MXPA00001596A - Latent reactive polymers with biologically active moieties - Google Patents

Abstract

A polybifunctional reagent having a polymeric backbone, one or more pendent photoreactive moieties, and two or more pendent bioactive groups. The reagent can be activated to form a bulk material or can be brought into contact with the surface of a previously formed biomaterial and activated to form a coating. The pendent bioactive groups function by promoting the attachment of specific molecules or cells to the bulk material or coated surface. Bioactive groups can include proteins, peptides, carbohydrates, nucleic acids and other molecules that are capable of binding noncovalently to specific and complimentary portions of molecules or cells.

Description

REACTIVE LATENT POLYMERS WITH BIOLOGICALLY ACTIVE PORTIONS FIELD OF THE INVENTION In one aspect, this invention relates to reagents that can be used to modify surfaces of bio-materials or to manufacture new bio-materials. In another aspect, the invention relates to bio-materials having surfaces that have been prepared or modified to provide a desired bio-active function. BACKGROUND OF THE INVENTION Biomaterials have long been employed to manufacture bio-medical devices for use in both in vitro and in vivo applications. The variety of bio-materials can be used for the manufacture of these devices, including ceramics, metals, polymers and their combinations. Historically, these materials were considered suitable for use in manufacturing bio-medical devices if they provide a convenient combination of basic properties such as being inert, low toxicity and the ability to be manufactured in the desired devices (Hanker, JS and BL Giammara, Science 242: 885-892, 1988). As the result of more recent advances, devices with surfaces having various convenient features can now be provided, for example in order to better interface with tissue or surrounding solutions. For example, approaches have been developed to promote the connection of specific cells or molecules to the device surface. A device surface, for example, can be provided with a bio-active group that is capable of attracting and / or connecting to various molecules or cells. Examples of these bioactive groups include antigens to bind with antibodies, ligands for binding to cell surface receptors and enzyme substrates for binding to enzymes. These bio-active groups have been provided on the surfaces of bio-materials in a variety of ways. In one approach, bio-materials can be made from molecules that themselves exhibit the desired bioactive groups on the surfaces of devices after manufacture. However, desirable bioactive groups are typically dihydrophilic and can not be incorporated into most of the hydrophobic polymeric metals or bio-materials at effective concentrations, without breaking the structural integrity of these bio-materials. An alternative approach involves adding bioactive groups to bio-material surfaces, for example after they have been manufactured in medical devices. These bio-active groups can occasionally be added by adsorption. However, groups that have been added by adsorption typically can not be retained on surfaces at high levels or for long periods of time. The retention of these bioactive groups on a surface can be improved by covalent attachment of these groups to the surface. For example, US patents. Nos. 4,722,906, 4,979,959, 4,973,493 and 5,263,992 relate to devices having biocompatible agents covalently linked by a photoreactive group and a chemical bonding portion with the biomaterial surface. The Patents of the U.S.A. Nos. 5,258,041 and 5,217,492 relate to the connection of bio-molecules to a surface through the use of long-chain chemical spacers. The Patents of the U.S.A. Nos. 5,002,582 and 5,263,992 relate to the preparation and use of polymeric surfaces wherein polymeric agents provide convenient properties are covalently linked by a photoreactive portion to the surface. In particular, the polymers themselves exhibit the desired characteristics, and in the preferred embodiment, they are substantially free of other groups (e.g., bio-active). Others have employed photochemistry to modify the surface of bio-medical devices, for example to coat vascular grafts (see Kita, H., et al., ASAIO Journal 39: M506-M511, 1993. See also Clapper, DL, et al., Trans. Soc. Biomat 16:42, 1993). Cholakis and Sefton synthesize a polymer that has a polyvinyl alcohol main structure (PVA) and bioactive heparin groups. The polymer was coupled to polyethylene tubing by non-latent reactive chemistry, and the resulting surface was evaluated for thromboresistance in a series of in vitro and in vivo assays. For any reason, the heparin in the polymer prepared by Cholakis and Sefton does not provide effective activity. (Cholakis, C.H. and M.V. Sefton, J. Biomed, Mater. Res. 23: 399-415, 1989. See also Cholakis, C.H., et al., J. Biomed, Mater.Res. 23: 417-441, 1989). Finally, Kinoshita et al. Describe the use of reactive chemistry to generate polyacrylic acid main structures in porous polyethylene, with collagen molecules subsequently coupled to carboxyl portions in the main structures of polyacrylic acid. (See Kinoshita, Y., and collaborators, Biomaterials 14: 209-215, 1993). In general, the resulting coating in the situations described above is provided in the form of bioactive groups covalently coupled with surfaces of bio-materials by short linear spacers. This approach works well with high molecular weight bioactive groups, such as collagen and fibronectin, where the use of short spacers is convenient and the size of the bioactive group is quite large compared to that of the spacer itself. The approaches described above, however, with the possible exception of Kinoshita et al., Are not optimal for coating bioactive groups of small molecular weight. Kinoshita seems to coat molecules of small molecular weight, although it describes a laborious process of multiple stages that can affect negatively both the performance and the ability to reproduce it. Bioactive groups of small molecular weight are typically provided in the form of either small regions derived from much larger molecules (for example fibronectin-derived cellular connection peptides) or as small molecules that normally diffuse in free form to produce their effects ( for example antibiotics or growth factors). It seems that short spacers can unduly limit the freedom of movement of these small bioactive groups and in turn impair activity when immobilized. What is clearly required are methods and compositions for providing improved concentrations of bioactive groups and particularly small molecular weight groups, to a biomaterial surface in a form that allows improved freedom of movement of the bioactive groups. SUMMARY OF THE INVENTION The present invention meets the needs described above by providing a "polybifunctional" reagent, comprising a polymer backbone containing one or more secondary photoreactive portions and one or more and preferably two or more secondary bioactive groups. The reagent preferably includes a polymer structure of high molecular weight, preferably linear, having an optimum density of both bioactive groups and photoreactive portions connected. The reagent allows useful densities of bioactive groups to be coupled to a biomaterial surface, by means of one or more photoreactive groups. The main structure, in turn, provides a spacer function of sufficient length to provide the bioactive groups with greater freedom of movement than would otherwise be achieved, for example by the use of individual spacers (as described above). As an added advantage, the present reagent allows for the formation of inter- and intra-molecular covalent bonds within and / or between polymer backbones and the biomaterial surface, thereby providing an optimal and controllable combination of properties such as backbone density. coating, freedom of movement, tenacity and stability. In addition to its use to modify a biomaterial surface, a reagent of the invention provides other benefits equally. The photoreactive portions allow individual polymer molecules to be efficiently coupled (e.g., entangled) with adjacent polymer molecules. This interlacing feature allows polymers to generate thick coatings on surfaces of biomaterials and / or generate independent films and bulk materials either in vi tro or in vivo. The present invention also discloses a method for synthesizing a polybifunctional reagent and for providing a coated surface such as the surface of a biomaterial, or biomedical device manufactured from this biomaterial. The coated surface has polybifunctional reagent molecules connected in order to provide the device with convenient properties or attributes. The photoreactive portions can be activated in order to connect the polybifunctional reagent to a surface that provides extractable hydrogen atoms so that the secondary bioactive group (s) retains its desired bioactive function. Preferably, the reagent is connected to the surface in a "one step" method, that is, by applying a reagent to the surface and activating there one or more of its photoreactive groups in order to form a coating. In contrast, a "two stage" method will involve a first step of immobilizing a polymeric backbone by photochemical means and a second step of connecting (eg thermochemically) one or more bioactive groups to the immobilized backbone. Preferred polybifunctional reagents of the invention can be used to coat the surfaces of existing biomaterials and / or to generate new biomaterials, for example by forming bulk materials. In any case, it can improve the surface properties of a biomedical device by providing bioactive groups covalently linked to the surface of the device. Preferred bioactive groups in turn act as either a non-covalent linkage with, or act on, specific complementary portions of molecules or cells that come in contact with these groups. In a preferred embodiment, a polybifunctional reagent of the invention is synthesized having a polymer backbone, one or more photoreactive portions and two or more bioactive groups. The polymeric molecule of the invention is contacted with the surface of a biomaterial previously formed or in contact with another polymeric molecule of the invention. The photoreactive portions are energized by external stimulus to form, by active species, a covalent bond between the reagent molecule and either the biomaterial surface or another reagent molecule. For example, a biomaterial can be moistened in a solution that has a convenient reagent (typically 0.1-5 minutes) and then exposed to light (typically 0.1-2 minutes) to achieve covalent coupling. Preferred bioactive groups work by promoting the connection of specific molecules or cells to the surface. Preferred bioactive groups include, but are not limited to, proteins, peptides, carbohydrates, nucleic acids and other molecules that are - able to bind non-covalently with specific and complementary portions of molecules or cells. Examples of this specific binding include cell surface receptors that bind to ligands, antigens that bind to antibodies and enzyme substrates that bind to enzymes. Preferably, the polymeric backbone comprises a synthetic polymer backbone selected from the group consisting of: addition type polymers, such as vinyl polymers. More preferably, of the photo-groups each comprises a reversibly photoactivatable ketone. DESCRIPTION OF THE PREFERRED MODALITIES As used herein, the following terms and words will have the following ascribed meanings: "Biomaterial" will refer to a material that is substantially insoluble in aqueous systems and that provides one or more surfaces for contact with fluids containing molecules biological, for example aqueous systems in vi tro or in vivo that contain tissues, cells or biomolecules; "Device" will refer to a functional object manufactured from a biomaterial; "Coating" when used as a pronoun will refer to one or more polymer layers on a biomaterial surface, and in particular to one or more layers immobilized on a biomaterial surface by the activation of a polybifunctional reagent of the present invention.; "Polybifunctional reagent", as used in the context of the reagent currently claimed, will refer to a molecule comprising a polymer backbone structure to which one or more photoreactive portions and two or more bioactive groups are covalently linked; "A photoreactive portion" will refer to a chemical group that responds to a specific applied external energy source in order to undergo generation of active species, resulting in covalent binding to an adjacent molecule or biomaterial surface; "Bioactive group" will refer to a molecule having desired specific biological activity, such as a binding or enzymatic (catalytic) activity; "Main polymer structure" will refer to a natural polymer or synthetic polymer, for example resulting from condensation or addition polymerization; Preferred reagents of the invention comprise a synthetic polymer that serves as a backbone, one or more secondary photoreactive portions which can be activated to provide covalent attachment to adjacent polymer surfaces or molecules and two or more secondary low molecular weight biologically active portions ( bioactive). Main structure: The polymer backbone may already be of natural or synthetic origin and preferably a synthetic polymer is removed from the group consisting of oligomers, homopolymers and copolymers resulting from addition or condensation polymerization. Polymers of natural origin such as polysaccharides and polypeptides can be used equally. Preferred main structures are biologically inert as they do not provide a biological function that is inconsistent with or harmful to their use in the manner described. These polymer backbones may include acrylics such as those polymerized from hydroxyethyl acrylate, hydroxyethyl methacrylate, glyceryl acrylate, glyceryl methacrylate, acrylic acid, methacrylic acid, acrylamide and methacrylamide; Vinyls such as polyvinyl pyrrolidone and polyvinyl alcohol; nylons such as polycaprolactam; derivatives of polylauryl lactam, polyhexamethylene adipamide and polyhexamethylene dodecanediamide, and polyurethanes; polyethers such as polyethylene oxide, polypropylene oxide and polybutylene oxide; biodegradable polymers such as polylactic acid, polyglycolic acid, polydioxanone, polyanhydrides and polyorthoesters. The polymeric backbone is also chosen to provide a spacer between the surface of the various photoreactive portions and bioactive groups. In this way, the reagent can be ligated to a surface or an adjacent reagent molecule, to provide the bioactive groups with sufficient freedom of movement to demonstrate optimal activity. The polymeric backbones are preferably water soluble, with polyacrylamide and polyvinyl pyrrolidone, as particularly preferred polymers. Photoreactive portions Polybifunctional reagents of the invention transport one or more secondary latent reactive portions (preferably photoreactive) linked to the polymer backbone. Photoreactive portions are defined here and preferred portions are sufficiently stable to be stored under conditions in which they retain these properties. See, for example, U.S. Pat. DO NOT. 5,002,582, the description of which is incorporated by reference. Latent reactive portions that respond to various portions of the electromagnetic aspect, with those that respond to the ultraviolet and visible portions of the spectrum (hereinafter referred to as "photoreactive"), can be selected as particularly preferred. Photoreactive portions respond to specific external applied stimuli to proceed to the generation of active species with resultant covalent binding with an adjacent chemical structure, for example as disposed by the same or a different molecule. Photoreactive portions are those groups of atoms in a molecule that retain their covalent bonds, without change under storage conditions but when activated by an external energy source of covalent bonds, with other molecules. The photoreactive portions generate reactive species such as free radicals and particularly nitrenes, carbenes and excited states of ketones before absorption of external electrical energy, electromagnetic or kinetic (thermal). Photoreactive portions can be selected to respond to various portions of the electromagnetic spectrum, and photoreactive portions responding for example to ultraviolet and visible portions of the spectrum are preferred and are occasionally referred to herein as the "photochemical" portion. Photoreactive arylketones are particularly preferred, such as acetophenone, benzophenone, anthraquinone, anthrone, and anthrone heterocycles (ie, heterocyclic anthrone analogs such as those having N, 0, or S at position 10), or their substituted derivatives (by substituted example of ring). The functional groups of this ketone are preferred since they are readily capable of undergoing the activation / inactivation / reactivation cycle described herein. Benzophenone is a particularly preferred photoreactive portion, since it is capable of photochemical excitation with the initial formation of an excited singlet state that undergoes inter-system crossing to the triplet state. The excited triplet state can be inserted into carbon-hydrogen bonds by extraction of a hydrogen atom (from a support surface, for example) thereby creating a radical pair. The subsequent crushing of the radical pair leads to the formation of a new carbon-carbon bond. If a reactive linkage (for example carbon-hydrogen) is not available for binding, the ultraviolet-light excited excitation of the benzophenone group is reversible and the molecule returns to the basal state energy level upon removal of the energy source. Aryl photoactivatable ketones such as benzophenone and acetophenone are of particular importance since these groups are subject to multiple reactivation in water and therefore provide increased coating efficiency. Therefore, photoreactive aryl ketones are particularly preferred. Azides constitute a preferred class of latent reactive moieties and include aryl azides (C6R5N3) such as phenyl azide and particularly 4-fluoro-3-nitrophenyl azide and acyl azides (-CO-N3) such as benzoyl azide and p-methylbenzoyl azide, azido formats (-0-CO-N3) such as ethyl azidoformate, phenyl azidoformate, sulfonyl azides (-S02-N3) such as benzenesulfonyl azide, and phosphoryl azides (RO) 2PON3 such as diphenyl phosphoryl azide and diethyl phosphoryl azide. Diazo compounds constitute another class of photoreactive portions and include diazoalkanes (-CHN2) such as diazomethane and diphenyldiazomethane, diazoketones (-CO-CHN2) such as diazoacetophenone and 1-trifluoromethyl-1-diazo-2-pentanone, diazoacetates (-0-CO) -CHN2) such as t-butyl diazoacetate and phenyl diazoacetate, and beta-keto-alpha-diazoacetates (-CO-CN2-CO-0-) such as t-butyl alpha-diazoacetoacetate. Other photoreactive portions include aliphatic azo compounds such as azobisisobutyronitrile, diazirines (-CHN2) such as 3-trifluoromethyl-3-phenyldiazirine, ketenes (-CH = C = 0) such as ketene and diphenyl ketene. Upon activation of the photoreactive portions, the coating adhesion molecule is covalently linked between s £ and / or to the material surface by covalent bonds through residues of the photoreactive groups. Exemplary photoreactive groups and their residues upon activation are shown as follows. Photoreactive Group Residual Functionality aryl azides amine R-NH-R 'acyl azides amide R-CO-NH-R' azido-forms carbamate R-0-CO-NH-R 'sulfonyl azides sulfonamide R-S02-NH-R' phosphoryl azides phosphoramide (RO) 2PO-NH-R 'diazoalkane new linkage CC diazo ketones new linkage CC and ketone Photoreactive Group Functionality of diazoacetate residues new linkage CC and ester β-keto- -diazoacetates new linkage CC and β-ketoester azo aliphatic new linkage CC diazirines new link CC cetenos new link CC photo-activated ketones new link CC and alcohol Bioactive groups. Low molecular weight bioactive groups of the present invention are typically those that are intended to improve or alter the function or performance of a particular biomedical device in a physiological environment. In a particularly preferred embodiment, the bioactive group is selected from the group consisting of cell connection factors, growth factors, antithrombotic factors, binding receptors, ligands, enzymes, antibiotics and nucleic acid. A reagent molecule of this invention includes at least one secondary bioactive group. The use of two or more secondary bioactive groups is currently preferred, however since the presence of several of these groups per reagent molecule tends to facilitate the use of these reagents. Convenient cell connection factors include connection peptides (defined below), as well as large proteins or glycoproteins (typically from 100 to 1000 kilodaltons in size) which in their native state can be firmly ligated to a substrate or an adjacent cell, ligated to a specific cell surface receptor and mechanically connect to a cell to the substrate or an adjacent cell. Connection factors of natural origin are primarily high molecular weight proteins, with molecular weights over 100,000 daltons. Connection factors bind to receptors on specific cell surfaces, and mechanically connect cells to the substrate (referred to as "adhesion, substrate molecules" here) or to adjacent cells (referred to as "cell-to-cell adhesion molecule" here) [ Albe &, B. et al., Molecular Bioloqy of the Cell, 2nd ed. , Garland Publ. , Inc., New York (1989)]. In addition to promoting cellular connection, each type of connection factor can promote other cellular responses, including cell migration and differentiation. Convenient connecting factors for the present invention include substrate adhesion molecules such as laminima, fibronectia, collagens, vitronectin, tenascin, fibrinogen, thrombospondin, osteopontin, von Willibrand factor, bone sialoprotein proteins. Other convenient connection factors include cell-to-cell adhesion molecules ("cadherins") such as N-cadherin and P-cadherin.

Connection factors useful in this invention typically comprise amino acid sequences or functional analogs thereof that possess the biological activity of a specific domain of an active connection factor, with the typical connection peptide being approximately 3 to about 20 amino acids of length. Connecting factors of native cells, typically have one or more domains that bind cell surface receptors and produce the connection, migration, and cell differentiation activities of parent molecules. These domains consist of specific amino acid sequences, several of which have been synthesized and reported to promote the connection, dispersion and / or proliferation of cells. These domains and functional analogues of these domains are referred to as "connecting peptides". Examples of fibronectin connecting peptides include, but are not limited to, RGD (arg-gly-asp) [Kleinman, H.K, et al., Vitamins and Hormones 47: 161-186, 19931, REDV (arg-glu-asp-val) [Hubbell, J.A. , and collaborators, Ann.

N.Y. Acad. Sci. & amp; 253-258, 19921, and CH-V (WQPPRARI or trp-gln-pro-pro-arg-ala-arg-ile) [Mooradian, D.L., et al., Invest. Ophth. & Vis. Sci. 34: 153-164, 1993].

Examples of laminin binding peptides include, but are not limited to, YIGSR (tyr-ile-gly-ser-arg) and SIKVAV (ser-ile-lys-val-ala-val) [Kleinman, H.K, et al., Vitamins and Hormones (Vitamins and Hormones) 47: 16 1- 186, 19931 and F-9 (RYWLPRPVCFEKGMNYTVR or arg-tyr-val -val-leu-pro-arg-pro-val -cys -phe -glu -lys-gly-met-asn-tyr-thr-val -arg) [Charonis, A. S. , and collaborators, J. Cell Biol. 107: 1253-1260, 1988]. Examples of type collagen connection peptides IV include, but are not limited to, HEP-111 (GEFYFDLRLKGDK or gly-glu-phe-tyr-phe-asp-leu-arg-leu-lys-20 gly-asp-lys) [Koliakos, G.G, et al., J. Biol.

Chem. 264: 2313-2323, 1989]. Conveniently, connection peptides employed in this invention have between about 3 and about 30 amino acid residues in their amino acid sequences. Preferably, connecting peptides have no more than about 15 amino acid residues in their amino acid sequences. Other suitable bioactive groups present in the invention include growth factors, such as fibroblast growth factors, epidermal growth factors, platelet-derived growth factors, transformation growth factors, vascular endothelial growth factors, bone morphogenic proteins and other bone growth factors, neural growth factors and the like. Still other suitable bioactive groups present in the invention include antithrombotic agents that inhibit the formation or accumulation of thrombi in devices that contact the blood. Suitable antithrombotic agents include heparin and hirudin (which inhibit coagulation cascade proteins such as thrombin) as well as lysine. Other suitable antithrombotic agents include prostaglandins such as PGI2, PGEX, and PGD2, which inhibit platelet adhesion and activation. Still other suitable antithrombotic agents include fibrinolytic enzymes such as streptokinase, urokinase, and plasminogen activator, which degrade fibrin clots. Another convenient bioactive group consists of lysine, which binds specifically to plasminogen which in turn degrades fibrin clots. Other suitable bioactive groups present in the invention include binding receptors, such as antibodies and antigens. Anticuperpos present on a biomaterial surface can be ligated to and remove specific antigens from aqueous media that come in contact with the immobilized antibodies. Similarly, antigens present on a biomaterial surface can be ligated to and withdraw specific antibodies from aqueous media that come into contact with the immobilized antigens. Other suitable bioactive groups consist of receptors and their corresponding ligands. For example, avidin and streptavidin bind specifically with biotin, with avidin and streptavidin which are receptors and biotin is a ligand. Similarly, fibroblast growth factors and vascular endothelial growth factor bind with high affinity to heparin, and transform the beta growth factor and certain morphogenic bone proteins bind to type IV collagen. Also included are immunoglobulin-specific binding proteins, derived from bacterial sources such as protein A and protein G and their synthetic analogues. Still other suitable bioactive groups present in the invention include enzymes that can bind to and catalyze specific changes in substrate molecules present in aqueous media that come in contact with immobilized enzymes. Other suitable bioactive groups consist of nucleic acid sequences (e.g., DNA, RNA and cDNA) that selectively ligate complementary nucleic acid sequences. Surfaces coated with specific nucleic acid sequences are used in diagnostic assays to identify the presence of complementary nucleic acid sequences in test samples. Still other suitable bioactive groups present in the invention include antibiotics that inhibit microbial growth on surfaces of biomaterials. Certain convenient antibiotics can inhibit biomicrobial growth by binding to specific components in bacteria. A particularly convenient class of antibiotics are antibiotic peptides that appear to inhibit microbial growth by altering the , permeability of the plasma membrane by mechanisms that at least in part may not involve specific complementary ligand-receptor binding [Zazloff, M., Curr.

Opinion Immunol. 4: 3-7, 1992]. Biomaterials Preferred biomaterials include those formed from synthetic polymers, including oligomers, homopolymers and copolymers resulting from either addition or condensation polymerizations. Examples of suitable addition polymers include but are not limited to acrylics, such as those polymerizations from methyl acrylate, methyl methacrylate, hydroxyethyl methacrylate, hydroxyethyl acrylate, acrylic acid, methacrylic acid, glyceryl acrylate, glyceryl methacrylate, methacrylamide, and acrylamide; vinyls such as ethylene-, propylene-, styrene-, vinyl chloride, vinyl acetate, vinyl pyrrolidone, and vinylidene difluoride. Examples of condensation polymers include but are not limited to nylons such as polycaprolactam, polylauryl lactam, polyhexamethylene adipamide, and polyhexamethylene dodecandiamide, and also polyurethanes, polycarbonates, polyamides, polysulfones, poly (ethylene terephthalate), polylactic acid, polyglycolic acid, polydimethylsiloxanes, and polyether ether ketone. Certain natural materials are also suitable biomaterials including human tissue such as bone, cartilage, skin and teeth; and other organic materials such as wood, cellulose, compressed carbon and rubber. Other suitable biomaterials are composed of substances that do not possess extractable hydrogens with which photographic groups can form covalent bonds. Such a class of biomaterials can be made convenient for coating by photochemistry by applying a convenient primer coating which binds to the surface of the biomaterial and provides a convenient substrate for ligating by the photo-groups. A subset of this group includes metals and ceramics that have oxide groups on their surfaces and are made convenient for coupling by photochemistry by adding a primer coating that binds the oxide groups and provides extractable hydrogens. The metals include, but are not limited to titanium, stainless steel and cobalt chromium. Ceramics include, but are not limited to, silicon nitride, silicon carbide, zirconium oxide, and alumina as well as silica and sapphire glass. A convenient class of primers for metals and ceramics consists of organosilane reagents, which binds to the oxide surface and provides hydrocarbon groups (Brzoska, J.B., et al., Lancrmuir 10: 4367-4373, 1994). Researchers have also discovered that -SiH groups are suitable alternatives for photographic union. A second class of biomaterials that require an organic primer are noble metals, which include gold, silver, copper and platinum. Functional groups with high affinity to noble metals include -CN, -SH, and -NH2, and organic reagents with these functional groups are used to apply organic monolayers on these metals (Record, K.C., and collaborators, 7Anal, Chem. 67: 735-743, 1995). Another class of biomaterials that do not possess extractable hydrogens are fibrous or porous. The polymers of the invention form covalently entangled polymer networks that fill the pores or form films around individual fibers and are therefore physically trapped. Expanded polytetrafluoroethylene is such a biomaterial. Biomaterials can be used to make a number of devices capable of being coated with bioactive groups using a polybifunctional reagent of the present invention. Implant devices are a general class of convenient devices including, but not limited to, vascular devices such as stent grafts, catheters, valves, artificial hearts, and heart aids; orthopedic devices such as joint implants, fracture repair devices and artificial tendons; dental devices such as dental implants and fracture repair devices; ophthalmic devices such as lenses and shunts for glaucoma discharge; and other catheters, synthetic prostheses and artificial organs. Other convenient biomedical devices include dialysis tubing and membranes, blood oxygenator tubing and membrane, blood bags, sutures, membranes, cell culture devices, chromatographic support materials, biodetectors and the like. Preparation of Reagents. Those skilled in the art given the present teachings will appreciate the manner in which the reagents of the present invention can be prepared using conventional techniques and materials. In a preferred method, a polymer backbone is prepared by the copolymerization of a base monomer, such as acrylamide or N-vinylpyrrolidone, with monomers having thermochemically reactive groups and / or secondary photoreactors. The polymers prepared by this copolymerization are then derivatized by the bioactive molecule by reaction through the thermochemically reactive groups. An example of this coupling is the reaction between an N-oxysuccinimide ester (NOS) in the polymeric backbone with an amine group in the bioactive molecule. An alternate preferred method involves the preparation of monomers containing the desired bioactive group as well as a polymerizable function such as a vinyl group. These monomers can then be copolymerized with monomers containing photoreactive groups and with a base monomer such as acrylamide or N-vinylpyrrolidone. A preferred method used to synthesize latent reactive peptide polymers involves the synthesis of N-substituted methacrylamide monomers containing each peptide (peptide monomer) and a methacrylamide monomer containing a substituted benzophenone (4-benzoylbenzoic acid, BBA). Peptide monomers were prepared by reacting the sulfhydryl portion of each peptide with the maleimide portion of N- [3- (6-maleimidylhexanamido) propyl] methacrylamide (Mal MAm). Then, each peptide monomer is copolymerized with acrylamide and the BBA-containing monomer (BBA-APMA) to produce the final latent reactive peptide polymer. Various parameters can be controlled to provide reagents that have a desired ratio (either on a molar or weight basis) of the polymeric main structure, photoreactive portions and bioactive groups. For example, the main structure itself will typically provide between about 4 and about 400 carbon atoms per photoreactive group, and preferably between about 60 and about 300 carbon atoms. With respect to the bioactive group, the length of the main structure can vary depending on factors such as the size of the bioactive group and the desired coating density. For example, for relatively small bioactive groups (MW less than 3000) the polymeric backbone will typically be in the range of about 5 to about 200 carbon atoms per bioactive group and preferably between about 10 and about 100. For larger bioactive groups , such as those having molecular weight between about 3000 and about 50,000, the preferred main structure provides on average between about 10 and about 5,000 carbon atoms between the bioactive group and preferably between about 50 and 1000 carbon atoms. In each case, those skilled in the art given the present disclosure will be able to determine the appropriate conditions to provide an optimal combination of density of bioactive groups and freedom of movement. Coating method. Reagents of the present invention can be coated on surfaces of biomaterials using techniques (e.g., dipping, spraying, brushing) within the skill of those in the relevant art. In a preferred embodiment, the polybifunctional reagent is first synthesized and then brought into contact (i.e. sufficient proximity to allow binding) with a previously formed biomaterial. The photoreactive group is energized by external stimulus (for example exposure to a convenient light source) to form, by free active spice generation, a covalent bond between the reagent and either another polybifunctional reactive molecule or biomaterial surface. This coating method is referred to herein as the "one-step coating method" since photo-reactive coupling chemistry connects a polymer of the invention with a biomaterial surface, and subsequent steps to add the bioactive group are not required. The external stimulus that is conveniently used is electromagnetic radiation, and is preferably radiation in the ultraviolet, visible or infrared regions of the electromagnetic spectrum. Photoactivatable polymers of the invention can also be used to immobilize bioporizations in standards on the surfaces of biomaterials, for example using techniques previously described to generate coating patterns with characteristics of 5 to 350 mm in size (see Matsuda, T. and T. Sugawara, J. Biomed, Mater. Res. 29: 749-756 (1995)). For example, hydrophilic patterns that inhibit the connection and growth of endothelial cells can be generated by: 1) synthesizing latent reactive hydrophilic polymers, 2) adding the latent reactive polymers to tissue culture polystyrene plates, 3) illuminating the polymers through a photomask with pattern and 4) remove non-immobilized polymers when washing with an appropriate solvent. This approach can be employed with polymers of the present invention in order to immobilize specific bioporization patterns. For example, micro sets of specific binding molecules (eg, antibodies, antigens / haptens, nucleic acid probes, etc.) can be immobilized on optical, electrochemical or semiconductor detector surfaces, to provide simultaneous multianalyte assay capabilities or assays. of multiple sensitivity range for simple analytes. Pattern immobilization also provides a useful tool to develop a "laboratory in a fragment" wherein sequential processing / reaction steps occur on a fluid movement path in a multi-stage micro volume assay system. The formation of patterns of cell connection factors for example, those that promote the connection of neural cells to electrodes, will allow the development of: 1) new generations of ultrasensitive biosensors and 2) artificial limbs controlled directly by the patient's nervous system. Reagents of the invention can be covalently coupled with biomaterials previously formed to serve as surface coatings. The present reagent molecules can also be covalently coupled with adjacent molecules in order to form bulk films or biomaterials. The surface coatings, films and bulk biomaterials that result from coupling through photoreactive portions provide useful densities of bioactive groups on the surface of resulting biomaterials.

Use of devices Bioactive polymers of the present invention are used to modify the surfaces of existing biomaterials or to generate new biomaterials. Biomedical devices containing the resulting biomaterials are used for a variety of applications in vi tro and in vivo. For example, biomedical devices that have cell connection groups or growth factors such as bio-promotions promote the connection and / or growth of cells from cell culture devices in vi tro and improve the integration of tissue with implant devices such as vascular grafts, orthopedic implants, dental implants, cornea lenses, and breast implants. Biomedical devices that have antithrombotic factors such as bioportions, prevent thrombosis on the surfaces of devices that contact the blood, such as catheters, heart valves, vascular, vascular grafts, stent grafts, artificial hearts and blood oxygenators. Medical devices such as resins or membranes that possess receptors or ligands as biogroups can be used for affinity purification of a wide range of biomolecules. For example, heparin (which is also an antithrombotic portion) is used to specifically bind and purify various coagulation factors, protease inhibitors, lipoproteins, growth factors, lipolytic enzymes, intracellular matrix proteins and viral coat proteins. Staphylococcal protein A specifically binds immunoglobulins and has been shown to be very useful for purification of antibodies. Streptavidin is a protein that binds specifically to biotin with extremely high affinity. Streptavidin and biotin are a very useful pair of reagents as a secondary binding pair in diagnostic assays. Many times signal amplification, improved sensitivity and faster test performance can be achieved using immobilized streptavidin. Biomedical devices that have surface-coated antigens or antibodies can be used in diagnostic tests that depend on the binding specificity for sensitive detection of the antigen or complementary antibody. Antigens or antibodies can be immobilized in membranes, plastic tubes, microplate wells or solid state biosensing devices. Immobilized antibodies are also important for purification of a variety of biopharmaceutical agents. Proteins produced in bacteria or fungi by genetic engineering techniques can be purified by affinity purification with immobilized antibodies. Blood fractions such as coagulation factor VIII (antihemophilic factor) are also purified by immobilized antibodies. Biomedical devices that have surfaces coated with nucleic acid sequences can be used to selectively ligate complementary nucleic acid sequences. These devices employ a diagnostic assay to identify the presence of complementary nucleic acid sequences and test samples. Devices that have surface-coated enzymes as bioporations can be used for a wide range of enzyme reactors that catalyze either synthetic processes (eg, producing chiral pharmaceuticals) or degradation / conversion processes (eg degrading starch and converting glucose into fructose to produce syrup) of high fructose corn). Antimicrobial coated agents can be employed to inhibit bacterial growth on the surfaces of the devices. These antimicrobial surfaces can reduce the proportion of infections associated with implant devices, including various types of catheters (intravascular, peripheral, hemodialysis, hydrocephalic and urological), arteriovenous shunts, heart valves, vascular grafts, tracheotomy tubes, orthopedic implants and peniles Several devices in vi tro can also benefit from these surfaces by inhibiting biofilm formation. These include containers for contact lenses, dental unit water lines, plumbing used in food and pharmaceutical industries, food packaging, table surfaces, and other surfaces used for food handling and air filters. EXAMPLES The invention will be further described reference to the following non-limiting examples. It will be appreciated by those skilled in the art that many changes can be made in the described embodiments out departing from the scope of the present invention. Unless otherwise indicated, all percentages are given by weight. EXAMPLE 1 Peptide Polymers A. Synthesis of 4-Benzoylbenzoyl chloride (BBA-C1) 4-Benzoylbenzoic acid (BBA), 200.0 g (0.884 moles), is added to a dry 2-liter round bottom flask, followed by addition of 273 ml of thionyl chloride. Dimethylformamide (DMF), 684 μl, then added and the mixture refluxed for 3 to 4 hours. After cooling, the excess thionyl chloride is removed in a rotary evaporator under pressure from a water aspirator. Any remaining thionyl chloride is removed by repeated evaporation 3 x 100 ml of toluene. The final product is then recrystallized from 5: 1 hexane: toluene typical yields of BBA-Cl to > 90% and a melting point of 92-94 ° C. B. Synthesis of N- [3- (4-benzoylbenzamido) propyl] methacrylamide (BBA-APMA) N- (3-aminopropyl) methacrylamide hydrochloride (APMA-HC1, 120 g, 0.672 mol), from Eastman Kodak Co., Rochester, NY) was added to a dry-bottom, three-neck, three-necked round-bottomed flask equipped an overhead stirrer. Phenothiazine, 23-25 mg, is added as an inhibitor, followed by 800 ml of chloroform. The suspension is cooled to below 10 ° C in an ice bath and 172.5 g (0.705 moles) of BBA-Cl are added as a solid. Triethylamine, 207 ml (1485 moles), in 50 ml chloroform are then added per drops for a period of time from 1 to 1.5 hours. The ice bath is removed and stirring at room temperature is continued for 2.5 hours. The product is then washed 600 ml of 0.3 N HCl and 2 x 300 ml of 0.07 N HCl. After drying over sodium sulfate, the chloroform is removed under reduced pressure and the product is recrystallized twice from 4: 1 toluene: chloroform using 23-25 mg of phenothiazine in each recrystallization to avoid polymerization. Typical yields of BBA-APMA were 90% a melting point of 147-151 ° C. C. Synthesis of N- [3- (6-Maleimidohexanamido) propyl] methacrylamide (Mal-MAm) 6-Maleimidohexanoic acid is prepared by dissolving 6-aminohexanoic acid (100.0 g, 0.762 mol) in 300 ml of acetic acid in a flask of 3 liters three necks, equipped an upper agitator and a drying tube. 7 Maleic acid 78.5 g (0.801 mol), dissolved in 200 ml of acetic acid and added to the solution of 6-aminohexanoic acid. The mixture is stirred for one hour while it is heated in a boiling water bath, resulting in the formation of a white solid. After cooling overnight at room temperature, the solid is harvested by fixation and rinsed 2 x 50 ml of hexane. Typical yield of (Z) -4-oxo-5-aza-2-undecendioic acid was 90 to 95% a melting point of 160-165 ° C. Acid (Z) -4-Oxo-5-aza-2-undecendioic, 150.0 g (0.654 mol), anhydrous acetic acid, 68 ml (73.5 g, 0.721 mol), and phenothiazine, 500 mg, are added to a bottom flask round three-necked two liters capacity, equipped a top agitator. Triethylamine (TEA), 91 ml (0.653 moles), and 600 ral of tetrahydrofuran (THF) are added and the mixture is heated to reflux while stirring. After a total of 4 hours of reflux, the dark mixture is cooled to < 60 ° C and empty in a solution of 250 ml of 12 N HCl in 3 liters of water. The mixture is stirred at 3 hours at room temperature and then filtered through a filter pad (Celite 545, J.T. Baker, Jackson, TN) to remove solids. The filtrate is extracted with 4 x 500 ml of chloroform and the combined extracts are dried over sodium sulfate. After adding 15 mg of phenothiazine to avoid polymerization, the solvent is removed under reduced pressure. The 6-maleimidohexanoic acid is recrystallized from 2: 1 hexane: chloroform to give typical yields of 50 to 60% with a melting point of 81-85 ° C. The N-oxysuccinimide ester (NOS) of 6-maleimidohexanoic acid is prepared by dissolving 1.0 g (4.73 mmoles) of 6-maleimidohexanoic acid and 0.572 g (4.97 mmole) of N-hydroxysuccinimide (NHS) in 10 ml of dry dioxane, followed by the addition of 1074 g (5.21 mmoles) of 1,3-dicyclohexylcarbodiimide (DCC). The reaction mixture is allowed to stir overnight at room temperature. The 1,3-dicyclohexylurea by-product is removed by filtration and the filter cake is rinsed with 3 x 10 ml of dioxane. Phenothiazine (0.2 mg) is added and the solution is evaporated under reduced pressure. The resulting solid is extracted with hexane to remove any excess DCC and this product is used without any further purification. The N-succinimidyl-6-maleimidohexanoate, 414 mg (1.34 mmol), and N- (3-aminopropyl) methacrylamide hydrochloride, 200 mg (1.12 mmol), were diluted with 10 ml of chloroform, followed by the addition of 153 pl ( 1.10 mmole) of TEA over a period of one hour at room temperature. The mixture is allowed to stir overnight at room temperature. The product is isolated by evaporation and purified by flash chromatography on silica gel using a gradient of 99: 1 followed by 97: 3 chloroform: methanol. Gathering the fractions, adding 10 mg of p-methoxyphenol, and evaporating the solvent gave 261 mg of product. 7 Mass spectral analysis of a mass gave M + = 335 (10.7%) and NMR showed few protons maleimide (6.6 ppm) and methyl allyl (2.0 ppm). D. Synthesis of peptide monomers Five peptides were used as bioporations. Each portion and peptides is synthesized by standard solid phase synthesis methods and is identified below by its common name, a representative literature citation, the main protein from which it is identified and the specific sequence used (indicated by simple letter notation). standard to identify amino acids.

Name Appointment of Protein Common Sequence Main literature used RGD Kleinman, et al .1 fibronectin CKKGRGDSPAF CH-V Mooradian, and col.1 fibronectin CKKWQPPRARI UH-11 McCarthy, and col.1 fibronectin CKNNQKSEPLIGRKKT F-9 Charonis, et al.4 laminin RYWLPRPVCFEKK HEP-111 Koliakos, and col.5 Type IV collagen CKGEFYFDLRLKGDK '' 'Kleinman, HK, et al., Vitamins and Hormones 47: 16 1- 186 (1993). 2 Mooradian, D.L., and collaborators, Invest. Ophth. & Vis. Sci. 34: 153-164 (1993). 3 McCarthy, J.B., et al., Biochem. 27: 1380-1388 (1988). 4 Charonis, A.S., et al., J. Cell Biol. 107: 1253-1260 (1988). 5 Koliakos, G.G, et al., J. Biol. Chem. 264: 2313-2323 (1989). For each peptide sequence, the portion of the sequence that is not underlined represents the native sequence that is present in the major protein. The portion of the sequence that is underlined represents amino acids that were added to provide specific functional groups. The lysine residue (K) was added to provide primary amines (amino epsilon groups) which were used for radiolabelling by reductive ventilation. Cysteine residue (C) were added to provide sulfhydryl groups which were used to couple each peptide with maleimide groups present in monomers that were subsequently polymerized to produce the peptide polymers. C / H-II contains sufficient lysine residues in its native sequence and does not require the addition of additional lysine residues; similarly F-9 contains a cysteine residue as part of its native sequence and does not require the addition of an additional cysteine residue. An appropriate amount of Mal-MAm is removed from a solution of Mal-MAm material in chloroform and placed in a dry reaction bulb under a stream of nitrogen and redissolved in dimethyl sulfoxide (DMSO). An equal molar amount of each peptide is dissolved in 50 mM acetate buffer (pH 5), added to the reaction ampule, and the mixture is stirred for 50 or 90 minutes at room temperature. Type Mal-MAm DMSO peptide buffer time of acetate reaction peptide (amol) (ml) (umol) (ml) (min.) RGD 53. 4 2 53. 4 10 90 F- 9 40. 4 1 40. 4 7 60 Type Mal-MAm DMSO peptide buffer time of acetate reaction peptide (μmol) (ml) (μmol) (ml) (min.) C / HV 8.6 0.2 8.6 1.3 90 C / H-II 38 2 38 10 90 HEP-III 6.4 0.3 6.4 2.7 90 E. Synthesis of photoreactive polyacrylamides using peptide monomers (peptide polymers) BBA-APMA is dissolved at a concentration of 10 mg / ml in DMSO, and acrylamide is dissolved in a concentration of 100 mg / ml in water The peptide monomers were not purified after being synthesized and were dissolved in acetate buffer solutions containing DMSO as described above. Appropriate molar amounts of BBA-APMA monomer and acrylamide were then added to each reaction ampule. Each mixture is degassed by water aspiration for 15 minutes. Ammonium persulfate (10% material solution in water) and N, N, N ', N'-tetramethylethylenediamine (TEMED) is added (in the amounts indicated below) to catalyze the polymerizations. Each mixture is gasified again and incubated overnight at room temperature in a desiccator. Each resulting peptide copolymer is dialyzed against water (using 50,000 MWCO dialysis tubes from Spectrum Medical Industries, Houston, TX) at 4 ° C to remove unincorporated reagents and then lyophilized. The following table indicates the amount of each reagent that is added for each copolymerization. RGD F-9 C / H-V C / H-II HEP-III Peptide monomer (μmol) 53.4 40.4 8.6 38.0 6.4 BBA-APMA monomer (μmol) 21.4 16.2 3.44 15.2 2.56 Acrylamide (μmol) 873 986 168 986 176 % ammonium persulphate (pl) 130 93 40 130 33 TEMED (pl) 26 19 8 26 6. 6 The amounts recovered from each peptide polymer after lyophilization were 72 mg of RGD, 135 mg of F-9, 100 mg of C / H-II, 15.2 mg of C / HV, and 17.8 mg of HEP-III. F. Coupling of peptide polymers to biomaterials. Three biomaterials were used: polystyrene (PS), polyurethane (PU), and silicone rubber (SR). Breakaway 96-well plate size polystyrene strips (Immulon 1 Removawell Strips, from Dynatech Laboratories, Inc., Chantilly, VA) were used to determine the immobilized levels of each peptide polymer in radiolabelling experiments. Both PS 24 to 48 well culture plates (which were not sterile and untreated with plasma) were obtained from Corning Costar Corp. (Cambridge, MA) and used to conduct cell growth bioactivity assays. Flat PU sheets (Pellethane 55-D) and flat SR sheets each were obtained from Specialty Silicone Fabricators, Inc. (Paso Robles, CA) and punched to produce discs with diameters of 6, 10, and 15 mm diameters respectively fit into wells of 96, 48 and 24 well plates. The discs were used both in radiolabelling assays and in bioactivity assays. Two different protocols were used to apply the peptides (peptide polymers or peptide reagent controls) to biomaterials, with the main difference being that the peptides were dried or not on the materials before being illuminated to activate the reactive latent groups. With the dry immobilization protocol, the peptides were diluted in 50% (v / v) isopropanol (IPA) in water, added to biomaterials as indicated below and dried before lighting. With the wet immobilization protocol, the peptides were diluted in water, added to biomaterials as indicated below and not allowed to dry prior to illumination. With each immobilization protocol, the final aggregate concentration of the peptide portions was 50 μg / ml, and the following volumes were added per well of each type of culture plate: 50 μl / well of 96-well plates, 100 μl / well of 48-well plates, 200 μl / well of 24-well plates. As indicated above, PU and SR discs were placed on the bottoms of the plates for the coating and evaluation procedures. The samples were illuminated with a Dymax lamp (model No. PC-2, Dymax Corporation, Torrington, CT) containing a Heraeus bulb (W.C. Heraeus GmbH, Hanau, Federal Republic of Germany) to activate the photo-groups present in each polymer and produce biomaterial covalent binding. The duration of illumination was for 1 to 2 minutes at an intensity of 1-2 mW / cm2 in the wavelength range of 330-340 nm. Adsorption controls were also generated with peptide polymers and peptide reagent controls that were not illuminated. Following the immobilization or adsorption photo, the peptide polymers and peptide reagent controls respectively were extensively washed in an orbital shaker ("150-200 rpm") to remove peptides that did not bind tenaciously to the substrate.The washing steps include: 1) a washing overnight with three changes in phosphate buffered saline (PBS), pH 7.3, containing 1% Tween 20 detergent, 2) a 30 minute wash / sterilization step in 70% (vol / vol) of ethanol in water and 3) four washes in sterile PBS.

G. Quantification of immobilized levels of peptides in biomaterials. Two peptide polymers (polymer RGD and polymer F-9) and their respective peptide reagent controls were radiolabelled with tritium by reductive methylation and used to determine the level of each peptide that is immobilized on each biomaterial. Peptide reagent controls were not incorporated into polymers and consisted of RGD (sequence GRGDSPKKC) and F-9. The four tritium-labeled peptides were respectively referred to as polymer [3 H] -RGD, polymer [3 H] -F-9 reagent control [3 H] -RGD, and reagent control [3 H] -F-9. Each tritium tagged peptide was coated on each biomaterial (Breakaway PS strips, 6 nm PU disks, or 6 mm SR disks) using the dry immobilization protocol and the washing procedure described herein. After the washing procedure, Breakaway PS strips were broken into individual wells, placed in blinking vials (1 well / ampule), dissolved in THF and counted in Aquasol-2 Fluor (DuPont NENMR, Boston, MA) to determine dpm / sample. PU discs were swollen in THF and counted in Aquasol-2. SR disks were dissolved in soluene-350 tissue solubilizer and counted in Hionic Fluor (each of Packard Instrument Co., Meriden, CT). After the biomaterials were counted by liquid flash spectrometry, the final charge densities of each peptide (ng / cm2) were calculated from the known specific activities (dpm / ng) of each tritiated reagent. A summary of the load density results is given in the table below. Each value is the average of three or more determinations. Immobilized refers to illuminated samples. Adsorbed refers to unlighted samples. ND = not determined.

In all cases, the immobilized peptide polymers (ie, the polymer coatings that have been illuminated) exhibit the highest charge densities.

The loading densities of the peptide polymers were also biomaterial dependent, with the largest retained levels of peptide polymers being in PS and SR, and the lowest levels retained in PU. In each case, the retained levels of immobilized photo peptide polymers were in the order of 1.5 to 9 times higher than the adsorbed peptide polymers and in the order of 4.5 to 39 times higher than the adsorbed peptide reagent controls. H. Activity of cellular connection of immobilized peptides. Vertebral pulmonary artery endothelial cells (CPAE) were acquired and cultured as indicated by ATCC (American Type Culture Collection, Rockville, MD). The connection assays were performed in 48-well PS culture plates. When PU and SR were evaluated, discs of each material (with or without coatings) were placed in the bottoms of the culture plate wells. When evaluating PS, well funds were coated. For each assay, peptide-coated and uncoated biomaterials (PS, PU, and SR) were seeded at 50,000 cells per well in serum-free medium containing 2 mg / ml bovine serum albumin (V BSA fraction from Sigma Chemical Company , St. Louis, MO). The cells were allowed to connect to each biomaterial for two hours. Then, the unconnected cells were removed by aspiration, and the wells were rinsed twice with Hank's balanced salt solution (Celox Corp., Hopkins, MN). Finally, the cells were quantified by the addition of culture medium containing a metabolic dye, MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide] which is converted from a salt of Yellow tetrazolium in a purple formazan insoluble in the presence of viable cells. After incubation for two hours in the medium containing the MTT, the medium is removed and the formazan dye that has been deposited inside the viable cells is solubilized (with acidic isopropanol) and read on a spectrophotometer at 570 nm. The absorbance of formazan is directly proportional to the number of viable connected cells present per well. The following table summarizes the results of the cell connection assays, comparing immobilized peptide polymers and adsorbed peptide reagent controls. In each case, the relative numbers of cells that are bound to biomaterials coated with peptide (as determined by MTT dye) were divided by the number of cells that are connected to uncoated biomaterials (UC) to obtain a connection rating. of relative cells. Each value represents the average of 1 to 4 different experiments, with each experiment being conducted with 3 to 4 replicates. The peptides were immobilized on PS using the wet immobilization protocol described herein and the peptides were immobilized on SR and PU by the dry immobilization protocol. Stationary Polymer Peptide refers to polymer peptide removed. Adsorbed peptide reagent refers to the unenlightened peptide reagent. ND = not determined.

These results show that the photoimmobilized peptide polymers improved the cellular connection to all three biomaterials. The greatest improvements were observed with photoimmobilized RGD polymer in all three biomaterials and polymer F-9 photoimmobilized in PS (4.3 to 10 times of improvement) with fewer improvements observed with polymer F-9 in polymer PU and C / H-V in SR and PU (1.3 to 1.9 times improvement). In each case where the adsorbed peptide reagent controls were compared with photoimmobilized peptide polymers, the cellular connection was higher in the peptide polymers. I. Cell growth activity of immobilized peptides. Growth assays were performed on 24-well PS culture plates. When PU and SR were evaluated, discs of each material (with or without coatings) were placed in the bottoms of the culture plate wells. When PS is evaluated, the bottoms of the wells were coated. For each assay, peptide-coated and uncoated biomaterials (PS, PU, and SR) were seeded with CPAE cells at 1500 cells per well and allowed to proliferate in vi tro for 4 to 7 days. Then, the medium is aspirated and cell growth is quantified using MTT dye. The following table summarizes the results comparing cell growth in uncoated substrates, adsorbed peptides and peptide polymers. As with the connection assays, the relative numbers of cells growing in each peptide-coated biomaterial are divided by the number of cells growing in uncoated biomaterials (UC) to obtain a relative cell growth score. Each value represents the average of 1 to 4 different experiments, with each experiment performed with 3 to 4 replicates. The dry immobilization protocol described herein is used to immobilize all the peptides evaluated. Immobile Peptide Polymer, refers to illuminated peptide polymer. Adsorbed peptide reagent refers to unenlightened peptide reagent ND = not determined.

In addition to the peptides shown in the above table, C / H-II and HEP-III were also evaluated for growth in PS. With these two peptides, growth in adsorbed peptides was 1.0 and 1.1 times that observed in uncoated PS, and growth in peptide polymers was 10.5 and 13 times that observed in uncoated PS. These two peptides (polymers and reagent controls) were immobilized by the wet immobilization protocol. The results with C / H-II and HEP-III are the averages of 1 and 2 experiments, respectively, with each experiment performed with four replicates. These growth assays show that all five peptide polymers photoimmobilized in PS promoted growth that was 10.5 to 17.5 times higher than the growth in uncoated PS. Also, the three peptide polymer improved cell growth in PU by 3.9 to 9.4 times. Only slight improvements in cell growth were observed in SR. EXAMPLE 2 Polymer of Hirudin A. Synthesis of N-Succinimidyl-6- (4-benzoylbenzamido) hexanoate (BBA-EAC-NOS) BBA-Cl (30.00 g, 0.123 mol), prepared as described in Example 1, is dissolved in 450 ml of toluene. An amount (16.1 g, 0.123 mol) of 6-aminohexanoic acid (which will alternatively be referred to herein as s-aminocaproic acid, or as its abbreviated form EAC) is dissolved in 375 ml of 1 N NaOH and this solution is added to the solution of the acid chloride. The mixture is stirred vigorously to generate an emulsion for 45 minutes at room temperature. The product is then acidified with HL1 and extracted with 3 X 450 ml of ethyl acetate. The combined extracts were dried over magnesium sulfate, filtered and evaporated under reduced pressure. The 6- (4-benzoylbenzamido) exanoic acid was recrystallized from toluene: ethyl acetate to give 36.65 g of product, m.p. 106-109 ° C. The 6- (4-benzoylbenzamido) hexanoic acid (25 g, 73.7 mmol) is added to a dry flask and dissolved in 500 ml of dry 1,4-dioxane. NHS (15.5 g, 0.135 moles) is added and the flask is cooled in an ice bath under a dry N2 atmosphere. DCC (27.8 g, 0.135 mol) in 15 ml of 1,4-dioxane is then added and the mixture is stirred overnight. After filtration to remove 1, 3-dicyclohexylurea, the solvent is removed under reduced pressure and the product is recrystallized twice from ethanol to give 23.65 g of a white solid, m.p. from 121-123 ° C. B. Synthesis of a photoreactive polyacrylamide containing hirudin ligands (hirudin polymer) Methacryloyl-EAC-BBA is prepared by reacting 112 mg (0.629 mmol) of APMA-HCl, 358 mg (0.818 mmol) BBA-EAC-NOS and 80 mg ( 104 pl, 0.692 mmol) TEMED in 22 ml of DMSO. The mixture is stirred for 4.5 hours, then the NOS polymer is prepared by adding this mixture 4.20 gm (59.1 mmole) of acrylamide, 532 mg (3.15 mmole) of N-acryloxysuccinimide (Eastman Kodak, Rochester, NY) and 64 mg (0.39). mmol) of 2, 2'-azobisisobutyronitrile (AIBN). The mixture is bubbled with helium and incubated at 50 ° C overnight. A portion of the resulting polymer solution (10 ml) is diluted with 10 ml of DMSO and slowly added to vigorously stirred acetone (200 ml) to precipitate the polymer. The polymer is collected, washed with acetone to remove impurities and dried under vacuum. A total of 1.47 g is recovered. Recombinant hirudin with a purity greater than 90% and an activity of 16,500 ± 12,000 ATU / mg is obtained from Transgene Laboratories (Strasbourg, France). (See below for definition of ATU.) Hirudin is an antithrombotic agent that acts by binding and inhibiting the proteolytic activity of thrombin. Hirudin (10.5 mg, 1.52 pmole) is dissolved in 1 ml of 0.1 M carbonate buffer. The NOS polymer (prepared as described above) is dissolved in DMSO at 4 mg / ml. Then 2 mg of the NOS polymer is added to the hirudin solution and mixed overnight. Half of the reaction mixture is dialyzed in a Spectra / Por 50,000 MWCO line against water followed by lyophilization. A total of 5.9 mg of hirudin polymer is recovered containing 5.0 mg of hirudin and 0.2 mole of BBA per mole of hirudin. Hirudin quantifies with a BCA protein assay kit (from Pierce Chemical Company, Rockford, IL) and the BBA content is determined spectrophotometrically. A control polymer ("ethanolamine polymer") is prepared by adding ethanol amine instead of hirudin to the NOS polymer. The resulting amine ethanol polymer is not charged and is used as a control in experiments comparing thrombin binding to a similar polymer containing amine ethanol instead of hirudin. C. Assay for hirudin and hirudin polymer activities in solution. The specific activities of hirudin and hirudin polymer were determined by standard protocols that are provided by Transgene and are expressed as antithrombin units (ATU) per mg of hirudin. An ATU is the amount of hirudin that is required to inhibit the proteolytic activity of an NIH unit of thrombin. For these assays, a known amount of bovine thrombin (175-350 NIH units / mg., From Sigma Chemical Co., St. Louis, MO) is preincubated with a series of dilutions of hirudin or hirudin polymer and thrombin activity. The remaining one is determined with a chromogenic substrate, Chromozym TH (from Boehringer Mannheim Corp, Indianapolis, IN). The specific activities of hirudin assayed before and after incorporation into the hirudin polymer were 11, 7, 10 and 10,204 ATU / mg, respectively. Therefore, the incorporation of hirudin in the product produced only a 13% decrease in this activity. D. Coupling of hirudin polymer and ethanolamine polymer to biomaterials. Flat sheets of three biomaterials were employed: 1) polyethylene (PE, primary reference materials from the National Institutes of Health, Bethesda, MD), 2) SR (SILASTICMR medical grade from Dow Corning Corporation, Midland, MI), and 3) PU (TecoflexMR from ThermoCardiosystems, Wobum, MA). Samples of each biomaterial were cut either in disks with diameter of 6 mm or squares of l x l cm. To remove surface contaminants before coating, the PU and PE samples were washed by brief immersion in IPA and SR was extracted 1 hour with hexane and dried overnight. In addition, the SR samples were treated with an argon plasma (3 min., 250 Watts, 250 mtorr) just before application of the hirudin polymer or ethanol amine polymer. The hirudin polymer is diluted to 1-25 pg / ml in 75:25 (v / v) water: IPA and added to one side of each sample of biomaterial that is washed or extracted as described above. The aggregated hirudin polymer solutions were allowed to dry and then illuminated with a Dymax lamp. To remove loosely adhering hirudin polymer remaining after the coating procedures, each sample is washed three times for 15 minutes and then overnight in a 1% solution of Tween 20 in PBS. Then, the Tween 20 is removed by rinsing the samples in deionized water. Three types of control samples were also prepared: 1) uncoated controls to which no hirudin polymer was added, 2) samples to which the hirudin polymer was added but not illuminated and 3) SR samples that were coated with the ethanolamine polymer. For further control, the ethanolamine polymer (prepared as described herein) is diluted in deionized water to 1 or 5 μg / 200 μl. Then, 200 μl aliquots of each material solution were added to one side of 1 cm 2 samples of SR, allowed to dry, photoactivated and washed in Tween 20 / PBS and deionized water as described herein for the hirudin polymer. E. Quantification of hirudin load in biomaterials. Hirudin is radiolabelled by reductive methylation, incorporated into a hirudin polymer as described herein, and used to quantify the amount of hirudin polymer that is immobilized in each of three biomaterials as described herein. To count the retained tritium, the samples are dissolved in THF, diluted in Aquasol, and counted in a Packard 1900CA liquid flash counter. The results presented in the table below show that the amount of retained hirudin polymer was proportional to the aggregate amount and the amount retained after photoactivation is 2.3 to 6.6 times that retained without photoactivation. Each result is the average of four determinations. N.A. = not tested.

F. Activity test of polymer coatings of hirudin in biomaterials. Polymer of hirudin that has not been labeled with tritium is coated in each biomaterial as described herein. The activity of the immobilized hirudin polymer is then assayed by quantifying the binding of thrombin labeled with added tritium (3H-Thr). 3H-Thr is prepared by labeling human thrombin (4000 NIH units / mg protein from Sigma Chemical Co.) by reductive methylation. Each coated sample is incubated in a 2 μg / ml solution of 3H-Thr in a Tris buffer (0.05 M Tris-HCl, 0.1 M NaCl, 0.1% PEG 3350, pH 8.5) for one hour and rinse with the same buffer that does not contain thrombin to remove unbound thrombin. The samples were then dissolved in THF, diluted in Aquasol and counted. The results presented in the table below show that the amount of thrombin that is retained by biomaterials coated with hirudin was proportional to the amount of immobilized hirudin. Each result is the average of four determinations. SR + EP1 and SR + EP5 indicate SR samples that were coated with the ethanolamine polymer added 1.0 and 5 μg / cm2, respectively. N.A. = not tested. Control experiments were performed where thrombin was added to uncoated biomaterials, and the amounts of thrombin bound to PE, PU, and uncoated SR were 0.01, 0.012, and 0.006 μg / cm2, respectively. Comparisons of thrombin bound to PE and uncoated PU against the same biomaterials coated with 25 μg / cm2 of hirudin polymer show that the latter promotes 200 to 23 times higher thrombin binding, respectively. Finally, results with SR coated with the ethanolamine polymer show that the hirudin moiety is essential for binding thrombin.

EXAMPLE 3 Heparin Polymer A. Synthesis of N-Succinimidyl &Maleimidohexanoate. 6-Maleimidohexanoic acid of 20.0 g (94.7 mmol) is dissolved in 100 ral of chloroform followed by the addition of 60.1 g (0.473 mol) of oxalyl chloride. The resulting solution is then stirred for two hours at room temperature. The excess oxalyl chloride is removed under reduced pressure and the resulting acid chloride is azeotroped with 4 x 25 ml of chloroform to remove excess oxalyl chloride. The acid chloride product is dissolved in 100 ml of chloroform followed by addition of 12.0 g (0.104 mmol) of NHS and a slow addition of 11.48 g (0.113 mol) of TEA. The mixture is stirred at room temperature overnight. After washing the reaction mixture with 4 x 100 ml of water, the chloroform solution is dried over sodium sulfate. Solvent removal gave 24.0 g of product for an 82% yield. Analysis in an NMR spectrometer was consistent with the desired product and was used without further purification.

B. Synthesis of a photoreactive polyacrylamide containing hydrazide ligands (hydrazide polymer). Acrylamide, 8.339 g (0.117 mol), dissolves in 112 ml of THF, followed by 0.241 g (1.50 mmol) of AIBN, 0.112 ml (0.74 mmol) of TEMED, 1284 g (3.70 mmol) of BBA-APMA (prepared as described herein) and 0.377 g (1.2 mmol) of N-succinimidyl-6-maleimidohexanoate (prepared as described herein). The solution is deoxygenated with a bubbling of helium for 4 minutes, followed by bubbling of argon for 4 minutes. The sealed container is then heated overnight at 55 ° C to complete the polymerization. The precipitated polymer is isolated by filtration and washed with stirring for 30 minutes with 100 ral of THF. The final product is recovered by filtration and dried in a vacuum oven to provide 9.64 g of a solid, a yield of 96%. The above polymer (1.0 g) is dissolved in 50 ml of 0.05 M phosphate buffer at pH 8 and this solution is added to a second solution containing 0.696 g (5.89 mmol) of oxalic dihydrazide in 50 ml of 0.05 M phosphate buffer. pH 8. The combined solutions were stirred overnight at room temperature. The product is subjected to dialysis against deionized water using dialysis tubing with molecular weight cut-off from 6,000 to 8,000. After 6 changes of water for two days, the polymer is isolated by lyophilization to give 850 mg of product. An analysis for hydrazide groups in this polymer gave a value of 0.0656 mmol of NH, / g of polymer, 55% theory.

C. Synthesis of a photoreactive polyacrylamide containing heparin ligands (heparin polymer). It is known that a controlled periodate oxidation of the uronic acid residues present in heparin will generate aldehyde functional groups, while retaining reasonable heparin activity. Heparin without leaching with an activity of 152 units / mg (from Celsus Laboratories, Cincinnati, OH) is dissolved at 250 mg / ml in 0.1 M acetate buffer (pH 5.5) and oxidized with sodium periodate at 20 mg / ml for 30 minutes to generate free aldehyde groups. The remaining periodate is inactivated by the addition of excess ethylene glycol. Then, the ethylene glycol and small molecular weight reaction products were removed by dialyzing overnight at 4 ° against 0.1 M acetate buffer (pH 5.5) using 6,000 Spectra / Por molecular weight cut dialysis tubing (from Spectrum Medical Industries ). The oxidized heparin retained 95 units / mg of activity. The concentration of oxidized heparin is adjusted to 15 mg / ml in 0.1 M acetate buffer (pH 5.5) and s reacted overnight with an equal volume of 20 mg / ml photopolyhydrazide in water at room temperature. The heparin polymer is used to coat biomaterials without further purification.

D. Immobilization of heparin polymer in hydrogenated cellulose membrane. The heparin polymer is synthesized as described herein and immobilized on regenerated cellulose (RC) membranes having a pore size of 0.45 mm. RC membrane with diameter of 2.54 cm (1") were incubated with heparin polymer for 15 minutes, they were air dried and then illuminated for 45 seconds on each side. The discs were first washed in lOx of PBS and then in PBS to remove unbound heparin polymer. E. Evaluation of thrombin inhibition by membranes coated with heparin. The antithrombotic activity of heparin is due to its inhibition of thrombin, which is a protease that is known to participate in the coagulation cascade. Heparin inhibits thrombin activity by first binding antithrombin III (ATIII). Then, the eparin / ATIII complex binds to and inactivates thrombin, after which the heparin is released and can be ligated to another ATIII. The assay for inhibition of thrombin by immobilized heparin is conducted by measuring the breakdown of a chromogenic peptide substrate by thrombin and using previously described methods. Each assay is conducted in 1 ml of PBS containing 0.85 mg of BSA (Sigma Chemical Co.), 10 mU human thrombin (Sigma Chemical Co.), 100 mU / ml of ATI11 (Baxter Biotech, Chicago, IL), and 0.17 μmol of chromogenic thrombin substrate S-2238 (Kabi Pharmacia, Franklin, OH). To this test solution is added either heparin coated or uncoated membranes (to assess heparin activity and membranes) or standard concentrations of heparin (to generate standard curves of heparin contents against absorbance). The amounts of heparin that were added were in the range of 2.5 to 25 mU. The color generated, measured as absorbance at 405 nm, by thrombin-mediated cleavage of S-2238 is read using a spectrophotometer after two hours of incubation at 37 ° C. Absorbance is directly related to thrombin activity and thus inversely related to the amount of ATIII activation induced by heparin in solution or immobilized on the surface of the substrate. The activity of surface-bound heparin is calculated by comparing the absorbance values generated with the absorbance values generated with known amounts of aggregated heparin. This assay is then used to evaluate the activity of heparin present in the coated and uncoated RC membranes. The coated membrane had heparin activity of 255 mU / cm2, while the uncoated membrane had <0.1 mU / cm2. Example 4 Lysine Polymers A. Synthesis of N-a- [6- (maleimido) exanoyl] lysine. 6-Maleimidohexanoic acid, 2.24 g (10.6 mmol) (prepared as described in Example 1) is dissolved in 10.76 g (84.8 mmol) of oxalyl chloride and stirred as a net solution for 4 hours at room temperature. The excess oxalyl chloride is then removed under reduced pressure and the resulting acid chloride is dissolved in 25 ml methylene chloride. This solution is added with stirring to a solution of 3.60 g (10.6 mmoles) of t-butyl ester hydrochloride of N-e-t-BOC lysine (Bachem California) in 25 ml of methylene chloride and 3.21 g (3 1.7 mmoles) of TEA. The resulting mixture is stirred overnight under nitrogen. After this time, the mixture is treated with water and the organic layer is separated and dried over sodium sulfate. The solvent is removed and the product is purified on a flash chromatography column on silica gel using a solvent gradient of 0 to 5% methanol in chloroform. The collection of desired fractions and evaporation of solvent gave 5.20 g of product (98% yield). Analysis in an NMR spectrometer was consistent with the desired product. The amino acid protected derivative, 0.566 g (1.14 mol) is dissolved in 5 ml of trifluoroacetic acid with stirring. After stirring for four hours at room temperature, the solvent is removed under reduced pressure. The resulting oil is triturated with ether to remove residual trifluoroacetic acid, giving 373 mg of product for a yield of 98%. 7 Analysis on a NMR spectrometer was consistent with the desired product. B. Synthesis of a photoreactive polyacrylamide containing e-amino lysine ligands (polymer lysine) Acrylamide (0.22 g, 3.10 mmol) BBA-APMA (0.014 g, 0.039 mmol), and N-30 a- [6- (maleimido) hexanoyl ] lysine (0.266 g, 0.784 mmol, prepared as described herein) is dissolved in 7.3 ml of dry DMSO. To initiate the polymerization, 8 mg (0.047 mmol) of AIBN and 4.0 μl of TEMED were added, followed by bubbling with nitrogen to remove all oxygen. The mixture is then heated at 55 ° C for 16 hours followed by evaporation of the DMSO under reduced pressure. The product is dissolved in DI water and dialyzed for three days using 6-8K molecular weight cutting line (MWCO) against DI water. The resulting solution is lyophilized to give 160 mg of product. C. Generation of lysine polymer coatings in polyurethane (PU). PU leaves are cut into 1 x 1 cm pieces, washed with IPA and air dried. To improve the wetting of the lysine polymer solution in PU, the PU pieces were treated with argon plasma at 250 Watts, 0.25 torr, for 1 minute. The PU pieces are then immersed in an aqueous solution of lysine polymer (prepared as described herein, 1 μg / ml) for 5 minutes, air-dried and illuminated for 30 seconds. The samples were then washed overnight (in three changes of phosphate buffered saline, pH 7.4, containing 1% Tween 20) to remove unbound lysine polymer. The coated PU pieces are stored in PBS containing 0.02% sodium azide until evaluated. D. Quantification of lysine polymer coatings The lysine polymer (prepared as described above) was radiolabelled by reductive methylation and used to quantify the levels immobilized in polyurethane. The tritiated lysine polymer is coated in PU pieces with or without illumination to determine the density of lysine polymer that was immobilized. After the washing procedure, samples were dissolved in Soluene-350 and counted in Hionic flour (each from Packard Instrument Co., Meriden, CT). The table below shows the immobilized levels (± SEM) expressed in terms of μg / cm2 and nmol / cm2 of lysine portion. Each level is the average of four replicates. The result shows that 1.51 μg / cm2 is immobilized after illumination that is more than sufficient to produce a monolayer coating and is 3.8 times as much polymer as retained with the adsorbed control. Treatment Polymer level Lysine lysine serving level (μg / cm2) (nraole / cra2) Adsorbed 0.40 + 0.03 0.359 + 0.027 Illuminated 1.51 + 0.23 1.36 + 0.21 E. Evaluation of plasminogen binding by lysine coated polyurethane. Others have described the covalent coupling of lysine in silane-derivatized crystal and the resulting derivatized lysine crystal is reported to promote plasminogen binding, with bound lysine exhibiting significant proteolytic activity. This surface is expected to demonstrate improved resistance to thrombus formation when placed in contact with blood. The coating chemistry used in the previous study uses a short spacer and is limited to glass as a surface, while the photoreactive lysine polymer can be applied at high densities with a wide range of biomaterials. The lysine portion in the lysine polymer is coupled via the a-amino group with the polymer backbone, and the e-amino group is free to bind plasminogen. Therefore PU that is coated with the lysine polymer is expected to inhibit thrombus formation by reversibly binding plasminogen to blood, with bound plasmin demonstrating protolitic activity that cleaves fibrin and prevents fibrin clot formation on the coated surface . Example 5 Prostaglandin Polymers A. Synthesis of a photoreactive polyacrylamide containing primary amine ligand (amine polymer). A solution of acrylamide (7.46 g, 105.1 mmol), APMA-HC1 (2.14 g, 11.9 mmol), and BBA-APMA (0.837 g, 2.39 mmol) is prepared in 170 mL of DMSO. To this solution is added AIBN (0.246 g, 1.50 mmole) TEMED (0.131 g, 1.13 mmole). The solution is then deoxygenated by bubbling helium gas for a period of 10 minutes and sealed and placed in an oven at 55 ° C for 18 hours to complete the polymerization. The polymer solution is diluted with water and dialyzed against deionized water using 12,000-14,000 MWCO dialysis tubing to remove the solvent, unreacted monomer and low molecular weight oligomers. The final product is isolated by lyophilization, and the photo-group charge is determined by UV absorbance at 265 nm. The amine content of the polymer is determined using a trinitrobenzensulfonate (TNBS) method.

The photo and amine load was changed by adjusting the amount of monomers used in the polymerization. B. Synthesis of a photoreactive polyacrylamide containing prostaglandin Ex ligands (Ex prostaglandin polymer). A solution of prostaglandin E1 (Sigma Chemical Co.) (30 mg, 0.0846 mmol) in 5 ml of dry 1,4-dioxane is prepared and NHS (10.7 mg, 0.093 1 mmol) and DCC (26.2 mg, 0.127 mraole) are prepared. add to the solution. The mixture is stirred overnight at room temperature with formation of the 1,3-dicyclohexylurea (DCU) byproduct. The solid is removed by filtration and the filter cake is rinsed with 1,4-dioxane. The solvent is removed under reduced pressure, and the resulting product is stored under dry conditions and used without further purification. The amine polymer (synthesized as described above) is dissolved in DMSO at a concentration of 10 mg / ml, followed by the addition of 1.5 equivalents of the NOS derived prostaglandin derived from the amine content of the amine polymer solution . Five equivalents of triethylamine are added to help catalyze the reaction. After overnight reaction, the polymer solution is placed in dialysis against deionized water using 12,000-14,000 MWCO dialysis tubing to remove excess low molecular weight reagent. The product is isolated by lyophilization. C. Synthesis of a photoreactive polyacrylamide containing carbacycline ligands (carbacycline polymers). A solution of carbacycline (Sigma Chemical Co.) (5 mg, 0.0143 mmol) in 2 ml of dry 1,4-dioxane is prepared, and NHS (1.8 mg, 0.0157 mmol) and DCC (4.4 mg, 0.0215 mmol) are added to the solution. The mixture is stirred overnight at room temperature with formation of the DCU by-product. The solid is removed by filtration and the filter cake is rinsed with 1,4-dioxane. The solvent is removed under reduced pressure and the resulting product is stored under dry conditions and used without further purification. The amine polymer (synthesized as described above) is dissolved in DMSO at a concentration of 10 mg / ml, followed by the addition of 1.5 equivalents of the derivatized NOS carbacycline relative to the amine content of the amine polymer solution. Five equivalents of TEA are added to help catalyze the reaction. After overnight reaction, the polymer solution is placed in dialysis against deionized water, using 12,000-14,000 MWCO dialysis tubing to remove excess low molecular weight reagents. The product is inflated by lyophilization. D. Prostaglandin polymer coatings.

Each prostaglandin polymer (synthesized as described above) is diluted to 5 mg / ml in 50% (v / v) IPA in water and added to samples of biomaterials (polyurethane, silicone rubber and polyethylene). The volume of polymer solution containing prostaglandin that is added to each polymer is barely sufficient to cover the surface of each biomaterial (approximately 100 ml / cm 2). The polymer solution is allowed to dry over each sample, after which each sample is illuminated for 1-2 minutes. Both prostaglandin Ex and carbacycline (which is a stable analogue of prostaglandin 1; PGI) are known to inhibit platelet activation and thrombus formation. Therefore, the generated protaglandin coatings are expected to inhibit platelet activation and thrombus formation in biomaterials. Example 6 Protein Polymer A A. Synthesis of N-Succinimidyl-6-methacrylamidohexanoate (MAm-EAC-NOS) E-aminoaminocaproic acid (EAC), 2.00 g (15.25 mmol), is added to a dry round bottom flask, followed by the addition of 2.58 g (16.73 mmol) of methacrylic anhydride. The resulting mixture is stirred at room temperature for two hours, followed by trituration with hexane. The hexane is decanted and the product is triturated an additional two times to give 3.03 g of the acylated product (yield >).99%). Without further purification, the product is dissolved in 50 ml of chloroform, followed by the addition of 1922 g (16.7 mmol) of NHS and 6.26 g (30.3 mmol) of DCC. The mixture is stirred overnight at room temperature with moisture protection. The resulting solid is removed by filtration and the filter cake is rinsed with chloroform. The solvent is removed under reduced pressure with 5 ppm of the hydroquinone monomethyl ether (MEHQ) to avoid polymerization. The residue, 4.50 g, is redissolved in 45 ml of dry THF and the solution is used without further purification. B. Synthesis of a photoreactive polyacrylamide containing protein A ligands (polymer protein A). To prepare the latent reactive NOS polymer, acrylamide (1.0 gm, 14.1 mmol) is dissolved in 15 ml of THF dry. To that solution is added 44 mg (0.149 mmol) of MAm-EAC-NOS (synthesized as described here) and 158 mg (0.45 mmoles) of BBA-APMA (synthesized as described in example 1). For the initiator, 500 mg (3.04 mmoles) of AIBN are added, followed by the addition of 50 pl of TEMED. The solution is bubbled with nitrogen and incubated at 550 ° C for 18 hours to allow polymerization. The insoluble polymer is collected by filtration, and then dissolved in dry DMSO. The polymer is precipitated by being added dropwise to stirred ethanol and then collected by filtration and dried to store until used. The product yield was 0.906 μm. To couple protein A with the latent reactive NOS polymer, recombinant Staphylococcus protein A (from Calbiochem-Novabiochem Corp., San Diego, CA) is dissolved in 10 mg / ml in 0.1 M carbonate buffer, pH 9.

The latent reactive NOS polymer is dissolved at 100 mg / ml in 50 mM phosphate buffer, pH 6.8. Then 200 pl (20 mg) of the NOS polymer is added to 1 ml (10 mg) of the protein A solution and the mixture is incubated overnight at 4 ° C. Evaluation by gel electrophoresis of sodium dodecyl sulfate polyacrylamide (SDS-PAGE) revealed that more than 80% of the aggregated protein A is incorporated into the resulting protein A polymer. The protein A polymer solution is used to coat biomaterials without further purification. C. Generation of protein A polymer coatings in biomaterials. The protein A polymer was then photo-coupled to two types of membranes, polysulfone (PSO) membranes with a pore size of 0.2 mm (HT-200 GeIman membranes).

Sciences, Ann Arbor, MI) and regenerated cellulose membranes (RC) with a pore size of 0.45 mm (No. SM1 8606, Sartorius, Edgewood, NY). Each membrane was in the form of a disk having a diameter of 2.54 cm (1"). Before the addition of protein A polymer, each membrane was first washed in 1: 1 (v / v) isopropanol: 0.1 N HCl and then in water The protein A polymer (estimated concentration of 8.67 mg / ml) is then added to each type of membrane (1 ml of polymer for 4-6 disks) and incubated overnight at 4 ° C. , the discs are removed from the polymer solution, air dried and illuminated for 1 minute on each side in a temperature controlled chamber (at 10 ° C) .The illumination is produced with a Dymax lamp as described here. They were washed to remove unbound protein A polymer.The washing is accomplished by placing the coated discs in membrane supports, (MAC-25 support from Amicon, Beverly, MA), with 2-4 membranes placed on each support. then they were sequentially washed with: 1) 10 ml of 0.1 M glycine in 2% acetic acid, 2) 10 ml of lOx PB S, and 3) 30 ml of PBS. The washed membranes were then stored in PBS containing 0.05% sodium azide until used. D. Activity evaluation of protein polymer coatings in biomaterials. Protein A is a bacterial protein that binds specifically to the Fc region of immunoglobin G (IgG) molecules. The activity of protein A coating on each type of membrane is calculated by assaying for binding by aggregated IgG. Uncoated membranes were used as controls. For this assay, 2 ml of rabbit serum is diluted 1: 5 in PBS and spread through the coated membranes at 2-3 ml / min. The membranes were then washed with PBS to remove unbound IgG. The bound IgG is then eluted with 0.1 M glycine in 2% acetic acid. The amount of eluted IgG is determined by measuring the absorbance of the eluent at 280 nm and using an extinction coefficient (e2β) of 1.4 ml / cm-mg to calculate the mg of eluted IgG. Also the IgG eluting from each type of membrane is evaluated by purity by SDS PAGE analysis. With the biomaterials coated with protein A polymer, the eluted protein was greater than 90% light and heavy IgG chains. In contrast, the main protein eluting from uncoated controls was albumin. Three discs of each type (PSO or RC) were placed on a MAC-25 support and evaluated using this procedure. The table below shows the average amounts of IgG that are eluted from each type of membrane; with each value that is the average of 10 determinations (10 cycles) for three PSO disks and the average of three determinations (3 cycles) for 3 RC disks. Type of membrane IgG coating eluted) Times higher IgG in (mg / disc) Protein coating A Polysulfone uncoated control 0.020 Polysulfone Protein polymer A 0.680 34 Controlled regenerated cellulose uncoated control 0.006 Regenerated cellulose Protein polymer A 0.383 64 These results show that each type of control membrane binds 34 to 64 times more IgG than its respective uncoated control. Also, protein A in the polymer has a stable conformation and binds tenaciously, since there is no decrease in IgG elution after 10 cycles of serum addition and IgG elution. Example 7 IgG Polymers A. Synthesis of a photoreactive polyacrylamide containing N-Oxisuccinimide ligands (NOS Polymer). Acrylamide, 3.897 g (0.0548 mol), dissolves in 53 ml of THF, followed by 0.115 g (0.70 mmol) of AIBN, 0. 053 ml of TEMED, 0.204 g (0.58 mmol) of BBA-20 APMA (prepared as described in Example 1) and 0.899 g (2.9 mmol) of N-succinimidyl-6-maleimidohexanoate (prepared as described in Example 3). The solution is deoxygenated with a bubbling of helium for 4 minutes, followed by a bubbling of argon for 4 minutes. The sealed container is then heated overnight at 55 ° C to complete the polymerization. The precipitated polymer is isolated by filtration and washed with stirring for 30 minutes with 100 ml of THF. The final product is recovered by filtration and dried in a vacuum oven to provide 4.688 g of solid, a yield of 94%. B. Synthesis and immobilization of a photoreactive polyacrylamide containing IgG ligands. IgG molecules are the class of antibody molecules that bind specific antigens. Tritiated rabbit IgG antiglucose is used in such a way that the tritium label can be used to quantify the immobilized level of IgG and glucose oxidase binding can be evaluated to test the IgG activity. Polymer NOS (50 mg) (prepared as described here) is added to 100 mg of [3 H] IgG (in 100 ml of 0.1 M sodium carbonate at pH 9) and allowed to react overnight at 4 ° C. The IgG polymer is used without further purification. Polyester membrane (Accuwick from Pall Corporation) is cut into 6 mm discs, and 4 μl aliquots of the IgG polymer are stained on each of 19 discs. Three discs were left as controls that were not illuminated or washed. Eight discs lit up for a minute and eight discs were left unlit. The last eight illuminated discs and eight unlighted discs were washed with 25 mM bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane (BIS-TRIS) at pH 7.2 containing 1% lactose, 1% BSA and Brij 35 at 0.1% . To quantify the immobilized levels of IgG polymer, the three control disks (uncoated, unwashed) and three of each of the illuminated and unlit conditions were dissolved in Soluene (0.5 ml) and counted in 5 ml of Hionic flour. . The results are reported in the table below. A comparison of the illuminated (washed) with the control (uncoated, unwashed) shows that 75% of the added IgG polymer is retained after illumination. In contrast to the unenlightened samples, only 12.5% of the added IgG polymer is retained.

To quantify the activity of the immobilized IgG, the remaining five illuminated and unenlightened discs were incubated with glucose oxidase at 0.1 mg / ml in PBS for 1 hour and washed 5 times with TNT (0.05 M Tris (hydroxymethyl) aminomethane, 0.15 M NaCl , 0.05% Tween-20). Each disk was then transferred to wells in a 96-well microtiter plate and assayed by adding 200 μl of 3, 3 ', 5, 5' -tetramethylbenzidine (TMB) chromogen mixture (100 μl of Kirkegaard & Perry Laboratories, Inc., 100 μl 0.2 M 0.2 M sodium phosphate, pH 5.5, 10 mg of glucose and 4 μg of horseradish peroxidase) and let the color reveal for 20 minutes. Aliquots (100 μl) were then transferred to a separate microtiter plate and the absorbance was read at 655 nm. A comparison of the illuminated and non-illuminated samples illustrates that 61% higher activity was expressed by the illuminated samples. Example 8 Streptavidin Polymer A. Synthesis of photoreactive polyacrylamide containing streptavidin ligands. Streptavidin (from InPerGene Company, Benicia, CA) is coupled to the NOS polymer (prepared as described in example 6). Streptavidin (15 mg) is dissolved in 1.5 ml of 0.1 M carbonate buffer (pH 9.0). The NOS polymer is prepared and dissolved in 5 mM acetate buffer (pH 5) at a final concentration of 100 mg / ml. The NOS polymer solution (0.3 ml) is added to the solution of setreptavidin (1.5 ml), and the mixture is stirred overnight at 4 ° C. The resulting streptavidin polymer is used without further purification or characterization. B. Generation of Streptavidin polymer on surfaces. Solid glass rods (diameter 3 mm x length 3 cm) were washed by sonication in 1: 1 (v / v) acetone in O.lN HCl for 30 minutes, rinsed in water, acetone, dried at 100 ° C for 1 hour cooled and stored dried up in class. Bis (trimethoxysilylethyl) benzene (from United Chemical Technologies, Inc., Bristol, PA) is diluted to 10% (v / v) in acetone. The rods were immersed in the silane reagent for 30 seconds, air dried, immersed in water for 30 seconds, removed and cured at 100 ° C for 15 minutes, and rinsed with acetone. The organosilane primed glass rods were immersed in a solution of streptavidin polymer for 30 seconds. The glass rods were removed from the solution, allowed to air dry and illuminated for 30 seconds with a Dymax lamp. Adsorption controls were prepared by the same protocol, except that they were not illuminated. Both types of coated rods were washed with PBS containing 0.05% Tween 20 to remove non-adherent streptavidin polymer. C. Evaluation of immobilized streptavidin polymer. Streptavidin is a receptor that binds strongly with biotin as its ligand. The streptavidin polymer coating activity is assessed by quantifying the binding of horseradish peroxidase derivatized with added biotin (biotin-HRP, which is obtained from Pierce Chemical Company, Rockford, IL). The glass rods were incubated for one hour in a 9 μg / ml solution of biotin-HRP. The binding of non-derivatized HRP (added at 9 μg / ml) is evaluated as a control for non-specific binding of HRP with the glass rods. The rods were then washed with PBS containing 0.05% Tween 20 to remove unbound HRP, and the relative activity of bound HRP is evaluated with a TMB peroxidase substrate system (Kirkegaard and Perry Laboratory, Inc., Gaithersburg, MD).

HRP catalyzes the oxidation of TMB and produces a color that is quantified spectrophotometrically at 405 nm. Each result is the average of 3 determinations.

The results show the expected trends, with the highest peroxidase activity observed in rods that were coated with photoimmobilized streptavidin polymer (covalent streptavidin polymer) and to which biotin-HRP has been added. The streptavidin polymer adsorbed (not illuminated) produces 3.5 times less peroxidase activity, and the remaining variants lacking streptavidin and / or biotin exhibit little peroxidase activity. Example 9 Biotin polymer The photoreactive amine polymer (80 mg) (synthesized as described in example 5) is dissolved in 2 ml of DMSO. To the polymer solution are added 40 mg of 3-sulfo-N-hydroxysuccinimide ester of biotinamidocaproic acid (Sigma Chemical Co.) and 0.05 ml. triethylamine. The solution is mixed for two hours at room temperature, then dialyzed against ionized water to remove any biotin that does not bind to the polymer. A solution of the biotin polymer (1.0 μg / ml in deionized water) is applied to wells of a polystyrene microtiter plate and incubated for one hour, after which the plate is illuminated for 1 or 2 minutes. The plate is then washed with deionized water to remove unbound biotin polymer. Biotin is a ligand that binds to streptavidin as its receptor. Polystyrene micro-crushing plates are coated with biotin polymer and evaluated by activity when assaying streptavidin binding. The streptavidin solution is added to the plates that are coated with biotin polymer and unbound streptavidin is removed to be washed with deionized water. The retained streptavidin is quantified by adding biotin-HRP and evaluating HRP activity. Example 10 Magainin Polymer A. Synthesis of Magainin peptide monomer. Magainin-2 is used in this example and was synthesized in the measure for BSI by Bachem, Inc. (Torrance, CA) with a cysteine added to the carboxyl terminus of the peptide. The resulting sequence of magainipa consists of GIGKFLHSAKKFGKAFVGEIMNSC. As described in Example 1, the underlined C (C) tes a non-native amino acid that is added to allow coupling by the sulfhydryl group. Magainin (2.36 μmol) is dissolved in 0.5 ml degassed water. To this solution is added 2.36 μmoles of Mal-MAm (dissolved in 20 μl chloroform) and 0.5 ml of ethanol. The reaction is stirred for 90 minutes at room temperature after which the solution is dried under nitrogen and resuspended in 1 ml of water. The recovered magainin monomer solution is determined to have 5.5 mg / ml magainin portion, as determined by the MicroBCA assay (equipment from Pierce Chemical Company, Rockford, IL). B. Synthesis of photoreactive polyacrylamide containing magainin ligand (magainin polymers) BBA-APMA is dissolved at a concentration of 10 mg / ml in DMSO, and acrylamide is dissolved at a concentration of 100 mg / ml in water. Magainin monomer (0.48 μmol in 220 μl of water) is not purified after being synthesized (as described above) . The appropriate molar amounts of BBA-APMA (0.25 pmol in 44 μl of THF) and acrylamide (6.9 μmol in 120 μl of water) were then added to the reaction ampule. 300 μl of additional THF are added, and the mixture is degassed by aspiration of water for 15 minutes. Ammonium persulfate (6.8 μl of 10% material solution in water) and TEMED (1.5 μl) were added to catalyze the polymerization. The mixture was gassed again and incubated overnight at room temperature in a sealed desiccator. The resulting magainin polymer is dialyzed against water (using Spectra dialysis tubing / Spectrum 50,000 MWCO, Houston, TX) at 4 ° C to remove unincorporated reagents and then lyophilize. Of the 1.2 mg of magainin peptide that is used to synthesize methacryloyl magainin, 0.35 mg is present in the solubilized magainin polymer. C. Evaluation of Immobilized Magainin Polymer. Magainin is an antibiotic cationic peptide that is originally from the skin of Xenopus Laevis. It is activated against a broad spectrum of pathogens and acts as on the surface of pathogens. The activity of the magainin polymer is evaluated by a standard solution test, which determines the minimum inhibitory concentration (MIC) of the magainin polymer, which is required to prevent the growth of bacteria. The MIC of magainin polymer was 50 μg / ml for both Escherichia coli (ATCC No. 25922) and Staphylococcus epidermidis (ATCC No. 12228), while native monomeric magainin (not incorporated in either a magainin monomer or the polymer of magainin) had a MIC of 6.25-12.5 μg / ml for E. coli and 25 μg / ml for S. epidermidis. The magainin polymer is diluted to 250 μg / ml in 50% (v / v) IPA in water and added to biomaterial samples (PU, SR and PE). The volume of magainin polymer solution that is added to each polymer is just enough to cover the surface of each biomaterial (approximately 100 μl / cm2). The polymer solution is allowed to dry over each sample, after which each sample is illuminated for one or two minutes. The coated samples are washed in 0.1 N HCl followed by PBS. The antimicrobial activity of immobilized magainin polymer is evaluated with a centrifugation assay. Biomaterial sheets are cut into discs with a diameter of 1.5 cm, coated with magainin polymer and placed in individual wells of 24-well culture plates. Bacteria (E. coli and S. epidemidis) are suspended at 200 to 400 colony forming units per ml (cfu / ml) in PBS. Aliquots of one ml of bacterial suspensions are placed in wells containing biomaterials coated with magainin, and the plates are centrifuged at 3500 xg at 4 ° C for 20 minutes to pellet the bacteria in the coated biomaterial discs. The discs are then placed on bacterial culture plates of tryptic soy agar (TSA) and covered with a thin layer of TSA. After incubation overnight at 37 ° C, the colonies of bacteria growing on the discs are counted. Magainin polymer coatings are expected to inhibit bacterial growth and support the growth of smaller bacterial colonies than uncoated controls. Example 11 β-Galactosidase polymer A. Synthesis of a photoreactive polyacrolamide containing β-Galactosidase (β-Galactosidase polymer) A mixture containing 50 mg / ml of NOS polymer (prepared as described in Example 6) and 6.4 mg / ml β-galactosidase (from Boehringer Mannheim, Indianapolis, IN) in 0.1 M sodium carbonate, pH 9, is prepared. The mixture is allowed to react at room temperature for one hour and stored at 4 ° C overnight. The resulting ß-galactosidase polymer solution is used without further purification for the generation of interlaced films.

B. Generation of interlaced films. Films were emptied by placing 40 ml aliquots of the β-galactosidase polymer solution (which is synthesized as described herein) in a Teflon block and letting each aliquot to dry. The resulting films were illuminated for 0.5 or 4 minutes as described above. C. Essay for the integrity of the films.

The integrity of the films is tested by determining whether they retain their shape after being placed in a PBS solution. Films illuminated for 0.5 minutes, dissolve before exposure in Salino; while movies illuminated for 4 minutes retain their form. These results are consistent with slight activation of the BBA groups that produce covalent entanglement of polymer molecules of the invention. The interlaced β-galactosidase polymer is washed three times with 1 ml of PBS to remove unincorporated enzyme. The last wash (0.2 ml) and the recovered film, each were analyzed by enzyme activity using o-nitrophenol-β-D-galactopyranoside (o-NPG) (from Pierce, Rockford, IL) at 1 mg / ml in water using the protocol described in the "Worthington Enzyme Manual" (Worthington Enzyme Manual) (Worthington Biochemical Corp., Freehold, NJ, 1977). The last wash showed no β-galactosidase activity while the film gave the yellow nitrophenol product. This result demonstrated that the β-galactosidase portion was active after the polymer of the invention was entangled to form an insoluble biomaterial. Example 12 DNA Polymer A model oligodeoxynucleotide probe (oligoDNA) with an exon 1 sequence of the H-2Kb gene of the major histocompatibility complex is synthesized and used as a capture probe. The sequence of the oligoDNA capture probe was 5'-GTCTGAGTCGGAGCCAGGGCGGCCGCCAACAGCAGGAGCA-3 'and was synthesized with an aliphatic Cl2 spacer at the 5' end which ended with a primary amine. The oligoDNA capture probe (80 μg, or 6 nmol) is coupled via the terminal amino group in the C12 spacer to 160 μg of NOS polymer described here in 50 mM phosphate buffer (pH 8.5, 1 mM EDTA, 0.24 ml final volume) at room temperature for 2.5 hours. The resulting DNA polymer is used without further purification or characterization. The DNA polymer is applied in wells of microplates (polypropylene plates from Corning Costar Corporation, Cambridge, MA) at 10 μmol (in solution 0.1 ml) per well and incubated for 10 to 30 minutes. Plates containing DNA polymer solutions were illuminated for 1.5 minutes with a Dymax lamp as described herein except that a filter that removes light of wavelengths shorter than 300 nm is employed. A control consists of the oligoDNA capture probe (10 μmoles in 0.1 ml 50 mM phosphate buffer, pH 8.5, 1 mM EDTA) that is added to wells, allowed to adsorb for 2.5 hours and does not light up. Plates were washed with phosphate buffered saline containing 0.05% Tween 20 in PBS to remove unbound DNA polymer or control oligoADN capture probe. A detection probe with a sequence complementary to the capture probe described herein is synthesized with a biotin at the 5 'end and used to evaluate the immobilized DNA polymer activity. The sequence of the detection probe was 5 '-CCGTGCACGCTGCTCCTGCTGTTGGCGGCCGCCCTGGCTCCGACTCAGAC-3'.

A control detection probe consisting of a non-complementary sequence of exon 2 of the H-2Kb gene is also synthesized with a portion of biotin at the 5 'end. The binding of each detection probe is tested by subsequently adding a conjugate of streptavidin and horseradish peroxidase (SA-HRP, available from Pierce, Rockford, IL) and measure the activity of bound HRP. For this test, the coated plates were blocked with a hybridization buffer (0.75 M NaCl, 0.075 M citrate, pH 7.0, 0.1% lauroyl sarcosine, 1% casein, and 0.02% sodium dodecyl sulfate) at 55 ° C. 30 minutes. Complementary and non-complementary detection probes were added at 50 fmol per 0.1 ml of hybridization buffer per well and incubated for one hour at 55 ° C. The plates were then washed with 0.3 M NaCl, 0.03 M citrate, pH 7.0 containing 0.1% SDS for 5 minutes at 55 ° C. SA-HRP is added at 0.5 μg / ml and incubated for 30 minutes at 37 ° C. Plates were then washed with 0.05% Tween 20 in PBS, followed by addition of peroxidase substrate (TMB Microwell Peroxidase substrate system from Kirkegarrd and Perry Laboratories, Gaithersburg, MD) and absorbance measurement at 655 nm in a microplate reader (model 3550 Bio-Rad Labs, Cambridge, MA). Since the polypropylene plates were opaque, the substrate solutions reacted were transferred to polystyrene plates to read the absorbance. Hybridization signals (A655) of polypropylene microwells coated with absorbed oligoADN capture probe or photoimmobilized DNA polymers (n = 3) Detection probe Complementary non-complementary detection probe Capture probe 0.037 + 0.005 0.033 + 0.001 adsorbed oligo / DNA DNA polymer .170 + 0.079 0.068 + 0.010 photoimmobilized The results in the previous table provide the hybridization signals of polypropylene microwells coated with photoimmobilized DNA polymers or oligoDNA capture probe adsorbed (n = 3). These results demonstrate that the photoimmobilized DNA polymer binds 32 times more the complementary detection probe than the adsorbed control, and no coating binds the non-complementary probe.

Claims (23)

1. CLAIMS 1.- A polybifunctional reagent comprising a plurality of molecules, each comprising a polymeric backbone containing (A) one or more secondary photoreactive portions capable of being activated by exposure to a convenient energy source, and (B) two or more secondary bioactive groups capable of specific non-covalent interactions with complementary groups, the reagent is capable, upon activation of the photoreactive portions, of forming a bulk material or surface coating in order to promote the attraction of these complementary groups.
2. 2. A polybifunctional reagent according to claim 1, characterized in that the photoreactive portions can be activated to form intermolecular covalent bonds between the adjacent reagent molecule and a biomaterial surface in order to form a coating.
3. 3. A polybifunctional reagent according to claim 1, characterized in that the photoreactive portions can be activated to form intermolecular covalent bonds between adjacent reactive molecules in order to form a bulk material.
4. 4. A polybifunctional reagent according to claim 1, characterized in that the bioactive groups participate in a specific binding reaction with complementary molecules or cell receptors.
5. 5. A polybifunctional reagent according to claim 4, characterized in that the bioactive groups each independently are chosen from the group consisting of proteins, peptides, amino acids, carbohydrates and nucleic acids, each is able to bind non-covalently with specific and complementary portions of molecules or cells.
6. 6. A polybifunctional reagent according to claim 1, characterized in that each of the bioactive groups has an identifiable or known complementary binding partner and each independently is selected from the group consisting of antithrombotic agents, cellular connection factors, receptors , ligands, growth factors, antibiotics, enzymes and nucleic acids.
7. 7. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise antithrombotic agents selected from the group consisting of heparin, hirudin, lysine, prostaglandins, streptokinase, urokinase and plasminogen activator.
8. 8. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise cell connection factors selected from the group consisting of surface adhesion molecules and cell-to-cell addition molecules. t
9. 9.- A polybifunctional reagent according to claim 8, characterized in that the bioactive groups comprise surface addition molecules selected from the group consisting of laminin, fibronictin, collagen, vitronectin, tenascin, fibrinogen, thrombospondin, osteopontin, von Willibrand factor. and bone cycloprotein, and its active domains.
10. 10. A polybifunctional reagent according to claim 8, characterized in that the bioactive groups comprise cell-to-cell addition molecules selected from the group consisting of N-cadherin P-cadherin and its active domains.
11. 11. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise growth factors selected from the group consisting of fibroblastic growth factors, epidermal growth factor, growth factors derived from platelets, transformation growth factors , vascular endothelial growth factor, morphogenic bone proteins and other bone growth factors, and neural growth factors.
12. 12. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise a ligand or receptor selected from the group consisting of antibodies, antigens, avidin, streptavidin, biotin, heparin, type IV collagen, protein A, and protein G.
13. 13. - A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise an antibiotic selected from the group consisting of antibiotic peptides.
14. 14. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise enzymes.
15. 15. A polybifunctional reagent according to claim 6, characterized in that the bioactive groups comprise nucleic acid sequences capable of selectively binding complementary nucleic acid sequences.
16. 16. A polybifunctional reagent according to claim 1, characterized in that the polymeric main structure comprises a synthetic polymer selected from the group consisting of addition type polymers such as vinyl polymers.
17. 17. - A polybifunctional reagent according to claim 1, characterized in that each of the photo-groups comprises a photoactivatable acetone.
18. 18.- A polybifunctional reagent comprising a synthetic polymeric main structure containing (a) one or more secondary photoreactive portions in the form of photoactivatable ketones; and (b) two or more secondary bioactive groups selected from the group consisting of proteins, peptides, amino acids, carbohydrates, nucleic acids and other molecules that are capable of binding. non-covalently with specific and complementary portions of molecules or cells.
19. 19.- Method for coating a biomaterial surface, the method is characterized in that it comprises the steps of: a) providing a polybifunctional reagent comprising a polymeric main structure containing (i) one or more secondary photoreactive portions capable of being activated by exposure to a convenient energy source and (ii) two or more secondary bioactive groups capable of specific, non-covalent interactions with complementary groups, (b) contacting the surface with the reagent and (c) activating the photoreactive portions in order to intertwine the reagent molecules with them and / or the surface.
20. 20. Method according to claim 19, characterized in that the reagent is coated on the surface by spraying, dipping or brushing.
21. 21.- Method for forming a bulk biomaterial, the method is characterized in that it comprises the steps of (a) providing a polybifunctional reagent comprising a polymeric backbone containing (i) one or more secondary photoreactive portions capable of being activated by exposure to a convenient energy source and (ii) two or more secondary bioactive groups capable of specific interactions, non-covalent with complementary groups, and (b) activating the reagent to form a bulk biomaterial.
22. 22. A coated biomaterial surface comprising the bound residues of an activated polyfunctional reagent that initially comprises a polymer backbone containing (a) one or more secondary photoreactive portions capable of being activated by exposure to a convenient energy source and (b) ) two or more secondary bioactive groups capable of specific, non-covalent interactions with complementary groups.
23. 23. - A bulk material comprising bound residues of an activated polybifunctional reagent that initially comprises a polymer backbone containing (a) one or more secondary photoreactive portions capable of being activated by exposure to a convenient energy source and (b) two or more secondary bioactive groups capable of specific, non-covalent interactions with complementary groups.