Patent Publication Number: US-2004048021-A1

Title: Surface modification of substrates

Description:
CROSS REFERENCE TO RELATED APPLICATIONS  
     [0001] This application is a continuation-in-part of U.S. patent application Ser. No. 09/430,857 filed on Oct. 29, 1999 and claims the benefit of U.S. Provisional Application No. 60/125,355 filed Mar. 19, 1999, the contents of which are incorporated herein by reference in their entirety. 
    
    
     
       BACKGROUND OF THE INVENTION  
       [0002] This invention relates to methods for inhibiting the adherence of platelets and certain cell types to the surfaces of medical devices and instruments which come into contact with platelets and cells, and for preventing mineralization of such devices and instruments. This is accomplished by chemically bonding a modified or derivatized hyaluronic acid molecule to the device or instrument, and by the addition of Fe +3  or Al +3  cations to the hyaluronic acid.  
       [0003] When a blood vessel is damaged and the normal endothelial-cell barrier is disrupted, platelets are quickly recruited from the circulating blood to form an occlusive plug. This occurs through a series of interactions between the platelets and macromolecules in the subendothelial matrix (platelet adhesion) and among the platelets themselves (platelet aggregation). The initial process of adhesion, in contrast to aggregation, does not require metabolic activity. It leads, however, to the activation of platelets which in turn secrete a number of factors which stimulate the activation of plasma coagulation factors, resulting in the generation of a fibrin clot that reinforces the platelet aggregate.  
       [0004] Under normal hemostatic conditions, the platelet aggregate and fibrin clot are degraded as healing of the injured area occurs. However, a platelet aggregate and/or a fibrin clot can occlude a blood vessel in a pathological process known as thrombosis. Venous thrombosis and pulmonary embolism are among the leading causes of morbidity and death in hospitalized patients.  
       [0005] Thrombosis that develops as a purely intravascular process may also be the primary factor in atherosclerosis. The formation of platelet aggregates on the surface of atheromatous plaques and subsequent organization of these white thrombi into fibrous occlusive intimal lesions may be one mechanism by which atherosclerotic lesions progress to severe obstruction and total occlusion; coronary artery thrombosis leading to myocardial infarction almost always occurs at the site of an atheromatous plaque. Percutaneous transluminal coronary angioplasty (PTCA) has become an important procedure for re-establishing blood flow to the heart through partially occluded blood vessels. Unfortunately, approximately 30% to 40% of patients that have coronary angioplasty suffer restenosis of the treated vessel within 6 months of treatment.  
       [0006] U.S. Pat. No. 5,585,361 describes the use of unmodified hyaluronic acid to inhibit platelet adherence and aggregation. The hyaluronic acid can be administered to a patient intravenously, or the hyaluronic acid can be used as a coating on a prosthetic device, such as a vascular graft, a heart valve, a vascular stent or a catheter. This patent does not disclose that the hyaluronic acid can be chemically attached to the surface of the prosthetic device, or, in fact, that the hyaluronic acid can be used in a form other than its native form, such as in a cross-linked or derivatized form.  
       [0007] Hyaluronic acid (“HA”) is a naturally occurring mucopolysaccharide found, for example, in synovial fluid, in vitreous humor, in blood vessel walls and the umbilical cord, and in other connective tissues. The polysaccbaride consists of alternating N-acetyl-D-glucosamine and D-glucuronic acid residues joined by alternating β1-3 glucuronidic and β1-4 glucosaminidic bonds, so that the repeating unit is -(1→4)-β-D-GlcA-(1→3)-β-D-GlcNAc-. In water, hyaluronic acid dissolves to form a highly viscous fluid. The molecular weight of hyaluronic acid isolated from natural sources generally falls within the range of 5×10 4  up to 1×10 7  daltons.  
       [0008] Hyaluronic acid, in chemically modified (“derivatized”) form, is useful as a surgical aid to prevent adhesions or accretions of body tissues during the post-operation period. The derivatized HA gel or film is injected or inserted into the locus between the tissues that are to be kept separate to inhibit their mutual adhesion. Chemically modified HA can also be useful for controlled release drug delivery. See U.S. Pat. No. 4,937,270 and U.S. Pat. No. 5,017,229 which disclosed derivatized versions of HA prepared, for instance, by reacting HA with a carbodiimide.  
       [0009] I. Danishefsky et al.,  Carbohydrate Res ., Vol. 16, pages 199-205, 1971, describe the modification of a mucopolysaccharide by converting the carboxyl groups of the mucopolysaccharide into substituted amides by reacting the mucopolysaccharide with an amino acid ester in the presence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (“EDC”) in aqueous solution. Danishefsky et al. react glycine methyl ester with a variety of polysaccharides, including HA. The resulting products are water soluble; that is, they rapidly disperse in water or in an aqueous environment such as is encountered between body tissues.  
       [0010] U.S. Pat. No. 4,582,865 and U.S. Pat. No. 4,636,524 describe HA compositions in which the HA is cross-linked by reaction with divinyl sulfone, and the use of these compositions for drug delivery applications. U.S. Pat. No. 5,676,964 describes the preparation of cross-linked polysaccharides, including HA, wherein the cross-linking occurs as a result of covalent bonds formed between carboxyl groups and hydroxyl groups of adjacent polysaccharide molecules. Johns et al.,  Fertility and Sterility , Vol. 68, pages 37-42 (1997), disclose hyaluronic acid formulations crosslinked with trivalent iron for use in reducing adhesion formation.  
       [0011] A heparin-like molecule is described in R. Barbucci et al.,  Society for Biomaterials Meetings , Abstracts (1998), which also describes the immobilization of the molecule on the surface of different materials, including polyethyleneterephthalate (“PET”), to study blood compatibility using various immobilization procedures, such as photolithography immobilization, glow discharge grafting, and PUPA immobilization. The heparin-like molecule contains the backbone of hyaluronic acid, with sulfate groups replacing the hydroxyl groups. The blood compatibility of the immobilized PET substrate was evaluated with respect to anticoagulant activity, platelet adhesion and thrombus formation, and coagulation factor activity. See also P. Favio et al.,  Immobilization of heparin and highly-sulphated hyaluronic acid onto plasma-treated polyeihylene , Plasmas and Polymers, Vol. 3, No. 2 (1998), which describes the immobilization of heparin and highly-sulphated hyaluronic acid onto plasma-processed polyethylene, using a diamine polyethylene glycol spacer molecule, to improve the compatibility of the polyethylene with human blood. Both a treatment and a deposition process is used to provide —COOH groups on the polyethylene surface.  
       [0012] The use of polyvinyl alcohol to coat polyethylene tubing, and the effect on platelet production and thrombogenicity was studied by W. E. Ip et al., in  Journal of Biomedical Materials Research , Vol. 25, pages 875-887 (1991); C. H. Cholakis et al., in  Journal of Biomedical Materials Research , Vol. 23, pages 417-441 (1989); and C. H. Gemmell et al., in  Journal of Biomedical Materials Research,  37(2), pages 176-181 (1997). These references disclose that polyvinyl alcohol is not thromboadherent, but it is thrombogenic. The use of immobilized heparin does not appear to have any additional effect on thrombogenicity.  
       [0013] U.S. Pat. No. 4,871,357 and U.S. Pat. No. 5,417,969 describe anti-thrombogenic surface coatings for plastic medical devices. The coatings are prepared by complexing heparin with cationic organic molecules, such as alkylbenzyl dimethyl ammonium salts. The coating can be fixed to the plastic substrate using ionizing radiation to form covalent bonds between the substrate and the coating. Other heparin coatings on medical devices are disclosed in U.S. Pat. No. 5,679,659 and U.S. Pat. No. 5,672,638, which describe the reaction of heparin with periodate, and contacting this reaction mixture with immobilized free amine groups on a blood-contacting substrate.  
       [0014] Implanted devices, and surrounding tissue, can also suffer from mineralization, and more particularly calcification, due to the reaction of the body to the implant. Calcification of heart valve leaflets is one of the main reasons for prostheses failure in humans. Calcification of heart valves can lead to incompetent valve performance that can result in congestive heart failure, thrombosis and/or stroke. Carpentier et al.,  The Society of Thoracic Surgeons , pages S332 to S338 (1995), describe the use of iron pretreatment of tissues preserved in glutaraldehyde to inhibit calcification. The calcification of porcine bioprosthetic valve tissue was found to correlate directly with the iron content within the tissue. Vyavahare et al.,  American Journal of Pathology,  155(3), pages 973-982 (1999), describe the pretreatment of elastin with AlCl 3  to provide resistance against calcification as a result of the binding of the aluminum ions to the elastin.  
       [0015] It will be appreciated that a need exists for modifying the surface of a medical device or instrument for improved, longer acting utility when contact with blood or plasma, as well as improved resistance against mineralization, following a medical procedure in which the device is installed in a subject.  
       SUMMARY OF THE INVENTION  
       [0016] In general terms, the present invention features a substrate having its surface modified with hyaluronic acid. The substrate can be a polymeric material, preferably a polyalkylene terephthalate, such as polyethylene terephthalate, or a metal such as stainless steel. The hyaluronic acid is chemically bound to the surface of the substrate to provide a hydrophilic, anti-platelet, anti-fouling surface. This is of particular advantage when the substrate is part of a medical device or instrument which contacts a biological fluid, such as blood or plasma, and the device or instrument is used in a surgical procedure performed on a patient. The surface of the substrate can be further modified to incorporate Fe +3  or Al +3  cations to prevent calcification of the device or instrument, and the surrounding tissue.  
       [0017] In one aspect, the modified substrate can be prepared by contacting the unmodified substrate with an activated hyaluronic acid in an aqueous solution under conditions sufficient to form a strong chemical bond between the substrate and the hyaluronic acid. The conditions can vary, but will generally include temperatures in the range of from about 4° C. to about 35° C., preferably from about 4° C. to about 25° C., and reaction time periods of from about 2 hours to about 24 hours.  
       [0018] In a further aspect, the activated hyaluronic acid is formed by reacting the hyaluronic acid with a carbodiimide in an aqueous medium under suitable reaction conditions. The preferred pH for carrying out the reaction is from about 3.5 to 8.0, and more preferably 4.0 to 5.1. The preferred concentration for the hyaluronic acid is from about 0.05% w/w to about 2% w/w, more preferably from about 0.1% w/w to about 1% w/w. The molar ratio of carbodiimide to hyaluronic acid is preferably in the range of from about 0.5:1 to about 2:1, with a ratio of 1.5:1 being particularly preferred. The molecular weight of the hyaluronic acid is typically in the range of 25,000 daltons to about 2 million daltons. The preferred carbodiimide is either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or 1-ethyl-3-(3 -dimethyl aminopropyl)carbodiimide methiodide.  
       [0019] In another aspect, the hyaluronic acid is modified to incorporate an amount of Fe +3  or Al +3  cations sufficient to inhibit the mineralization, and particularly calcification, of the substrate when in contact with a source of calcium ions. The hyaluronic acid can be modified by contacting a solution of the hyaluronic acid with a soluble ferric or aluminum salt. Preferred salts include ferric chloride, aluminum chloride, ferric citrate, aluminum citrate, and the like. Alternatively, the hyaluronic acid can be contacted with the ferric or aluminum salt after immobilization onto the surface of a substrate.  
       [0020] In yet another aspect, this invention relates to medical devices and instruments which have been modified as provided herein to produce surfaces which have improved hydrophilic, anti-fouling and anti-platelet adhesion characteristics, as well as improved resistance to calcification. These medical devices and instruments can be prosthetic devices, typically synthetic or bioprosthetic devices, including stents, grafts, sutures, catheters, tubings, and guidewires. Optionally, the hyaluronic acid surface layer can include a pharmaceutically active substance dispersed throughout the hyaluronic acid, making the device or instrument useful for drug delivery applications. Suitable pharmaceutically active substances include proteins, such as growth factors and enzymes, drugs, antibodies, biopolymers, and biologically compatible synthetic polymers.  
       [0021] It has been discovered that a medical device or instrument having a modified surface as provided herein has several advantages in comparison to an unmodified device or instrument, particularly when used in an environment in which contact with a biological fluid, such as blood or plasma, is necessitated. Such modified devices and instruments have enhanced hydrophilicity, as well as improved anti-platelet and anti-fouling characteristics, and improved resistance to calcification. Since the hyaluronic acid is chemically bound to the surface of the device or instrument, it forms a durable and stable coating, as contrasted to a simple physical coating. Further, HA is a biopolymer, and is less likely to exhibit an unfavorable biological response than a synthetic polymer, such as polyvinyl alcohol. Suitable devices and instruments include prosthetic devices, such as coronary valves and vascular grafts, bioprosthetic devices, stents, catheters, tubes, sutures, guidewires, films, and fabrics.  
       [0022] The devices and instruments of this invention, when used in a surgical procedure performed on a human patient, can result in a reduction in thrombosis, as well as a reduction in complications resulting from thrombosis, a reduction in tissue damage, increased resistance to bacterial adhesion, a resistance to cell binding, and a reduction in adhesion formation. Moreover, the reduction in platelet adhesion and aggregation can be accomplished without interfering with other hemostatic events, such as thromboxane production, which is a potent regulator of normal platelet function, and fibrinolytic activity, which induces lysis of clots in the general circulation system.  
       [0023] The devices and instruments of the invention have been found to decrease the risk of pathological thrombus formation associated with a diseased state or a medical procedure including cardiovascular surgery, cardiopulmonary bypass, catheterization (e.g., cardiac catheterization, or angioplasty) with a substantially reduced risk of affecting overall hemostasis. Resistance to calcification allows the devices and instruments to function effectively for a prolonged period of time without adverse biological consequences.  
       [0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any method and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder, including published patent applications, and issued or granted patents, are incorporated herein by reference in their entirety. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not intended to be limiting.  
       [0025] Other features and advantages of the invention will be apparent from the following description of the preferred embodiments, and from the claims. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0026]FIG. 1 illustrates the Fourier transformed infrared spectroscope (FT-IR) of an unmodified polyethylene terephthalate surface.  
     [0027]FIG. 2 illustrates the Fourier transformed infrared spectroscope (FT-IR) of a polyethylene terephthalate surface modified by reaction with hyaluronic acid.  
     [0028]FIG. 3 illustrates the HPLC trace of a glucosamine standard (RT of 15.5 and 15.8 minutes) and aminopropanol (RT of 18.7 minutes).  
     [0029]FIG. 4 illustrates the HPLC trace of an acid hydrolysate from a polyethylene terephthalate surface modified by reaction with hyaluronic acid showing a glucosamine peak.  
     [0030]FIG. 5 illustrates the glucosamine analysis using an HPLC trace of a polyethylene teraphthalate surface modified by reaction with hyaluronic acid at different time points.  
     [0031]FIG. 6 illustrates the electron spectroscopy for chemical analysis (ESCA) of a polyethylene teraphthalate surface modified by reaction with hyaluronic acid at different time points.  
     [0032]FIG. 7 illustrates the Fourier transformed infrared spectroscope (MIR-FTIR) of a polyethylene terephthalate surface modified by reaction with hyaluronic acid at Day 0.  
     [0033]FIG. 8 illustrates the Fourier transformed infrared spectroscope (MIR-FTIR) of a polyethylene terephthalate surface modified by reaction with hyaluronic acid at Day 62.  
     [0034]FIG. 9 is a bar graph depicting cell attachment for unmodified polyethylene terephthalate, aminated polyethylene terephthalate, and a polyethylene terephthalate surface-modified by reaction with hyaluronic acid.  
     [0035]FIG. 10 is a scanning electron microscope (SEM) image of an unmodified polyethylene terephthalate surface after exposure to whole blood.  
     [0036]FIG. 11 is a scanning electron microscope (SEM) image of an aminated polyethylene terephthalate surface after exposure to whole blood.  
     [0037]FIG. 12 is a scanning electron microscope (SEM) image of a polyethylene terephthalate surface-modified by reaction with hyaluronic acid after exposure to whole blood.  
     [0038]FIG. 13 is a graphic representation of platelet deposition on unmodified stainless steel tubes an on hyaluronic acid-modified stainless steel tubes.  
    
    
     DETAILED DESCRIPTION  
     [0039] The medical devices and instruments of the present invention can be prepared generally as described herein. The surface portion of the device or instrument, or a part thereof, is modified to present reactive sites which are capable of reacting with hyaluronic acid which has been suitably activated. The preferred reactive sites are primary amino groups which are formed on the substrate by amination techniques which are known in the art. Such techniques can be used to aminate plastic films and fabrics, as well as stainless steel tubes and wires. Suitable aminated substrate materials of this general description are also available commercially from various sources, such as Advanced Surface Technologies of Billerica, Mass. Substrate materials include metallic materials typically used in surgical procedures, such as stainless steel, and the like, as well as polymeric materials, such as polyalkylene therephthalates, polypropylene, polyurethane, and the like.  
     [0040] By “polyalkylene terephthalates” is generally meant polyethylene terephthalate, polypropylene terephthalate, polybutylene terephthalate, and the like. “PET” refers to polyethylene terephthalate.  
     [0041] As used herein, and unless otherwise indicated, the terms “HA” and “hyaluronic acid” denote hyaluronic acid and any of its hyaluronate salts, including, for example, sodium hyaluronate (the sodium salt), potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate.  
     [0042] A “modified substrate” is a substrate which has its surface modified to be capable of chemically bonding with hyaluronic acid. An example of a modified substrate is a substrate which has been “aminated” and subsequently reacted with an “activated” hyaluronic acid molecule.  
     [0043] A substrate is “aminated” when it is chemically modified to present a reactive amino group on its surface. The amino group can be a primary or secondary amine, although primary amines are preferred. As an example of an amination technique within the scope of this invention, a substrate can be treated with radio frequency plasma deposition followed by adsorption with polyethyleneimine.  
     [0044] Hyaluronic acid is “activated” by reaction with an activating agent, such as a carbodiimide, or by reaction with a suitable crosslinking agent.  
     [0045] An “activating agent” is a substance that, in an aqueous mixture including hyaluronic acid, renders the carboxyl groups on the hyaluronic acid vulnerable to nucleophilic attack.  
     [0046] The terms “chemically bound” or “chemical bond” mean a covalent bond which is formed between the modified substrate and the hyaluronic acid. “HA-PET” refers to hyaluronic acid chemically bound to polyethylene terephthalate; “HA-Dacron™” refers to hyaluronic acid chemically bound to DacronTM; “HA-S.S.” refers to hyaluronic acid chemically bound to stainless steel.  
     [0047] An “acyl derivative,” as may be used herein, is a compound produced by the displacement of the hydroxyl group bound to the acyl carbon atom of a carboxylic acid moiety by either the reaction of the hydroxyl group with a nucleophilic group of another compound, or by the rearrangement of the O-acylisourea formed by reaction of the hydroxyl group with a carbodiimide. Examples of acyl derivatives include acylureas, acylisoureas, amides, thioesters, and phenolates.  
     [0048] The term “mineralization” means the formation of mineral deposits on the surfaces of medical devices or instruments, or in tissue surfaces adjacent to the device after implantation in a subject. As used in a biomedical context, the term “mineralization” generally denotes “calcification”, or the formation of calcium salt deposits on a surface.  
     [0049] The term “platelet aggregation” as used herein means the amassing together of individual platelets through specific interactions between platelets.  
     [0050] The term “platelet adhesion” as used herein means the amassing of platelets onto a surface (e.g., a vascular wall, prosthetic device) through interactions of the platelets with the surface.  
     [0051] The substrate can be a polymeric material or a metal, preferably a polyalkylene terephthalate, such as polyethylene terephthalate, or a metal such as stainless steel. The hyaluronic acid is chemically bound to the surface of the modified substrate to provide a hydrophilic, anti-platelet, anti-fouling surface. This is of particular advantage when the substrate is part of a medical device or instrument which contacts a biological fluid, such as blood or plasma. The finished substrate can be prepared by contacting the modified substrate with an activated hyaluronic acid in an aqueous solution under conditions sufficient to form a strong chemical bond between the modified substrate and the hyaluronic acid. The conditions can vary, but will generally include temperatures in the range of from about 4° C. to about 35° C., preferably from about 4° C. to about 25° C., and reaction time periods of from about 2 hours to about 24 hours.  
     [0052] The activated hyaluronic acid can be prepared by treating a hyaluronic acid with a suitable carbodiimide in the presence or absence of a nucleophile. The resulting product may be water soluble or water insoluble, depending on the reaction conditions and the relative proportions of ingredients in the reaction mixture.  
     [0053] The reaction of the carbodiimide with the carboxyl group of the hyaluronic acid proceeds through the addition of the free carboxylate to one of the double bonds of the diimide to give the O-acylisourea derivatives of hyaluronic acid and the carbodiimide. In the presence of a nucleophile, such as a primary amine, the amide derivative of the hyaluronic acid forms, as well as the unimolecular O→N rearrangement of the O-acylisourea derivative, to give the more stable N-acylurea derivative of the hyaluronic acid and the carbodiimide. In the absence of a nucleophile, the intramolecular rearrangement from the O-acylisourea id derivative to the N-acylurea derivative is the predominant reaction.  
     [0054] The hyaluronic acid, or a salt of hyaluronic acid, such as sodium hyaluronate, is dissolved in water to make an aqueous mixture. HA from any of a variety of sources can be used. As is well known to those skilled in the art, HA can be extracted from animal tissues or harvested as a product of bacterial fermentation. Hyaluronic acid can be produced in commercial quantities by bioprocess technology, as described for example in PCT Publication No. WO 86/04355. Preferably the concentration of HA in this first aqueous mixture is in the range of between 0.05% to 2.0% weight/weight (“w/w”), and more preferably from 0. 1% to 1%. Subsequent reactions are slower and less effective at significantly lower concentrations, while significantly higher concentrations are difficult to handle due to their high viscosity. The aqueous HA mixture should initially be acidic, preferably with a pH between pH 3.5 and pH 7.01, more preferably between a pH 4.0 and 5.1. As the reaction proceeds, it becomes more basic over time. At lower pH values, the preferred activating agent, EDC, is unstable, and at higher values the reaction rate is diminished. Preferably hydrochloric acid is added to adjust the pH, although other known acids can be used.  
     [0055] After the addition of the carbodiimide to the aqueous solution of HA, the pH of the reaction mixture is adjusted. Preferred carbodiimides include EDC (in some references this substance is termed 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide or “DEC”) or ETC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide methiodide).  
     [0056] We have found that the best results are obtained when the ratio of carbodiimide to HA ranges from about 0.5:1 to 2:1, and preferably is about 1.5:1.  
     [0057] Suitable procedures for preparing coatings of the general type as those employed herein, and for using HA to inhibit platelet aggregation, can be found in U.S. Pat. Nos. 5,527,893 and 5,585,361, respectively, the disclosures of which are incorporated by reference herein.  
     [0058] The activated hyaluronic acid can then be immobilized onto a solid substrate, preferably in a one step procedure, by contacting the substrate with the activated hyaluronic acid in an aqueous solution. The substrate has been modified, such as by amination, to accommodate the reactive carboxylic acid sites on the hyaluronic acid. The following illustrates the reaction of the aminated substrate, ETC and HA:  
                 
 
     [0059] wherein R is  
                 
 
     [0060] and R′ represents the substrate.  
     [0061] The chemical bonding of HA onto the surface of the substrate can be illustrated as follows:  
                 
 
     [0062]FIG. 1 shows the FT/IR spectrum of the unmodified PET substrate. The surface-bound HA has attached to some of the carboxyl groups an N-acylurea group as shown by the FT/IR spectrum in FIG. 2. The peak denoted by the arrow in FIG. 2 has been assigned to the asymmetric stretch of the N-acylurea carbonyl as referenced in the literature. DeLos et al.,  J. Am. Chem. Soc ., Vol. 88, page 1013 (1967).  
     [0063] The immobilized hyaluronic acid can also include an amount of Fe +3  or Al +3  cations sufficient to impair or retard the mineralization of the implanted device and surrounding tissues. The amount of Fe +3  or Al +3  cations contained in the hyaluronic acid is generally in the range of from about 0.1% to about 15.0% by weight. The Fe +3  or Al +3  cations can be incorporated in the hyaluronic acid by the addition of soluble salts of these metals to a solution of the hyaluronic acid prior to or during the addition of the activating agent to the solution. Alternatively, the trivalent metal cations can be incorporated into the hyaluronic acid after immobilization onto the surface of the substrate by contacting the substrate with a solution of the trivalent metal salt. Trivalent metal salts which are particularly useful and well suited for this purpose include ferric chloride, aluminum chloride, ferric citrate and aluminum citrate. Combinations of trivalent iron and aluminum salts can also be used, and are effective.  
     [0064] The substrate of this invention is preferably part of a medical device or instrument. Typical of such devices are stents, grafts, prosthetic devices, bioprosthetic devices, vascular grafts, tubes, natural or synthetic heart valves, films, fabrics, catheters, sutures, blood dialysis membranes, guidewires, and the like. These devices are provided with a coating of hyaluronic acid which is chemically bound to the surface of the device in accordance with the methods described herein. Upon exposure to blood, platelets will be less likely to adhere to the surface compared to non-HA coated surfaces.  
     [0065] The efficacy of any device may be tested prior to use by standard cell adhesion assays well known to those skilled in the art. For example, a small sample containing a platelet suspension is incubated with a device coated with HA at physiological temperatures, and then the percentage of platelets bound to the surface of the device is calculated.  
     [0066] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications to the invention to adapt it to various usages and conditions.  
     [0067] In addition, the invention also includes the use of HA to deliver therapeutic drugs directly to desired sites within the body. For example, drugs can be incorporated into the HA by admixing or by immobilizing the drug by chemical attachment to the HA molecule, or by ionic interaction between the drug and the HA molecule (e.g., see Sparer et al., 1983, Chapter 6, pp. 107-119,  In Controlled Release Delivery Systems , Roseman et al. (ed), Marcel Dekker, Inc.: New York). The HA-drug complex, or HA derivative complex, can then deliver the drug in a site-specific manner, for example to a damaged vessel wall.  
     [0068] As one skilled in the art will appreciate, the invention can be practiced using protocols that are within the method of the invention yet are different in particulars from those described herein.  
     [0069] The following examples of this aspect of the invention are given by way of illustration and are not intended to limit the invention except as set forth in the claims.  
     EXAMPLE 1  
     [0070] This example describes the preparation of HA-modified PET, HA-modified Dacron™, HA-modified stainless steel tubes.  
     [0071] Aminated substrates, for example, aminated PET films, aminated Dacron fabric and stainless steel (316W) tubes, are prepared by Advanced Surface Technology (Billerica, Mass.). In a typical experiment, a 0.5% w/w hyaluronic acid solution was prepared by dissolving 500 mg of hyaluronic acid (sodium salt) in 100 gm of distilled water. To the HA solution was added, dropwise with stirring, an aqueous solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide methiodide (EDCI, 750 mg in 150 ml water). Sufficient 0.5N hydrochloric acid was added to adjust the pH to about 4.5, in this example about one ml. The aminated substrate was then immersed in the hyaluronic acid coating solution, and the reaction was allowed to continue for 16 hrs at 4° C.  
     [0072] At the end of this period, the coated substrate was removed from the coating chamber and transferred to a washing chamber filled with distilled water. The coated substrate was washed for at least 4 hours with 2 to 3 changes of water. The coated substrate was then air-dried in a laminar flow hood and characterized for HA content by an HPLC procedure, and by standard spectrophotometric techniques, including electron spectroscopy for chemical analysis (ESCA), Fourier transformed spectroscopy (FT-IR), and scanning electron microscopy (SEM), as shown in FIGS.  1  to  4 , and in the table below.  
                              ESCA Analysis                                                 HA-coated           Element   PET   Aminated Substrate   Substrate                       C   70.4%   67.4 ± 0.42%   63.8 ± 2.8%           N    0   29.6 ± 1.3%     8.5 ± 1.22%           O   26.2%    2.6 ± 0.12%   26.4 ± 3.3%           Other    3.5%   —    1.4 ± 1.2%                      
 
     [0073] The hydrophilicity of the surface was also measured by captive bubble contact angle measurements as shown in the table.  
                              Contact Angle Measurements                             Sample   Contact Angle                       Unmodified PET   112 degrees +/− 1.9           Aminated PET   128 degrees +/− 7.7           HA-PET   152 degrees +/− 1.1                      
 
     EXAMPLE 2  
     [0074] This example illustrates the stability of HA-PET in phosphate buffered saline solution (“PBS”).  
     [0075] Samples of HA-PET (˜1″×3″) were immersed in 45 ml centrifuge tubes filled with PBS buffer. The tubes were placed on a Nutator mixer and incubated in a 37° C. oven. At 1, 3, 7, 14, 21 and 62 days, at least one sample was retrieved and analyzed. The analysis included a glucosamine analysis by HPLC, ESCA and FT-IR. The results are shown in FIGS.  5  to  8 .  
     EXAMPLE 3  
     [0076] This example describes the attachment of cells on HA-PET films.  
     [0077] HA-PET films, prepared as described in Example 1, were cut and placed in wells of 6-well plates (well diameter 35 mm). Prior to cell seeding, cell culture media with serum was added to each well and the entire plate was incubated at 37° C. for two hours. Human smooth muscle cells were seeded into each well at a density of 1×10 6  per well, and the plate was again incubated for two hours at 37° C. The films were removed and rinsed three times with Hanks Balanced Salt Solution to remove all non-adherent cells. Following rinsing, 1 ml of trypsin was added to each of the films and the total number of adherent cells counted by Coulter Counter.  
     [0078] Rat fibroblasts and mouse mesothelial cells were also tested.  
     [0079] For comparison, the same procedure was repeated for non-treated PET films and aminated PET films. FIG. 9 shows that the HA surface bound less cells than either the aminated or unmodified surfaces.  
     EXAMPLE 4  
     [0080] This example describes the in vitro platelet deposition on unmodified, aminated and HA-PET films.  
     [0081] Samples of unmodified, aminated and HA-PET films were placed in individual wells of a 24-well plate. To each well was added 1 ml of freshly drawn, citrated whole blood. The plate was rotated gently on a rotator at 400-600 rpm for 45 minutes at 37° C.  
     [0082] The PET samples were removed and thoroughly washed with PBS buffer. The samples were fixed with a 1.25% aqueous solution of glutaraldehyde, dehydrated with increasing concentrations of ethanol, critical point dried, and examined under scanning electron microscope. The results are shown in FIGS. 10, 11 and  12 .  
     EXAMPLE 5  
     [0083] This Example describes the effect of bovine hyaluronidase digestion on HA-PET films.  
     [0084] HA-PET film was incubated in a 1 mg/ml solution of bovine testes hyaluronidase (Hydase) in 30 mM sodium succinate buffer containing 120 mM of sodium chloride (pH 4) at 37° C. for 24 hours. The sample was rinsed with distilled water and analyzed by ESCA. The results are shown in the table below.  
                                                       Element   Pre-Hydase (% atom)   Post-Hydase (% atom)                          C   63.4   59.0           N    7.0    8.9           O   29.6   30.0           Other        0.63 (C1), 1.5 (Si)                      
 
     [0085] The glucosamine content was also determined by HPLC analysis. The results are shown in the table below.  
                                                   Sample   [HA]                          Pre-Hydase HA-PET   100 ug/cm 2             Post-Hydase HA-PET    30 ug/cm 2                        
 
     EXAMPLE 6  
     [0086] This example describes platelet deposition on an ex-vivo AV Shunt. Indium-labeled autologous baboon platelets were re-infused into a normal male baboon equipped with a surgically implanted, exteriorized silicone rubber shunt between the femoral artery and vein. HA-S.S. tubes (316 medical grade stainless steel, 3.5 mm i.d., at least 18 mm in length) were inserted into 3.2 mm i.d. Silastic tubing and interposed into the shunt system. Radio labeled platelet deposition was monitored by gamma scintillation camera, and the total number of deposited platelets was calculated by dividing the deposited platelet activity (counts/min) by the whole blood activity (counts/min/ml), and multiplying by the circulating platelet count (platelets/ml). The platelet deposition was monitored for a total of 2 hours. The results are shown in FIG. 13.