Patent 6,617,142

Methods are provided for forming a coating of an immobilized biomolecule on a surface of a medical device to impart improved biocompatibility for contacting tissue and bodily fluids. A biomolecule such as a glycoprotein having an unsubstituted amide moiety is combined with an amine forming agent to form an amine-functional biomolecule. The amine-functional biomolecule is combined with a medical device surface having a chemical moiety such as aldehyde, epoxide, isocyanate, 1,2-dicarbonyl, phosphate, sulphate or carboxylate to form a chemical bond immobilizing the biomolecule on the surface. The chemical bond may be combined with a reducing agent or a stabilizing agent. The aldehyde moiety may be formed by combining a periodate with a 2-aminoalcohol moiety or a 1,2-dihydroxy moiety. Alternatively, an amine-functional medical device surface is combined with a biomolecule having a chemical moiety that reacts with an amine moiety. In another embodiment, the amine-functional biomolecule is converted to a guanidino-functional biomolecule and is combined with a medical device surface having a chemical moiety that reacts with a guanidino moiety. Alternatively, an amine-functional medical device surface is converted to a guanidino-functional surface and is combined with a biomolecule having the chemical moiety. Biomolecules may be crosslinked by combining the amine-functional or guanidino-functional biomolecule with a biomolecule having a chemical moiety that forms a chemical bond with an amine moiety or a guanidino moiety.

SUMMARY OF THE INVENTION

The present invention provides methods for attaching a biomolecule to a substrate surface and corresponding medical devices. The present invention provides methods for making a medical device having at least one biomolecule immobilized on a biomaterial surface. One method of the present invention includes converting a biomolecule comprising an unsubstituted amide moiety (RCONH₂) into an amine-functional material (RNH₂); combining the amine-functional material with a medical device biomaterial surface comprising a chemical moiety (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the amine-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface.

The present invention provides another method for making a medical device having at least one biomolecule immobilized on a biomaterial surface. The method includes converting a medical device biomaterial surface comprising an unsubstituted amide moiety (RCONH₂) into an amine-functional material (RNH₂); combining the amine-functional material with a biomolecule comprising a chemical moiety (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the amine-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface.

The present invention also provides a method for making a medical device having at least one biomolecule immobilized on a biomaterial surface. The method includes converting a biomolecule comprising an unsubstituted amide moiety (RCONH₂) into an amine-functional material (RNH₂); converting the amine-functional material into a guanidino-functional material (RNHC(NH)NH₂); combining the guanidino-functional material with a medical device biomaterial surface comprising a chemical moiety (such as, for example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the guanidino-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface.

Additionally, the present invention provides a method for making a medical device having at least one biomolecule immobilized on a biomaterial surface. The method includes converting a medical device biomaterial surface comprising an unsubstituted amide moiety (RCONH₂) into an amine-functional material (RNH₂); converting the amine-functional material into a guanidino-functional material (RNHC(NH)NH₂); combining the guanidino-functional material with a biomolecule comprising a chemical moiety (such as, for example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) which is capable of forming a chemical bond with the guanidino-functional material, to bond the two materials together to form an immobilized biomolecule on a medical device biomaterial surface.

Another method of the present invention may be employed to crosslink biomolecules, located in solution or on biomaterial surfaces, comprising both an unsubstituted amide moiety and a chemical moiety capable of forming a chemical bond with an amine moiety. Such a method comprises converting a biomolecule comprising an unsubstituted amide moiety into an amine-functional material; allowing the amine-functional material to combine with the chemical moiety capable of forming a chemical bond with an amine moiety to form a chemical linkage and a crosslinked material. This crosslinked material may be employed as a biomaterial or as a biomaterial coating. In addition, such crosslinked materials may be further modified to contain additional biomolecules. For example, biomolecules comprising a chemical moiety capable of forming a chemical bond with an amine moiety may be attached to residual amine moieties present in or on the surface of the crosslinked material. Alternatively, biomolecules comprising an amine moiety may be attached to residual chemical moieties capable of forming chemical bonds with amine moieties present in or on the surface of the crosslinked material. Additionally, biomolecules coated onto a biomaterial surface may be crosslinked according to still another method of the present invention.

Another method of the present invention may be employed to crosslink biomolecules, located in solution or on biomaterial surfaces, comprising both an unsubstituted amide moiety and a chemical moiety capable of forming a chemical bond with a guanidino moiety. Such a method comprises converting a biomolecule comprising an unsubstituted amide moiety into an amine-functional material; converting the amine-functional material into a guanidino-functional material (RNHC(NH)NH₂); allowing the guanidino-functional material to combine with the chemical moiety capable of forming a chemical bond with an guanidino moiety to form a chemical linkage and a crosslinked material. This crosslinked material may be employed as a biomaterial or as a biomaterial coating. In addition, such crosslinked materials may be further modified to contain additional biomolecules. For example, biomolecules comprising a chemical moiety capable of forming a chemical bond with an guanidino moiety may be attached to residual guanidino moieties present in or on the surface of the crosslinked material. Alternatively, Piomolecules comprising a guanidino moiety may be attached to residual chemical moieties capable of forming chemical bonds with guanidino moieties present in or on the surface of the crosslinked material. Additionally, biomolecules coated onto a biomaterial surface may be crosslinked according to still another method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As used in the specification and claims hereof, the following terms have the particular meanings and definitions set forth below.

We define the term "chemical bond" appearing herein to be interpreted a broadly to encompass not only covalent bonding and ionic bonding but also interactions, such as, for example, van der Waals forces and hydrogen bonding.

We define the term "biomolecule" appearing herein as a material that engages in a biological activity or which is effective in modulating a biological activity such as eliminating, reducing or enhancing various biological reactions that typically accompany the exposure of human or animal bodily tissues or fluids to a biomaterial. Biomaterial-associated reactions include thrombosis, tissue death, tumor formation, allergic reaction, foreign-body reaction (rejection), inflammatory reaction, infection and cellular attachment and growth. Biomolecules suitable for use in the present invention comprise either an unsubstituted amide moiety, a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, a 1,2-dicarbonyl moiety, a guanidino moiety, a chemical moiety capable of forming either a covalent bond with an amine moiety (such as, for example, an aldehyde moiety, an epoxide moiety or an isocyanate moiety) or a chemical moiety capable of forming an ionic bond with an amine moiety (such as, for example, a phosphate moiety, a sulphate moiety or a carboxylate moiety), or any possible combination of any one or more of these moieties alone or in combination. In addition, the term "biomolecule" appearing herein may mean any one or more of a biomolecule alone or a combination of different biomolecules.

Generally, biomolecules used according to this invention may be, for example an anticoagulant agent such as heparin and heparan sulfate, an antithrombotic agent, a clotting agent, a platelet agent, an anti-inflammatory agent, an antibody, an antigen, an immunoglobulin, a defense agent, an enzyme, a hormone, a growth factor, a neurotransmitter, a cytokine, a blood agent, a regulatory agent, a transport agent, a fibrous agent, a protein such as avidin, a glycoprotein, a globular protein, a structural protein, a membrane protein and a cell attachment protein, a peptide such as a glycopeptide, a structural peptide, a membrane peptide and a cell attachment peptide, a proteoglycan, a toxin, an antibiotic agent, an antibacterial agent, an antimicrobial agent such as penicillin, ticarcillin, carbenicillin, ampicillin, oxacillian, cefazolin, bacitracin, cephalosporin, cephalothin, cefuroxime, cefoxitin, norfloxacin, perfloxacin and sulfadiazine, hyaluronic acid, a polysaccharide, a carbohydrate, a fatty acid, a catalyst, a drug, biotin, a vitamin, a DNA segment, a RNA segment, a nucleic acid, a lectin, a ligand and a dye (which acts as a biological ligand). The biomolecules may be found in nature (naturally occurring) or may be chemically synthesized.

Biomolecules may be chemically synthesized by a number of methods well known to those skilled in the art. For example, a number of methods are know for synthesizing proteins or peptides from amino acids including solution (classical) synthesis methods and solid phase (e.g., SPPS) synthesis methods. Peptides of varying length may also be formed by the partial hydrolysis of very long polypeptide chains of proteins. In addition, proteolytic enzymes such as trypsin, chymotrypsin, and pepsin may be used to cleave specific peptide bonds in proteins and peptides. Furthermore, site-specific oxidative cleavage of peptide bonds using either cupric ions or ferric ions may be used to create peptide and/or polypeptide chains comprising unsubstituted amide moieties. Peptides are short chains constructed of two or more amino acids covalently joined through substituted amide linkages, termed peptide bonds. Two amino acids joined by a peptide bond forms a dipeptide. Three amino acids joined by two peptide bonds forms a tripeptide; similarly, there are tripeptides and pentapeptides. When there are many amino acids joined together, the structure is termed a polypeptide. In general, polypeptides contain less than 100 amino acid residues and proteins contain 100 or more amino acid residues.

Some biomrolecules are susceptible to conformational changes when brought into contact with a hydrophobic substrate surface. These conformational changes can lead to the exposure of internalized nonpolar groups which may lead to hydrophobic interactions between the biomolecule and the surface. These hydrophobic interactions may 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 may 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 such cases, it is preferred to create a hydrophilic environment on the biomaterial surface, thereby preventing any unwanted hydrophobic interactions between the biomolecule and the surface which may 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.

We define the term "glycoprotein" appearing herein as a conjugated protein which contains at least one carbohydrate group which may comprise a 1,2-dihydroxy moiety. A typical glycoprotein contains one or more oligosaccharide units linked to either asparagine amino acid residues by N-glycosidic bonds or serine or threonine amino acid residues by O-glycosidic bonds. The saccharide unit directly bonded to asparagine is typically N-acetylglucosamine, whereas N-acetylgalactosamine tends to be the saccharide unit bonded to serine or threonine residues. Oligosaccharides bound to glycoproteins may contain a variety of carbohydrate units. They tend to be located at sites away from the biologically active site of the protein. Thus, oligosaccharide moieties of glycoproteins can typically be modified with little or no effect on the biological properties of the protein

We define the term "glycopeptide" appearing herein as a conjugated peptide which contains at least one carbohydrate group which may comprise a 1,2-dihydroxy moiety. As mentioned earlier, peptides are short chains constructed of two or more amino acids covalently joined through substituted amide linkages, termed peptide bonds. Two amino acids joined by a peptide bond forms a dipeptide. Three amino acids joined by two peptide bonds forms a tripeptide; similarly, there are tetrapeptides and pentapeptides. When there are many amino acids joined together, the structure is termed a polypeptide. In general, polypeptides contain less than 100 amino acid residues and proteins contain 100 or more amino acid residues.

Glycoproteins and glycopeptides can be chemically synthesized by a number of methods well known to those skilled in the art. For example, glycoproteins and/or glycopeptides can be formed from natural or chemically synthesized proteins and/or peptides by glycosylation, which is the addition of carbohydrate side chains. There are a number of methods well known to those skilled in the art for glycosylating proteins or peptides. For example, side-chain glycosylation can be performed chemically with glycosylbromides for serine (Ser, S) and threonine (Thr, T) amino acid residues and glycosylamines for aspartic acid (Asp, D) amino acid residues, thereby producing glycosylated asparagine (Asn, N) amino acid residues. In addition, glycosylating enzymes can be used to attach carbohydrate side chains to proteins or peptides.

Proteins or peptides, chemically synthesized or naturally occurring, also suitable for use in the present invention comprise an asparagine (Asn, N) amino acid residue or a glutamine (Gln, Q) amino acid residue, both of which comprise an unsubstituted amide moiety. In addition, proteins or peptides, again chemically synthesized or naturally occurring, which are also suitable for use in the present invention comprise a N-terminal serine (Ser, S) amino acid residue, a N-terminal threonine (Thr, T) amino acid residue, or a 5-hydroxylysine (5-hydroxylysine is only known to occur naturally in collagen, but in principal may be placed anywhere in a synthetic peptide or protein) amino acid residue, all of which comprise a 2-aminoalcohol moiety.

Biomolecules or biomaterials of the present invention comprising an unsubstituted amide moiety may be converted into an amine-functional material via a Hofmann rearrangement reaction, also known as a Hofmann degradation of amides reaction. For example, Wirsen et al., "Bioactive heparin surfaces from derivatization of polyacrylamide-grafted LLDPE", Biomaterials, 17, 1881-1889 (1996), demonstrated the conversion of unsubstituted amide moieties of a polyacrylamide-low density polyethylene film into primary amine moieties using a Hofmann rearrangement reaction. In another example, Sano et al., "Introduction of functional groups onto the surface of polyethylene for protein immobilization", Biomaterials. 14, 817-822 (1993), demonstrated the conversion of unsubstituted amide moieties of a polyacrylamide-high density polyethylene film into primary amine moieties using a Hofmann rearrangement reaction. In another example, Fuller et al., "A new class of amino acid based sweeteners", J. Am. Chem. Soc., 107, 5821-5822 (1985), demonstrated the conversion of an unsubstituted amide moiety of an amino acid into a primary amine moiety using a Hofmann rearrangement reaction. In another example, Loudon et al., "Conversion of aliphatic amides into amines with [l,l-bis(trifluoroacetoxy)iodo]benzene. 1. Scope of the reaction", J. Org. Chem., 49, 4272-4276 (1984), demonstrated the conversion of various unsubstituted amide moieties, including the amide side chain in a glutamine amino acid residue, into primary amine moieties using Hofmann rearrangement reactions.

The Hofmann rearrangement reaction which converts an unsubstituted amide moiety into a primary amine moiety may be carried out with chemical reactants such as, for example, bromine, bromide, bromite, hypobromite, chlorine, chloride, chlorite, hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide and hypervalent organoiodine compounds such as, for example, [bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene and iodosylbenzene. A general discussion of Hofmann rearrangement reactions is contained in Comprehensive Organic Synthesis, Volume 6, 800-806, Pergamon Press. For example, Kajigaeshi et al., "An efficient method for the Hofmann degradation of amides by use of benzyltrimethylammonium tribromide", Chemistry Letters, 463-464 (1989), described various methods for obtaining amines from amides using the Hofmann rearrangement reaction. These methods include, for example, the use of bromine or chlorine in an alkaline solution, the use of lead tetraacetate in an alcohol solution, the use of [bis(trifluoroacetoxy)iodo]benzene in an aqueous acetonitrile solution, the use of sodium bromite in an alkaline solution, and the use of benzyltrimethylammonium tribromide in an alkaline solution. Catalysts such as, for example, triethylamine, tin(IV) chloride, dibutylstannyl dilaurate or pyridine are sometimes used in a Hofmann rearrangement reaction. Typical solvents include, for example, water, hydroxides, methoxides, alcohols, dimethylformamide, acetonitrle, benzene, carboxylic acids or combinations thereof.

Depending on the chemical reactants, the Hofmann rearrangement reaction may be carried out under acidic, neutral or basic conditions. Although applicants do not wish to be bound by any single theory, it is generally believed that when the reaction is carried out with particular amine forming agents in an aqueous base a N-halo amide intermediate is formed. An isocyanate is then formed from the N-halo amide intermediate. The formed isocyanate then readily hydrolyzes into a primary amine. In contrast, when the reaction is carried out in an alcohol a carbamate is generally formed. The carbamate may then be hydrolyzed into a primary amine. For example, when the reaction of an amide with bromine is carried out in methanol containing sodium methoxide instead of in aqueous base, the product is a carbamate which is easily converted to an amine via hydrolysis. Depending on the reaction conditions, side-reactions such as chain scission, hydrolysis and/or urea formation may occur. However, side-reactions may be minimized by changes in the reaction conditions. For example, changes in the reaction conditions such as pH, time, temperature and/or the amount of amine forming agent may minimize various side-reactions.

In general, the Hofmann rearrangement reaction is carried out with an amine forming agent in amounts ranging from about 0.5 eq. to about 2 eq. based on the amide content of the biomolecule or biomaterial. In addition, the reaction is generally carried out at a temperature between about -10 and about 100 degrees Celsius, preferably from about 0 and about 50 degrees Celsius. Depending on the material, a Hofmann rearrangement reaction may be carried out for as short as a few minutes to as long as many hours. Time, temperature and pH limitations of the present invention are generally governed by the stability of the materials imparted by the Hofmann rearrangement process. Wide latitude may be employed in determining the optimum conditions for a particular system. Such conditions may be determined readily by one skilled in the art by routine experimentation upon examination of the information presented herein.

We define the term "amine forming agent" appearing herein to include any chemical agent or combination of chemical agents capable of forming an amine moiety upon its or their reaction with an unsubstituted amide moiety. Examples of amine forming agents include, for example, bromine, bromide, bromite, hypobromite, chlorine, chloride, chlorite, hypochlorite, lead tetraacetate, benzyltrimethylammonium tribromide and hypervalent organoiodine compounds such as, for example, [bis(trifluoroacetoxy)iodo]benzene, hydroxy(tosyloxy)iodobenzene and iodosylbenzene. Amine forming agents include any of the many possible Hofmann rearrangement reactants. As mentioned above, the term "amine forming agent" appearing herein may mean any one or more of an amine forming agent or a combination of different amine forming agents.

Biomolecules or biomaterials of the present invention comprising, in addition to an unsubstituted amide moiety, a primary amine moiety of which is desired to be left intact and unreacted following coupling may be protected or blocked prior to the Hofmann rearrangement reaction. For example, a protein may comprise both a lysine amino acid residue and, for example, an asparagine amino acid residue. As mentioned earlier, there are a number of established coupling procedures which may couple a protein comprising a lysine amino acid residue to a substrate surface through the protein's lysine amino acid residue. However, a protein's lysine amino acid residues are typically associated with the protein's biologically active site. Therefore, coupling a protein to a substrate surface via a protein's primary amine moiety in the side chain of its lysine amino acid residue may destroy the biological properties of the attached protein. However, a protein's lysine amino acid residue may be protected or blocked by a number of methods well known to those skilled in the art. For example, the amine moiety may be protected using, for example, a tert.butyloxycarbonyl (Boc) group which is typically cleaved with acid, a benzyloxycarbonyl (Z) group which is typically cleaved by hydrogenolysis, a biphenylisopropyloxycarbonyl (Bpoc) group which is typically cleaved with acid, a triphenylmethyl (trityl) group which is typically cleaved with acid, a 9-fluoroenylmethyloxycarbonyl (Fmoc) group which is typically cleaved with base or a blocking group which is pH stable but is cleaved by enzyme-catalyzed hydrolysis. The appropriate amine-blocking group to use to protect the amine moiety will depend highly on the entire sequence of reaction conditions chosen for biomolecule attachment or crosslinking. Following blocking of the amine moiety of the lysine residue, the amide moiety of the asparagine residue may then be converted into an amine moiety via a Hofmann rearrangement reaction. The protein may then be coupled to the substrate via one method of the present invention through the newly formed amine moiety. Following coupling, the amine-blocking group may then be removed, thereby preserving the protein's biological activity. This type of blocking scheme may be employed on biomolecules and/or biomaterials of the present invention which contain amine moieties which are desired to be left intact and unreacted.

Biomaterials of the present invention not comprising unsubstituted amides on their surface may be amidated readily through a number of methods well known in the art. For example, unsubstituted amides may be provided by ceric ion grafting acrylamide to a biomaterial surface as set forth in U.S. Pat. No. 5,344,455 to Keogh et al. Alternatively, for example, a grafted acrylamide-containing polymer may be attached by radiation grafting as set forth in U.S. Pat. No. 3,826,678 to Hoffman et al. There are a number of surface-derivatization techniques (e.g., grafting techniques) well known in the art for creating substrate surfaces comprising unsubstituted amide moieties. 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. In addition, amides can generally be prepared by reaction of ammonia with acid chlorides. This reaction is commonly known as ammonolysis. Acid chlorides are prepared by substitution of --Cl for the --OH group of a carboxylic acid. Reagents commonly used to form acid chlorides from carboxylic acids include thionyl chloride (SOCl₂), phosphorus trichloride (PCl₃) and phosphorus pentachloride (PCl₅). Two amino acids comprising carboxylic acid moieties which may be converted into acid chlorides are aspartic acid (Asp, D) amino acid and glutamic acid (Glu, E) amino acid. The acid chloride moieties may then be converted into amide moieties followed by conversion into amine moieties. In addition, treatment of an ester moiety with ammonia, generally in ethyl alcohol solution, will yield an amide moiety.

Biomolecules or biomaterials suitable for use according to one method of the present invention may comprise at least one negatively charged moiety (also known as an anionic moiety) at physiological pH, such as a phosphate moiety, a sulphate moiety or a carboxylate moiety. A negatively charged moiety is capable of interacting electrostatically with a positively charged moiety (also known as a cationic moiety) thereby forming an ionic chemical bond or linkage.

Biomaterials that do not contain a negative charge on their surfaces may be furnished with a net negative and may be modified readily through a number of methods well known in the art. For example, polyethylene may be exposed to sulfuric acid comprising potassium permanganate thereby creating a negative charge. Other examples of furnishing biomaterials with negatively charged surfaces are taught in U.S. Pat. Nos. 5,344,455 to Keogh et al. and 5,429,618 to Keogh.

Biomolecules or biomaterials suitable for use according to one method of the present invention may comprise at least one positively charged moiety at physiological pH, such as an amine moiety or a guanidino moiety. Biomolecules or biomaterials that do not comprise a positive charge may be furnished with a net positive charge by one method of the present invention. As mentioned earlier, a positively charged moiety is capable of interacting electrostatically with a negatively charged moiety thereby forming an ionic chemical bond or linkage.

Biomolecules or biomaterials suitable for use according to one method of the present invention may comprise at least one epoxide moiety. Epoxide moieties are three-membered rings comprising two carbon atoms and an oxygen atom. An epoxide ring is also known as an oxirane ring. There are a number of techniques well known in the art to produce epoxides. Epoxide moieties are highly reactive due to the ease of opening of the highly strained three-membered ring. An epoxide moiety will react readily with an amine moiety, thereby forming a covalent bond or linkage. The reaction of an epoxide moiety with an amine moiety is well known in the art.

Biomolecules or biomaterials suitable for use according to one method of the present invention may comprise at least one isocyanate moiety. An isocyanate moiety (RNCO) will react readily with an amine moiety, thereby forming a covalent bond or linkage called a urea. There area number of techniques well known in the art to produce isocyanates. In addition, the reaction of an isocyanate moiety with an amine moiety is well known in the art.

We define the term "1,2-dihydroxy moiety" appearing herein as a carbon-carbon bond bearing two adjacent hydroxyl moieties.

We define the term "2-aminoalcohol moiety" appearing herein as a carbon-carbon bond bearing an amine moiety adjacent to a hydroxyl moiety.

The 1,2-dihydroxy moiety and the 2-aminoalcohol moiety are both oxidizable with periodate, which may 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 desired number of 1,2-dihydroxy moieties or 2-aminoalcohol moieties to form aIdehyde moieties, however less than a stoichiometric amount or more than a stoichiometric amount may be used.

Periodate oxidation of a 1,2dihydroxy moiety or a 2-aminoalcohol moiety is generally carded out in an aqueous solution, preferably an aqueous buffered solution, at a temperature that does not destroy the desired properties of the material. Generally, buffers having a pH in a range between about 4 and about 9 can be used, with a pH between about 6 and about 8 desired for certain pH sensitive materials. Generally, the oxidation is carried out at a temperature between about 0 and about 50 degrees Celsius, and preferably at a temperature between about 4 and about 37 degrees Celsius. Depending on the material, oxidation reactions can be carried out for as short 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 "overoxidation."

Treatment times and temperatures for the oxidation process tend to be inversely related. That is, higher treatment temperatures require relatively shorter treatment times. Time and temperature limitations of the present invention are generally governed by the stability of the materials imparted by the oxidation process. Wide latitude may be employed in determining the optimum conditions for a particular system. Such conditions may be determined readily by one skilled in the art by routine experimentation upon examination of the information presented herein.

Subsequent to oxidation, the reaction solution may be stored prior to use at about 4 degrees Celsius. Typically, the storage stability of the reaction solution at a neutral pH or slightly acidic pH may extend between about one and about fourteen days and sometimes even months when stored in the dark.

In general, an aldehyde moiety (RCHO) will react chemically with a primary amine moiety (R'NH₂) to form a relatively unstable imine moiety (R'N=CHR). The reaction of an aldehyde moiety with a primary amine moiety which is commonly referred to as a Schiff base reaction may be carried out under the same conditions as described above for the periodate oxidation reaction, which is generally designed to protect a biomolecule from damage. To stabilize the relatively unstable imine linkage, subsequent reductive,alkylation of the imine moiety is carried out using reducing agents (i.e., . stabilizing agents) such as, for example, sodium borohydride, sodium cyanoborohydride, and amine boranes, to form a secondary amine (R'NH--CH₂ R). This reaction can also be carried out under the same conditions as described above for the periodate oxidation reaction. Typically, however, the coupling and stabilizing reactions are carried out in a neutral or slightly basic solution and at a temperature between about 0 and about 50 degrees Celsius. Preferably, the pH is between about 6 and about 10, and the temperature is between about 4 and about 37 degrees Celsius, for the coupling and stabilizing reactions. These reactions (coupling and stabilizing) may 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.

We define the term "guanidino moiety" appearing herein to include guanidine, guanidinium, guanidine derivatives such as (RNHC(NH)NHR'), monosubstituted guanidines, monoguanides, biguanides, biguanide derivatives such as (RNHC(NH)NHC(NH)NHR"), and the like. In addition, the term "guanidino moiety" appearing herein may mean any one or more of a guanide alone or a combination of different guanides.

Guanidine is the imide of urea, or the amidine of carbamic acid. It is a very strong base with a pK_a of 13.5 in water. The great basicity of guanidine is a result of the stability of the conjugated acid (guanidinium) in water. The positive charge on the guanidinium ion can be spread equally among the three nitrogens by resonance. The guanidinium ion is also quite hydrophilic and is well solvated in aqueous media due to the extensive hydrogen bonding of six potential hydrogen bond donors to the solvent. The partial positive charge of the hydrogen bond donors increases their strength for donation to the negative dipole of water. Crystal structures of simple guanidinium derivatives have revealed several common features. First, the C--N single bond length in an alkyl guanidine is typically shorter than the usual C--N single bond length. Usually, the three C--N bonds in the guanidinium group itself are nearly equal in length with an average of 1.33 A. The three N--C--N bond angles are almost always near 120^o.

The guanidinium group's features make it a very attractive moiety. For example, its high basicity (a pK_a of 13.5 for guanidinium itself) allows it to remain protonated over a much wider range of pH than does the ammonium group. In fact, at physiological pH, all but a small fraction of the guanidine molecules will exist as positively charged species. The guanidinium group's enhanced hydrogen bonding capabilities, typically two linear hydrogen bonds, allow it to form tighter complexes with anions that are capable of hydrogen bonding. In fact, the guanidinium group may form characteristic pairs of zwitterionic hydrogen bonds which provide binding strength by their charge and structural organization by their arrangement.; Another feature of guanidines are their ability to react with 1,2-dicarbonyl moieties under mild alkaline conditions to form covalent bonds. The reaction of a guanidino moiety and a 1,2-dicarbonyl moiety is similar to a Schiff base reaction (the reaction between an amine moiety and an aldehyde moiety). In some cases, it may be desirable to use a stabilizing agent such as borate ion (BO₃) to stabilize the resultant compound.

We define the term "1,2-dicarbonyl moiety" appearing herein as two carbonyl (C=O) groups located on adjacent carbon atoms. A carbonyl group contains a carbon-oxygen double bond.

Biomolecules or biomaterials of the present invention comprising an unsubstituted amide moiety may be modified to comprise guanidino moieties.

The method of the present invention includes converting an unsubstituted amide moiety (RCONH₂) into an amine-functional material (RNH₂). The amine-functional material is then modified to comprise guanidino moieties by reaction with compounds such as S-ethylthiouronium bromide, S-ethylthiouronium chloride, O-methylisourea, O-methylisouronium sulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea, 2-methyl-1-nitroisourea, aminoiminomethanesulfonic acid, cyanamide, cyanoguanide, dicyandiamide, 3,5-dimethyl-l-guanylpyrazole nitrate and 3,5-dimethyl pyrazole. For example, reaction of amines with O-methylisourea, S-methylisourea, S-ethylthiouronium bromide or S-ethylthiouronium chloride, thereby yielding guanidino moieties, are generally completed after 8 hours at 70 degrees Celsius in a solution of sodium hydroxide (NaOH) at pH 10. Reactions of amines with aminoiminomethanesulfonic acid or cyanamide are generally performed at room temperature. Another example is the reaction of an amine with 2-methyl-1-nitroisourea in water to form a nitroguanidine. The nitro group is then easily removed to form a guanidino moiety by hydrogenolysis.

We define the term "guanidino forming agent" appearing herein to include any chemical agent capable of forming a guanidino moiety upon its reaction with a non-guanidino moiety. Examples of guanidino forming agents include S-ethylthiouronium bromide, S-ethylthiouronium chloride, O-methylisourea, O-methylisouronium sulfate, O-methylisourea hydrogen sulfate, S-methylisothiourea, 2-methyl-i-nitroisourea, aminoiminomethanesulfonic acid, cyanamide, cyanoguanide, dicyandiamide, 3,5-dimethyl-1-guanylpyrazole nitrate and 3,5-dimethyl pyrazole. In addition, the term "guanidino forming agent" appearing herein may mean any one or more of a guanidino forming agent or a combination of different guanidino forming agents.

We define the term "biomaterial" appearing herein as a material that is substantially insoluble in human or animal bodily 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; may be purified, fabricated and sterilized easily; 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 present invention comprise either an unsubstituted amide moiety, a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, a 1,2-dicarbonyl moiety, a guanidino moiety, a chemical moiety capable of forming either a covalent bond with an amine moiety (such as an aldehyde moiety, an epoxide moiety or an isocyanate moiety) or a chemical moiety capable of forming an ionic bond with an amine moiety (such as a phosphate moiety, a sulphate moiety or a carboxylate moiety), or any possible combination of any one or more of these moieties alone or in combination. Additionally, biomaterials comprising both an unsubstituted amide moiety and a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, or a chemical moiety capable of forming a chemical bond with an amine moiety may by crosslinked, according to one method of the present invention. Also, biomaterials may be fabricated by crosslinking biomolecules, comprising both an unsubstituted amide moiety and a 1,2-dihydroxy moiety, a 2-aminoalcohol moiety, or a chemical moiety capable of forming a chemical bond with an amine moiety, according to another method of the present invention.

Biomaterials or substrates that may be modified according to one method of the present invention include metals such as titanium, titanium alloys, TiNi alloys, shape memory alloys, super elastic alloys, aluminum oxide, platinum, platinum alloys, stainless steels, stainless steel alloys, MP35N, elgiloy, haynes 25, stellite, pyrolytic carbon, silver carbon, glassy carbon, polymers such as polyamides, polycarbonates, polyethers, polyesters, polyolefins including polyethylenes or polypropylenes, polystyrenes, polyurethanes, polyvinylchlorides, polyvinylpyrrolidones, silicone elastomers, fluoropolymers, polyacrylates, polyisoprenes, polytetrafluoroethylenes, 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, a string, a suture, a fiber, a mesh and other materials such as glass and the like. Biomaterials of the present invention made using these materials may be coated or uncoated, porous or nonporous, permeable and nonpermeable, derivatized or underivatized. We define the term "medical device" appearing herein as a device having surfaces that contact human or animal bodily tissue and/or fluids in the course of their operation. This definition includes within its scope, 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. The definition includes within its scope endoprostheses implanted in blood contact in a human or animal body such as vascular grafts, stents, pacemaker leads, heart valves, and the like that are implanted in blood vessels or in the heart. The definition also includes within its scope 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.

One method of the invention may be used to modify substrates of any shape or form including tubular, sheet, rod and articles of proper shape 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.

The present invention has an object of solving a number of problems associated with the use of medical devices. The present invention includes within its scope methods for attaching biomolecules to biomaterial surfaces for use in medical devices. The present invention further provides methods for fabricating crosslinked biomaterials or crosslinked biomaterial coatings comprising biomolecules.

One preferred method of the present invention may be employed to immobilize a least one biomolecule comprising at least one 2-aminoalcohol moiety on a biomaterial surface comprising at least one unsubstituted amide moiety. The method comprises the steps of: combining a periodate with a biomolecule comprising a 2-aminoalcohol moiety to form an aldehyde-functional material in an aqueous solution having a pH between about 4 and about 9 and a temperature between about 0 and about 50 degrees Celsius;. providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the aldehydefunctional material with the amine-functional material to chemically bond the two materials together through an imine moiety; and reacting the imine moiety with a reducing agent to form an immobilized biomolecule on a medical device biomaterial surface through a secondary amine linkage.

Another preferred method of the present invention may be employed to immobilize a least one glycoprotein or glycopeptide comprising at least one 1,2-dihydroxy moiety on- a biomaterial surface comprising at least one unsubstituted amide moiety. The method includes the steps of: combining a periodate with a glycoprotein or glycopeptide comprising a 1,2-dihydroxy moiety to form an aldehyde-functional material in an aqueous solution having a pH between about 4 and about 9 and a temperature between about 0 and about 50 degrees Celsius; providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the aldehyde-functional material with the amine-functional material to chemically bond the two materials together through an imine moiety; and reacting the imine -moiety with a reducing agent to form an immobilized glycoprotein or glycopeptide on a medical device biomaterial surface through a secondary amine linkage.

Still another preferred method of the present invention may be employed to immobilize a least one biomolecule comprising at least one epoxide moiety on a biomaterial surface comprising at least one unsubstituted amide moiety. The method includes the steps of: providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the epoxide-functional biomolecule with the amine-functional material to chemically bond the two materials together through a covalent linkage.

Yet another preferred method of the present invention may be employed to immobilize a least one biomolecule comprising at least one isocyanate moiety on a biomaterial surface comprising at least one unsubstituted amide moiety. The method includes the steps of: providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the isocyanate-functional biomolecule with the amine-functional material to chemically bond the two materials together through a covalent linkage.

Another preferred method of the present invention may be employed to immobilize at least one biomolecule comprising at least one negatively charged moiety, such as a phosphate moiety, a sulphate moiety or a carboxylate moiety, on a biomaterial surface comprising at least one unsubstituted amide moiety. The method includes the steps of: providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the negatively charged biomolecule with the amine-functional material to chemically bond the two materials together through an ionic linkage.

One preferred method of the present invention may be employed to immobilize at least one biomolecule comprising at least one chemical moiety which is capable of forming a chemical bond with a guanidino-functional material (such as, for example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety) on a biomaterial surface comprising at least one unsubstituted amide moiety. The method includes the steps of: providing a biomaterial surface comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; converting the amine-functional material into a guanidino-functional material using a guanidino forming agent; combining the guanidino-functional material with a biomolecule capable of forming a chemical bond with a guanidino-functional material to chemically bond the two materials together through a chemical linkage.

Still another preferred method of the present invention may be employed to immobilize a least one biomolecule comprising a least one unsubstituted amide moieity on a biomaterial surface comprising a least one chemical moiety which is capable of forming a chemical bond with an amine-functional material (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety). The method includes the steps of: providing a biomolecule comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; combining the amine-functional material with a biomaterial surface capable of forming a chemical bond with an amine-functional material to chemically bond the two materials together through a chemical linkage.

Yet another preferred method of the present invention may be employed to immobilize a least one biomolecule comprising a least one unsubstituted amide moiety on a biomaterial surface comprising a least one chemical moiety which is capable of forming a chemical bond with a guanidino-functional material (such as, for example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety). The method includes the steps of: providing a biomolecule comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional material; converting the amine-functional material into a guanidino-functional material using a guanidino forming agent; combining the guanidino-functional material with a biomaterial capable of forming a chemical bond with a guanidino-functional material to chemically bond the two materials together through a chemical linkage.

One preferred method of the present invention may be employed to crosslink biomolecules, located in solution or on biomaterial surfaces, comprising at least one unsubstituted amide moiety and at least one chemical moiety which is capable of forming a chemical bond with an amine-functional material (such as, for example, an aldehyde moiety, an epoxide moiety, an isocyanate moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety). This method comprises the steps of: providing a biomolecule comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional biomolecule; allowing the amine-functional biomolecule to combine with a biomolecule capable of forming a chemical bond with an amine-functional biomolecule to chemically bond the two biomolecules together through a chemical linkage, thereby forming a crosslinked material. This crosslinked material may be employed as a biomaterial or as a biomaterial coating. In addition, such crosslinked material may be further modified to contain additional biomolecules. For example, biomolecules comprising aldehyde moieties may be attached to residual amine moieties present in or on the surface of the crosslinked material.

Another preferred method of the present invention may be employed to crosslink biomolecules, located in solution or on biomaterial surfaces, comprising at least one unsubstituted amide moiety and at least one chemical moiety which is capable of forming a chemical bond with a guanidino-functional material (such as, for example, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety or a carboxylate moiety). This method comprises the steps of: providing a biomolecule comprising an unsubstituted amide moiety, converting the amide moiety into an amine moiety using an amine forming agent to form an amine-functional biomolecule; converting the amine-functional biomolecule into a guanidino-functional biomolecule using a guanidino forming agent; allowing the guanidino-functional biomolecule to combine with a biomolecule capable of forming a chemical bond with an guanidino-functional biomolecule to chemically bond the two biomolecules together through a chemical linkage, thereby forming a crosslinked material. This crosslinked material may be employed as a biomaterial or as a biomaterial coating. Additionally, such crosslinked material may be further to modified to contain additional biomolecules. For example, biomolecules comprising 1,2-dicarbonyl moieties may be attached to residual guanidino moieties present in or on the surface of the crosslinked material.

One example of a biomolecule of the present invention is collagen. Collagen, which is found in connective tissue, has special amino acids, one of which is 5-hydroxylysine which may be oxidized with a source of periodate, which may be provided as periodic acid or salts thereof, such as sodium periodate, potassium periodate, or other alkali metal periodates, to form a pendant aldehyde moiety. The resultant aldehyde moieties may be used to crosslink the collagen through bonds formed between the aldehydes and amines, for example, lysine amino acid residues or modified asparagine amino acid residues, contained on neighboring collagen molecules. The resultant imine bonds may then be reduced using a mild reducing agent like sodium borohydride, sodium cyanoborohydride, or amine boranes. These crosslinks may endow the collagen biomaterial or biomaterial coating with desirable biological and/or physical properties such as mechanical strength, anti-immunogenicity, biostability, among others, without the use of a coupling agent. Thus, the method of the present invention eliminates the need for using glutaraldehyde, a commonly used cytotoxic coupling agent, to crosslink the collagen to control its physical and biological properties.

The aldehyde moieties formed by oxidation of collagen may also be used to couple a variety of amine-containing biomolecules to the crosslinked collagen biomaterial or biomaterial coating. Also, the ability to create aldehyde moieties along collagen molecules enables them to be covalently attached to amine containing biomaterial surfaces. Such collagen-coated biomaterial surfaces may be used, for example, as cell seeding surfaces, cell binding surfaces, cell separating surfaces, tissue fixation, collagen-coated stents, collagen-coated vascular grafts or collagen glues.

Other biomolecules, such as structural proteins, may be crosslinked to form a material that may be used as a biomaterial or a biomaterial coating. Also, additional biomolecules, as described herein, may be attached to residual amine moieties contained in or on a fabricated crosslinked biomaterial or biomaterial coating, as described herein. Alternatively, amine containing biomolecules may be attached to residual aldehyde moieties contained in or on a fabricated crosslinked biomolecule biomaterial or biomaterial coating, as described herein.

An example of a glycoprotein that can be used in a number of aspects of the present invention is fibrin(ogen). Fibrin(ogen), which is a structural protein, has oligosaccharides which can be oxidized with a source of periodate, which can be provided as periodic acid or salts thereof, such as sodium periodate, potassium periodate, or other alkali metal periodates, to form a pendant aldehyde moiety. The resultant aldehyde moieties can be used to crosslink the fibrin(ogen) through bonds formed between the aldehydes and amines contained on neighboring fibrin(ogen) molecules. The resultant imine bonds can then be reduced using a mild reducing agent like sodium borohydride, sodium cyanoborohydride, or amine boranes. These crosslinks can endow the fibrinogen and/or fibrin (thrombin polymerized fibrinogen) biomaterial or biomaterial coating with desirable biological and/or physical properties such as mechanical strength, anti-immunogenicity, biostability, among others, without the use of a coupling agent. Thus, the method of the present invention eliminates the need for using glutaraldehyde, a commonly used cytotoxic coupling agent, to crosslink the fibrinogen and/or fibrin to control its physical and biological properties.

The aldehyde moieties formed by oxidation of fibrin(ogen) can also be used to couple a variety of amine-containing biomolecules to the crosslinked fibrin(ogen) biomaterial or biomaterial coating. Also, the ability to create aldehyde moieties along fibrin(ogen) molecules enables them to be covalently attached to amine containing biomaterial surfaces. Such fibrinogen/fibrin coated biomaterial surfaces can be used, for example, as cell seeding surfaces, cell binding surfaces, cell separating surfaces, fibrinogen/fibrin-coated stents, fibrinogen/fibrin-coated vascular grafts or fibrinogen/fibrin glues.

Claim 1 of 53 Claims

We claim:

1. A method of forming a coating on a surface of a medical device, the coating imparting improved biocompatibility characteristics to the surface, the method comprising the steps of:

(a) providing the medical device, the device having a biomaterial disposed on or forming a surface thereof, the biomaterial comprising an unsubstituted amide moiety;

(b) combining the amide moiety with an amine forming agent to form an amine-functional surface;

(d) providing a biomolecule, the biomolecule comprising a chemical moiety selected from the group consisting of an aldehyde moiety formed by combining a periodate with a 2-aminoalcohol moiety, an aldehyde moiety formed by combining a periodate with a 1,2-dihydroxy moiety, an epoxide moiety, an isocyanate moiety, a 1,2-dicarbonyl moiety, a phosphate moiety, a sulphate moiety and a carboxylate moiety; and

(e) combining the chemical moiety with the guanidino-functional surface to form a chemical bond, the chemical bond immobilizing the biomolecule on the surface, the immobilized biomolecule forming the coating.

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