WO2006127712A2 - Compositions and methods for inducing, stimulating and directing neuronal growth - Google Patents

Abstract

Provided are nerve growth guidance devices, compositions and methods for inducing, enhancing and stimulating neuronal growth and for repair and regeneration of damaged nerves in both the central nervous system and the peripheral nervous system of mammals. The compositions are composed of cationic, aromatic polyheterocyclic substrates electrostatically bound by with anionically charged dopants able to append alone or through a linker nerve growth guidance cues. The compositions may be formed into devices and used in methods for stimulating nerve growth or nerve regeneration.

Description

COMPOSITIONS AND METHODS FOR INDUCING, STIMULATING AND DIRECTING NEURONAL GROWTH

FIELD OF THE INVENTION

The invention relates to compositions, devices and methods for inducing, stimulating and directing neuronal growth. More specifically, in one aspect the invention relates to the regenerative repair of damaged nerve tissue in mammals. In another aspect, the invention relates to inducing nerve growth. BACKGROUND

Nerve growth in mammals in vivo is modulated by the presence of growth- promoting and growth-inhibitory cues. Injury to the central nervous system (CNS) leads to the formation of a glial scar, which, along with the production of inhibitory substances prevents regenerative repair of the damaged nerve tissue. Regenerative repair of damaged nerve tissue in the peripheral nervous system (PNS) is possible due to the presence of growth-promoting "cues". Such cues are typically biological molecules such as polypeptides, small peptides, polysaccharides, glycoproteins and the like that are produced and secreted by various glial cells, i.e., Schwann cells, macrophages and monocytes. The high molecular weight glycoprotein, laminin, produced by Schwann cells and widely dispersed in the peripheral nervous system; and fibronectin, the major constituent of the extracellular matrix produced by fibroblasts and collagen, are exemplary. See, Woolley et al., Fibronectin-laminin combination enhances peripheral nerve regeneration across long gaps, Otolaryngol Head Neck Surg 103:509-18 (1990); Chen et al., Peripheral ner-ve regeneration using silicon rubber chambers filled with collagen, laminin and fibronectin, Biomaterials 21: 1541-47 (2000); Longo et al., Neurite-promoting factors and extracellular matrix components accumulating in vivo within nerve regeneration chambers, Brain Res 309: 105-17 (1984); Lander et al., Purification of a factor that promotes neurite outgrowth: isolation of laminin and associated molecules, J. Cell Biol. 101:898-913 (1985); Cornbrooks et al., In vivo and in vitro observations on laminin production by Schwann cells, Proc. Nat. Acad. Sci. USA 80: 3850-54 (1983); and Linsenmayer, Collagen. In: Hay, E. D., ed., Cell Biology of Extracellular Matrix: Plenum Press, New York, 1991, pps. 7-44. Injury to peripheral nerves can be repaired by juxtaposing the severed nerve ends endings. This results in some degree of regenerative repair if the ends of the severed nerve are less than 10 mm apart. Other damage requires surgical repair with autologous nerve grafts harvested from elsewhere in the body. This procedure has disadvantages: loss of function at the donor nerve site, the requirement of multiple procedures and the failure to achieve return of function due to dimensional mismatching between the donor nerve and the severed nerve. Autologous nerve grafting, using embryonic spinal cord or peripheral nerve tissue, has met with limited success when applied to injuries to the CNS. Thus, biological control over neuronal growth induction, stimulation and inhibition is a fundamental objective in neuroscience and neurological and surgical medicine.

One approach has involved the use of weak optical forces to guide the direction of the growth cone of the nerve cell. In extending the growth cone, which is the leading edge of the cell where new growth takes place, a laser spot is placed in front of a specific area and this serves to stimulate growth in a guided manner into the beam. See, for example, Eltrlicher, Proc Natl Acad Sd USA 99: 16024-28 (2002).

The use of nerve guidance channels to bridge gaps between severed nerves has been under investigation for some time. In this approach, natural or synthetic tubes are inserted to help direct axons sprouting off the regenerating nerve end, to provide a conduit for diffusion of neurotrophic and neurotopic factors secreted by the damaged nerve stump and to minimize the infiltration of fibrous tissue. Natural tubes investigated include veins and muscles. Synthetic tubes investigated include derivatives of collagen, laminin and fibronectin, and silicone and polymeric materials such as polyhydroxybutyric acid (PHB). See for example United States Patent Publication Nos. 2002/0137706 and 2005/0069525 for a discussion of prior art nerve guidance channels or conduits made of polymeric materials.

Past work demonstrates that electrical charge stimulates the proliferation or differentiation of various types of cells. To this end, researchers have incorporated electrical signals into biomaterials. Certain piezoelectric polymers, for example polyvinylidine fluoride, generate transient surface charges. Other polymers,

"electrets" such as polytetrafluoroethylene, possess a permanent surface charge. A third class of polymer generates electrical signals by electron transfer between different polymer chains. In recent years such organic conducting polymers have shown promise as energy storage devices, in electrochemical displays, in corrosion prevention, in molecular electronic devices, as electrocatalytic structures, in drag delivery and as ion gates.

Known conducting polymers include organic polyheterocyclics, such as polypyrrole, polythiophene, polyaniline and their derivatives. The aromatic heterocyclic polypyrrole is a polymer of repeating C₄H₃N units. Polythiophene is a polymer of repeating C₄H₂S units. Polyaniline is a polymer of repeating C₆HsN units. One advantage of using electrically conducting polymers is that such materials can be modified with negatively charged "dopant" ions tailored to specific applications. The monomer is electropolymerized anodically to generate polarons and subsequently bipolarons, with one positive charge for every three to four monomeric units: in the case of pyrrole (C₄H₆N)⁺ _{3 or4}- To compensate the positive charge that develops along the polymeric backbone, a counter anion, the dopant, becomes electrostatically bound to the polymer during electrodeposition: [(C₄H₆N)⁺ _{3 O}r4(X^~)]n- If the counter anion is large or polyanionic, it actually becomes physically entangled within the conducting polymer 'matrix'. However, these materials have poor mechanical properties; they are brittle at high conductivities. United States Patent No. 4,898,921 discloses conducting polymeric films comprising a polysaccharide matrix with intercalated, bonded conducting organic heterocyclic polymers. In the past, polypyrrole (pPy) has been doped with transition metal complex ions, hyaluronan, adhesive peptides, heparin and nucleic acids. See, United States Patent Nos. 4,818,646 and 4,617,353; Garner et al., J Mater Sd: Mat Med 10: 19-27 (1999); Wang et al., Langmuir 16: 2269-74 (2000); and Garner et al., JBiomedMater Res 44: 121-29 (1999). In addition, polypyrrole has been doped with a fibronectin fragment and a nonapeptide fragment of laminin for use as neural probes. Cui, J

Biomed Mater Res 56: 261-272 (2001). Although glial or neuroblastoma cells of the rat have been cultured on pPy doped with fragments of fibronectin or laminin, their low densities indicate that most of the chemical cue is embedded within the bulk of the pPy film and thus, is inaccessible to the cultured cells. Oxidized pPy invokes little adverse tissue response compared with poly(lactic acid-co-glycolic acid) in animal implantation studies. Schmidt, Proc Natl Acad Sd U SA 94: 8948-53 (1997). The addition of ester linkages to the pyrrole monomer renders it biodegradable. Rivers et al., Adv Fund Mater 12: 33-37 (2002). An applied constant current or constant voltage enhances axonal extension in vitro from nerve cells adhered to substrates coated with oxidized pPy. Schmidt et al., supra; Kotwal, Biomaterials 22: 1055-64 (2001). Films of polypyrrole doped with a complex of dextran sulfate and nerve growth factor have been shown to exhibit a voltage- induced release of nerve growth factor for promoting neurite extension in PC 12 cells. Hodgson et al., Proc SPIE 2716: 164-76 (1996). Although these previous studies indicate that polypyrrole has some advantages as a biomaterial, we are unaware of any studies to date that take advantage of polypyrrole 's ability to bind dopants electrostatically to which nerve guidance cues are appended. Despite the existence of these various materials, there remains a need in the art for generalized methods and materials for stimulating nerve growth or regeneration.

SUMMARY

A. Definitions The following definitions are used throughout, unless otherwise indicated.

The term "administering at the site" means applying at or proximal to a given site to produce a desired therapeutic effect in a localized manner, e.g. to stimulate nerve growth at the site. The "administration" or application of a compound or device "to" or "near" a tissue or cell refers to the delivery of the device or compound to a location proximal to, or in direct contact with, the tissue or cell to produce the desired localized therapeutic effect.

The term "amino acid" means residues of the natural amino acids, e.g. the D or L forms of alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine (Cys), glutamic acid (GIu), glutamine (GIn), glycine (GIy), histamine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (VaI), and non-natural amino acids, e.g. phosphoserine, phosphothreonine, phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid, octahydroindole-2-carboxylic acid, statine, l,2,3,4-tetrahydroisoquinoline-3- carboxylic acid, penicillamine, ornithine, citruline, .alpha.-methyl-alanine, para- benzoylphenylalanine, phenylglycine, propargylglycine, sarcosine, and tert- butylglycine, among many others. The term "amino acid" also comprises natural and non-natural amino acids bearing a conventional amino protecting group e.g. acetyl or benzyloxycarbonyl, as well as natural and non-natural amino acids protected at the carboxy terminus, e.g. as a Ci-C₆ alkyl, phenyl or benzyl ester or amide; or as an .alpha.-methylbenzyl amide. Other suitable amino and carboxy protecting groups are known to those skilled in the art, and are included within the context of this invention. See, for example, Greene, T. W. and Wutz, P. G. M. "Protecting Groups In Organic Synthesis", Second Edition, New York, John Wiley & Sons, Inc., 1991, and references cited therein.

"Appended" means in relation to a nerve guidance cue that the cue is bonded or complexed to the substrate as a side chain or side group, but is not part of the backbone of the substrate. The cue is bonded to or complexed with the substrate via the dopant directly or through a linkage or linker sequence that may optionally release it when applied or administered according to the methods of the invention. For example, a cue may be linked through a hydrolyzable linkage such as a carboxyl, anhydride or ester linkage. Other linkages are also suitable. "Biocompatible" means being compatible with a living system or entity and not detrimental to the general existence and functioning of the system or entity, e.g. neither toxic to, nor causes a detrimental reaction (e.g. immunological reaction) in a living system, so that it would make it undesirable to continue its use.

"Biodegradable" means able to be broken down into components smaller than its original size when present in the living system or entity.

"Dispersed through or throughout" means that a compound is located within a matrix by mixing, spreading, sprinkling, thoroughly mixing, physically admixing, or dispersing.

"Formed into" includes a polymer, compound, composition, formulation, matrix of the invention that may be physically placed into various shapes, geometries, structures and configurations including, but not limited to films, fibers, rods, coils, spheres, cones, pellets of various shapes, tablets, tubes (smooth or fluted), discs, membranes, sleeves, cuffs, free-standing films, sheaths, wraps, tubes, etc.

"Layered" or "layer" means superposed so as to be substantially one above or below the other.

"Nerve" and "neuron" are used interchangeably and refer to the signaling cells of the nervous system. They are described in detail in Chapter 2: Nerve Cells and Behavior, Principles of Neural Science, 3d ed., Kandel, E.R., Schwartz, J.H. and Jessell, T.M. eds., Elsevier, NY, 1991.

"Nerve growth guidance device" means a structure that is formed from a composition of the invention and that can be used in a living system or entity to induce, enhance, stimulate or control the growth of neuronal cells and tissues.

"Nerve growth guidance cue" refers to a chemical entity that is able to induce, enhance, stimulate or control the growth of neurons. Any biological molecule that stimulates, induces, enhances or controls the growth of neurons, either by direct or indirect activation or by repression or inhibition of nerve growth inhibitor substances, and is able to bind, either by itself or through a linker sequence, to the functional group of the dopant may be employed.

"Peptide" refers to sequences of about 2, 3, or 5 to about 15, 20, or 35 and more amino acids, or peptidyl residues that may be linear or cyclic, such as those that may be prepared or result from the formation of disulfide bridges between two cysteine residues. Peptide derivatives may be prepared as disclosed in U.S. Pat. Nos. 4,612,302; 4,853,371; 4,684,620. Peptide sequences are written with the amino terminus on the left and the carboxy terminus on the right. B. Invention summary

The invention provides devices, compositions and methods for stimulating neuronal growth and for repair and regeneration of damaged nerves in both the central and peripheral nervous systems.

The nerve growth guidance composition of the invention includes three components.

The first component is a substrate composed of a cationic, aromatic polyheterocyclic organic compound. The cationic, aromatic polyheterocyclic must have the ability to incorporate a dopant into its structure, preferably by electrostatic interaction (electrostatic binding) or by physical entanglement. Exemplary cationic polyheterocyclic organic compounds include polypyrroles, polythiophenes, polyanilines, polyfuran, polyselenophene, polyindole, polypyridazine and derivatives thereof, for example the polythiophene derivatives disclosed in United States Patent No. 6,872,801 herein incorporated by reference and the polythiophene derivative, poly(2,3-dihydrothieno-l,4-dioxin) or PEDOT. The cationic, aromatic polyheterocyclic may be employed alone or in combination with a polymeric compound as a laminate or as a composite to enhance mechanical strength or conductivity. An example of a laminate would be to electrodeposit a layer of polypyrrole doped with polysulphonate [pS] followed by the electrodeposition of a layer of the invention (e.g., pPy[pGlu]). Very thick films of pPy[pS] are possible, which would improve the mechanical strength of the laminate. An example of a composite would be to electrodeposit polypyrrole in the presence of both pS and pGlu. Similar improvement in mechanical strength would be observed. Such polymeric compounds may be biodegradable and should be biocompatible. Exemplary polymeric compounds include polysaccharides, polyesters, polyhydroxybutyric acids, polyglycolic acids, polylactic acids (PLLA), poly(lactic-co- glycolic) acids (PLGAs) and poly-L-lactide/poly-L-caprolactone copolymers. PLGAs are specifically described in United States Patent Publication No. 2004/0102793 incorporated by reference herein. United States Patent Publication No. 2002/0137706 incorporated by reference herein describes a variety of polymeric compounds which can be employed. In addition, cationic polyheterocyclic organic compounds described as the first component herein can be used as the polymeric compound when they are synthesized with inorganic dopants such as chloride and perchlorate; and organic dopants such as polystryrene sulfonate, p-toluenesulfonic acid, dodecylbenzenesulfonic acid, bis(2-ethylhexyl)phosphate, dinonylnaphthalenesulfonic acid and camphor- 10-sulfonic acid.

The second component is an anionic dopant. The anionic dopant is electrostatically bound to the first component, the substrate. The dopant enables the attachment of a nerve growth guidance cue to the cationic aromatic polyheterocyclic organic compound. The dopant should provide at least one functional group to which a nerve guidance cue can be attached and also provide negative charges to neutralize the positive charge of the cationic aromatic polyheterocyclic. The critical aspect is that the dopant be able to form a covalent bond by reacting a specific functional group of the dopant with another specific functional group of the guidance cue or with a linker molecule to which the guidance cue is covalently attached. As is known in the art, the chemistry to form such covalent bonds includes the reactions of amines (- NH₂) with succinimidyl esters (-CsH₄O₄N), isothiocyanates (-N=C=S), 4-sulfo- 2,3,5,6-tetrafluorophenol esters or sulfonyl chlorides (-SO₂Cl); the reactions of sulfhydryls (-SH) with alkyl halides or haloacetamides (-CH₂X, where X = I, Br, Cl), maleimϊdes (-C₄H₂O₂N) and symmetric disulfides (-S-S-R, where R = alkyl); and the reaction of carbonyls or aldehydes (-COH) with hydrazines (-NH-NH₂). Carboxyls are converted to succinimidyl esters by using l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

Homofunctional or heterofunctional linkers that include the functional groups described above can be used to connect two specific functional groups, one from the dopant and the other from the guidance cues. For example, a homofunctional linker including two succinimdyl esters is used to connect amine groups of dopants to the same groups of guidance cues; or a heterofunctional linker including one succinimidyl ester and one maleimide is used to connect between amine and sulfhydryls. Materials and methods for forming these covalent bonds are known in the art.

Polyglutamic acid, an especially preferred dopant, has numerous anionic groups for balancing the positive charge of polypyrrole and excess carboxylic acid groups for reaction with and attachment to guidance cues. Other acidic polyamino acids, for example polyaspartic acid, may be employed for the same reasons. A polyamino acids with non-polar R groups, such as polyglutamine, polyserine and polyalanine, also may be employed as a dopant. In such a case, the polyamino acid becomes entangled within the polycationic matrix of the cationic aromatic polyheterocyclic instead of being electrostatically bound as a polyanion would be. Any excess carboxylic acid groups would be available for reaction with and attachment to the guidance cues. In addition to the homopolymers of amino acids, heteropolymers of amino acids can be used if they have negative charges and multiple number of functional groups that can be used for the attachment of the guidance cues. An example is a polymer consisting of the repeating unit of glycine and glutamic acid.

With regard to the molecular weight of the anionic dopant, the dopant chosen should be soluble in water or any other solvents employed in the electrodeposition process and be as long as possible (the longer the molecule, the more carboxylic groups it has to bind the nerve guidance cues) given the solubility parameter. For pGlu for example, an exemplary range of molecular weight is about 1,000 to about 100,000 as measured by multi-angle laser light scattering, more preferably about 5,000 to about 50,000 and most preferably about 8,000 to about 20,000. The selection of the appropriate molecular weight of the anionic dopant is within the level of skill in the art.

The corresponding monomeric amino acids can be used, especially if they have an anionic functional group to bind electrostatically with the backbone of cationic aromatic polyheterocyclic. However, it is believed that monomeric amino acids will not work as well because only a single ion-pair holds each of the individual monomers to the substrate. Employing polymeric amino acids provides, in addition to multiple ion pairs for electrostatic binding to the substrate, entanglement of the polymeric amino acid within the tertiary structure of the polyheterocyclic, which lessens or prevents its loss into solution.

As dopant, preferred are polyglutamic acid and polyaspartic acid. Especially preferred is polyglutamic acid.

The third component comprises at least one nerve growth guidance cue. The at least one nerve growth guidance cue is appended to the dopant, either directly or through a linker molecule via the available carboxyl groups of the dopant. The linker sequence may be a divalent, branched or unbranched, saturated or unsaturated hydrocarbon chain, a biological molecule such as a saccharide, carbohydrate, polysaccharide, fatty acid, lipid, nucleic acid, peptide, amino acid or a combination thereof. The critical characteristics in choosing an appropriate linkage or linker sequence are: the linker must have the ability to bind with the dopant and append the desired nerve growth guidance cue to the dopant. The selection and construction of appropriate linkers is within the level of skill in the art.

Exemplary nerve growth guidance cues, which may be employed singly or in mixtures, include proteins encoded by neuronal regeneration-associated genes (RAG) for example, cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, cytokines, neurotrophins and neurotrophin receptors. RAGs are highly expressed during nervous system development and there is evidence for a coordinated neuronal gene program involved in the repair process. See United States Patent Publication No. 2005/0054094, incorporated by reference herein. Accordingly, axotomy-induced neuropeptides are candidates for attachment as nerve guidance cues and employment in the invention. Such neuropeptides include vasoactive intestinal peptide, galanin and neuropeptide Y. In addition neurotrophic factors and their receptors are known to play roles in nervous development and can be employed as cues. There exist more than 20 known neurotrophic factors, the most studied subgroup being the neurotrophins. Of the neurotrophins, exemplary are nerve growth factor (including the entire three chain molecule or the bioactive β chain alone), brain-derived neurotrophic factor and neurotrophin-3 and neurotrophin-4/5. See United States Patent Publication No. 2005/0048606 incorporated by reference herein.

Also included are the neurotrophic factors acidic fibroblast growth factor, platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), VEGF, neuregulin, activin, nerve growth factor receptor p75, GAP-43, CAP-23 and the SPRRlA polypeptides disclosed in detail in United States Patent Publication 2005/0054094. In addition RICH proteins (Regeneration Induced CNPase Homologs), CNPases, NF-kappa-β, P134K, GSK-3β, APC, Nogo receptor inhibitors, extracellular matrix molecules including glycoproteins, for example, laminin, fibronectin and collagen (types I and/or IV), glycosaminoglycans such as hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate, proteoglycans such as phosphacan, NG2 proteoglycan, agrin, receptor-type protein tyrosine phosphatase, neurocan and brevican may be employed as nerve growth guidance cues. Also included are inhibitory cues, for example, the inhibitory protein ephrin, which binds to the Eph receptor on the growth cone of the axon. Ephrin binding activates the Vav2 protein which induces engulfment of the ephrin-Eph complex and causes axon inhibition. Ephrin binding also induces, either directly or indirectly, the chemical modification of ephexin 1, which in turn modifies the neural cell's internal scaffolding a causes the growth cone to collapse. This results in growth termination or in the modification of the direction of growth.

In addition to nerve growth guidance cues, the compositions and devices of the invention may optionally include Schwann cells or other glial cells, or neuronal stem cells. Such cells provide a permissive environment in the nerve lesion or gap by secreting permissive extracellular growth inducing factors and by removing growth inhibitory factors. Schwann cells or neuronal stem cells can be plated onto the composition or device of the invention. The cells may be adhered to the composition and devices of the invention in, for example, an alginate matrix as describe in United States Patent Publication No. 2005/0069525 herein incorporated by reference. Schwann cells were plated onto an ITO/substrate coated with the invention. The specific composition of the invention examined consisted of four components: polypyrrole, polyglutamic acid, polylysine, laminin (pPy[pGlu]-pLys-Lmn). Preliminary results indicate Schwann cells show preference within 24 hr after culturing to attach to an ITO/glass substrate coated with the invention compared to an uncoated ITO/glass substrate.

The compositions of the invention (e.g., pPy[dopant] or pPy[dopant]- (guidance cue)) may be formed into implantable devices of any shape suitable for the application. For example, the compositions may be formed into devices that are films, sheets, membranes, solid rods, rods with longitudinally disposed internal channels, tubes, holes or tunnels, cylinders, discs, cubes, channels, conduits, fibers, coils, spheres, cones, pellets of various shapes, tablets, tubes (smooth or fluted), discs, sleeves, cuffs, free-standing films, sheaths, wraps. Rods with longitudinally disposed internal channels, tubes, holes or tunnels are preferred. The various forms may be solid, porous or perforate i.e., they may contain holes, pores, grooves, recesses, notches, or slots to aid in nerve growth and axonal extension. Suitable pore forming agents are disclosed in United States Patent Publication No. 2002/0137706. The preparation of such polymers is well known in the art. Solvent casting, extrusion and particulate leaching techniques are well known in the art and may be advantageously employed. It is preferred that the composition used to make a device contains dopant prior to fabrication of device. Guidance cues subsequently can be introduced by immersion of the device into a reactive solution containing guidance cue.

The shape, geometry, structure or configuration of the device will vary upon the use of the device. For example, for treatment of a brain or spinal cord injury the composition of the invention may be formed into a medical implant in the shape of a disc for placement under the dura or dura mater or a film, membrane or sheet for covering the spine. In another example, the composition of the invention may be formed into an implant in the shape of a tube, a rod, with or without longitudinally disposed internal channels, tubes, holes or tunnels, or a channel for use in the treatment or injury or damage to the peripheral nervous system.

In another aspect, the invention includes a method of inducing nerve growth or regeneration in a mammal. The method comprises administering to a mammal one or more of the nerve growth or regeneration compositions described above. In one embodiment, the nerve growth or regeneration is induced in the peripheral nervous system. In another embodiment, the nerve growth or regeneration is induced in the central nervous system. Preferably the mammal in which nerve growth or regeneration is induced is a human.

Administration will preferably be by surgical implantation. The methodology is analogous to the methodology used in nerve graft procedures and is well known in the art. In such procedures, harvested donor nerves are placed within a defect in the damaged nerve to bridge a gap using microsurgical techniques and microsutures. The sutures pass through the outer layer of the nerve and the graft, allowing for axons to travel down the bridging graft and make contact with the severed portion of the nerve bundle. Exemplary techniques and methods are described in detail in United States Patent Publication Nos. 2002/0137706, 2004/0102793 and 2005/0069525 incorporated by reference herein. The compositions and methods of the invention may be employed to treat a variety of diseases, conditions, injuries and states that cause injury or damage to nerves. Nerve pathology can arise from, for example, trauma, mechanical damage, thermal damage, electrical trauma, congenital defects or acquired disease states. The devices compositions and methods of the invention may be employed in the treatment of any neurodegenerative disease or condition, or injury, that involves retraction or impedance of neurite connections. The devices, compositions and methods of the invention may be employed in the treatment of acquired neuropathies such as olfactory groove meningioma, otic neuritis, Leber's disease, optic nerve glioma, ischemic optic neuropathy, microvessel ischemia, trigeminal neuralgia, scleroderma, Bell's palsy, Lyme disease, sarcoidosis, vestibular neuronitis, acoustic schwanoma, glossopharyngeal neuralgia, motor neuron disease, tumor caused neuropathies and drug cause neuropathies. They may also be employed in the treatment of inherited neuropathies such as Charcot-Marie Disease, Werdnig-Hoffman Disease, Kennedy Syndrome and neuropathies caused by mutations in PMP-22, connexin-32, the Po gene and the androgen receptor gene. Any condition or state in a mammal in which nerve growth is needed or desired, or in which regeneration or reconnection of injured nerves is needed or desired is an appropriate candidate for treatment with the compositions and methods of the invention. Also disclosed is a method of inducing or enhancing nerve growth or nerve regeneration in a mammalian subject comprising administering a composition of the invention to the subject by surgical implantation in a site in the body of the subject where nerve growth or nerve regeneration is desired or needed to alleviate or treat the state, condition or defect in need of treatment. In the case of severed nerves, an appropriately sized and shaped device of the invention is surgically implanted in the gap at the site of the injury in the same manner as an autograft would be to provide a scaffold for nerve regeneration. To accomplish this the composition should be sized and shaped so as to be substantially contiguous with the severed nerve ends once implanted; appropriate tubes, flat, thin films and other implantable structures are well known to the skilled artisan.

DESCRIPTION OF THE DRAWINGS

Fig. 1 illustrates the procedure for fabricating microscale patterns of poly-L- glutamic acid doped polypyrrole and poly-L-glutamic acid (PP-pGlu) doped polypyrrole chemically modified with poly-Lysine (PP-pGlu-pLys) or Laminin (PP- pGlu-Lmn).

Fig. 2: (a) is a graphic representation of the cyclic voltammogram (CV) results obtained upon electropolymerization of pyrrole in the presence of poly-L-glutamic acid. The current increases with each subsequent cycle. Only the first, tenth, twentieth, thirtieth, fortieth and fiftieth CVs are shown, (b) is a graphic representation of the CV results obtained polypyrrole doped with poly-L-glutamic acid in 0.2 KCl, taken at scan rates of 25, 50 and 100 mV/s. (c) is a graphic representation of current density at 150 mV plotted as a function of scan rate for bare ITO (open circles) and for ITO coated with poly-L-glutamic acid doped polypyrrole (closed circles). The slope of the data for the coated ITO is 30 times steeper than that for the bare ITO.

Fig. 3 illustrates the procedure for the covalent attachment of poly-L-lysine or laminin to the surface of poly-L-glutamic acid doped polypyrrole.

Fig. 4 is a graphic representation of CVs of poly-L-glutamic acid doped polypyrrole (solid line), poly-L-glutamic acid doped polypyrrole-NHS (dashed line) and poly-L-glutamic acid doped polypyrrole-poly-L-lysine (dotted-dashed line) in 0.2

M KCl at 25 mV/s. Fig. 5: (a) is a graphic representation of the infrared spectra of poly-L- glutamic acid doped polypyrrole films (pPy-pGlu) electrodeposited onto gold-coated silicon wafers. Fig 5(b) is a graphic plot of the intensity of peaks at 1675 and 1400 cm^"1 for films of pPy-pGlu, values taken from 5(a). Fig. 5(c) is a reproduction of the infrared spectra of pPy prior to surface activation and formation of pPy-pGlu, subsequent to surface activation and formation of pPy-pGlu-NHS and subsequent to covalent attachment of pLys and formation of pPy-pGlu-pLys.

Fig. 6 are xerographic reproductions of the phase contrast and fluorescent images of dorsal root ganglia adhered to a surface of PP-pGlu-pLys as described in Example 6.

Fig. 7 are xerographic reproductions of phase contrast (a) and fluorescent (b) images of dorsal root ganglia adhered to a surface of PP-pGlu-Lmn. Cells were fixed twelve days post plating. Magnification 1OX. Neurites stained positive for GAP-43 (green fluorescence). Fig. 8 are xerographic reproductions of phase contrast (a) and fluorescent (b)-

(c) images of dorsal root ganglia neuronal cells on PP-pGlu-Lmn. Cells were fixed two days post plating. Magnification: 1OX for (a)-(b) and 4X for (c). Neurites stained positive for GAP-43 (green fluorescence).

DETAILED DESCRIPTION

Micropatterned films of polypyrrole (pPy) doped with poly glutamic acid (pGlu) are prepared and subsequently modified with laminin (Lmn) alone or with polylysine (pLys) alone via carbodiimide-ccoupling chemistry. In addition to individual layers of pLys or Lmn, pPy [GIu] was modified with multiple layers of guidance cues produced by sequential reaction of pLys and Lmn, resulting in the production of patterned compositions of pPy-pGlu-pLys-Lmn. Dorsal root ganglia were cultured in the presence of these compositions and shown to adhere preferentially to the areas of positive guidance cues and to extend neurites within these areas.

Example 1: Substrate Preparation

A glass slide coated with a thin film of indium tin oxide (ITO/glass) was used as the electrically conductive substrate for film deposition. ITO was coated on glass substrates using dc magnetron sputtering with a flow of mixture of argon and oxygen. The thickness of ITO layer was 0.1 um. Positive photoresist (PR), available from Shipley Microposit Sl 813, Marlborough, MA, was spin-coated onto the indium tin oxide coated glass slide at 3500 rpm for 30 sec. with a ramp of 500 rpm/sec. The PR- coated glass slide was then soft-baked at 110 deg.C for 2 min. on a hotplate. The PR- coated glass slide was then covered with a photomask bearing the desired pattern and exposed to UV light for 45 sec. Subsequent to pattern transfer, the slide was developed in a mixture of MF312 developer (Shipley Co., Marlborough, MA) and water (1 : 1 by vol.) for 30 sec, followed by rinsing with water for 15 sec. The patterned slide was then hard-baked at 110 deg.C for 30 min.

Example 2: Electrodeposition And Chemical Modification

Fig. 1 illustrates the procedure for fabricating patterns of poly-L-glutamic acid doped polypyrrole and poly-L-glutamic acid (pPy-pGlu) doped polypyrrole chemically modified with poly-Lysine (pPy-pGlu-pLys) or Laminin (PP-pGlu-Lmn). 2 mM poly-L-glutamic acid (pGlu, MW = 8,853 by multi-angle laser light scattering or 17,000 by viscosity; from Sigma- Aldrich, sιipra)wειs added to 200 mM pyrrole (Sigma- Aldrich Fine Chemicals, St. Louis, MO) at room temperature to form a solution. Because the concentration of pGlu was based on the monomelic unit of glutamic acid and because one cationic charge develops for every three to five pyrrole monomers in the polymerization process, an influx of anions from the electrolyte is necessary to maintain charge neutrality during the electrodeposition. Consequently, pGlu with a molecular weight of 10K, equivalent to about 100 monomers was employed. The patterned ITO/glass slides made as described in example 1 were used as working electrodes. The counter and reference electrodes were platinum gauze and a saturated silver/silver chloride electrode (Ag/AgCl, 0.197 V vs. NHE), respectively. The electrodes were attached to an EG&G Potentiostat/Galvanostat, Model 263 and the working electrode was placed in the polypyrrole polyglutamic acid solution and a potential of between 0.0 V and 1.0 V for ten cycles at a scan rate of 100 mV/sec was applied. The working electrode was then rinsed thoroughly with sterile water. The polypyrrole is electrodeposited on the portion of the ITO pattern exposed to electrolyte. During electrodeposition, polyglutamic acid is doped within the polypyrrole matrix to form a conductive film. The film may be removed from the underlying ITO substrate by chemical etching with hydrochloric acid.

The exposed carboxylic acids group of the poly-L-glutamic acid were then activated chemically with N-hydroxysuccinimide (NHS) as follows. The patterned electrode with the deposited pPy-pGlu film was incubated in an aqueous solution containing 0.5 mg/ml l-ethyl-3-(3-dimethylaminopropyl)carboiimide hydrocchloride (EDC), 0.5 mg/ml N-hydroxysuccinimide (NHS), 0.1 M 2-[N-morpholino]ethane sulfonic acid (MES) (all from Sigma-Aldrich) and 0.5 M NaCl at pH 6.0 for 30 min. After rinsing again with sterile water, the electrodes with their resulting activated surfaces, pPy-pGlu-ΝHS, were placed in an aqueous solution containing either 100 μg/ml poly-L-lysine (pLys, MW = 70,0000 - 150,000; from Sigma- Aldrich) or 100 μg/ml laminin (Lmn, from Invitrogen Life Technologies, Carlsbad, CA) for three hours at room temperature to allow formation of an amide bond between the activated carboxylic groups of the poly-L-glutamic acid and the amine groups of pLys or Lmn, resulting in the product, either pPy-pGlu-pLys or pPy-pGlu- Lmn, on the patterned surface substrate, the ITO/glass slide. The substrate was then removed from the reaction medium and rinsed with sterile water.

The process was then repeated with the electrode having the film pPy-pGlu- pLys to attached Lmn to the pLys to form a film composed of polyglutamic acid doped polypyrrole to which a polylysine linker is used to bind laminin to the dopant.

Example 3: Characterization of Micropatterned, Coated Substrates

The pPy-pGlu, pPy-pGlu-pLys, pPy-pGlu-pLys-Lmn compositions made as described in Example 2 were analyzed by scanning electron micrographic analysis and by infrared spectral analysis as follows.

1. Scanning Electron Micrographic (SEM) Analysis

Fig. Ia is a xerographic reproduction of a scanning electron micrograph of a composition prepared in Examples land 2. The dark areas in the image correspond to film compositions formed on the electrode surface and the light areas correspond to the unmodified ITO/glass.

2. Infrared Spectral Analysis

Films of pPy-pGlu, pPy-pGlu-pLys and pPy-pGlu-pLys-Lmn were made by electrodeposition onto gold-coated silicon wafers as described in Examples 1 and 2 above. Infrared spectra of the electrodeposited films were collected on a Nicolet Nexus 670 FTIR spectrometer, equipped with a MCT/A detector and a SAGA (Smart Aperture Grazing Angle) accessory. A clean, gold-coated silicon wafer was used to obtain a baseline spectrum. Specular reflectance (i.e., transmittance) was measured and subsequently converted to absorbance.

Illustrated in Fig. 5a are the results of the infrared spectra analysis. The seven characteristic absorbance peaks are labeled in bold numerals: 1 = v(NH) at 3320 cm^"1, 2 = V₃₅(CH₂) at 2950 cm^'1, 3 = v(C=O) for COOH at 1710 cm^"1, 4 = v(C=O) for amide I at 1675 cm^"1, 5 = v_as(COO^~) at 1590 cm^"1, 6 = v(CN) + δ(NH) for amide II at 1550 cm^"1, 7 = V_s(COO^") + δ(NH) at 1400 cm^"1. Peak 1 represents NH stretching mode at 3320 cm^"1 corresponding to the amine in pyrrole monomers and the α-amine, ε-amine and amide groups of the polypeptides poly-glutamic acid, poly-Lysine and lamellin. Peak 2 represents the asymmetric stretching modes of methylene groups at 2950 cm^"1 corresponding to the amino acid residues in the polypeptides. Peak 3 represents the carbonyl stretching mode at 1710 cm^"1 corresponding to the carboxylic acid groups (- COOH) of poly-glutamic acid. Peak 4 represents the carbonyl stretching mode at 1675 cm^" corresponding to the amide I bonds in the polypeptides. Peak 5 represents the asymmetric stretching mode at 1590 cm^"1 corresponding to the deprotonated carboxylic acid groups (-COO^") on poly-glutamic acid. Peak 6 represents a combination of N-H bending and C-N stretching modes at 1550 cm^"1 corresponding to the amide II bonds in the polypeptides. Peak 7 shows a combination of a relatively weak scissor mode of α-CH₂ groups and a symmetric stretching mode of deprotonated carboxylic acid groups (-COO^") of poly-glutamic acid at 1400 cm^"1. A broad absorption band at 3320 cm^"1 (Peak 1) was observed in the FTIR (Fourier transform infrared) spectra for all samples of pPy-pGlu. The intensity of this band did not change significantly with the covalent attachment of the polypeptides pLys and Lmn, because the most abundant amine in the sample corresponds to pPy. Peak 2, assigned to the methylene stretching modes, become more prominent after covalent attachment of the polypeptides. Three absorbance peaks related to the carboxylic acid groups apparent in the FTIR spectrum of pPy-pGlu. Peak 3 corresponds to the carbonyl stretching modes of the carboxylic groups in pGlu. Peaks 5 and 7 correspond to the carboxylate stretching modes of deprotonated carboxylic acid groups in pGlu. In addition, peak 6, the amide II peak, was observed in the FTIR spectrum of the same polymer while peak 4, the amide I peak, overlaps the strong peak 3 carbonyl stretching modes of the carboxylic acids groups. Thus, direct evidence that pGlu is present in the electrodeposited polypyrrole films made in Examples 1-2 is provided by the presence of carboxylate and amide peaks in the infrared spectra.

Fig. 5(b) is a graphic plot of the intensity of peaks at 1675 and 1400 cm^"1 for films of PP-pGlu, values taken from (a). Changes to the FTIR spectra resultant from the covalent attachment of pLys, Lmn, or pLys-Lmn are an increase in the intensity of amide I and amide II peaks 4 and 6 respectively (solid line) and a relatively small change in intensity of peaks 5 and 7 (dashed line) corresponding to the carboxylate stretching modes of deprotonated carboxylic acid groups in pGlu. Fig. 5b illustrates these changes. In Fig. 5b, the intensity of peaks 4 and 7 is compared for all samples of pPy-pGlu. The intensity of the amide peaks is expected to increase upon covalent attachment of polypeptides to pPy-pGlu because the relative concentration of amide groups increases. The fact that the intensity of the peaks corresponding to the deprotonated carboxylic acid groups in pGlu changed only slightly is evidence that the added polypeptides chemically react with carboxylate groups that are NHS- activated, namely those protruding from the surface of pPy-pGlu, to form new amid bonds. In contrast, the FTIR spectrum of pLys physically adsorbed onto pGlu exhibits a decrease in the intensity of peaks associated with free carboxylic acid groups and an increase in peaks associated with carboxylate groups. This reflects the formation of ion pairs between GIu and Lys residues upon physical adsorption instead of covalent attachment through the formation of amide bonds. Frey, supra.

Fig. 5(c) is a reproduction of the infrared spectra of PP prior to surface activation and formation of PP-pGlu (dotted line), subsequent to surface activation and formation of pPy-pGlu-NHS (solid line) and subsequent to covalent attachment of pLys and formation of pPy-pGlu-pLys (dashed line). Peaks related to the NHS substituent on pPy-pGlu-NHS are labeled: Nl = v(C=O) of NHS ester at 1815 cm^"1, N2 = V₈(C=O) of NHS carbonyls at 1785 cm^"1, N3 = v_as(C=O) for NHS carbonyls at 1785 cm^"1.

Fig. 5c illustrates the infrared spectra of pPy films prior to the electrostatic binding pGly, subsequent to the electrostatic binding (the form pPy-pGlu) and subsequent to the appending of pLys to form pPy-pGlu-Lys. Four peaks characteristic of an NHS ester appeared subsequent to activation of carboxylic acid groups (X = NHS, solid line in Fig. 5c). IN Fig. 5c, peak Nl represents the carbonyl streetch of NHS ester at 1815 cm^"1. PeakN2 represents the symmetric stretch of NHS carbonyls at 1785 cm^'1. PeakN3 represents the asymmetric stretch of NHS carbonyls at 1785 cm^"1. PeakN4 representing the asymmetric CNC stretch {v_as(C-N-C)} of NHS at 1218 cm^"1 is not shown. These peaks were not observed in the FTIR spectra of pPy prior to the surface modification or after the covalent attachment of pLys. The disappearance of the NHS-related peaks after the addition of pLys indicates that the reaction between the activated carboxylic acid groups of pPy[pGlu]-NHS and the amino groups of pLys proceeded to completion and resulted in the formation of amide bonds.

The percentage of carboxylic acid groups protruding from the surface relative to the total amount present in the film can be determined from the ratio of the intensities of the peaks that correspond to the NHS groups in pPy-pGlu-NHS to the intensities of the peaks that correspond to the carboxylic acid groups in pPy-pGlu. 2. Cyclic Volammetric (CV) Studies

The cyclic voltammograms of the electrodeposited pPy films were measured during the electrodeposition detailed in Example 2 with an EG & G Potentiostat/Galvanostat, Model 263. The counter electrode used was a platinum gauze and the reference electrode used was a saturated silver/silver chloride electrode (Ag/ AgCl, 0.197 V versus NHE). All potentials were reported versus Ag/ AgCl. Fig. 2a illustrates the results. Faradaic current at voltages more positive than 0.6 V (oxidation or pyrrole) and non-faradaic charging current at voltages of between 0.2 and 0.6 V both increased with each cycle. This increase is attributed to an increase in the surface area of the electrode, which provides indirect evidence that the conductive polymer pPy[Glu] was deposited. The increase in surface area of the electrode is determined from the relation:

Electrode Area (in m²) = C x C_df¹ where C is capacitance (in F or CV^"1) and Cd₁ is the double layer capacitance per unit area (Fm^"2). Capacitance is a measure of the amount of charge stored at the interface between an electrode and the electolyte and is related to two experimental parameters in cyclic voltammerty by the relation: C = IV -" where i is current ( in A or Cs^"1) and v is scan rate (Vs^"1). The double layer capacitance is an intrinsic property of a given combination of electrode surface and electrolyte and is approximately the same value for both an ITO/glass electrode coated with pPy-pGlu and a clean ITO/glass electrode immersed in the same electrolyte. Song, Electrochimica Acta 45: 2241-057 (2000). Consequently, the area of an electrode can be determined from the non-faradaic charging current in cyclicc voltammograms obtained at different scan rates:

Electrode Area (m²) = iv ^~l x C_di^{" 1} Illustrated in Fig. 2b are CVs of an ITO/glass electrode coated with pPy-pGlu. Three different scan rates were used to generate the CVs. The values of current at 150 mV from these three CVs are plotted as a function of scan rate in Fig. 2c. Data generated from a clean ITO/glass electrode is included in Fig. 2c for comparison. The current data for both samples are proportional to scan rate with a slope of 255 uF cm^"2 for an ITO/glass electrode coated with pPy-pGlu and a slope of 8.84 uF cm^"2 for a clean ITO/glass electrode using the final equation above, where the values per unit area are based on the geometric area of the electrodes. Based on the ratio of these two slopes, the surface area of the ITO/glass electrode increased 30-fold subsequent to the electrodeposition of pPy-pGlu.

Example 4: Preparation of Dorsal Root Ganglion

Dorsal root ganglion (DRG) from postnatal P0-P5 rat pups were dissected and cleaned of axons, blood and connective tissue. The ganglia were incubated in 0.05% trypsin-EDTA in Hank's Balanced Salt solution at 37 deg. C for 45 min. Digestion was terminated by the addition of an equal volume of media with serum: Dulbecco's Modified Eagle Medium, 10% fetal bovine serum, 4 mM L-glutamine, penicillin (100 μg/ml) and 50 ng/ml nerve growth factor (NGF, from Sigma- Aldrich). DRG were dissociated by titration in medium with serum and the DRG cells were added to the film compositions made in Example 2 in a 24-well plate at a density of 80,000 cells/well and incubated in media with serum at 37 deg.C and 5% CO₂ for 3 hours at 37 ⁰C. The cells were then cultured in serum-free medium containing Neurobasal medium 0.5 mM GlutaMAX, B27 supplement and 50 ng/ml NGF (all from Invitrogen Life Technologies) at 37 deg C and 5% CO₂. As a control, samples consisting of DRG cultured on acid-washed glass cover slips coated with a 0.1% solution of poly- L-lysine followed by a solution containing 50 μg/ml lamellin were cultured in the same medium.

Example 5: DRG Immunocytochemistiy Immunocytochemistry was performed on the DRG preparations made in

Example 4. The cells were fixed in 2% paraformaldehyde at room temperature for 15 min. and rinsed in phosphate buffered saline (PBS). Non-specific binding of secondary antibody was blocked with a solution of 5% normal goat serum, 1% bovine serum albumin (BSA) and 0.1% Triton-XlOO in PBS for 60 minutes at room temperature. Samples were incubated overnight at 4 deg. C with rabbit anti-GAP43 (from Chemicon, Temecula, CA) in a ratio of 1:500, diluted in 5% normal goat serum, 1% BSA and PBS. After washing with PBS, the cells were incubated in Cy2- coηjugated goat anti-rabbit secondary antibody (from Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 60 minutes at room temperature and washed again with PBS. Control cells were incubated only in secondary antibody. The cells were mounted onto glass slides using a SlowFade Antifade Kit with DAPI nuclear counter stain (from Molecular Probes, Eugene, OR) prior to optical analysis.

Example 6. Use ofMicropatterned Substrates to Control DRG Location The dissociated DRG prepared as described in Example 4 above were cultured in the presence of the micropatterned ITO/Glass electrodes coated with either the electrodeposited doped substrate pPy-pGlu film or the electrodeposited doped substrates bound to the nerve guidance cue laminin alone and bound through the polylysine linker, pPy-pGly-pLmn and pPy-pGlu-pLys-Lmn. The cultures contained a mixed population of cells, composed mainly of neurons, glia and fibroblasts. In general, the cells showed good viability, adhesion and neurite extension. Very few cells were observed on either untreated ITO/glass substrates or ITO/glass substrates plated with the control pPy-pGlu doped substrate. In contrast, DRG cell adherence was strongly preferential in areas of the electrodes containing the attached guidance cues. These results are shown in Figs. 6-8.

Fig. 6 shows xerographic reproductions of phase contrast and fluorescent images of DRG cells fixed two days post-plating. Photos (a), (c) and (e) are the phase contrast images and photos (b), (d) and (f) are the fluorescent images. The magnification is 5X for (a)-(b) and 2OX for (c)-(f). Neurites stained positive for GAP-43, which, in these reproductions, shows as dark in the phase contrast plates and as light in the fluorescent plates. Cell nuclei are labeled with DAPI (blue fluorescence, cannot be seen). In Fig. 7, xerographic reproductions of phase contrast fluorescent images of dorsal root ganglia adhered to a surface of PP-pGlu-Lmn are shown. Plate (a) is the phase contrast image and plate (b) is the fluorescent image. The cells were fixed twelve days post-plating. The magnification is 1OX. Neurites stained positive for GAP-43, which, in these reproductions, shows as dark in the phase contrast plates and as light in the fluorescent plates.

In Fig. 8, xerographic reproductions of phase contrast and fluorescent images of dorsal root ganglia neuronal cells on PP-pGlu-pLys-Lmn are shown. Plate (a) is the phase contrast image and plates (b) and (c) are the fluorescent images. This time, the cells were fixed two days post-plating. The magnification is 1OX for (a)-(b) and 4X for (c). Neurites stained positive for GAP-43, which is expressed by developing and regenerating neurons and used to score neuronal regeneration.

In all plates, cellular extensions developed within the areas of the positive guidance cues, with many neurite extensions co-aligned. The cellular extensions were indirectly confirmed to be neurites by the immunofluorescent GAP-43 staining. Cells adhering to areas outside the substrate developed very few cellular extensions, which did not stain positive for GAP-43, and these extensions were unaligned with neurites on the on the substrates.

In sum, the foregoing examples demonstrate a general method for fabricating doped substrates having the ability to attach biological molecules to serve, for example, as nerve growth guidance cues. Besides positive cues, the method may be employed to attach biological molecules which act as negative, inhibitory, cues and to fabricate doped substrates having the ability to attach both positive and negative cues. Although the invention has been described in detail with reference to certain, embodiments, one skilled in the art will appreciate that it can be practiced by others than the explicitly described embodiments, which are presented for the purpose of illustration and not limitation.

Claims

I claim:

1. A nerve growth guidance composition to stimulate nerve growth or nerve regeneration comprising: i) a cationic, aromatic polyheterocyclic substrate; ii) an anionic dopant, electrostatically bound to said substrate; iii) a nerve growth guidance cue bound to said anionic dopant.

2. The composition of claim 1 wherein said substrate comprises a matrix composed of a cationic, aromatic polyheterocyclic compound and compound selected from the group consisting of a polysaccharide, a polyester, a polyhydroxybutyric acid, a polyglycolic acid, a polylactic acid, a polylactic-co-glycolic acid, a polystyrene sulfonate and mixtures thereof. Enhanced mechanical strength enhanced conductivity

3. The composition of claim 1 wherein said cationic polymeric substrate is selected from the group consisting of polypyrrole, polythiophene, polyaniline and derivatives thereof.

4. The composition of claim 2 wherein said cationic polymeric substrate is selected from the group consisting of polypyrrole, polythiophene, polyaniline and derivatives thereof.

5. The composition of claim 3 wherein said anionic dopant is selected from the group consisting of acidic amino acids, amino acids with uncharged polar R groups, acidic polyamino acids, and polyamino acids with uncharged polar R groups.

6. The composition of claim 4 wherein said anionic dopant is selected from the group consisting of acidic amino acids, amino acids with uncharged polar R groups, acidic polyamino acids, and polyamino acids with uncharged polar R groups.

7. The composition of claim 5 wherein said anionic dopant is an acidic polyamino acid.

8. The composition of claim 6 wherein said anionic dopant is an acidic polyamino acid.

9. The composition of claim 7 wherein said polyamino acid is polyglutamic acid.

10. The composition of claim 8 wherein said polyamino acid is polyglutamic acid.

11. The composition of claim 3 wherein said nerve growth guidance cue is selected from the group consisting of proteins and peptides encoded by neuronal regeneration-associated genes, cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, cytokines, neurotrophins, vasoactive intestinal peptide, galanin and neuropeptide Y, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, acidic fibroblast growth factor, platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), VEGF, neuregulin, activin, nerve growth factor receptor p75, GAP-43, CAP-23, SPRRlA polypeptides, Regeneration Induced CNPases, CHPases, NF-kappa-β, P134K, GSK-3β, APC, Nogo receptor inhibitors, extracellular matrix molecules, glycoproteins, laminin, fibronectin and collagen (types I and/or FV), glycosaminoglycans, hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate, proteoglycans, phosphacan, NG2 proteoglycan, agrin, receptor-type protein tyrosine phosphatase, neurocan and brevican.

12. The composition of claim 4 wherein said nerve growth guidance cue is selected from the group consisting of proteins and peptides encoded by neuronal regeneration-associated genes, cytoskeletal proteins, neurotransmitter metabolizing enzymes, neuropeptides, cytokines, neurotrophins, vasoactive intestinal peptide, galanin and neuropeptide Y, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, neurotrophin-4/5, acidic fibroblast growth factor, platelet-derived growth factor (PDGF), glial cell line-derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), VEGF, neuregulin, activin, nerve growth factor receptor p75, GAP-43, CAP-23, SPRRlA polypeptides, Regeneration Induced CNPases,

CHPases, NF-kappa-β, P134K, GSK-3β, APC, Nogo receptor inhibitors, extracellular matrix molecules, glycoproteins, laminin, fibronectin and collagen (types I and/or IV), glycosaminoglycans, hyaluronic acid, dermatan sulfate, chondroitin sulfate, heparin, heparan sulfate, and keratan sulfate, proteoglycans, phosphacan, NG2 proteoglycan, agrin, receptor-type protein tyrosine phosphatase, neurocan and brevican.

13. A nerve growth guidance composition to stimulate nerve growth or nerve regeneration comprising: i) a cationic, aromatic polyheterocyclic substrate selected from the group consisting of polypyrrole, polythiophene, polyaniline and derivatives thereof alone or in a matrix forming combination with a compound selected from the group consisting of a polysaccharide, a polyester, a polyhydroxybutyric acid, a polyglycolic acid, a polylactic acid, a polylactic-co-glycolic acid, a polystyrene sulfonate and mixtures thereof; ii) an acidic polyamino acid dopant electrostatically bound to said substrate; and iii) a nerve growth guidance cue bound to said acidic polyamino acid dopant.

14. The composition of claim 13 wherein said substrate is polypyrrole and said dopant is polyglutamic acid.

15. An implantable nerve growth guidance device for inducing or stimulating nerve growth or nerve regeneration in mammals comprising a composition of claim 1.

16. An implantable nerve growth guidance device for inducing or stimulating nerve growth or nerve regeneration in mammals comprising a composition of claim 1.

17. An implantable nerve growth guidance device for inducing or stimulating nerve growth or nerve regeneration in mammals comprising a composition of claim 13.

18. A method for stimulating nerve growth or nerve regeneration in mammalian subjects comprising administering at the site where nerve growth or nerve regeneration is desired the device of claim 15.

19. A method for stimulating nerve growth or nerve regeneration in mammalian subjects comprising administering at the site where nerve growth or nerve regeneration is desired the device of claim 16.

20. A method for stimulating nerve growth or nerve regeneration in mammalian subjects comprising administering at the site where nerve growth or nerve regeneration is desired the device of claim 17.