WO2009006538A2 - Modified polymers and methods for making and using the same - Google Patents

Abstract

The present invention provides block copolymers, e.g., thermoplastic elastomers, having at least one monomer that is covalently bonded to a sulfonate moiety. The present invention also provides block copolymers, e.g., thermoplastic elastomers, having at least one monomer that is covalently bonded to a peptide. The present invention also provides methods for making and using (e.g., in articles of manufacture such as medical devices) the copolymers of the present invention.

Description

MODIFIED POLYMERS AND METHODS FOR MAKING AND USING THE SAME

RELATED APPLICATIONS

[0001] This application is related and claims priority to U.S. Provisional Application Serial No. 60/947,619, filed July 2, 2007 and entitled "MODIFIED POLYMERS AND METHODS FOR MAKING AND USING THE SAME" which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0002] Copolymers have numerous commercial applications, for instance, unique properties in pure form, blends, melts, solutions, etc. For example, polymer-based medical devices have been developed for the delivery of therapeutic agents to the body. Polymers have typically been successful when used in medicine; however, there is a need for substrates that are able to interact favorably with the body.

[0003] For example, in individuals suffering from circulatory disease caused by a blockage of the blood vessels that perfuse the heart and other major organs, treating such blockages is limited by the tendency of the treatment to damage the endothelial layer of the vessels.

SUMMARY OF THE INVENTION

[0004] Typically, cell adhesion to biomaterials (e.g., polyethylene, polytetrafluoroethylene, silicone) depends on proteins from the body fluids being adsorbed nonspecifically to the surface of the biomaterial. Some of the adsorbed proteins, (e.g., fibronectin, fibrinogen, vitronectin) promote adhesion of cells by interacting with adhesion receptors. The present invention provides a more direct approach, where protein or peptide-modified copolymers allow control of the adhesion by not relying on nonspecific adsorption.

[0005] Accordingly, in one aspect the present invention is directed to a modified block copolymer comprising a first block and a second block, wherein each block comprises a plurality of monomer species and wherein at least one internal monomer species is covalently bonded to a sulfonate moiety or a bioactive molecule. In some embodiments, the block copolymer is a thermoplastic elastomer. [0006] The sulfonate moiety can be any of the sulfonate moieties described herein, e.g., a 4-fluorobenzenesulfonate moiety. The bioactive molecule can be any of the bioactive molecules described herein, e.g., peptides and proteins such as peptides containing the YIGSR sequence, peptides containing the RGD sequence, peptides containing the REDV sequence, P- 15, Heparin, and Platelet activation peptides.

[0007] In some embodiments, the first block comprises at least one methacrylate monomer species. In some embodiments, the first block comprises at least one A- fluorobenzenesulfonate ethyl methacrylate monomer species. In some embodiments, the first block comprises a methylmethacrylate monomer species and a A- fluorobenzenesulfonate ethyl methacrylate monomer species. In some embodiments, the first block comprises at least one peptidylethyl methacrylate monomer species. In some embodiments, the first block comprises a methylmethacrylate monomer species and a peptidylethyl methacrylate monomer species.

[0008] In some embodiments, the second block comprises at least one monomer selected from the group consisting of isobutylene, 2-methylbutene, 3-methyl-l-butene, 4-methyl-l-pentene and beta-pinene. In some embodiments, the second block comprises isobutylene.

[0009] In some embodiments, the block copolymer further comprises a third block, wherein the third block comprises a plurality of monomers. In some embodiments, at least one internal monomer of the third block is covalently bonded to a sulfonate moiety or a peptide. In some embodiments, the third block comprises at least one methacrylate monomer species. In some embodiments, the third block comprises at least one A- fluorobenzenesulfonate ethyl methacrylate monomer species. In some embodiments, the copolymer is an A-B-A block copolymer and wherein A represents the first and third blocks and B represents the second block.

[0010] In some embodiments, the number average molecular weight of the copolymer ranges from about 10,000 to about 1,000,000. In some embodiments, copolymer is a linear copolymer.

[0011] In some embodiments, at least 1% of the internal monomers of the first block are covalently bonded to a sulfonate moiety or a peptide. In some embodiments, at least 10% of the internal monomers of the first block are covalently bonded to a sulfonate moiety or a peptide. In some embodiments, at least one surface monomer is covalently bonded to a sulfonate moiety or a peptide. [0012] In some embodiments, the copolymer is a medical device or a coating on a medical device. In some embodiments, the medical device is a stent. In some embodiments, the medical device is a graft or stent graft. In some embodiments, the medical device is an electrical lead. In some embodiments, the medical device is designed for neurological implantation. In some embodiments, a therapeutic agent is also incorporated in the device or coating, through covalent attachment or through incorporation into the coating or device.

[0013] In some aspects, the present invention is directed to a method for making sulfonate-modified block copolymers. The method generally includes dissolving a copolymer comprising at least one modifiable monomer with a suitable solvent; and contacting the dissolved copolymer with a sulfonyl chloride, under suitable conditions such that a sulfonate-modified block copolymer is formed. In some embodiments, the block copolymer is a thermoplastic elastomer. In some embodiments, the sulfonate- modified block copolymer is modified both internally and on the surface. In some embodiments, the modifiable monomer comprises a pendant hydroxyl moiety. In some embodiments, the sulfonyl chloride is a fosylchloride.

[0014] In some aspects, the present invention is directed to a method for making bioactive molecule-modified block copolymers. The method generally includes dissolving a sulfonate-modified block copolymer with a suitable solvent; and contacting the dissolved copolymer with a bioactive molecule, under suitable conditions such that a bioactive molecule-modified block copolymer is formed.

[0015] The bioactive molecule can be any of the bioactive molecules described herein, e.g., peptides and proteins such as peptides containing the YIGSR sequence, peptides containing the RGD sequence, peptides containing the REDV sequence, P- 15, Heparin, and Platelet activation peptides.

[0016] In some aspects, the present invention is directed to an article of manufacture comprising at least one copolymer described herein. In some embodiments, the article of manufacture is an insertable or implantable medical device. In some embodiments, the implantable medical device is a stent.

DESCRIPTION OF THE FIGURES

[0017] Figure Ia is a ¹H NMR spectra of exemplary fosylated polymers (PF1-PF4) of the present invention. Figure Ib is a ¹³C NMR spectra of an exemplary fosylated polymer of the invention. Figure Ic shows the surface topography of exemplary polymer films of the invention. [0018] Figure 2 is a confocal micrograph image of an exemplary dye labeled fosylated polymer of the present invention, PF3. [0019] Figure 3 depicts a number of graphs showing the thickness of an exemplary fosylated polymer of the present invention, PF3, coated on a steel surface by the draw down technique. [0020] Figure 4 is a Mgα CIs XPS spectra of PFl, PF2, PF3 and PF4 polymer films acquired at a 90° TOA. The results of deconvoultion of the spectra into component peaks are shown as dashed lines. [0021] Figure 5 is a MgKa S2p XPS spectra of PFl , PF2, PF3 and PF4 polymer films acquired at a 90° TOA. [0022] Figure 6 is a MgKa NIs XPS spectra of PGG2, PYR2, PGG4 and PYR4 polymer films acquired at a 90° TOA. The spectra have not been corrected for surface charging. [0023] Figure 7 depicts the swelling profile of PFl, PF2, PF3 and PF4 showing the percent weight gain with increase in time. [0024] Figure S is a FT-IR spectra showing the variation in peak intensities at 1600 and 3560 cm^"1 before and after tris reaction.

DETAILED DESCRIPTION

[0025] In one aspect, the present invention is based, at least in part, on the discovery of novel modified copolymers (e.g., surface- and internally-modified copolymers) which are able to promote endothelialization, e.g., when used in insertable or implantable medical devices. Without wishing to be bound by any particular theory, it is believed that covalent attachment of sulfonate moieties and/or bioactive molecules, e.g., peptides or proteins, to an insertable or implantable medical device (or coating thereof) is desired in order to enhance endothelialization of the biomaterial surface or to improve overall biocompatibility, e.g., imparting no to limited inflammatory or thrombogenic response. For example, when a biologically active peptide, e.g., YIGSR peptide, is not covalently attached, spreading of endothelial cells is not observed due to de-adsorption of the peptide from the device. (See, e.g., Hubbell, J.A. et al., Polym. Mater. ScL Eng. 1992, 66, 30-31). Moreover, the residue at the carboxylic acid end of the peptide is often essential for biological activity. For example, with respect to YIGSR, the R residue is essential for activity and direct attachment to a polymer via the R residue may hinder development of the secondary structure that is important for receptor recognition. Accordingly, attachment via the amino terminus is preferred for maintaining activity of the peptide.

[0026] Bioactive peptides or proteins can be chosen from a wide range of molecules which are capable of binding to epithelial cells (e.g., endothelial cells) via cell surface molecules, such as integrins, displayed on the surface of epithelial cells, stem cell populations or endothelial progenitor cells having a role in vascular biology. Exemplary cell types can be found, for example, in Urbich et al. (2004) Circ. Res. 95:343-353.

[0027] Typically bioactive peptides or proteins are any of the peptides or proteins of the extracellular matrix which are known to play a role in cell adhesion, including fibronectin, vitronectin, laminin, elastin, fibrinogen, and collagens, such as types I, II, and V. Additionally, the bioactive peptides or proteins may be any peptide derived from any of the aforementioned proteins, including derivatives or fragments containing the binding domains of the above-described molecules. Example peptides include those having integrin -binding motifs, such as the RGD (arginine-glycine-aspartate) motif, the YIGSR (tyrosine-isoleucine-glycine-serine-arginine) motif, and related peptides that are functional equivalents. For example, bioactive peptides or proteins containing RGD sequences (e.g., GRGDS) and WQPPRARI sequences are known to direct spreading and migrational properties of endothelial cells. See V. Gauvreau et al., Bioconjug Chem., 2005 Sep-Oct, 16(5), 1088-97. REDV tetrapeptide has been shown to support endothelial cell adhesion but not that of smooth muscle cells, fibroblasts, or platelets, and YIGSR pentapeptide has been shown to promote epithelial cell attachment, but not platelet adhesion. More information on REDV and YIGSR peptides can be found in U.S. Patent No. 6,156,572 and U.S. Patent Application No. 2003/0087111. See also, Boateng et al., RGD and YIGSR Synthetic Peptides Facilitate Cellular Adhesion Identical to That of Laminin and Fibronectin But Alter the Physiology of Neonatal Cardiac Myocytes, Am. J. Physiol. - Cell Physiol. 288:30-38 (2005), which is incorporated by reference herein. A further example of a cell-adhesive sequence is NGR tripeptide, which binds to CD13 of endothelial cells. See, e.g., L. Holle et al., "In vitro targeted killing of human endothelial cells by co-incubation of human serum and NGR peptide conjugated human albumin protein bearing alpha (1-3) galactose epitopes," Oncol. Rep. 2004 Mar; ll(3):613-6. The bioactive peptides or proteins may also be any of the peptides described in U.S. Patent Publication No. 20060067909 (West et al.), which is incorporated by reference herein. Alternatively the bioactive peptides or proteins can be obtained by screening peptide libraries for adhesion and selectivity to specific cell types (e.g. endothelial cells) or developed empirically via Phage display technologies. The bioactive peptides or proteins may also impart improved general biocompatibility, e.g. no to limited inflammatory or thrombogenic response. Non limiting examples include coagulation pathway modulators including heparinoids such as heparin, low molecular weight heparin, dextran sulfate and β- cyclodextrin tetradecasulfate.

Definitions

[0028] In order to more clearly and concisely describe the subject matter of the claims, the following definitions are intended to provide guidance as to the meaning of specific terms used herein.

[0029] It is to be noted that the singular forms "a," "an," and "the" as used herein include "at least one" and "one or more" unless stated otherwise. Thus, for example, reference to "a pharmacologically acceptable carrier" includes mixtures of two or more carriers as well as a single carrier, and the like.

[0030] Numerous values and ranges are recited in connection with various embodiments of the present invention, e.g., number of constitutional units present in a block. It is to be understood that all values and ranges which fall between the values and ranges listed are intended to be encompassed by the present invention unless explicitly stated otherwise. Additionally, it is also to be understood that all numerical values listed herein are implicitly modified by the term "about" unless specifically stated otherwise.

[0031] As used herein, the term "polymer" refers to a molecule that contains one or more chains, each containing multiple copies of one or more constitutional units. An

example of a common polymer is

, where n is an integer, typically an integer of 10 or more, more typically on the order of 10' s, 100' s, 1000' s or even more, in which the constitutional units in the chain correspond to styrene

{i.e., they originate from, or have the appearance of originating from, the polymerization of styrene monomers— in this case the addition polymerization of styrene monomers). Copolymers are polymers that contain at least two dissimilar constitutional units.

[0032] As used herein a polymer "block" refers to a grouping of 10 or more constitutional units, commonly 20 or more, 50 or more, 100 or more, 200 or more, 500 or more, or even 1000 or more units, and can be branched or unbranched. A "chain" is a linear (unbranched) grouping of 10 or more constitutional units {i.e., a linear block). In the present invention, the constitutional units within the blocks and chains are not necessarily identical, but are related to one another by the fact that that they are formed in a common polymerization technique, e.g., a cationic polymerization technique or anionic polymerization technique.

[0033] As used herein, "alkyl" groups include saturated hydrocarbons having one or more carbon atoms, including straight-chain alkyl groups {e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), cyclic alkyl groups (or "cycloalkyl" or "alicyclic" or "carbocyclic" groups) {e.g., cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, etc.), branched-chain alkyl groups (isopropyl, tert-butyX, sec-butyl, isobutyl, etc.), and alkyl-substituted alkyl groups {e.g., alkyl-substituted cycloalkyl groups and cycloalkyl-substituted alkyl groups). In certain embodiments, a straight-chain or branched-chain alkyl group may have 30 or fewer carbon atoms in its backbone, e.g., C₁-C₃O for straight-chain or C3-C30 for branched- chain. In certain embodiments, a straight-chain or branched-chain alkyl group may have 20 or fewer carbon atoms in its backbone, e.g., C₁-C_2O for straight-chain or C₃-C₂₀ for branched-chain, and more preferably 18 or fewer. Likewise, preferred cycloalkyl groups have from 4-10 carbon atoms in their ring structure, and more preferably have A- 7 carbon atoms in the ring structure. The term "lower alkyl" refers to alkyl groups having from 1 to 6 carbons in the chain, and to cycloalkyl groups having from 3 to 6 carbons in the ring structure. The term "C₁-C₆" as in "C₁-C₆ alkyl" means alkyl groups containing 1 to 6 carbon atoms. [0034] Moreover, unless otherwise specified the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls," the latter of which refers to alkyl groups having substituents replacing one or more hydrogens on one or more carbons of the hydrocarbon backbone. Such substituents may include, for example, alkenyl, alkynyl, halogeno, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclic, alkylaryl, or aromatic (including heteroaromatic) groups.

[0035] It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with the permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

[0036] As used herein, the term "peptide" refers to a compound that consists of two or more amino acid residues joined by a peptide bond. The term "peptide bond" means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid. Accordingly, peptides are compounds in which the α-carboxyl group of one amino acid is joined by an amide bond to the main chain (α- or β-) amino group of the adjacent amino acid. Peptide sequences specifically recited herein are written with the amino terminus on the left and the carboxy terminus on the right.

[0037] The term "amino acid" as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group. The term "amino acid residue" as used herein means that portion of an amino acid (as defined herein) that is present in a peptide. [0038] As used herein, the term "modifiable monomer" refers to monomers which are able to react with electrophiles via nucleophilic attack, e.g.,:

H X

Modifiable monomers include nucleophiles, e.g., monomers with pendant nucleophilic groups. Electrophiles are reagents that are attracted to electrons. Typically, electrophiles participate in chemical reactions by accepting an electron pair in order to bond to a nucleophile. Nucleophiles are reagents that form chemical bonds with electrophiles by donating both bonding electrons. Suitable modifiable monomers include, but are not limited to, monomers having pendant hydroxyl moieties, e.g., hydroxyalkyl methacrylate monomers.

[0039] As used herein, the term "internally-modified copolymer" refers to a copolymer which includes at least one internal monomer that is covalently bonded to at least one nucleophilic moiety or at least one active agent. As used herein, the term "surface-modified copolymer" refers to a copolymer which includes at least one internal monomer that is covalently bonded to at least one nucleophilic moiety or at least one active agent. Accordingly, in the presence of a sufficiently strong nucleophile (e.g., an amine moiety on a peptide), the nucleophilic moiety on the monomer will undergo an S_N2 reaction with the amine moiety, allowing the attachment of the new nucleophile to the polymer. In some embodiments, the electrophilic moiety is a sulfonate moiety, e.g., a 4-fluorobenzenesulfonate moiety. In some embodiments, the active agent is a peptide.

[0040] As used herein, the term "surface monomer" refers to a monomer which is present on the surface of the polymer. As used herein, the term "internal monomer" refers to a monomer not present on the surface of the polymer.

Copolymers of the Present Invention

[0041] In some aspects, the present invention is directed to a modified block copolymer (e.g., an internally-modified block copolymer) which includes a first block and a second block. Each block, in turn, includes a plurality of monomer species wherein at least one internal monomer species is covalently bonded to a sulfonate moiety or a bioactive molecule, e.g., a protein or a peptide. As used herein, the term "monomer species" refers to the portion of a polymer that is attributed to a single

monomer. That is, if the monomer is isobutylene,

, then the monomer species is

represented by:

[0042] Examples of monomers include the following: (a) olefins, including isomonoolefins with 4 to 18 carbon atoms per molecule and multiolefins with 4 to 14 carbon atoms per molecule, for example, isobutylene, 2-methylbutene, isoprene, 1,3- butadiene, 3-methyl-l-butene, 4-methyl-l-pentene, beta-pinene, and the like, (b) vinyl aromatics such as styrene, alpha-methyl styrene, para-chlorostyrene, para- methylstyrene, and the like, and (c) vinyl ethers such as methyl vinyl ether, isobutyl vinyl ether, butyl vinyl ether, N-vinyl carbazole, and the like, and (d) acrylates or methacrylates, e.g., compounds having the formula CH₂=CHCO₂R or CH₂=C(CH₃)CO₂R where R is a substituted or unsubstituted, branched, unbranched or cyclic alkyl groups containing 1 to 20 carbons. Substituents for the alkyl groups include hydroxyl, amino and thiol functional groups, among others. In embodiments where monomers are utilized that have functional groups, proper protection of the functional group may be needed during the course of anionic polymerization. Specifc examples of nonfunctional and protected functional methacrylate monomers include ethyl methacrylate, methyl methacrylate, tert-butyl methacrylate, isodecyl methacrylate, dodecyl methacrylate, stearyl methacrylate, glycidyl methacrylate, 2- [(trimethylsilyl)oxy] ethyl methacrylate, 2- [(te/t-butyldimethylsilyl)oxy] ethyl methacrylate, and 2-[(methoxymethyl)oxy]ethyl methacrylate. [0043] In some embodiments, monomers include an olefin or a multiolefin, e.g., isobutylene. In some embodiments, the monomers include acrylates or methacrylates, e.g., methylmethacrylate, hydroxyethylmethacrylate.

[0044] In some embodiments, the block copolymer is a thermoplastic elastomer. As used herein, the term "thermoplastic elastomer" refers to a class of copolymers which consist of materials with both thermoplastic and elastomeric properties. Whether an amorphous polymer is a thermoplastic or an elastomer typically depends on its glass transition temperature (T_g), which is the temperature above which the polymer is soft and pliable, and below which it is hard and glassy. A T_g below room temperature delineates an elastomer, which is soft and rubbery at room temperature. A T_g above room temperature delineates a thermoplastic, which is hard and glassy at room temperature.

[0045] A thermoplastic material is plastic or deformable, melts to a liquid when heated and freezes to a brittle, glassy state when cooled sufficiently. Many thermoplastics are polymers with high molecular weight, and have chains that associate through interactions such as van der Waals forces, dipole-dipole interactions, hydrogen bonding, and/or pi-pi stacking of aromatic rings. Thermoplastic polymers can be remelted and remoulded, which makes them relatively easy to use in manufacturing, for example, by injection molding. Thermoplastic materials include, but are not limited to polypropylene, acrylonitrile butadiene styrene (ABS), polyalkyl methacrylate, celluloid, cellulose acetate, ethylene-vinyl acetate (EVA), ethylene vinyl alcohol (EVAL), fluoroplastics (PTFEs, including FEP, PFA, CTFE, ECTFE, ETFE), liquid Crystal Polymer (LCP), polyacetal (POM or Acetal), polyacrylonitrile (PAN or Acrylonitrile), polyamide (PA or Nylon), polyamide-imide (PAI), polyaryletherketone (PAEK or Ketone), polybutylene terephthalate (PBT), polyethylene terephthalate (PET), polycyclohexylene dimethylene terephthalate (PCT), polycarbonate (PC), polyhydroxyalkanoates (PHAs), polyketone (PK), polyester, polyethylene (PE), polyetheretherketone (PEEK), polyetherimide (PEI), polyethersulfone (PES), polyethylenechlorinates (PEC), polyimide (PI), polylactic acid (PLA), polymethylpentene (PMP), polyphenylene oxide (PPO), polyphenylene sulfide (PPS), polyphthalamide (PPA), polystyrene (PS), polysulfone (PSU), polyvinyl chloride (PVC), polyvinylidene chloride (PVDC) and mixtures thereof.

[0046] An elastomer, also called a rubber, refers to a material that cures, generally through the addition of energy (e.g., heat typically above 200⁰C, chemical reaction, or irradiation, to a stronger form. Elastomers are amorphous polymers, generally liquid, powder, or malleable materials, prior to curing. The long polymer chains of elastomers cross-link during curing. However, once crosslinking occurs, the resultant polymers do not melt, because the crosslinks secure all the polymer chains together, and thus the material is not able to flow. Elastomeric materials include, but are not limites to natural rubber (NR), polyisoprene (IR), polyisobutylene, butyl rubber (copolymer of isobutylene and isoprene, HR) halogenated butyl rubbers (chloro butyl rubber: CIIR; bromo butyl rubber: BIIR), polybutadiene (BR), styrene-butadiene rubber (copolymer of polystyrene and polybutadiene, SBR), nitrile rubber (copolymer of polybutadiene and acrylonitrile, NBR), hydrated nitrile rubbers (HNBR), e.g., Therban® and Zetpol®, chloroprene rubber (CR), polychloroprene, neoprene, baypren, ethylene propylene rubber (EPM, a copolymer faeces of polyethylene and polypropylene), and ethylene propylene diene rubber (EPDM, a terpolymer of polyethylene, polypropylene and a diene-component), epichlorohydrin rubber (ECO), polyacrylic rubber (ACM, ABR), silicone rubber (SI, Q, VMQ), fluoro silicone rubber (FVMQ), fluoroelastomers (FKM, FPM), e.g., Viton®, Tecnoflon®, Fluorel®, Aflas and Dai-El®, perfluoroelastomers (FFKM), e.g., Kalrez®, polyether block amides (PEBA), tetrafluoro ethylene/propylene rubbers (FEPM), chloro sulfonated polyethylene (CSM), e.g., Hypalon®, ethylene- vinyl acetate (EVA), polyurethane rubber, Resilin, Elastin, polysulfide rubber and mixtures thereof.

[0047] Thermoplastic elastomers show advantages typical of both rubbery materials and plastic materials, such as recyclability, elasticity and ability to absorb shock. Thermoplastic elastomers can also be molded, extruded and reused, if desired.

[0048] The copolymers of the invention include a monomer species covalently

bonded to either a sulfonate moiety (e.g., Covalent

bonding of a sulfonate moiety or bioactive molecule typically occurs through a modifiable monomer or monomer species. As discussed further herein, modifiable monomer refers to a monomer which is able to react with a sulfonyl chloride (e.g.,

to form a sulfonate-modified monomer (i.e., a monomer covalently bonded to

a sulfonate moiety). In some embodiments, the modifiable monomer is a monomer with a pendent hydroxyl group. Pendent hydroxyl groups are -OH moieties which do not react to form the backbone of the polymer and are not attached to a carbon (or other atom) which forms the backbone of the polymer. In some embodiments, the modifiable monomer is a methacrylate monomer. In some embodiments, the modifiable monomer is a methacrylate monomer having the formula CH2=C(CH3)CO2R where R is a branched, unbranched or cyclic alkyl group containing 1 to 20 carbons and substituted with at least one nucleophile, e.g., at least one hydroxyl moiety. One example of a modifiable methacrylate monomer is 2-hydroxyethyl methacrylate. The modifiable methacrylate monomer can be activated with a sulfonyl chloride as a monomer or as a monomer species contained within a copolymer. Accordingly, activation with a sulfonyl chloride provides both monomers covalently bonded to a sulfonate moiety or copolymers which include at least one monomer species covalently bonded to a sulfonate moiety.

[0049] Exemplary sulfonyl moieties include, but are not limited to, methanesulfonyl, 2-propanesulfonyl, 1-butanesulfonyl, benzenesulfonyl, 1- naphthalenesulfonyl, 2-naphthalenesulfonyl, p-toluenesulfonyl, α-toluenesulfonyl, A- acetamidobenzenesulfonyl, 4-amidinobenzenesulfonyl, 4-tert-butylbenzenesulfonyl, A- bromobenzenesulfonyl, 2-carboxybenzenesulfonyl, 4-cyanobenzenesulfonyl, 3,4- dichlorobenzenesulfonyl, 3,5-dichlorobenzenesulfonyl, 3,4-dimethoxybenzensulfonyl, 3,5-ditrifluoromethylbenzenesulfonyl, 4-fluorobenzenesulfonyl (fosyl), A- methoxybenzenesulfonyl, 2-methoxycarbonylbenzene- sulfonyl, A- methylamidobenzenesulfonyl, 4-nitrobenzenesulfonyl, 4-thioamidobenzenesulfonyl, A- trifluoromethylbenzenesulfonyl, 4-trifluoromethoxybenzenesulfonyl, 2,4,6- trimethylbenzene- sulfonyl, 2-phenylethanesulfonyl, 2-thiophenesulfonyl, 5-chloro-2- thiophenesulfonyl, 2,5-dichloro-4-thiophenesulfonyl, 2-thiazolesulfonyl, 2-methyl-4- thiazolesulfonyl, l-methyl-4-imidazolesulfonyl, l-methyl-4-pyrazolesulfonyl, 5-chloro- l,3-dimethyl-4-pyrazolesulfonyl, 3-pyridinesulfonyl and 2-pyrimidinesulfonyl. In some embodiments, the sulfonyl moiety is a fosyl moiety.

[0050] In some embodiments, the block copolymers of the present invention comprise at least one sulfonate-alkyl methacrylate monomer species. In some embodiments, the block copolymers comprise at least one sulfonate-ethyl methacrylate monomer species. In some embodiments, the block copolymers comprise at least one 4-fluorobenzenesulfonate-alkyl methacrylate monomer species. In some embodiments, the block copolymers comprise at least one 4-fluorobenzenesulfonate ethyl methacrylate monomer species. In some embodiments, the block copolymers of the present invention include at least one monomer species covalently bonded to a sulfonate moiety or a bioactive molecule, e.g., a peptide and at least one monomer species not covalently bonded to either a sulfonate moiety or a bioactive molecule. For example, in some embodiments, the block copolymers of the present invention include a first block and a second block. The first block can comprise at least one monomer species covalently bonded to a sulfonate moiety or a bioactive molecule. The first block can comprise at least one monomer species covalently bonded to a sulfonate moiety or a bioactive molecule and at least one monomer species not covalently bonded to either a sulfonate moiety or a bioactive molecule. The second block can comprise at least one monomer species covalently bonded to a sulfonate moiety or a bioactive molecule. The second block can comprise at least one monomer species covalently bonded to a sulfonate moiety or a bioactive molecule and at least one monomer species not covalently bonded to either a sulfonate moiety or a bioactive molecule. The second block can comprise only monomer species not covalently bonded to either a sulfonate moiety or a bioactive molecule. An exemplary copolymer of the present invention includes a first block comprising a methylmethacrylate monomer species and a A- fluorobenzenesulfonate-ethyl methacrylate monomer species. Another exemplary copolymer of the present invention includes a first block comprising a methylmethacrylate monomer species and a peptidylethyl methacrylate monomer species.

[0051] The copolymers of the present invention can be synthesized to include any bioactive molecule, e.g., protein or peptide, without limitation. Bioactive molecules include, but are not limited to peptides and proteins such as peptides containing the YIGSR sequence, peptides containing the RGD sequence, peptides containing the REDV sequence, P- 15, Heparin, and Platelet activation peptides. In some embodiments, the peptide is a biologically active peptide. In some embodiments, the peptide promotes endothelialization. In some embodiments, the peptide is at least one peptide selected from the group consisting of peptides containing the YIGSR sequence.

[0052] In some aspects, the present invention is directed to a modified block copolymer wherein at least one internal monomer species is covalently bonded to a buffer moiety and at least one surface monomer is covalently bonded to a bioactive molecule, e.g., a peptide. That is, in some aspects, the present invention is directed to a copolymer with internal buffering capacity. A copolymer having internal monomers covalently bonded to fosyl groups can be swelled, at which point the fosyl groups may be replaced, e.g., with an active agent as described herein or with a buffering group, e.g., 2-amino-2-hydroxymethyl-l,3-propanediol (TRIS). Without wishing to be bound by any particular theory, it is believed that this is beneficial in the use of the copolymer in living subjects. Polymers tend to organize themselves to minimize surface free energy in response to changes in the local environment of the interface. Accordingly, a copolymer with internal buffer modifications may reorganize in the presence of a polar substance (e.g., plasma) to exhibit the buffer groups on the surface of the copolymer. Because the thermodynamic cost of replacing the water attracted to the buffer groups is high, it is believed that ordinary cells will not adhere to the copolymer. However, the same copolymer is modified on the surface with a bioactive molecule, e.g., a peptide such as YIGSR, which will still adhere to their targeted cells, e.g., endothelial cells. Such modification would provide an article of manufacture which is targeted to specific cells.

[0053] Accordingly, in some embodiments, the present invention is directed to a modified block copolymer wherein at least one internal monomer species is covalently bonded to a buffer moiety and at least one surface monomer is covalently bonded to a peptide which comprises the YIGSR sequence.

[0054] In some aspects, the present invention is directed to a surface-modified block copolymer. The copolymer includes a first block and a second block which each include a plurality of monomer species wherein at least one surface monomer species is covalently bonded to a sulfonate moiety or a bioactive molecule, e.g., a peptide. In some embodiments, the all of the surface monomer species of one of the blocks (e.g., the first block or the second block) are modified. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the surface monomer species of one of the blocks are modified. In some embodiments, the copolymer is modified on the surface with one or more sulfonate moieties or bioactive molecules in a pattern. Block copolymers phase separate on the nanoscale, which produces a pattern of blocks on the surface. When one type of block is able to covalently bond to a sulfonate moiety or a bioactive molecule and one type of block is not able to bond, a surface patterned with modifications is provided. Without wishing to be bound by any particular theory, it is believed that the modified surface of a patterned block copolymer behaves differently than a modified surface of a random copolymer. For example, in some embodiments, the size of the patterns on the surface of the copolymer are the same size as the cells that are targeted.

[0055] In some embodiments, the plurality of monomer species in the first block comprises a plurality of constitutional units that correspond to a single monomer species. In other embodiments, the plurality of monomer species in the first block comprises a plurality of constitutional units that correspond to two or more monomer species.

[0056] In some embodiments, the plurality of monomer species in the second block comprises a plurality of constitutional units that correspond to a single monomer species. In other embodiments, the plurality of monomer species in the second block comprises a plurality of constitutional units that correspond to two or more monomer species.

[0057] In some embodiments, the block copolymers of the present invention include more than two blocks. In such embodiments, the additional blocks can have the same characteristics as the first block or the characteristics as the second block. For example, the copolymers of the present invention can include a third block which comprises a plurality of monomer species. In some embodiments, at least one internal monomer apecies of the third block is covalently bonded to a sulfonate moiety or a bioactive molecule, e.g., a peptide. In some embodiments, the third block comprises at least one methacrylate monomer species, e.g., a 4-fluorobenzenesulfonate ethyl methacrylate monomer species and/or a methylmethacrylate monomer species. In some embodiments, the block copolymers of the present invention are A-B-A block copolymers, where A represents the first and third blocks and B represents the second block.

[0058] The copolymers of the present invention may be block copolymers. The copolymers of the present invention can include any number of polymer blocks, e.g. , can be a diblock copolymer, a triblock copolymer, or may have four or more (e.g., 5, 6, 7, 8, 9 or 10) blocks. In some embodiments, the copolymers of the present invention include more than 10 blocks. In some embodiments, the copolymers of the present invention are diblock copolymers. In other embodiments, the copolymers of the present invention are triblock copolymers.

[0059] The copolymers of the present invention also embrace a variety of configurations, including linear and branched configurations. Branched configurations include radial configurations, star-shaped configurations (e.g., configurations in which three or more chains emanate from a single region), comb configurations (e.g., graft copolymers having a main chain and a plurality of side chains), and dendritic configurations (e.g., arborescent or hyperbranched copolymers). [0060] In some embodiments, the copolymers of the present invention have a number average molecular weight ranging from about 200 to about 2,000,000. In other embodiments, the polymers of the present invention have a number average molecular weight ranging from about 500 to about 500,000. In still other embodiments, the polymers of the present invention have a number average molecular weight ranging from about 10,000 to about 100,000.

[0061] The ratio of monomer species corresponding to the first block (e.g., hydroxyethyl methacrylate) relative to the monomer species corresponding to the second block (e.g., isobutylene) in the copolymer usually ranges from 1/99 to 99/1 w/w, preferably from 70/30 to 5/95 w/w. In some embodiments, copolymers are provided which have a narrow molecular weight distribution such that the ratio of weight average molecular weight to number average molecular weight (MwMn) (i.e., the polydispersity index) of the polymers ranges from about 1 to about 10. In some embodiments, the polydispersity index of the copolymers of the present invention range from about 1 to about 2.

[0062] In some embodiments, the copolymers of the present invention contain no silyl moieties.

[0063] In some embodiments, the polymers of the present invention also include a therapeutic agent. The therapeutic agent may or may not also be linked to the polymers of the present invention. For example, therapeutic agents may be linked via covalent linkages, e.g., as described in US Application Publication No. 20070020308, the entire contents of which is incorporated herein by reference, or by ionic forces. In other embodiments, therapeutic agents may be linked to the polymer in the same manner as the peptides of the present invention. As used herein "therapeutic agents" are compounds which can result in an improvement in the growth or health of the subject, when administered to the same at an effective dosage level. Therapeutic agents include genetic therapeutic agents, non-genetic therapeutic agents and cells. In addition to covalently linked therapeutic agents, the medical articles of the present invention may also include one or more optional non-covalently bound therapeutic agents. In some embodiments, non-coupled therapeutic agents, can be physically compound with the polymer prior to use, applied to the surface with various means or absorbed into the polymer bulk. [0064] Specific examples of therapeutic agents, which may be covalently coupled to the polymer (e.g., where appropriate linking groups such as hydroxyl and amine groups are present, either inherently or by modification of the therapeutic agent) or combined with the polymer in a non-coupled manner, include, but are not limited to, those listed below.

[0065] Non-genetic therapeutic agents for use in conjunction with the present invention include: (a) anti-thrombotic agents such as heparin, heparin derivatives, urokinase, and PPack (dextrophenylalanine proline arginine chloromethylketone); (b) anti-inflammatory agents such as dexamethasone, prednisolone, corticosterone, budesonide, estrogen, sulfasalazine and mesalamine; (c) antineoplastic/antiproliferative/anti-miotic agents such as paclitaxel, 5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones, endostatin, angiostatin, angiopeptin, monoclonal antibodies capable of blocking smooth muscle cell proliferation, and thymidine kinase inhibitors; (d) anesthetic agents such as lidocaine, bupivacaine and ropivacaine; (e) anti-coagulants such as D-Phe-Pro-Arg chloromethyl ketone, an RGD peptide-containing compound, heparin, hirudin, antithrombin compounds, platelet receptor antagonists, anti-thrombin antibodies, anti-platelet receptor antibodies, aspirin, prostaglandin inhibitors, platelet inhibitors and tick antiplatelet peptides; (f) vascular cell growth promoters such as growth factors, transcriptional activators, and translational promotors; (g) vascular cell growth inhibitors such as growth factor inhibitors, growth factor receptor antagonists, transcriptional repressors, translational repressors, replication inhibitors, inhibitory antibodies, antibodies directed against growth factors, bifunctional molecules consisting of a growth factor and a cytotoxin, bifunctional molecules consisting of an antibody and a cytotoxin; (h) protein kinase and tyrosine kinase inhibitors (e.g., tyrphostins, genistein, quinoxalines); (i) prostacyclin analogs; (j) cholesterol-lowering agents; (k) angiopoietins; (1) antimicrobial agents such as triclosan, cephalosporins, aminoglycosides and nitrofurantoin; (m) cytotoxic agents, cytostatic agents and cell proliferation affectors; (n) vasodilating agents; (o) agents that interfere with endogenous vasoactive mechanisms; (p) inhibitors of leukocyte recruitment, such as monoclonal antibodies; (q) cytokines; (r) hormones; (s) inhibitors of HSP 90 protein (i.e., Heat Shock Protein, which is a molecular chaperone or housekeeping protein and is needed for the stability and function of other client proteins/signal transduction proteins responsible for growth and survival of cells) including geldanamycin, (t) beta-blockers, (u) bARKct inhibitors, (v) phospholamban inhibitors, (w) Serca 2 gene/protein, and (x) other non-genetic therapeutic agents, including arginine, 2-nitrorethanol, sirolimus, everolimus, tacrolimus, Epo D, estradiol, halofuginone, cilostazole, geldanamycin, ABT-578 (Abbott Laboratories), trapidil, liprostin, Actinomcin D, Resten-NG, Ap-17, abciximab, clopidogrel and Ridogrel.

[0066] Genetic therapeutic agents for use in conjunction with the present invention include anti- sense DNA and RNA as well as DNA coding for the various proteins (as well as the proteins themselves): (a) anti-sense RNA, (b) tRNA or rRNA to replace defective or deficient endogenous molecules, (c) angiogenic and other factors including growth factors, (d) cell cycle inhibitors including CD inhibitors, and (e) thymidine kinase ("TK") and other agents useful for interfering with cell proliferation.

[0067] Cells for use in conjunction with the present invention include cells of human origin (autologous or allogeneic), including whole bone marrow, bone marrow derived mono-nuclear cells, progenitor cells (e.g., endothelial progenitor cells), stem cells (e.g., mesenchymal, hematopoietic, neuronal), pluripotent stem cells, fibroblasts, myoblasts, satellite cells, pericytes, cardiomyocytes, skeletal myocytes or macrophage, or from an animal, bacterial or fungal source (xenogeneic), which can be genetically engineered, if desired, to deliver proteins of interest.

[0068] Additional therapeutic agents, not necessarily exclusive of those listed above, have been identified as candidates for vascular treatment regimens, for example, as agents targeting restenosis. Such agents are useful for the practice of the present invention and include one or more of the following: (a) Ca-channel blockers including benzothiazapines such as diltiazem and clentiazem, dihydropyridines such as nifedipine, amlodipine and nicardapine, and phenylalkylamines such as verapamil, (b) serotonin pathway modulators including: 5-HT antagonists such as ketanserin and naftidrofuryl, as well as 5-HT uptake inhibitors such as fluoxetine, (c) cyclic nucleotide pathway agents including phosphodiesterase inhibitors such as cilostazole and dipyridamole, adenylate/Guanylate cyclase stimulants such as forskolin, as well as adenosine analogs, (d) catecholamine modulators including α-antagonists such as prazosin and bunazosine, β-antagonists such as propranolol and α/β-antagonists such as labetalol and carvedilol, (e) endothelin receptor antagonists, (f) nitric oxide donors/releasing molecules and precursors including organic nitrates/nitrites such as nitroglycerin, isosorbide dinitrate and amyl nitrite, inorganic nitroso compounds such as sodium nitroprusside, sydnonimines such as molsidomine and linsidomine, nonoates such as diazenium diolates and NO adducts of alkanediamines, S-nitroso compounds including low molecular weight compounds (e.g., S-nitroso derivatives of captopril, glutathione and N-acetyl penicillamine) and high molecular weight compounds (e.g., S- nitroso derivatives of proteins, peptides, oligosaccharides, polysaccharides, synthetic polymers/oligomers and natural polymers/oligomers), as well as C-nitroso-compounds, O-nitroso-compounds, N-nitroso-compounds and L-arginine, (g) ACE inhibitors such as cilazapril, fosinopril and enalapril, (h) ATII-receptor antagonists such as saralasin and losartin, (i) platelet adhesion inhibitors such as albumin and polyethylene oxide, (O) platelet aggregation inhibitors including cilostazole, aspirin and thienopyridine (ticlopidine, clopidogrel) and GP Ilb/IIIa inhibitors such as abciximab, epitifibatide and tirofiban, (k) coagulation pathway modulators including heparinoids such as heparin, low molecular weight heparin, dextran sulfate and β-cyclodextrin tetradecasulfate, thrombin inhibitors such as hirudin, hirulog, PPACK(D-phe-L-propyl-L-arg- chloromethylketone) and argatroban, FXa inhibitors such as antistatin and TAP (tick anticoagulant peptide), Vitamin K inhibitors such as warfarin, as well as activated protein C, (1) cyclooxygenase pathway inhibitors such as aspirin, ibuprofen, flurbiprofen, indomethacin and sulfinpyrazone, (m) natural and synthetic corticosteroids such as dexamethasone, prednisolone, methprednisolone and hydrocortisone, (n) lipoxygenase pathway inhibitors such as nordihydroguairetic acid and caffeic acid, (o) leukotriene receptor antagonists, (p) antagonists of E- and P-selectins, (q) inhibitors of VCAM-I and ICAM-I interactions, (r) prostaglandins and analogs thereof including prostaglandins such as PGEl and PGI2 and prostacyclin analogs such as ciprostene, epoprostenol, carbacyclin, iloprost and beraprost, (s) macrophage activation preventers including bisphosphonates, (t) HMG-CoA reductase inhibitors such as lovastatin, pravastatin, fluvastatin, simvastatin and cerivastatin, (u) fish oils and omega-3-fatty acids, (v) free -radical sc avenger s/antioxidants such as probucol, vitamins C and E, ebselen, trans-retinoic acid and SOD mimics, (w) agents affecting various growth factors including FGF pathway agents such as bFGF antibodies and chimeric fusion proteins, PDGF receptor antagonists such as trapidil, IGF pathway agents including somatostatin analogs such as angiopeptin and ocreotide, TGF-β pathway agents such as polyanionic agents (heparin, fucoidin), decorin, and TGF-β antibodies, EGF pathway agents such as EGF antibodies, receptor antagonists and chimeric fusion proteins, TNF- α pathway agents such as thalidomide and analogs thereof, Thromboxane A2 (TXA2) pathway modulators such as sulotroban, vapiprost, dazoxiben and ridogrel, as well as protein tyrosine kinase inhibitors such as tyrphostin, genistein and quinoxaline derivatives, (x) MMP pathway inhibitors such as marimastat, ilomastat and metastat, (y) cell motility inhibitors such as cytochalasin B, (z) antiproliferative/antineoplastic agents including antimetabolites such as purine analogs (e.g., 6-mercaptopurine or cladribine, which is a chlorinated purine nucleoside analog), pyrimidine analogs (e.g., cytarabine and 5-fluorouracil) and methotrexate, nitrogen mustards, alkyl sulfonates, ethylenimines, antibiotics (e.g., daunorubicin, doxorubicin), nitrosoureas, cisplatin, agents affecting microtubule dynamics (e.g., vinblastine, vincristine, colchicine, Epo D, paclitaxel and epothilone), caspase activators, proteasome inhibitors, angiogenesis inhibitors (e.g., endostatin, angiostatin and squalamine), rapamycin, cerivastatin, flavopiridol and suramin, (aa) matrix deposition/organization pathway inhibitors such as halofuginone or other quinazolinone derivatives and tranilast, (bb) endothelialization facilitators such as VEGF and RGD peptide, and (cc) blood rheology modulators such as pentoxifylline.

[0069] Numerous additional therapeutic agents useful for the practice of the present invention are also disclosed in U.S. Pat. No. 5,733,925 assigned to NeoRx Corporation, the entire disclosure of which is incorporated by reference.

[0070] The amount of therapeutic agent present in the polymers of the present invention is not limited. In some embodiments, however, the amount of therapeutic agent present in the polymers of the present invention is a therapeutically effective amount. The language "therapeutically effective amount" is that amount necessary or sufficient to produce the desired physiologic response. The effective amount may vary depending on such factors as the size and weight of the subject, or the particular compound. The effective amount may be determined through consideration of the toxicity and therapeutic efficacy of the compounds by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it may be expressed as the ratio LD50/ED50. Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to unaffected cells and, thereby, reduce side effects.

Synthesis of Copolymers of the Present Invention

[0071] In some aspects, the present invention is directed to methods of making the copolymers (e.g., block copolymers) described herein, e.g., sulfonate- and/or bioactive molecule-modified copolymers. The method for making a sulfonate-modified block copolymer generally includes contacting a modifiable monomer species with a sulfonyl chloride, under suitable conditions such that a sulfonate-modified block copolymer is formed. The method for making a bioactive molecule-modified block copolymer generally includes contacting a sulfonate-modified block copolymer with a peptide, under suitable conditions such that a peptide-modified block copolymer is formed.

[0072] In some embodiments, the method for making a sulfonate-modified block copolymer includes dissolving a copolymer comprising at least one modifiable monomer species with a suitable solvent; and contacting the dissolved copolymer with a sulfonyl chloride, under suitable conditions such that a sulfonate-modified block copolymer is formed. The modifiable monomer species can be any of the modifiable monomer species described herein, e.g., a monomer which includes a pendant hydroxyl moiety. The sulfonyl chloride can be any of the sulfonyl chlorides described herein, e.g., fosyl chloride.

[0073] In some embodiments, the method for making a sulfonate-modified block copolymer includes contacting a copolymer comprising at least one modifiable monomer species with a sulfonyl chloride, under suitable heterogeneous conditions such that a sulfonate-modified block copolymer is formed. The modifiable monomer species can be any of the modifiable monomer species described herein, e.g., a monomer which includes a pendant hydroxyl moiety. The sulfonyl chloride can be any of the sulfonyl chlorides described herein, e.g., fosyl chloride. As used herein, the term "heterogeneous conditions" refers to the fact that the copolymer comprising at least one modifiable monomer species is not dissolved prior to contact with the sulfonyl chloride.

[0074] The monomer species covalently bonded to a sulfonate moiety can be a monomer species which has been modified with any of the sulfonyl chlorides described herein, e.g., can be a 4-fluorobenzenesulfonyl ethyl methacrylate monomer species. The copolymer can additionally include any of the monomers described herein, e.g., isobutylene. In some embodiments, the sulfonate-modified block copolymer is modified internally. In some embodiments, the sulfonate-modified block copolymer is modified on the surface. In some embodiments, the sulfonate-modified block copolymer is modified both internally and on the surface.

[0075] In some embodiments, the method for making a bioactive molecule- modified block copolymer generally includes dissolving a sulfonate-modified block copolymer with a suitable solvent; and contacting the dissolved copolymer with a bioactive molecule, e.g., a peptide, under suitable conditions such that a peptide- modified block copolymer is formed.

[0076] The monomer species covalently bonded to a peptidyl moiety can be a monomer species which has been modified with any of the peptidyl chlorides described herein, e.g., the peptide can be any peptide known in the art, e.g., GYIGSR or a peptide which includes the YIGSR sequence. The copolymer can additionally include any of the monomers described herein, e.g., isobutylene. In some embodiments, the peptidyl- modified block copolymer is modified internally. In some embodiments, the bioactive molecule-modified block copolymer is modified on the surface. In some embodiments, the bioactive molecule-modified block copolymer is modified both internally and on the surface.

[0077] The copolymers used in the syntheses of the present invention may be any of the polymers provided herein, e.g., may be thermoplastic elastomers, provided that they include at least one modifiable monomer species.

[0078] Various methods have previously been used for chemically coupling biologically active ligands to hydroxyl bearing polymeric carriers. All of the methods involve activation of the -OH groups for subsequent coupling via the amino terminus. The most widely use method is the cyanogen bromide (CNBr) method that yields the active cyanate ester precursor that readily reacts with the amino group. This method has several limitations including the poor stability of the linking group and the high toxicity of CNBr. The method used in the present invention, the use of highly reactive sulfonyl chlorides, e.g., 4-fluorobenzenesulfonyl chloride (fosyl chloride), effect rapid, and close to quantitative activation with excellent bond stability and low toxicity of coupling agents.

[0079] In some embodiments, periodic, random, statistical or gradient copolymers are used as starting materials for the synthesis of activated polymers of the present invention. In such cases, the density of the activating groups (e.g., the sulfonyl moieties) within the resulting activated copolymer (and ultimately the density of the covalently attached peptides), can be varied by varying the ratio of monomers that contain activating groups relative to those that do not.

[0080] In other embodiments, block copolymers are used as starting materials for the synthesis of activated polymers of the present invention. In some embodiments, the copolymers which act as starting materials for synthesizing the activated polymers of the present invention can be prepared via the combination of living cationic polymerization and living anionic polymerization. Hence, copolymers containing one or more cationically polymerized blocks and one or more anionically polymerized blocks can be formed. Such polymerization can be carried out using techniques such as those described in U.S. Patent No. 7,056,985, the entire contents of which are incorporated herein by this reference.

[0081] In some embodiments, diblock copolymers, triblock copolymers and/or radial- shaped block copolymers are prepared using monofunctional, difunctional or multifunctional polymers, respectively. In some embodiments, end-functionalized polymers are used to synthesize multifunctional polymers, e.g., star polymers such as polyisobutylene stars, for example, by reacting the polymer (e.g., a polyisomonoolefin) with coupling molecules such as unhindered chlorosilanes. Chlorosilanes have been used previously to couple living anionic chain ends to form star polymers in Roovers, J. E. L. and S. Bywater, Macromolecules 1972, 5, 385 and in U.S. Application Publication No. 20050143526. Triblock copolymers and radial-shaped block copolymers typically exhibit elastomeric properties which are dependant upon the composition of polymer segments in the blocks.

[0082] With respect to block copolymers having modifiable monomer species, the blocks containing such modifiable species may also contain monomer species which are not able to react with sulfonyl chloride (e.g., ones which possess no pendant hydroxyl moieties). Regarding such blocks, the number of modifiable species within the polymer (and ultimately the number covalently attached peptides), can be varied by varying the length of the blocks containing the modifiable species and/or the density of the modifiable species within such blocks (e.g., where the block also contains monomers devoid of modifiable species). [0100] In other embodiments of the invention, multiblock copolymers can be formed using coupling molecules such as (di- or trichloromethyl)benzene or (di- or tribromomethyl)benzene (See, e.g., U.S. Application Publication No. 20050143526).

[0083] Regardless of the synthesis of the starting materials, once obtained, the copolymer comprising at least one modifiable monomer species can be reacted with a sulfonyl chloride to form a sulfonate-modified copolymer of the present invention.

[0084] Sulfonyl chlorides employed in connection with forming the activated copolymers of the present invention are typically either known compounds or compounds that can be prepared from known compounds by conventional synthetic procedures. Such compounds are typically prepared from the corresponding sulfonic acid, using phosphorous trichloride and phosphorous pentachloride.

[0085] Examples of sulfonyl chlorides suitable for use in this invention include, but are not limited to, methanesulfonyl chloride, 2-propanesulfonyl chloride, 1- butanesulfonyl chloride, benzenesulfonyl chloride, 1-naphthalenesulfonyl chloride, 2- naphthalenesulfonyl chloride, p-toluenesulfonyl chloride, α-toluenesulfonyl chloride, A- acetamidobenzenesulfonyl chloride, 4-amidinobenzenesulfonyl chloride, 4-tert- butylbenzenesulfonyl chloride, 4-bromobenzenesulfonyl chloride, 2- carboxybenzenesulfonyl chloride, 4-cyanobenzenesulfonyl chloride, 3,4- dichlorobenzenesulfonyl chloride, 3,5-dichlorobenzenesulfonyl chloride, 3,4- dimethoxybenzensulfonyl chloride, 3,5-ditrifluoromethylbenzenesulfonyl chloride, A- fluorobenzenesulfonyl chloride, 4-methoxybenzenesulfonyl chloride, 2- methoxycarbonylbenzenesulfonyl chloride, 4-methylamidobenzenesulfonyl chloride, A- nitrobenzenesulfonyl chloride, 4-thioamidobenzenesulfonyl chloride, A- trifluoromethylbenzenesulfonyl chloride, 4-trifluoromethoxybenzenesulfonyl chloride, 2,4,6-trimethylbenzenesulfonyl chloride, 2-phenylethanesulfonyl chloride, 2- thiophenesulfonyl chloride, 5-chloro-2-thiophenesulfonyl chloride, 2,5-dichloro-4- thiophenesulfonyl chloride, 2-thiazolesulfonyl chloride, 2-methyl-4-thiazolesulfonyl chloride, l-methyl-4-imidazolesulfonyl chloride, l-methyl-4-pyrazolesulfonyl chloride, 5-chloro-l,3-dimethyl-4-pyrazolesulfonyl chloride, 3-pyridinesulfonyl chloride, 2- pyrimidinesulfonyl chloride and the like. If desired, a sulfonyl fluoride, sulfonyl bromide or sulfonic acid anhydride may be used in place of the sulfonyl chloride in the reactions of the present invention. [0086] The various reactions of the present invention are typically carried out in the presence of a diluent or a mixture of diluents. For example, the polymerization of the block copolymers used as starting materials for the synthesis of activated polymers of the present invention is typically carried out in a diluent or a mixture of diluents, which include (a) halogenated hydrocarbons which contain from 1 to 4 carbon atoms per molecule, such as methyl chloride and methylene dichloride, (b) aliphatic hydrocarbons and cycloaliphatic hydrocarbons which contain from 5 to 10 carbon atoms per molecule, such pentane, hexane, heptane, cyclohexane and methyl cyclohexane, or (c) mixtures thereof. In some embodiments, such polymerization is carried out in a mixture of a polar solvent and a non-polar solvent. The activation of the starting material (i.e., to form an activated copolymer of the present invention) is typically carried out in the presence of diluent or mixture of diluents, including amines such as dimethylaminopyridine, aliphatic hydrocarbons such as hexanes, acetonitrile, ethers such as tetrahydrofuran, and N,N-dimethylformamide. Additionally, the conversion of an activated copolymer of the present invention to a bioactive molecule-modified copolymer of the present invention is typically carried out in the presence of a diluent or mixture of diluents including, but not limited to, halogenated hydrocarbons which contain from 1 to 4 carbon atoms per molecule, such as chloroform, methyl chloride and methylene dichloride, alcohols such as methanol, water, and acetonitrile.

[0087] The various reactions of the present invention are typically carried out under varying temperatures for varying periods of time. For example, in some embodiments, temperatures employed in the polymerization of the monomers range from 0⁰C to - 150⁰C. In other embodiments, temperatures employed in the polymerization of the monomers range from -10⁰C to -90⁰C. In some embodiments, the reaction time for the polymerization of the monomers ranges from a few minutes to 24 hours. In other embodiments, the reaction time for the polymerization of the monomers ranges from 10 minutes to 10 hours.

[0088] In some embodiments, temperatures employed in the sulfonate modification range from -50⁰C to 50⁰C. In other embodiments, temperatures employed in the sulfonate modification range from -25°C to 25°C. In some embodiments, the reaction time for the sulfonate modification ranges from 10 minutes to 48 hours. In other embodiments, the reaction time for the sulfonate modification ranges from 6 hours to 18 hours. [0089] In some embodiments, temperatures employed in the bioactive molecule modification range from 0⁰C to 75°C. In some embodiments, temperatures for the bioactive molecule modification range from 10⁰C to 40⁰C. In some embodiments, reaction time for the bioactive molecule modification ranges from 1 hour to 96 hours. In other embodiments, reaction time for the bioactive molecule modification ranges from about 12 hours to about 36 hours.

[0090] In addition to the above techniques, in which copolymers containing sulfonate moieties are reacted with bioactive molecule, bioactive molecule-modified copolymers in accordance with the present invention may also be formed by polymerizing monomers that contain one or more bioactive molecule covalently linked via a covalent bond. That is, the bioactive molecule-modified copolymers of the present invention may also be formed by polymerizing bioactive molecule-modified monomer species.

[0091] In some embodiments, copolymers are formed, for example, by polymerizing one or more monomers, each containing one or more covalently linked bioactive molecules, with one or more additional monomers, each devoid of a covalently linked bioactive molecule. Such monomers may be reacted simultaneously (leading, for example, to periodic, random, statistical or gradient copolymers) or sequentially (leading, for example, to block copolymers). Depending on the bioactive molecule, however, various groups may require protection prior to such polymerization.

[0092] Specific examples of monomers with covalently linked bioactive molecules include those formed by contacting modifiable monomers, such as the methacrylate monomers described above, with a sulfonyl chloride followed by contacting the resultant sulfonate-modified monomer with a bioactive molecule that contains primary and/or secondary amine groups (e.g., peptides).

[0093] Where addition polymerization techniques are employed (e.g., cationic polymerization reactions, such as those described above in connection with copolymers used as starting materials), polymer chains are commonly created which contain a saturated (e.g., in the case of olefin or vinyl polymerization) or unsaturated (e.g., in the case of diolefin polymerization) carbon backbone. Depending on the monomer(s) employed, the carbon backbones can have a wide range of pendant groups in addition to the pendant groups of the modifiable monomer species. Specific examples include pendant substituted and unsubstituted alkyl groups (e.g., where various aliphatic olefins and dienes are employed), pendant substituted and unsubstituted aromatic groups (e.g., where various vinyl aromatic monomers are employed), pendant ethers (e.g., where various vinyl ethers are employed), pendant silane group (e.g., where various silane monomers are employed), and so forth.

[0094] Regardless of the technique employed, it is apparent from the above description that a wide variety of bioactive molecule-modified copolymers in accordance with the present invention can be formed.

Articles of Manufacture

[0095] In some aspects, the present invention is directed to articles of manufacture which include the copolymers described herein. For example, the block copolymers of the present invention can be employed as new biomaterials.

[0096] Bioactive molecule-modified copolymers of the present invention can be administered to a wide variety of subjects, including, but not limited to mammals such as humans and domestic mammals such as cattle, sheep, pigs, goats, horses, camels, buffalo, dogs, cats, and birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, for a variety of therapeutic purposes. [0097] By "therapeutic purpose" is meant an improvement in the size or health of the subject, including the treatment of one or more diseases, pests or conditions. As used herein, "treatment" refers to the prevention of the disease or condition, the reduction or elimination of symptoms associated with the disease or condition, or the substantial or complete elimination of the disease or condition. [0098] In some embodiments, the article of manufacture is an insertable or implantable medical device. Examples of implantable or insertable medical devices include, for example, endotracheal tubes, tracheostomy tubes, wound drainage devices, wound dressings, implants, intravenous catheters, medical adhesives, shunts, gastrostomy tubes, medical tubing, cardiovascular products, heart valves, urine collection devices, catheters (e.g., renal or vascular catheters such as balloon catheters), guide wires, balloons, filters (e.g., vena cava filters), stents (including coronary vascular stents, peripheral vascular stents, cerebral, urethral, ureteral, biliary, tracheal, gastrointestinal and esophageal stents), stent grafts, vascular grafts, vascular access ports, embolization devices including cerebral aneurysm filler coils (including Guglilmi detachable coils and metal coils), myocardial plugs, patches, pacemakers and pacemaker leads, left ventricular assist hearts and pumps, total artificial hearts, heart valves, vascular valves, biopsy devices, and any coated substrate (which can comprise, for example, glass, metal, polymer, ceramic and combinations thereof) that is implanted or inserted into the body and from which therapeutic agent is released. Examples of medical devices further include patches for delivery of therapeutic agent to intact skin and broken skin (including wounds); sutures, suture anchors, anastomosis clips and rings, tissue staples and ligating clips at surgical sites; cannulae, metal wire ligatures, orthopedic prosthesis such as bone grafts, bone plates, joint prostheses, orthopedic fixation devices such as interference screws in the ankle, knee, and hand areas, tacks for ligament attachment and meniscal repair, rods and pins for fracture fixation, screws and plates for craniomaxillofacial repair; dental devices such as void fillers following tooth extraction and guided-tissue-regeneration membrane films following periodontal surgery; tissue bulking devices, and tissue engineering scaffolds for cartilage, bone, skin and other in vivo tissue regeneration. Specific examples of implantable or insertable medical devices for use in conjunction with the present invention include vascular stents, such as coronary stents and cerebral stents.

[0099] In some aspects, the present invention is directed to a targeted stent. In general targeted stents allow for the adhesion of certain cells, e.g., endothelial cells, while excluding other cells, e.g., smooth muscle cells. Such stents can be formulated using the modified block copolymers of the present invention, e.g., block copolymers having bioactive molecule modification on the surface and an internal buffer modification. Without wishing to be bound by any particular theory, it is believed that the ability to target cells, e.g., endothelial cells, would be advantageous because other cells may interact poorly with the stent. For example, adhesion of smooth muscle cells to a stent may cause the holes in the stent to close.

[00100] In some embodiments, one or more layers of the copolymers of the present invention are formed over all or a portion of an underlying medical device substrate. Layers can be provided over an underlying substrate at a variety of locations, and in a variety of shapes. Materials for use as underlying medical device substrates include ceramic, metallic and polymeric substrates. The substrate material can also be a carbon- or silicon-based material, among others. As used herein a "layer" of a given material is a region of that material whose thickness is small compared to both its length and width. As used herein a layer need not be planar, for example, taking on the contours of an underlying substrate. Layers can be discontinuous (e.g., patterned). Terms such as "film," "layer" and "coating" may be used interchangeably herein.

[00101] In some embodiments, the articles of manufacture include medical devices from which a therapeutic agent is released. Such therapeutic agents may be, e.g., trapped within the polymer system or attached to the polymer itself as indicated above. In such embodiments where the therapeutic agent is attached to the polymer itself, it is understood that the therapeutic agent will have a release profile (e.g., is able to be released via, for instance, hydrolysis or enzymatic cleavage. Accordingly, in some embodiments, compositions of the present invention include a therapeutic agent and exhibit an appropriate release profile. Such compositions and materials are also useful as medical drug eluting articles and drug eluting coatings.

[00102] In some embodiments, copolymers of the invention can be dried and melt processed, for example, by injection molding and extrusion. Compositions used for this method can be used alone or compounded with any other melt-proces sable material for molding and extrusion of antimicrobial articles.

[00103] For example, where the copolymers of the present invention and/or any other supplemental materials to be processed have thermoplastic characteristics, and so long as the copolymers and any other supplemental materials are sufficiently stable (e.g., so as to avoid substantial reaction/degradation during processing, including degradation of the above described covalent linkages between the polymer and the bioactive molecule), a variety of standard thermoplastic processing techniques may be used to form the articles of manufacture, including compression molding, injection molding, blow molding, spinning, vacuum forming and calendaring, as well as extrusion into sheets, fibers, rods, tubes and other cross-sectional profiles of various lengths. Using these and other thermoplastic processing techniques, entire devices or portions thereof can be made.

[00104] The copolymers of the invention can also be coated onto preformed articles. When used as a coating, the copolymers can be applied by any means, including those methods known in the art. For example, a composition comprising the copolymers of the invention can be brushed or sprayed onto the article from a solution, or the article can be dipped into the solution containing the copolymers of the invention.

[00105] In other embodiments, solvent-based techniques are used to coat articles of manufacture with the copolymers of the present invention. Using these techniques, coatings can be formed by first providing solutions that contain the copolymers of the present invention (and/or any other supplemental materials to be processed), and subsequently removing the solvents to form the coating. The solvents that are ultimately selected will contain one or more solvent species, which are generally selected based on their ability to dissolve the materials that form the coating, as well as other factors, including drying rate, surface tension, etc. Moreover, as above, the solutions and processing conditions that are employed are generally selected to ensure the stability of the copolymers and any other supplemental materials that are present. Preferred solvent-based techniques include, but are not limited to, solvent casting techniques, spin coating techniques, web coating techniques, solvent spraying techniques, dipping techniques, techniques involving coating via mechanical suspension including air suspension, ink jet techniques, electrostatic techniques, and combinations of these processes.

EQUIVALENTS

[00106] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

[0100] The contents of all references, patents, and patent applications cited throughout this application are hereby incorporated herein by this reference.

EXEMPLIFICATION

Materials

[0101] 4-Dimethylaminopyridine (Aldrich, 99%), Pyridine, anhydrous (Aldrich,

99.8%), 4-Fluorobenzenesulfonyl chloride (Fluka, >98%), Dansylhydrazine (Sigma, 90- 95%), tris borate-EDTA buffer concentrate {Fluka, 1.3 M), Gly-Gly (Aldrich, 99%), GYIGSR (Creosalus Inc, 95%), Acetonitrile, anhydrous (Aldrich, 99.8%) and Chloroform (Aldrich, >99.8%) were used as received. Stainless Steel-AISI 316L (Fe/Crl8/Nil0/Mo 3) Foils and Stainless Steel Strips (APT SF&SP, WR 825-00) were purchased from Goodfellow Cambridge Limited and Arrow Cryogenics Inc. respectively.

Measurements

[0102] Nuclear Magnetic Resonance (NMR) spectroscopy was carried out on a

Bruker 500 MHz spectrometer using CDCl₃ as a solvent. ¹H NMR spectra of solutions in CDCI₃ were calibrated to tetramethylsilane (TMS) as internal standard (5_H 0.00). Confocal micrographs were recorded using Olympus Fluo View FVlOOO Confocal Laser Scanning Microscope. A JOEL model JSM 7401F field emission scanning electron microscope was used for SEM imaging. Magnifications of x35 and x350 were used to record the micrographs. The FT-IR spectra of the films were recorded in AVATAR 370 FT-IR ThermoNicolet spectrometer using sodium chloride pellets.

[0103] Surface analysis of the polymer films was performed with X-ray

Photoelectron Spectroscopy (XPS) using a Vacuum Generators ESCALAB MK II photoelectron spectrometer having base pressures in the low 10^"10 mbar range. The spectrometer was equipped with a Mg KR X-ray source (hv = 1253.6 eV) and a concentric hemispherical analyzer operating in constant pass energy mode and detecting photoelectrons approximately normal to the sample plane. The photoelectrons were energy-analyzed with a concentric hemispherical analyzer in fixed analyzer transmission mode using a pass energy of 20 eV. All specimens for surface analysis were prepared by spin coating from chloroform solutions (5 wt %) onto steel alloy disks. No corrections for surface charging (which results in the XPS peaks shifting to higher binding energies) have been made. To a first approximation, all peaks and peak components shift by the same amount due to this effect. Data processing and curve fitting were performed using Avantage software for surface chemical analysis.

[0104] Thickness of the films was measured using Veeco White-Light

Interferometer under VSI mode. Molecular weights and molecular weight distributions of polymers were measured at room temperature using a Waters HPLC system equipped with a model 510 HPLC pump, model 410 differential refractometer, model 486 UV/visible detector, model 712 sample processor, and five Waters ultraStyragel^® columns connected in the series (500, 10³, 10⁴,10⁵ and 100 A). THF was used as an eluent at a flow rate of 1 niL/min. The films were coated on steel strips using a Gardeners Co. 5 mil blade. The films on circular discs were made by spin coating using 450 revolutions per minute.

Synthesis of block copolymers

[0105] The syntheses of block copolymers, P[HEMA(25)-co-MMA(75)]-b-PIB- b-P[MMA(75)-co-HEMA(25)] (Pl), P[HEMA(50)-co-MMA(50)]-b-PIB-b- P[MMA(50)-co-HEMA(50)] (P2), P[HEMA(75)-co-MMA(25)]-b-PIB-b-P[MMA(25)- co-HEMA(75)] (P3) and PHEMA-b-PIB-b-PHEMA (P4) were synthesized as described in, e.g., "Synthesis, Properties and Surface Characterization of Polyisobutylene-Based Thermoplastic Elastomers Containing Poly(methyl methacrylate-co-hydroxyethyl methacrylate) as Hard Blocks," Feng, D.; Chandekar, A.; Whitten, J.; Faust, R. Polym. Prepr. 48(2):1017-18 (2007), and "Synthesis and Characterization of Poly(methyl methacrylate-co-hydroxyethyl methacrylate)-b- polyisobutylene-b-poly(methyl methacrylate-co-hydroxyethyl methacrylate) Triblock Copolymers," D. Feng, A. Chandekar, J. E. Whitten, R. Faust, J. Macromol. Sci. Pure Appl. Chem. A, 44, 1141-1150 (2007). These publications are incorporated herein in their entirety by this reference, including, but not limited to, the descriptions of the synthesis of the block copolymers. The polymers exhibited the following characteristics: Pl: M_n= 110,800; PD = 1.21; PIB wt% = 55.5; P2: M_n = 96,000; PD = 1.19; PIB wt% = 63.9; P3: M_n = 98,400; PD = 1.22; PIB wt% = 67.2; P4: M_n = 101,300; PD = 1.19; PIB wt% = 62.6.

4

Scheme 1: Fosylation of Pn (n = 1-4) followed by peptide attachment [0106] The activation reactions were performed in pyridine in the presence of dimethylaminopyridine (DMAP) and fosylchloride at O⁰C. See, e.g., scheme 2, below:

PF_n, n = 1 ,2,3 and 4

Scheme 2: Fosylation of Pn (n = 1-4)

[0107] For example, P4 (109 mg, 0.001 mmol) was dissolved in anhydrous pyridine (3.5 mL, 43 mmol) by stirring the mixture at room temperature for 72 hours under nitrogen atmosphere. To the mixture, DMAP (464 mg, 3.8 mmol) was added. Once a clear solution was obtained, the temperature of the solution was decreased to 0⁰C. The stirred mixture was charged with fosyl chloride (800 mg, 4.1 mmol) and was further stirred for 12 hours. The reaction was quenched by adding 2 mL of distilled water and the solid precipitate was filtered. The residue was washed repeatedly with water, methanol and acetonitrile to remove low molecular weight organic impurities. The activated polymer (PF4) obtained was dried in vacuum for 3 hours at room temperature. ¹H NMR (CDCl₃, ppm, δ): 8.0 (s, 2H), 7.25 (s, 2H), 4.2 (s, 2H), 4.15 (s, 2H), 3.8 (s, 2H), 3.6 (s, 3H), 1.55, 1.4, 1.1. ¹³C NMR (CDCl₃, ppm, δ): 177.5, 167.3, 165.2, 132.2, 131.3, 117.3, 62.6, 60.0, 54.5, 52.3, 45.2, 38.6, 31.7. FT-IR (thin film, cm^{" 1}Y. 2985.0, 1739.0, 1592.5, 1495.6, 1363.5, 1237.0, 919.6, 841.1, 747.8, 685.9.

[0108] The same reaction was carried out with Pl, P2, and P3. The extents of activation were: PFl: 81%, PF2: 83%, PF3: 83%, PF4: 90%.

[0109] As can be seen in Figure Ia, ¹H NMR spectra showed new resonances at

8.0, 7.3 and 4.3 ppm for phenyl and fosyloxymethyl protons indicating the product formation. Additionally, ¹H NMR spectroscopy suggested 80-90% activation of the hydroxyl groups with an overall yield of 70-80% for the different block copolymers. The ratio of peaks at 4.3 (-CH₂OSO₂C₆H₄F) and 3.8 (-CH₂CH₂OH) ppm were considered to determine the extent of activation. The results showed 80-90% activation for different block copolymers with 70-80% of overall yield. In the ¹³C NMR spectra, peaks around 130 and 60 ppm further confirmed the fosylation (Figure Ib).

[0110] The fosylated polymers (PF1-PF4) were soluble in chloroform, tetrahydrofuran and dichloromethane. Their chloroform solutions were used to coat thin films on smooth steel strips and discs. SEM analysis indicated that the films are continuous and replicate the morphology of the steel strip as shown in Figure Ic. Figure Ic (a and b) shows the surface topography of polymer films at different magnifications and Figure Ic (c and d) shows that of the steel strip at different magnification.

[0111] As shown in Figure 3, the thickness of the drawn down films, determined by a white light interferometer, was in the range of 3-3.3 μm as predicted (theoretical value=3.5 μm). To assess the distribution and homogeneity of the activating groups present on the film surface, a fluorescent dye (dansyl hydrazine) was attached to the surface of the film. The PF3 film on a steel strip was dipped in a solution of dye in methanol/water 4/1, v/v mixture (pH 9) for 24 h. The morphology of the polymer films analyzed using an optical microscope were found to replicate the surface morphology of the steel strip. Fluorescence microscopy revealed uniform distribution of the dye on the surface, however, rapid photo bleaching was observed under the mercury lamp. Confocal fluorescence microscopy also showed uniform and effective attachment of the dye all over the polymer surface, as bright fluorescence through out the film was observed when irradiated with a laser of 350 nm (see, e.g., Figure 2a). Interestingly, the dye penetrated through the whole film as indicated by Z-stacking (see, e.g., Figure 2c), which may be attributed to the swelling of the film and similarity in the polarity of the hard block and the dye. Fluorescence was not detected on films, which were not dipped in the dye solution (Figure 2b). Fluorescence microscopy revealed uniform distribution of the dye on the surface, however, rapid photo bleaching was observed under the mercury lamp.

[0112] NMR spectroscopy was further employed to further analyze the presence of activating group. For this purpose, the film was dipped in a solution of n-butylamine in methanol/water (pH 9) for 16 hours at room temperature. The film was then dissolved in CDCl₃ and the ¹H NMR spectrum was recorded. The spectrum showed complete removal of activating groups and the attachment of n-butylamine, which also indicated that small organic molecules can easily penetrate the film and react with the activating groups not only on the surface but also in the bulk.

[0113] The presence of activating group on the surface was investigated using X- ray photoelectron spectroscopy. The surface elemental compositions of the copolymer films were calculated from the CIs, S2p and Ols XPS peak areas using appropriate sensitivity factors, and the results are summarized in Table 1. Figures 4 and 5 show CIs and S2p spectra, respectively, of the polymers PFl, PF2, PF3 and PF4 at a 90° take-off angle. These polymers differ in their MMA/FEMA molar ratios. Deconvolution of the CIs spectra indicates four distinct peaks corresponding to the four different carbon species present in the polymers (Figure 4). In increasing order of binding energy, these peaks may be assigned to carbon bonded to hydrogen (C-H), to carbon single-bonded to oxygen (C-O), to carbon single bonded to sulfonate (C-SO₃) and to carbon double -bonded to oxygen (C=O) at observed binding energies of 287.3, 288.8, 290.0 and 291.5 eV respectively. The new peak at 290.0 eV confirms the attachment of fosylate group to the parent polymer (PFn). The peaks appear at higher binding energies than reported in the literature because no correction have been made for surface charging. The peak due to the carbon attached to the fluorine (C-F), which is present in the fosylate group, overlaps with the peak due to C-O bond at 288.8 eV so this peak is not visible in the CIs spectra after the fosylate group attachment. S2p spectrum of these polymers shows the peak at (uncorrected) binding energies of 171.4 eV (Figure 5). This originates from the sulfonate group of the 4-fluorobenzenesulfonyl moiety. S2p region also shows a shoulder on the high energy side of the peak at 172.3 eV in case of PF3 and PF4 which arose due to spin orbit coupling in the S2p level. This spin- splitting is an indication of homogeneity of the sulfur on the surface confirming the homogenous attachment of the fosylate group on the surface. This spin- splitting is not clearly visible in case of PFl. The reason may be the amount of fosylate group, which is less on the surfaces of PFl due to lower initial percentage of FEMA in these polymers, the sulfur is not homogenously distributed over the surface. Based on the measured C/S ratios, a composition gradient was observed for the four samples. It was observed that, the amount of the activating groups present on the surface varied directly with the HEMA percentage in the triblock copolymers as expected.

Table 1. The element composition on the surface of PFl, PF2, PF3 and PF4 derived by

XPS Measurements.

Element PFΪ PF2 PF3 PF4

Surface^a Overall Surface Overall Surface Overall Surface Overall

C 90.59 79.19 92.16 79.40 91.40 79.12 91.68 76.06

S 0.56 1.79 0.79 2.49 0.88 2.80 1.96 3.77

O 8.85 19.02 7.05 18.11 7.72 18.08 6.36 20.17

S/C 0.0062 0.022 0.0085 0.031 0.0096 0.035 0.021 0.049 ratio a Atomic weight percent derived from XPS data using 90 ° TOA, Theoretical atomic composition of activated polymers

Example 1: Preparation of peptide-modified copolymers- solvent method

[0114] The peptides (GG and GYIGSR) were attached to the polymer according to the following procedures: 50 mg of the polymers (PF_n, n=l-4) were dissolved in 1 mL of chloroform. Three drops of the solution were placed on the surface of an electropolished stainless steel strip and was drawn down using a 5 mil blade (P. N. Gardner Co.). The film was kept at room temperature for 10 minutes to evaporate the excess solvent. In the second procedure the polymer was spin coated on a stainless steel disc using 2.5% solution at a speed of 450 rpm for 1 min. Both films were dried under vacuum for 2 h at room temperature to remove final traces of solvent. The films were then dipped in a 5 mL standard solution of peptide (15 mg) in carbonate-buffered solution (pH = 10) for 24 hours at room temperature. The films were then rinsed with distilled water four times and dried.

[0115] The atomic compositions of the activated and peptide bound surfaces were determined using XPS data recorded at 90 ° takeoff angle. Take-off angle (TOA) has been defined in different ways; here it refers to the angle between the plane of the sample and the normal to the entrance of the electron energy analyzer. Detection depth depends on the TOA (θ) and the inelastice mean free path (λ) of the escaping photoelectrons, with 95% of the photoelectron signal originating from a depth of 3 λ sin λ. CIs electrons ejected by MgKa X-rays have kinetic energy of about 970 eV, which corresponds to a mean free path (λ) of approximately 24A for electrons traversing through organic material. Therefore, for 90° and 30° TOAs, 95% of the XPS signal originates from less than about 7 and 4 nm, respectively. This is an approximate depth since the mean free path is material dependent, with mean free path values in the range of 30A also commonly reported for organic films.

[0116] Representative data for PF4, PGG4 and PYR4 are shown in Table 2, below. The difference between the surface and bulk composition of the PF4 films is expected due to the lower surface tension of PIB. The presence of sulfur (S) is related to the fosylate group attached to the HEMA segment. The presence of nitrogen (N) and higher level of oxygen (O) concentration for PGG4 and PYR4, indicated the successful attachment of the peptides. The decrease in sulfur (S), however, is relatively small, which suggests the attachment of peptides only to the surface.

Table 2: XPS data o the polymers recorded at 90 ° take o angle

a represents the theoretical atomic composition of PF4 in the bulk

Example 2: Preparation of peptide-modified copolymers - surface method [0117] A 5% solution of PFn was made in chloroform by dissolving 50 mg of the polymer in 1 mL of the solvent. The solution was filtered through 0.45 micron pore size filter paper to get a clear homogeneous solution. Three drops of the polymer solution was placed on a steel strip and the solution was drawn down using a 5 mil blade. The solvent was then allowed to evaporate at room temperature. The strip was dried under vacuum at room temperature for two hours to remove traces of solvent. When circular discs were used, three drops of the solution was placed on top of it and the disc was rotated at a speed of 450 revolutions per minute for one minute on a spin coater. The polymer film was dried in vacuum for 2 hours prior to use. GYIGS

Scheme 3: Peptide attachment

[0118] A solution of Gly-Gly (20 mg, 0.15 mmol) and 4 niL of distilled water was made at room temperature. The pH of the solution was maintained at 10 using carbonate buffer. To the solution, thin film of activated polymer (PFn) coated on steel strip was dipped at room temperature. The reaction was carried out for 24 hours. The film was then removed from the solution, rinsed with dilute hydrochloric acid followed by distilled water four times. The strip was finally washed with methanol and dried in vacuum over for 6 h at room temperature.

[0119] A solution of GYIGSR (12 mg, 0.02 mmol) and 5 mL of distilled water was made at room temperature. The pH of the solution was maintained at 10 using carbonate buffer. The polymer (PFn) coated strips or discs were dipped in the solution. The reaction was carried out at room temperature for 24 hours. The strip or disc was then removed from the solution, rinsed with dilute hydrochloric acid followed by distilled water four times. Finally the coatings were rinsed with methanol and dried in vacuum over for 6 h at room temperature.

[0120] The films showed partial solubility in chloroform indicating the attachment at the surface only. To further clarify the observation, XPS studies on the peptide modified polymers were performed. The atomic compositions of the activated and peptide bound surfaces were determined using XPS data recorded at 90 ° takeoff angle. Figure 6 shows NIs regions of XPS spectra of PGG2 and PYR2. As it can be seen clearly, peaks at 402.6 eV and 404.1 eV appear. The peak at a lower binding energy (402.6 eV) can be assigned to -NH-, -NH2, and -NHC=O whereas the peak at a higher binding energy (404.1 eV) can be assigned to the protonated nitrogen (-NH₂ ⁺) present due to the zwitterionic form of the peptide present on the surface of the polymer.²¹ This confirms the successful attachment of the peptides. The surface elemental composition data for PF2, PGG2 and PYR2 are shown in Table 3. The presence of nitrogen (N) and higher level of oxygen (O) concentration for PGG2 and PYR2 also indicate the attachment of the peptides as shown in Figure 6. The decrease in sulfur (S), however, is relatively small, which suggests the attachment of peptides only to the surface. In case of PGG4 and PYR4 a similar trend was observed as the peptides were attached to the surface of the polymer film with higher concentration. This may be because of the higher percentage of activating groups present on the surface of PF4 compared to PF2 as studied from XPS analysis (Figure 5).

A study of swelling behavior and Tris replacement

[0121] A swelling study of the polymers (PF1-PF4) was carried out in methanol and water mixture to estimate the time period needed for each polymer to swell completely. The swelling of the polymers is important in the replacement of the fosylate groups by tris buffer (Scheme 4).

PF2 or PF4

PF2-tris or PF4-tris Scheme 4: Replacement of Fosyl groups

[0122] Figure 7 shows the increase in weight of the films versus time. All the activated triblock copolymers possessed similar swelling kinetics as the solvent uptake reached a plateau in -10 hours. However, the extent of solvent uptake was different and was proportional to the number of activating groups present in the polymer. T his indicated that the fosyl groups are mainly responsible for solvent uptake. The PMMA- PIB-PMMA triblock copolymer did not show any significant solvent uptake even after 24 hours. [0123] Based on the above observation, it was concluded that a 3:2 mixture of methanol and water can be used as a medium to replace all the activating groups. The thin films of activated polymers were reacted with tris buffer in methanol/water mixture to further estimate the time period and extent of replacement. The tris reaction was carried out by incubating the films (PFn) for 12 hours at 45 ⁰C in tris solution in methanol/water. The progress of the reaction was monitored by FT-IR spectroscopy. The peak at 1600 cm^"1 assigned to the C=C stretching frequency of the A- fluorobenzylsulfonate group decreased significantly as shown in Figure 8 and the peak at 3350 cm-1 increased indicating more than 90% replacement of activating group. For all the polymers, more than 90% replacement was observed after 12 hours incubation. The tris reaction was further confirmed by the XPS analysis using PF2 and PF4 as representative samples. As shown in Figure 6, a peak appears at 401.8 eV in NIs spectra of PF2-Tris which corresponds to the amine group. This confirms the attachment of tris to PF2. As shown in Table 3, the sulfur concentration on the surface decreased from 1.96 to 0.08 atomic weight percent in PF4-tris and completely disappeared on PF2-tris surface. This indicates that 94.6% and 100% of the fosylate group has been replaced by tris group of PF4 and PF2, respectively.

Table 3. The element composition on the surface ofPF4, PGG4, PYR4 and PF2, PGG2,

PYR2 derived by XPS measurements.

Element^* PF2 PGG2 PYR2 PF2^ PF4 PGG4 PYR4 PFA^~-

Tris Tris

C 92.16 89.24 86.75 89.75 91.68 86.93 77.77 90.82

O 7.05 9.52 11.04 9.72 6.36 10.37 16.41 7.65

S 0.79 0.66 0.64 0.00 1.96 1.63 1.53 0.08

N 0.00 0.58 1.57 0.53 0.00 1.07 4.29 1.45 ^a The atomic weight percent is measured at 90 ° take off angle

Claims

CLAIMSWe claim:

1. A modified block copolymer comprising a first block and a second block, wherein each block comprises a plurality of monomer species and wherein at least one internal monomer species is covalently bonded to a sulfonate moiety or a bioactive molecule.

2. The block copolymer of claim 1, wherein the block copolymer is a thermoplastic elastomer.

3. The block copolymer of any of the preceding claims, wherein the sulfonate moiety is a 4-fluorobenzenesulfonate moiety.

4. The block copolymer of any of the preceding claims, wherein the bioactive molecule is at least one molecule selected from the group consisting of peptides and proteins.

5. The block copolymer of claim 4, wherein the peptide is at least one peptide selected from the group consisting of peptides containing the YIGSR sequence, peptides containing the RGD sequence, peptides containing the REDV sequence, P- 15, Heparin, and Platelet activation peptides.

6. The block copolymer of any of the preceding claims, wherein the first block comprises at least one methacrylate monomer species.

7. The block copolymer of any of the preceding claims, wherein the first block comprises at least one 4-fluorobenzenesulfonate ethyl methacrylate monomer species.

8. The block copolymer of any of the preceding claims, wherein the first block comprises a methylmethacrylate monomer species and a A- fruorobenzenesulfonate ethyl methacrylate monomer species.

9. The block copolymer of any of the preceding claims, wherein the first block comprises at least one peptidylethyl methacrylate monomer species.

10. The block copolymer of any of the preceding claims, wherein the first block comprises a methylmethacrylate monomer species and a peptidylethyl methacrylate monomer species.

11. The block copolymer of any of the preceding claims, wherein the second block comprises at least one monomer selected from the group consisting of isobutylene, 2-methylbutene, 3-methyl-l-butene, 4-methyl-l-pentene and beta- pinene.

12. The block copolymer of any of the preceding claims, wherein the second block comprises isobutylene.

13. The block copolymer of any of the preceding claims, further comprising a third block, wherein the third block comprises a plurality of monomers.

14. The block copolymer of claim 13, wherein at least one internal monomer of the third block is covalently bonded to a sulfonate moiety or a peptide.

15. The block copolymer of any of claims 13-14, wherein the third block comprises at least one methacrylate monomer species.

16. The block copolymer of any of claims 13-16, wherein the third block comprises at least one 4-fluorobenzenesulfonate ethyl methacrylate monomer species.

17. The block copolymer of any of claims 13-16, wherein the copolymer is an A-B- A block copolymer and wherein A represents the first and third blocks and B represents the second block.

18. The block copolymer of any of the preceding claims, wherein the number average molecular weight of the copolymer ranges from about 10,000 to about 1,000,000.

19. The block copolymer of any of the preceding claims, wherein said copolymer is a linear copolymer.

20. The block copolymer of any of the preceding claims, wherein at least 1% of the internal monomers of the first block are covalently bonded to a sulfonate moiety or a peptide or a buffer.

21. The block copolymer of any of the preceding claims, wherein at least 10% of the internal monomers of the first block are covalently bonded to a sulfonate moiety or a peptide or a buffer.

22. The block copolymer of any of the preceding claims, wherein at least one surface monomer is covalently bonded to a sulfonate moiety or a peptide.

23. The block copolymer of any of the preceding claims, wherein the copolymer is a medical device or a coating on a medical device.

24. The block copolymer of claim 23, wherein the medical device is a stent.

25. The block copolymer of any of claims 23-24, wherein the medical device is a graft or stent graft.

26. The block copolymer of any of claims 23-25, wherein the medical device is an electrical lead.

27. The block copolymer of any of claims 23-26, wherein the medical device is designed for neurological implantation.

28. The block copolymer of any of claims 23-27 ', wherein a therapeutic agent is also incorporated in the device or coating, through covalent attachment or through incorporation into the coating or device.

29. A method for making a sulfonate-modified block copolymer comprising: dissolving a copolymer comprising at least one modifiable monomer with a suitable solvent; and contacting the dissolved copolymer with a sulfonyl chloride, under suitable conditions such that a sulfonate-modified block copolymer is formed.

30. The method of claim 29, wherein the block copolymer is a thermoplastic elastomer.

31. The method of any of claims 29-30, wherein the sulfonate-modified block copolymer is modified both internally and on the surface.

32. The method of any of claims 29-31, wherein the modifiable monomer comprises a pendant hydroxyl moiety.

33. The method of any of claims 29-32, wherein the sulfonyl chloride is a fosylchloride.

34. A method for making a bioactive molecule-modified block copolymer comprising: dissolving a sulfonate-modified block copolymer with a suitable solvent; and contacting the dissolved copolymer with a bioactive molecule, under suitable conditions such that a bioactive molecule-modified block copolymer is formed.

35. The method of claim 34, wherein the bioactive molecule is at least one molecule selected from the group consisting of peptides and proteins.

36. The method of any of claims 34-35, wherein the peptide is at least one peptide selected from the group consisting of peptides containing the YIGSR sequence, peptides containing the RGD sequence, peptides containing the REDV sequence, P- 15, Heparin, and Platelet activation peptides.

37. An article of manufacture comprising at least one copolymer of claim 1.

38. The article of manufacture of claim 37, wherein the article of manufacture is an insertable or implantable medical device.

39. The article of manufacture of claim 38, wherein the implantable medical device is a stent.