CA2262643A1 - Biocompatible medical article and method - Google Patents

Abstract

A medical article having a metal or glass surface with the surface having an adherent coating of improved biocompatibility. The coating is made by first applying to the surface a silane compound having a pendant vinyl functionality such that the silane adheres to the surface and then, in a separate step, forming a graft polymer on the surface with applied vinylsilane such that the pendant vinyl functionality of the vinylsilane is incorporated into the graft polymer by covalent bonding with the polymer. Biomolecules may then be covalently attached to the base layer.

Description

CA 02262643 1999-02-0=, W O 98/08551 PCT~US96/13783 BIOCOMPATIBLE MEDICAL ARTICLE AND METHOD
BACKGROUND OF INVENTION
The present invention generally relates to medical devices for il~lp~ AIion in ahuman or animal body which are provided with improved tissue and blood bioco~ )a~il,i!;Ly.
S More specifically, metal or glass portions of the medical device are provided with a surface which has been ch~.mir,Ally modified with covalently ~tt~çhed bioactive molecules.
Medical devices which serve as substitute blood vessels, synthetic and intraocular lenses, electrodes, catheters and the like in and on the body or as extracorporeal devices int~n~led to be connected to the body to assist in surgery or dialysis are well known.
However, the use of such biomAt~riAl~ in medical devices can stim~ te adverse body responses, inr.l~lding rapid thrombogenic action. Various plasma proteins play a role in initiAting platelet and fibrin deposition on plastic surfaces. These actions lead to vascular constriction to hinder blood fiow, and the illnAI~llllAlory reaction that follows can lead to the loss of function of the medical device.
A "biomaterial" may be defined as a material that is subsL~ ;ally insoluble in body fluids and that is d~ign~d and constructed to be placed in or onto the body or to contact fluid ofthe body. Ideally, a biomaterial will not induce undesirable reactions in the body such as blood clotting, tissue death, turnor forrnation, allergic reaction, foreign body reaction (rejection) or ;"ni-".,.~lc..y reaction; will have the physical properties such as strength, elasticity, permeability and flexibility required to function for the int~nded purpose; can be purified, fabricated and sterilized easily; will substantially ~ - its physical properties and function during the time that it remains ;~ ~ ~p~ ed in or in contact with the body.
As used herein, the solid surface of a biomaterial is characterized as "bioco~ ible" if it is capable of functioning or existing in contact with biological fluid andlor tissue of a living organism with a net beneficial effect on the living organism. Long term biocompatibility is desired for the purpose of reducing disturbance of the host organism. One approach to improved bioco.~ l;bilily for biolll~l~l;als is to attach various "biomolecules" which can promote the ~tt~shm~nt and growth of a normal cell or protein layer such that the body accepts the device as a normal part ofthe body. Biomolecules such as growth factors and cell ~tt~chm~nt proteins which have been ~tt~r.hed to the device CA 02262643 1999-02-0~

surface could be used for this purpose. In addition, biomolecules such as al.LiLl~ ol~lbogenics, antiplatelets, anti-infl:~mm~tories and the like have also been used to improve the biocompatibility of surfaces.
A number of approaches have been suggested to attach such biomolecules. One S such approach is set forth in Dekker et al., "Adhesion of endothelial cells and adsorption of serum ploteills on gas plasma-treated polytetrafiuoroethylene", Biomaterials, vol. 12 March 1991. In that approach, PTFE SUI~ eS were modified by radio frequency plasma to improve the wettability of the surface. Human serum albumin, human fibronectin, human immlln~globulin and human high-density lipoprotein were adsorbed to the plasma-treated substrates followed by seeding with human endothelial cells. Another approach isdescribed in U. S. Patent 5,055,316 to Hoffman et al in which serum proteins such as albumin, immunoglobulins, fibrinogen or fibronectin, or proteins from di~erellL sources such as protein-A or glycopl oteh~s are bound to a surface by first using plasma gas discharge in the pl~sence of a plasma-poly.lleliGable fluorinated hydrocarbon gas to provide a plasma-deposited surface, followed by exposure to a solution ofthe protein. Covalent att~r.hm~nt of such biomolecules can be found in Ito et al., "Materials for Fnh~nc.in~ Cell Adhesion by Immobilization of Cell-Adhesive Peptide", Journal of Biomedical Materials Research, 25: 1325-1337 (1991) in which ~ ,ne~,Lill or RGD peptide are bonded to the hydrogel by the use of a water soluble carbodiimide. Although this method allows coupling of the biomolecule extended from the surface, the fact that the biomolecule is immobilized throughout the gel layer reduces the availability of the biomolecule for interaction with, for example, cells int~ntled to adhere to the biomolecule.
Spacer molecules have been used to address this problem. A spacer molecule is a molecule or compound which is capable of ~tt~rhm~nt to a solid surface, is large enough to extend from the surface of said surface and is capable of immobilizing a biomolecule and/or biomolecl lles The spacer insures that the active site of the biomolecule is held outward away from the support so as to contact the body fluid ~.fficiently. The spacers are derived from organic molecules having at least two reactive functional groups, or more, generally situated at opposing ends of the molecule. Such groups serve as ~tt~r.hment vehicles capable of coupling the spacer to the solid surface and to the biomolecule. For example, in U. S . Patent 5,132,108 to Narayanan et al., a copolymer surface was subjected to radio I

CA 02262643 1999-02-0~

frequency plasma Ll ~LIllellL by subjecting it to a radio frequency electric field in the presence of a water vapor plasma m~di~lm An aqueous solution of polyethylçneirnine (PEI) and 1-(3-dimethylpropyl)-3-carbodiimide (EDC) coupling agent was applied to the radio frequency plasma discharge modified polyurethane surface. An aqueous solution of heparin and EDC was then applied to the PEI-treated surface in order to provide a polymeric surface having an anti-thrombogenic agent secured to its surface. However, considering the heterogeneity of the polyurethane surface even coating with the multi-functional spacer molecule is not guaranteed.
Additional coverage can be provided, for example, according to U. S. Patent 4,565,740 to Golander et al. or U.S. Patent 5,049,403 to Larm et al. In the first ofthese patents, a complex of a polymeric cationic surfactant (e.g. a polyalkyleneimine) and a dialdehyde (e.g. glutaraldehyde) is adsorbed onto a substrate material. In the second of these patents, a polyarnine is adsorbed onto the surface of a substrate and crosslinked with croton~ldehyde. Multiple coatings, inr~ ling intermediate layers of anionic material are then applied to obtain an effective coating. However, these crosslinked coatings rely on adsorption onto the surface and ionic bonding to the surface, which may not provide good bonding ofthe coating to the surface.
The inventors of the present invention have contributed to improvements in biocompatibility of biomaterials through the use of multilayer coatings in their U.S. Patents 5,229,172; 5,308,641 and 5,350,800. For example, in U.S. Patent S,229,172, we discovered a method for modifying the surface characteristics of a polymeric material by providing a base layer of grafted acrylamide on the polymeric surface which can be used to attach various spacers and biomolecules. Or, in U.S. Patent 5,308,641, we discovered an improved spacer material which inr~ lçs a polyalkyçn-qimine covalently attached to an ~min~ted substrate and crosslinked with a cros~linking agent which is difunctional in aldehyde groups. Or, in U.S. Patent 5,350,800, we discovered a method for ~tt~r.hin~ a biomolecule having carboxyl groups to an ~min~ted solid surface by a carbodiimide and then selectively I ~LO~ , the bio-functionality of the carboxyl groups.
On metal or glass surfaces, the binding of the base layer of such multi-layer coatings can be a problem since there is no organic structure to provide covalent bonds between the metal or glass substrate and the grafted base layer. Others have addressed the problem of CA 02262643 1999-02-0=7 W O 98/08551 PCTrUS96/13783 binding to metals and glass by applying aminosilanes to adhere to the surface and then ~tt~hing the biomolecule to the aminosilane through the amine functionality ofthe aminosilane. This can be seen in U.S. Patent 5,355,433 issued to Rowland et al in which an aminosilane is used to adhere a heparin molecule to an oxidized t~nt~ m surface.Aminosilanes are also disclosed for ~tt~hm~nt of a heparin molecule to glass or metal s~ .es in U.S. Patent 4,1 18,485 issued to Eriksson et al. However, the use of aminosilanes in coatings of this sort has not been very good in producing a surface with a high level of both bioeffectiveness and stability.
It is th~lt;role an object ofthe invention to provide a base for the ~tt~rhm~nt of biomolecules and/or spacer molecules with improved stability on metal or glass substrates.
It is also an object of the invention to provide a combined base/spacer which presents a stable pla~ for the attachrnent of the biomolecule and thereby prevents the ~tt~rh~d biomolecule from being buried in the spacer layer.
Summary of the Invention The present invention therefore includes a medical article having a metal or glass surface with the surface having an adherent coating. The coating inrl~ld~ a silane compound having a vinyl functionality such that the silane adheres to the surface with the vinyl functionality pendant from the surface and then forming a graft polymer on the surface with applied silane such that the pendant vinyl functionality of the silane is incorporated into the graft polymer by covalently bonding it to the graft polymer.
The preferred silane is generally a compound of the structure C2H3-Si- X3 where X is a halogen, methoxy or ethoxy groups. Compounds of this type include the p,t;r~lled compound trichlorovinylsilane. Other silanes can also be used as set forth in greater detail below.
The base layer which incorporates the vinyl functionality ofthe silane also includes a thin but densely formed graft polymer. Pl~rel~bly, the graft polymer is formed by free radical reaction from an ethylenically unsaturated monomer. Thus the reaction which forms the graft polymer also activates the pendant vinyl group of the silane and incorporates it into the graft polymer during its formation. An oxidizing metal such as ceric ion can be used to initiate the polymeli~Lion reaction. Ceric ion grafting is known to work best when , . . .

CA 02262643 1999-02-0~

W O98/085Sl PCTAJS96/13783 s the monomer does not have a tendency to plecil,;taLe with the ceric ion, for in.~t~nce, when used with acrylamide. Acrylic acid is also a good c~n~ te monomer for use with ceric ion grafting provided that polyl~ alion inhibitors present in eommercial supplies of acrylic acid are first removed by tli~till~tion. Blends of aerylic acid, acrylamide and other monomers can also be used depending on the desired properties of the graft.
The biofunetional molecules ~tt~r,hed to grafted surfaces ean be biomolecules sueh as antiCo~ nt~ (e.g. heparin, heparin sulfate, dermatan sulfate, glycos~minoglycan sequ~nr.es and analogs, hirudin, Llllolllbill inhibitors), thrombolytie agents (e.g.
streptokinase, tissue pl~.cminogen activator), procoagulant agents, platelet adhesion inhibitors, platelet aetivity inhibitors, cell attachrnent proteins, growth faetors/cytokines, wound healing agents, antimicrobial agents, antic~n~.~r agents, hormones, ~n~lgçsicc~
det:oxification agents and the like. These biomolecules can be covalently bonded to the grafted surface by pendant functional groups such as amine and carboxyl groups in the grafted polymer which are reacted with corresponding groups on the biofunctionalmolecules accoldillg to methods which are well known by those skilled in the art.
P~ ~;îel ~tbly, a medical article according to the present invention includes a spacer as a means for ~tt~hm~nt for the biomoleeule. Such spacers are well known in the art as set forth above in the baekground of the invention. A preferred spaeer ean be a polyamine spacer as set forth in our U.S. Patent 5,308,641 whieh is hereby ineorporated by referenee in its entirety.
Examples of devices which may be provided with biocompatible surfaees in aeeordanee with this invention inelude prosthetie devices and components of implanted prosthetic devices such as in vascular grafts or hard tissue prosthetics, invasive devices sueh as indwelling catheters or in devices for extraeorporeal blood h~n~lling such as dialysis equipment or blood circulation or oxygenation eq~.irmçnt used in cardiovascular surgery.
In a particular vascular prosthesis embodiment of the invention, the base layer deseribed above can be ~tt~che(l to a metallic medical device which undergoes movement during impl~nt~tion and/or use, since the bioaetive coating is able to withstand flexure without cracking or d~l~min~tion Exemplary in this regard are metallic radially expandable generally tubularly shaped endoprostheses which are generally known as stents. An exemplary stent in this regard is described in U.S. Patents 4,886,062 and 5,133,732 issued CA 02262643 1999-02-0~

W O 98/0~551 PCT~US96/13783 to Wiktor, the subject matter thereofbeing illcull.ol~Led by reference. Stents such as these are made of very fine gauge metallic wire, typically t~nt~ m wire or stainless steel wire.
During impl~nt~tion, these stents are mol-nted onto the balloon of an angioplasty catheter or the like until a partially occluded location along a blood vessel or the like is reached, at which time the balloon and the stent are radially and circumr~ ially expanded for purposes of opening the occlusion and supporting the vessel at that location. This nece.c~rily involves rather extensive bending ofthe t~nt~ m wire. Many coaLillgs do not have the llexibility and/or adherence properties which are needed to avoid cracking and/or loss ofthe coating when subjected to this type of flexure. Further, a stent provided with heparin as a biomolecule according to the present invention and implanted within a blood vessel can prevent thrombus formation on the metallic Ill~lllber that may otherwise occur as a result of the stent impl~nt~tion procedure.
Detailed Description of the Invention The base layer ofthe multilayer coating is made by first applying to the surface a silane having a pendant vinyl functionality such that the silane adheres to the surface. The silane used includes compounds ofthe structure C2H3-Si- X3 or, optionally C2H3 - R - Si - X3 where X is a halogen, methoxy or ethoxy group, and R is a short chain alkyl group. A
pl erel I ed silane compound is trichlorovinylsilane.
Those skilled in the art will recognize that the s~lcces~fi 1l applicalion of the silane to a surface includes preCle~ning of the surface and the control of moisture at the surface during application ofthe silane. Multistep r.le~ning and drying operations are th~l~rol~
used to provide a clean surface and to control moisture. An c~.enl~ y method for cleaning and drying can be found herein in the examples.
The base layer which incorporates the vinyl functionality of the silane inr.llldes a thin but densely formed graft polymer. Pl~r~l~ly, the graft polymer is formed by free radical reaction from an ethylenically unsaturated monomer. Thus the reaction which forms the graft polymer also activates the pendant vinyl group ofthe silane and incorporates it into CA 02262643 1999-02-0~

W O 98/08551 PCT~US96/13783 the graft polymer during its fomlation. An oxidizing metal such as ceric ion can be used to initiate the poly~ .alion reaction.
The plefell~d grafting process is carried out on the substrate in an aqueous solution (20 to 40 wt% of monomer) as contrasted with other solvent polymerization processes such as organic solvent polymerization or even bulk pOlyl~ alion The composition of the pl~r~lled monomer solution is predonl~ Lly acrylic acid and acrylamide in order to provide a desired density of carboxyl groups on the grafted surface which can be used to attach the biomo}ecule or a spacer layer. Altematively, acrylamide can be used exclusively followed by hydrolysis ll~allllel~l on the resulting polymer to provide the desired density of carboxyl groups.
The grafting reaction ofthe present invention may be carried out at lelllpe~ resbetween about 1 8~C and 25~C. The present invention may be carried out under pressure or under partial vacuum, but it is ~ r~ d to utilize atmospheric pressure in~cmll~h as the reaction proceeds very favorably at this pressure. The pH of a grafting solution with ceric ~lllllvni~lm nitrate is typically about 1.4.
The amount of ceric ion utili~d in the practice of the process of the present invention can be varied over fairly wide limits. For CA~IIPIC~ one may utilize from about 0.0001 to 0. 002 mole of ceric ion per mole of polyllleliGable mGnolllel . Preferably one would use between 0.0002 to 0.0005 mole of ceric ion per mole of acrylamide. Ceric ion is preferably introduced into the reaction mixture in the fomm of a ceric salt. Among the cerium salts adapted for use in the present invention are ceric nitrate, ceric sulfate, ceric ammonium nitrate, ceric ~mmr)ni~lm sulfate, ceric ammonium pyrophosphate, ceric iodate, ceric salts of organic acids, such as cerium n~ and cerium linoleate and the like.
These compounds may be employed singly or in com~ ~lion with one another.

2~ In general, the time required to achieve a desired degree of polymerization may be dete~nined empirically. Thus, for C~ le, acrylamide may be grafted at di~lelll time intervals and the extent of grafting d~Lel ll~illed by staining of functional groups introduced in the graft by chemical modifica~ion. The length of the polymeric chain and graft density may be varied by varying the acrylamide conc~ lion, ceric ion concenllalion, temperature and oxygen conct~ ion.

CA 02262643 1999-02-0~

W 098tO8551 PCTrUS96/13783 Biofunctional molecules (biomolecules) such as antico~ ntc (e.g. heparin, heparin sulfate, dermatan sulfate, glycosaminoglycan sequences and analogs, hirudin, ~h~u---l)in inhibitors), thrombolytic agents (e.g. streptokin~cç~ tissue plasmogen activator), proco~ nt agents (e.g. Factor VIII, von Willebrand's Factor, collagen), platelet adhesion inhibitors (e.g. albumin, albumin adsorbing surfaces, hydrophilic hydrogels, phospholipids), platelet activity inhibitors (e.g. aspirin, dipyrim~lole, forskolin), cell ~tt~hm~nt proteins (fibronectin, villollecLh~, di~l~ collagen types, larninin, elastin, b~c~.m~nt membrane proteins, fibrin, peptide seqUçnres), growth factors/cytokines (e.g. I~ansr~",llillg growth factor, basic fibroblast growth factor, platelet derived growth factor, endothelial cell growth factor, gamma interferon), hydrogels, collagens, epidermal growth factor, ~ lfi~lobial agents (e.g. ge.l~ n, lir~m~ , silver salts), anticancer agents (e.g. 5-fluorouracil), hormones (insulin, vasopressin proge~ Jne, human growth hormone), analgesics, detoxification agents (e.g. çh~l~ting agents) and the like can be ionically or covalently bonded to a metallic substrate by first applying the grafling method of the present invention to provide a suitable surface to which to attach the biomolecule. Such molecules can be covalently ~tt~ched to a grafted surface made according to the present invention in which pendant functional groups such as amine and carboxyl groups introduced in the gel by r.h~qmir.~l modification are reacted with corresponding groups on the biofunctional molecules accold;ng to methods which are well known by those skilled in the art.Preferably, a medical article according to the present invention inçludt?.~ a spacer m~lec~]le as a means for ~tt~r.hmrnt for the biomolec~le. Such spacer molecules are well known in the art as set forth above in the background of the invention.
The preferred spacer molecule is a polyalkyl~n~imine or other branched polyamines. By polyalkyl~nPimine we lhel~rol~ mean to include the water soluble,hydrophilic, polyamines evolving from aziridine and ~etifline monomers such as 1-unsubstituted imines, 1-substituted basic imines, activated imines (1-acyl substituted imines), isomeric oxazolinesloxazines and the like. The polyalkyl~ e~ employed in the present invention are preferably highly branched, thereby possessing primary, secondary, and tertiary amine groups. Thus, ethylP.~ ;n~. polylll~ ed by classical cationic chain-growth polymerization, either alone or with other monomers suitable for copolyl.. cli~dlion with ethylrn~.iminr, could be used in the present invention.

CA 02262643 1999-02-0~

W O 98/08551 PCTrUS96/13783 A cro~ nkin~ agent can be employed in the present invention in order to provide additional stability of the polyamine spacer. The cros.clinking agent can be any cros~linking agent which is at least difunctional in groups which are reactive with the amine groups present in the polyamine spacer. The crosslinking agent may thel ~rOI ~ have an aldehyde functionality. For example, glutaraldehyde, croton~l~lP.hyde, goxal, m~on ld~hyde, suc~.in~ldP.hyde, ~ ldPhyde, and dialdehyde starch could be used. Other suitablecros~linking agents are cyanuric chloride and derivatives, divinyl sulfone, epoxy compounds, imidate esters and other cro.~linking agents reactive toward amines.
The spacer of the present invention can therefore be made by applying a polyalkylPnPiminp to the grafted surface and then treating the applied polyalkylPnPimine with the cro~linking agent. Pl ~r~- ~bly, the cros.clinking agent used to crosslink the po]yalkylPnPimin~: is applied in dilute solution and at a suitable pH to accomplish light cro~.~linking For example, an aldehyde solution that has a concentration in the range of about 0.0005 M to about 0.05 M could be used while a concel~ lion in the range of about 0.0005 M to about 0.005 M would be pr~f~ d. Also, for example, a pH for the aldehyde solution in the range of about 7 to about 10 would be pl~r~ d. The time required to complete the light cro~ g reaction is typically just a few minutes in the case of dialdehydes or longer times for other crosslinkers. Pler~l~bly, the cro~ nking reaction with the polyamine is undertaken before applying it to the grafted surface.
The polyalkyl~n~imine is covalently bonded to the gra~ed surface by cont~cting the grafted surface with an activating agent which will activate the carboxyl groups on the grafted surface and cause them to bind to the polyalky~i-f ~ The covalent bonding agent used is preferably a water soluble carbodiirnide of the structure RIN=C=NR2 where R, can be an allcyl or cycloalkyl group and R2 can be an alkylarnine or cycloalkylamine group such as 1-ethyl-3-(3-dimethyl-arninopropyl) carbodiimide hydrochloride, orl-cyclohexyl-3-(2-morpholinoethyl) carbodiimide. The reaction with the carbodiimide is undertaken in a cold solution (04~C) at a pH of about 5 although a room tenlp~l ~lure reaction is also acceptable. The grafted surface can be pl~LI~led with the carbodiimide and then contacted with the polyamine or, pl ~r~l al)ly, the grafted surface can be coated with the polyamine and then treated with the carbodiimide. Pre~ly, the polyamine and carbodiirnide are previously mixed together at a pH of a~Jpl oxilllately 9 before applying to CA 02262643 1999-02-0~

the graft. In the reaction, the carbodiimide activates the carboxyl groups of the graft after reaction with the polyamine which leads to the formation of a suitable amide bond, resulting in effective immobilization of the polyamine on the grafted surface.
The immobilized polyamine can then be used as a plalr~ ll for the immobilizationof various biofunctional mol~ For example, in the case of heparin, the heparin can be modified to contain a reactive aldehyde moiety which does not inhibit the bioactivity of the heparin but does react with the amine groups of the polyamine to covalently attach the heparin to the polyamine in the pl esellce of a suitable reducing agent such as NaCNBH3.
The aldehyde groups can be formed on heparin by controlled periodate oxidation. Part of the saccharide molecules in the heparin contain unsubstituted glycol structures (c(2)-OH
AND C(3)-OH), which react with periodatem splitting the C(2)-C(3) bond, gene~ g a dialdehyde structure and leaving the poly~ac~,h~ide main chain intact. AfLer oxidation (under the exclusion of light) the solution colll~;lling the activated heparin can be diluted into a proper buffer to a suitable conc~ Lion. The solution is then applied to the polyamine immobilized on the surface. The aldehyde functional groups on the heparin are then reacted with the free amine groups to give a Schiffbase formation that may be reduced to provide stable secondary amines. Exemplary reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane and tetrahydrofuran-borane.
Upon completion of the coupling reaction, the surface may be washed with water and solutions of sodium chloride to remove loosely bound or unreacted heparin.
Example 1 A piece of coiled t~nt~l-lm wire was ultrasonically cleaned in 2% Micro-clean for 30 minutes followed by ultrasonic lle~ in deionized water for 30 mimltes. This last step was repeated after which the coil was rinsed in isopropanol and dried at 50~C for 20 minl.t~s The cleaned coil was swirled in a 2% solution of trichlorovinylsilane (Merck Darmstadt, FRCi) in xylene for 60 seconds followed by rinsing for 60 seconds in xylene, 60 seconds in isoplopanol, 60 seconds in water and finally in acetone. The coil was then allowed to air dry overnight.
The dried coil was then placed into a glass tube which was filled with 15 ml of an aqueous solution of 35 wt% of freshly distilled acrylic acid and 5 wt % acrylamide. To the CA 02262643 1999-02-0=, 15 rnl of monomer solution 0.9 ml of a solution of ceric ~mmoni~Im nitrate (0.1M) in nitric acid (0. lM) was added. Deaeration was pe~ "ed for 3-5 minutes at about 18 mm Hgfollowed by ultrasonic l,e~l,,,c,,l for 10 minutes and an additional incubation of 3540 mimlt~s all at room te"")t;~ re. The grafted samples were then rinsed 10 times with deionized water at 50~C followed by an overnight incl1b~tion at 50~C. Samples taken showed a deep stain when soaked in toluidine blue sol--ti- n.
A solution of 375 ml crotonaldehyde in 0. lM sodium borate (pH=9. 1) was made and after 10 minutes stirring polyethyl~ in.; ~ (PEI, Polymin SN from BASF with a Mw of 60,000) was added. After an additional mixing of 5 min.ltec the coil was incubated in the crosslinked PEI solution for one hour while shaking. After rinsing with deionized water the coil was ct-nt~r.ted with a solution of 0. 5 wt% PEI (Polymin SN) in 0. lM sodium borate (pH=9. 1) for 10 min--t~s. Water soluble carbodiimide (1-(3-diethylarninopropyl)-3-ethylcarbodiimide.HCI) at a COllC~ lion of 0.05M was added. Coupling was allowed to proceed for one hour while shaking followed by rinsing with deionized water for 10 1 5 minutes.
Oxidized heparin was p,epa,ed by adding 0.165 mg NaIO4/ml to 5 mg native heparin (Akzo)/ml 0.05M phosphate buffer (pH=6.88; 0.025M K2HPO4 +
NaH2PO~*2H2O) under the exclusion of light. After overnight oxidation the resulting heparin solution was diluted in 0.4M acetate pH~.6 at a ratio of 1:20. 0.1 mg ofNaCNBH3 /ml was added to the diluted heparin and the coil was in~llhated in this solution for 2 hours at 50~C. A~er rinsing with de;oni~.ed water lM NaCI and water again to remove loosely bonded heparin, the coil was incubated with toluidine blue which provided an even lilac stain, il~iç ~ g s~lccescfill hep~ ll An additional bioactivity test was also succ~ccfillly p~,r~ ed to determine the ability ofthe hep~il~i~ed surface to deactivate IhlUn~;n via activation of previously adsorbed ~llill"on~ l III. The bioactivity was also tested s-lcceq.~fi~lly after an overnight c.h~lle~e with 1% sodium dodecylsulfate at 50~C
in~liç~ting excellent stability of the coating on the metal substrate.
Co"".a. ~ te Exarnples 2-8 Co~n~ e tests were conducted on a variety of variables. Different methods to aminate the metal surface and their ability to bind heparin via the reductive ~min~ti~n process descnbed in Example I were evaluated. Variables tested inclufled the use of no W O 98/08551 12 PCT~US96/13783 silane, an aminosilane, ~~ opl~lJyl triethoxysilane (APTES), and a vinylsilane, trichlorovinylsilane (TCVS); the use of grafted polymers (acrylamide (AAm) and acrylic acid (Ac)) and copolymers as a base layer; adsorbed or grafted PEI layer; and various crosclinkin~ of the PEI layer (crotonaldehyde (Ca), glutaraldehyde (Gda) and divinylsulfone (DVS)). For Example 8, coils were ple~aied with a grafted hydrogel cS~nt~ y as described in Example 1. A solution of 0.1 wt% PEI (Polymin SN from Basf with a Mw of 60,000) in 0. lM borate pH=9.0 was pl~ared to which water soluble carbodimide (1-(3-dimeth~ldln,noplo~yl)-3-ethylcarbodiimide.HCI, Aldrich) up to a concentration of 0.05M was added. Imm~ t(?ly after dissolution ofthe carbodiimide, the grafted coils were contacted with the solution for 50 minutes while gently ~h~king After copious rinsing with water, heparin was coupled to the coils as described in Example 1.
The test results for Examples 2-8 are given below in Table 1.
Table 1 Exarnple Silane Process PEI Layer Bioactivity Stainin~
2 None Adsorbed Basf/Ca 0.0465 0 3 APTES Adsorbed Basf/Ca 0.0864 0 4 APTES Adsorbed Fluka~DVS 0.1512 0 APTES Adsorbed FlukalGda 0.0090 0 6 TCVS GraftlAc Basf/Ca 0.2192 4 7 TCVS Graft Basf/Ca 0.330 4+
Ac+AAm 8 TCVS Graft Basf 0.323 4+
Ac+AArn The coll~pa~ dLi~re test results in Table 1 indicate that the bioactivity of the adsorbed PEI layer (in IU of DEA/cm2)was less than that for a covalently bonded layer as present in Ex~unrlel, 7 and 8. Further, the staining following the overnight challenge with 1% sodium dodecylsulfate (on a scale of 0=no stain to 5=dark stain) showed that the heparin layer was not effectively m~int~in~d on test samples with the aminosilane and adsorbed PEI whereas the heparin layer was retained on the covalently bonded PEI.
It will be d~ cid~ed by those skilled in the art that while the invention has been described above in connection with particular embodiments and examples, the invention is CA 02262643 l999-02-OF7 WO 98/08551 PCT/US96tl3783 not nPc~s~rily so limited and that numerous other embo(lim~nt~, examples, uses, modifications and departures from the embo~im~nt~, examples and uses may be madewithout departing from the inventive collcepts.

Claims

We Claim:
1. An endoprosthesis having a metal surface contacting body fluids, the metal surface having a coating thereon comprising:
(a) a silane which includes a vinyl functionality, the silane adherent to the metal surface such that the vinyl functionality is pendant from the surface; and (b) a graft polymer on the silane adherent surface such that the pendant vinyl functionality of the silane is covalently bonded with the graft polymer 2. The endoprosthesis of claim 1 also comprising:
(a) a polyamine spacer covalently attached to the graft polymer; and (b) a biomolecule covalently attached to the spacer.
3. An endoprosthesis as in claim 1, in which the silane also includes a functional group selected from the group consisting of halogen, methoxy and ethoxy groups.
4. An endoprosthesis as in claim 3, in which the silane is trichlorovinylsilane.5. An endoprosthesis as in claim 1, in which the graft polymer is formed by freeradical reaction from an ethylenically unsaturated monomer.
6. An endoprosthesis as in claim 5, in which the monomer is selected from acrylamide and acrylic acid.
7. An endoprosthesis as in claim 2, in which the polyamine spacer is a polyalkyleneimine.
8. An endoprosthesis as in claim 2, in which the biomolecule is an antithrombotic.
9. An endoprosthesis as in claim 8, in which the antithrombotic is selected from the group consisting of heparin and heparin derivatives.
10. A medical device having metal or glass surface in contact with body fluids, the surface having a coating thereon comprising:
(a) a silane having the structure C2H3 - R- Si - X3 where X is a halogen, methoxy or ethoxy and R is an optional short chain alkyl group; the silane adherent to the surface such that the vinyl functional group is pendant from the surface; and (b) a graft polymer formed by free radical reaction from an ethylenically unsaturated monomer, the graft polymer formed on the silane adherent surface such that the pendant vinyl functionality of the silane is incorporated into the graft polymer.
11. A medical device as in claim 10 also comprising:
(a) a polyalkyleneimine spacer covalently attached to the graft polymer; and (b) a biomolecule covalently attached to the spacer.
12. A medical device as in claim 10, in which the silane is trichlorovinylsilane13. A medical device as in claim 10, in which the monomer is selected from acrylamide and acrylic acid.
14. A medical device as in claim 11, in which the biomolecule is an antithrombotic 15. A medical device as in claim 14, in which the antithrombotic is selected from the group consisting of heparin and heparin derivatives.
16. In a radially expandable stent comprising a hollow cylindrical shape made of a low-memory level metal such that the metal can controllably deform to effect radial expansion of the stent from a first diameter to a second, expanded diameter, the metal coated with a biologically compatible coating, the improvement wherein the coating includes:
(a) a silane adherent to the metal; and (b) a graft polymer covalently attached to the silane such that the coating is retained on the wire as the stent is expanded.
17. A stent as in claim 16 wherein the silane has a pendant vinyl functionality which is incorporated into the graft polymer.
18. A stent as in claim 17 wherein the biocompatible coating also comprises a biomolecule covalently attached to the spacer.
19. A method of providing a medical article having a glass or metal surface with a biocompatible coating on the glass or metal surface comprising the steps of:
(a) applying to the surface a silane having a vinyl functionality such that the silane adheres to the surface;
(b) forming a graft polymer on the silane adherent surface such that the vinyl functionality of the silane is incorporated into the graft polymer.
20. A method as in claim 19 wherein the silane has the structure C2H3 - R - Si - X3 where X is a halogen, methoxy or ethoxy and R is an optional short chain alkyl group.
21. A method as in claim 20, in which the silane is trichlorovinylsilane applied to the surface in dilute solution.
22. A method as in claim 19, in which the graft polymer is formed on the silane adherent surface by applying to the silane adherent surface an ethylenically unsaturated monomer in the presence of a free radical initiator.
23 . A method as in claim 19 also comprising the step of covalently attaching a biomolecule to the graft polymer.
24. A method as in claim 23, in which the biomolecule is covalently attached by the steps of:
(a) covalently attaching a spacer molecule to the graft polymer; and (b) covalently attaching the biomolecule to the spacer molecule.
25. A method of providing a medical article having a glass or metal surface with a bioactive heparin coating on the glass or metal surface comprising the steps of:(a) applying to the surface a silane having a vinyl functionality such that the silane adheres to the surface;
(b) forming a graft polymer on the silane adherent surface such that the vinyl functionality of the silane is incorporated into the graft polymer.
(c) covalently attaching a polyamine spacer to the graft polymer;
(d) treating heparin to form aldehyde functional groups on the heparin; and (e) covalently attaching the heparin to the polyamine spacer.