and N-(3-aminopropyl) - methacrylamide (APMA) monomers using Ce IV ion. The grafting solution consisted of 40 g AAm, 10 g APMA, 50 g deionized water solution, and 20 g Ce IV ion solution. The Ce IV ion solution consisted of 2.74 g ceric ammonium nitrate and 3.15 g nitric acid in 50 ml deionized water. The plates were allowed to graft for 3 hours in a 65°C nitrogen purged oven. Following grafting the plates were rinsed vigorously with deionized water. The grafted plates were then tested with ponceau S dye. Following staining, the ponceau S dye was released from the surface using a 1% sodium dodecyl sulphate (SDS) solution and quantified spectrophotometrically at 520 nm. Sample absorbances obtained at 520 nm were 0.00 for nonderivatized plates and 1.44 for surface-derivatized plates. As the results demonstrate, the surface-derivatized plates contained primary amines on their surfaces. The 2-aminoalcohol moiety of a peptide can be oxidized using the procedure of Example 1. Sodium cyanoborohydride (1mg/ml) is then added to the oxidized peptide solution. The resultant solution is then immediately added to each of the amine containing surface-derivatized tissue culture plate wells (approximately 1 ml solution/well). The oxidized peptide is then incubated in the derivatized tissue culture plate wells overnight at ambient temperature. Following incubation, the wells are vigorously rinsed with phosphate buffered saline (PBS) solution. Polyurethane film samples were cut into 1.4 cm diameter disks. Sample disks were grafted with Aam and APMA monomers using Ce IV ion. The sample disks were allowed to graft for 1 hour at ambient temperature. Following grafting, the sample disks were rinsed vigorously with deionized water. Again, the 2-aminoalcohol moiety of a peptide can be oxidized as previously described. Sample disks are then exposed to the oxidized peptide solution. Sodium cyanoborohydride is then added (1 mg/ml) and the resultant solution and sample disks are incubated overnight at ambient temperature. Following incubation, the polyurethane sample disks are vigorously rinsed with PBS.

Claims: A method for linking a protein or a peptide biomolecule comprising a 2-aminoalcohol moiety to a substrate surface comprising a primary amine moiety, said method comprising the steps of: (a) treating said protein or peptide with a periodate whereby to form an aldehyde-functional protein or peptide; (b) reacting said aldehyde-functional protein or peptide with said substrate surface whereby to link the substrate surface thereto via an imine moiety; and (c) treating the resulting material with a reducing agent whereby to form a secondary amine linkage. A method as claimed in claim 1 wherein said substrate is selected from the group comprising titanium/titanium alloys, TiNi (shape memory/super elastic), aluminum oxide, platinum/platinum alloys, stainless steels, MP35N, elgiloy, haynes 25, stellite, pyrolytic carbon, silver or glassy carbon; polyamides, polycarbonates, polyethers, polyesters, polyethylenes or polypropylenes, polystyrenes, polyurethanes, polyvinyl chlorides, polyvinylpyrrolidones, silicone elastomers, fluoropolymers, polyacrylates, polyisoprenes, polytetrafluoroethylenes, and rubber; hydroxapatite; bone, skin, teeth, collagen, laminin, elastin or fibrin; wood, cellulose, or compressed carbon; and glass. A method as claimed in claim 2 wherein said substrate is in tubular, sheet or rod form. A method as claimed in any one of claims 1 to 3 wherein said substrate surface comprises the surface of a medical device. A method as claimed in claim 4 wherein said medical device is selected from the group comprising vascular grafts, aortic grafts, arterial, venous, or vascular tubing, vascular stents, dialysis membranes, tubing, or connectors, blood oxygenator tubing or membranes, ultrafiltration membranes, intra-aortic balloons, blood bags, catheters, sutures, soft or hard tissue prostheses, synthetic prostheses, prosthetic heart valves, tissue adhesives, cardiac pacemaker leads, artificial organs, endotracheal tubes, contact or intraocular lenses, blood handling equipment, apheresis equipment, diagnostic and monitoring catheters and sensors, biosensors, dental devices, drug delivery systems, or bodily implants of any kind. A method as claimed in any one of claims 1 to 5 wherein said protein or peptide is naturally occurring. A method as claimed in any one of claims 1 to 6 wherein said protein or peptide comprises an N-terminal serine, an N-terminal threonine or a 5-hydroxylysine group. A method as claimed in any one of claims 1 to 7 wherein said substrate comprises a metal, polymer, ceramic or glass. A method as claimed in any preceding claim wherein said periodate is periodic acid, sodium periodate or potassium periodate. A method as claimed in any preceding claim wherein said reducing agent is sodium borohydride, sodium cyanoborohydride or an amine borane. A method as claimed in any preceding claim wherein step (a) is effected in an aqueous solution at a pH of from 4 to 9. A method as claimed in any preceding claim wherein step (a) is effected in an aqueous solution at a temperature of from 0 to 50°C. A method as claimed in any preceding claim wherein step (b) is effected in an aqueous solution at a pH of from 6 to 10. A product, obtainable by a method as claimed in any preceding claim.

Inventors: KEOGH JAMES R, KEOGH, JAMES R.
Applicants: MEDTRONIC INC, MEDTRONIC, INC.
Application year: 1997
Main classification: A61L33
IPC codes: A61L27, A61L31, A61L29, A61L33