| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | February 18, 2003 |
| PATENT TITLE |
Biodegradable cross-linkers having a polyacid connected to reactive groups for cross-linking polymer filaments |
| PATENT ABSTRACT |
Biodegradable cross-linkers are provided having a polyacid core with at least two acidic groups covalently connected to reactive groups usable to cross-link polymer filaments. Between at least one reactive group and an acidic group of the polyacid is a biodegradable region which preferably consists of a hydroxyalkyl acid ester sequence having 1, 2, 3, 4, 5 or 6 hydroxyalkyl acid ester groups. The polyacid may be attached to a water soluble region that is attached to the biodegradable region having attached reactive groups. The hydroxyalkyl acid ester group is preferably a lactate or glycolate. Polyacids include diacids, triacids, tetraacids and pentaacids, and the reactive group may contain a carbon-carbon double bond. A network of cross-linked polymer filaments having adefined biodegradation rate can be formed using the cross-linkers. The network may contain biologically active molecules, and can be in the form of a microparticle or nanoparticle, or hydrogel. The polymer filaments may be preformed polymer filaments of polynucleic acids, polypeptides, proteins or carbohydrates. The cross-linkers may be copolymerized with charged monomers such as acrylic monomers containing charged groups. Applications of the cross-linkers and network include controlled release of drugs and cosmetics, tissue engineering, wound healing, hazardous waste remediation, metal chelation, swellable devices for absorbing liquids and prevention of surgical adhesions. |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | June 22, 1999 |
| PATENT REFERENCES CITED |
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| PATENT CLAIMS |
What is claimed: 1. A monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly being covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of hydroxyalkyl acid ester groups selected from the group consisting of 1, 2, 3, 4, 5 and 6; the cross-linker being usable for crosslinking polymer filaments to form a network of cross-linked polymer filaments with a defined biodegradation rate. 2. The cross-linker of claim 1 wherein the polyacid is a polycarboxylic acid. 3. The cross-linker of claim 1 wherein a water soluble region is between at least one of said acidic groups and said reactive groups. 4. The cross-linker of claim 1 wherein the cross-linked polymer filaments are those of a hydrogel. 5. The cross-linker of claim 1 wherein the polymer filaments are hydrophobic. 6. The cross-linker of claim 1 wherein the polyacid comprises at least one acidic group attached to a water soluble region. 7. The cross-linker of claim 1 wherein the polyacid is a diacid. 8. The cross-linker of claim 1 wherein the polyacid is a triacid. 9. The cross-linker of claim 1 wherein the polyacid is a pentaacid or tetraacid. 10. The cross-linker of claim 1 wherein the polyacid is ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DTPA). 11. The cross-linker of claim 1 wherein when polymer filaments are cross-linked, the biodegradable sequence contains at least two hydroxyalkyl acid ester groups. 12. The cross-linker of claim 1 wherein when polymer filaments are cross-linked, the biodegradable sequence contains one hydroxyalkyl acid ester group. 13. The cross-linker of claim 1 wherein the hydroxyalkyl acid ester sequence comprises an alpha hydroxyalkyl acid ester group. 14. The cross-linker of claim 1 wherein the biodegradable sequence comprises a hydroxyalkyl acid ester group selected from the group consisting of at least one of lactate and glycolate. 15. The cross-linker of claim 1 wherein the hydroxyalkyl acid ester sequence contains a hydroxyalkyl acid ester group selected from the group consisting of glycolic ester, DL-lactic acid ester, L-lactic acid ester, and combinations thereof. 16. The cross-linker of claim 1 further comprising at least one member selected from the group consisting of ethylene glycol oligomer, poly(ethylene) glycol, poly(ethylene oxide), poly(vinylpyrolidone), poly(ethylene oxide)-co-poly(propylene oxide), and poly(ethyloxazoline). 17. The cross-linker of claim 1 wherein the reactive group contains a carbon-carbon double bond. 18. The cross-linker of claim 1 wherein the reactive group is an end group. 19. The cross-linker of claim 1 wherein the reactive group contains a carbonate, carbamate, hydrazone, hydrazine, cyclic ether, acid halide, acyl azide, succinimidyl ester, imidazolide or amino functionality. 20. The cross-linker of claim 1 wherein cross-linking of polymer filaments can be started by thermal, catalytic or photochemical initiation. 21. The cross-linker of claim 1 wherein cross-linking of polymer filaments can be initiated by pH change. 22. The cross-linker of claim 1 wherein crosslinking of polymer filaments can be by free radical addition or Michael addition. 23. The cross-linker of claim 1, or network of claim 25 wherein the polyacid has a molecular weight between 60 and 400 Da; the hydroxyalkyl acid ester sequence has a molecular weight between 70 and 500 Da and the reactive group has a molecular weight between 10 and 300 Da. 24. The cross-linker of claim 1 where the polyacid is selected from the group consisting of succinic acid, adipic acid, fumaric acid, maleic acid, sebacic acid, malonic acid, tartaric acid and citric acid. 25. A network of cross-linked polymer filaments with a defined biodegradation rate and cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of hydroxyalkyl acid ester groups selected from the group consisting of 1, 2, 3, 4, 5 and 6. 26. A network of cross-linked polymer filaments with a defined biodegradation rate under in vivo mammalian conditions formed of preformed polymer filaments of polynucleic acids, polypeptides, proteins or carbohydrates and cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of hydroxyalkyl acid ester groups selected from the group consisting of 1, 2, 3, 4, 5 and 6. 27. The network of claim 26 comprising biologically active molecules. 28. A network of cross-linked polymer filaments with a defined biodegradation rate under mammalian in vivo conditions, cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of hydroxyalkyl acid ester groups selected from the group consisting of 1, 2, 3, 4, 5 and 6; said network comprising an organic molecule, inorganic molecule, protein, carbohydrate, poly(nucleic acid), cell, tissue or tissue aggregate. 29. A network of cross-linked polymer filaments with a defined biodegradation rate under mammalian in vivo conditions cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of hydroxyalkyl acid ester groups selected from the group consisting of 1, 2, 3, 4, 5 and 6, and the network comprising an organic radioisotope, inorganic radioisotope or nuclear magnetic resonance relaxation reagent. 30. A microparticle or nanoparticle cross-linked polymer composition with a defined biodegradation rate under mammalian in vivo conditions and containing polymer filaments cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid with at least two acidic groups directly or indirectly covalently connected to reactive groups usable to cross-link polymer filaments wherein between at least one reactive group and an acidic group of the polyacid is a biodegradable sequence consisting of a hydroxyalkyl acid ester sequence having a number of -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION The present invention relates to novel cross-linking agents, more particularly to novel biodegradable cross-linking agents. Earlier use of cross-linking agents in a variety of fields involving proteins, carbohydrates or polymers is well established. Even biodegradable cross-links have previously been prepared and utilized. However, none before have utilized particular and advantageous cross-linker designs of the present invention. Within the pharmaceutical, agricultural, veterinary, and environmental industries, much attention has been directed to the applications of biodegradable polymers. The Oxford English dictionary defines biodegradable as: "susceptible to the decomposing action of living organisms especially bacteria or broken down by biochemical processes in the body." However, due to the advent of the widespread use of polyhydroxyacids as degradable polymers, this definition should be extended to include non-enzymatic chemical degradation which can progress at an appreciable rate under biologically relevant conditions (the most relevant condition being water at pH 7; 100 mM salt and 37.degree. C.). Thus, the meaning of the term biodegradation can be broadened to include the breakdown of high molecular weight structures into less complicated, smaller, and soluble molecules by hydrolysis or other biologically derived processes. In the biomaterials/pharmaceutical area, there is great interest in the use of biodegradable materials in vivo, due to performance and regulatory requirements. However, most of the reports on biodegradable materials have focused on linear water-insoluble hydroxyacid polyesters. Much less work has been done on biodegradable network polymers which are cross-linked. Therefore, due to the unique properties of network polymers, it is to be expected that biodegradable networks will find many new and important applications Biodegradable Polymers Much work has been accomplished in the last 20 years in the area of hydrophobic biodegradable polymers, wherein the biodegradable moieties comprise esters, lactones, orthoesters, carbonates, phosphazines, and anhydrides. Generally the polymers made of these biodegradable linkages are not water soluble and therefore in themselves are not amenable for use in systems where water is required, such as in hydrogels. Since the mechanism of biodegradation in these polymers is generally through the hydrolytically-active components of water (hydronium and hydroxide ions), the rate of hydrolytic scission of the bonds holding a polymer network together is generally pH sensitive, with these moieties being susceptible to both specific-acid catalyzed hydrolysis and base hydrolysis. Other factors affecting the degradation of materials made of these polymers are the degree of polymer crystallinity, the polymer volume fraction, the polymer molecular weight, the cross-link density, and the steric and electronic effects at the site of degradation. Degradable Network Structures Biodegradable network structures are prepared by placing covalent or non-covalent bonds within the network structure that are broken under biologically relevant conditions. This involves the use of two separate structural motifs. The degradable structure is either placed into (i) the polymer backbone or (ii) into the cross-linker structure. The method described herein creates a degradable structure through placing degradable regions in the cross-linking domain of the network. One of the first occurrences of degradable hydrogels was published in 1983 by Heller. This system contains a water soluble linear copolymer containing PEG, glycolylglycolic acid and fumaric acid linkages. The fumaric acid allowed the linear polymer to be cross-linked through free radical polymerization in a second network forming polymerization step, thus creating a polymer network which could degrade through hydrolysis of the glycolic ester linkages. This is an example of creating degradable linkages in the polymer backbone. Biodegradable Cross-linkers The first truly degradable cross-linking agents were made from aryl diazo compounds for delivery of drugs in the digestive tract. The diazo moiety is cleaved by a bacterial azoreductase which is present in the colon. This has been used to create colon specific delivery systems (Brondsted et al. & Saffan et al.). Another biodegradable cross-linking agent appears in the work of Ulbrich and Duncan where a bis-vinylic compound based on hydroxyl amine was synthesized. Hydrogels made from this degradable cross-linker were shown to undergo hydroxide induced hydrolysis of the nitrogen-oxygen bond. Hubbell et al. have made hydrogels composed of macromonomers composed of a central PEG diol which was used as a bifunctional alcohol in the tin octanoate catalyzed transesterifying ring opening polymerization of lactide to give a bis-oligolactate PEG. This compound was then reacted with acryloyl chloride to give a macromolecular cross-linker which could be formed into a homo-polymer interpenetrating network of PEG and oligolactylacrylate through free radical polymerization (Pathak et al.). Hubbell mostly intended these compounds for use as photopolymerizable homo-polymers useful to prevent surgical adhesions. A second solution to this problem has been recently reported in the work of Van Dijk et al. which is the first report of a biodegradable cross-linking macromonomer composed of alpha-hydroxy esters (Van Dijk-Wolthius et al.). This work combines natural polymers with synthetic polymers in an interpenetrating network. This group functionalized dextran with oligo-alpha hydroxy acid domains which were end capped with vinyl regions that were polymerized into biodegradable networks via free radical polymerization. The most recent report of a biodegradable cross-linking agent was one designed to undergo enzymatic degradation. This cross-linker is composed of a centro-symmetric peptide terminated by acrylamide moieties with a central diamine linking the two ends (Kurisawa et al.). This report is related to the invention described herein in that the property of biodegradability is built into the polymer network by first synthesizing a small symmetrical cross-linker which can undergo cleavage, then incorporating this in a polymer network. Properties of Degradable Gels: Swelling and Porosity Since degradability is a kinetic effect, the properties of degradable gel networks are the similar to those standard gel networks, except they change with time. The two main properties that are exhibited by degradable hydrogel networks are swelling and network porosity that increase with time as the network degrades. The main feature observed with degradable cross-linked polymer networks in solvents which cause them to swell is that the polymer network swells as it degrades. This is because network degradation results in a decrease in cross-link density. As the cross-link density decreases there is more available volume for solvent within the network. The solvent increasingly permeates the network structure, driven by a favorable thermodynamic mixing of solvent with the polymer network. Important uses envisioned for degradable gels are as controlled drug delivery devices and as degradable polymers for other in vivo uses. These devices are able to change from a high viscosity material (gel) to a lower viscosity soluble material (sol). The resulting water soluble linear polymer can then be readily transported and excreted or degraded further. Degradable hydrogel networks offer the opportunities to effect the diffusitivity of materials bound in the hydrogel network, because as the network degrades the diffusion coefficient of molecules in the network increases with time thus facilitating the release of materials locked within the polymer network (Park). Moreover, because the hydrogel network structure itself is of such a high molecular weight, transport of the hydrogel network out of the body or environment is slow. This is especially true in vivo where non-degradable implanted hydrogel networks can remain in the body for many years (Torchilin et al.). Therefore, such devices would be more useful if they could be made of a high molecular weight polymer that would degrade into smaller molecular weight components after the device has performed its task and then could be excreted through normal routes of clearance. Since excretion of polymers is molecular weight-dependent (Drobnik et al.), with the preferred route being through the renal endothelia (Taylor et al. & Tomlinson), the chains making up the polymer backbone should be between 10 and 100 kDa. Because the material is engineered to degrade into excretable parts, biodegradable hydrogel networks offer increased biocompatibility. Biodegradable Network Polymers as Controlled Release Depots Biodegradable network polymers can be used as carriers for biologically active substances. These include proteins, peptides, hormones, anti-cancer agents antibiotics, herbicides, insecticides and cell suspensions. The hydrophilic or hydrophobic polymer network can act as a stabilizing agent for the encapsulated species and as a means to effect a controlled release of the agent in to the surrounding tissue or systemic circulation. By changing the size of the depot, the degree of porosity, and the rate of degradation (through modification of the degradable regions in the polymer network) controlled release depots with a variety of release characteristics can be fashioned for application in the medical and diagnostic areas. Biodegradable Network Polymers as Water Adsorbents Owing to the ability of hydrophilic network polymers to adsorb water, biodegradable versions of these networks may prove to have many uses in items for example, sanitary napkins, wound dressings, and diapers. When these materials are used in consort with other degradable materials a completely biodegradable and disposable product could be produced. Although a literature search in the Chemical Abstracts database for biodegradable adsorbents produced no citations, the use of degradable adsorbents in the above mentioned products would be very desirable. Biodegradable Network Polymers as Adhesives There is a great need for biodegradable adhesives and sealers in surgery and elsewhere. Synthetic polymers have been used as adhesives in surgery with the cyano acrylate esters being the most commonly cited. Recent reports using biodegradable networks as sealants in dentistry and orthopedics have displayed the utility of biodegradable polymers (Burkoth). Here the use of a biodegradable cross-linking monomer (bis-methacrylated diacid anhydride) which has been photopolymerized is envisioned for use in dentistry. Here a hydrophobic network-forming monomer is photopolymerized in situ to form a mechanically stable and non-swellable bonding material. Degradability would be a desirable property for any short term application and of course would be undesirable for long term applications. Use of Biodegradable Polymers in Drug Delivery Since most biodegradable polymers are not soluble in water, a hydrophilic drug is formulated in these polymers by a dispersion method using a two phase system of water (containing drug) and organic solvent (containing the polymer). The solvent is removed by evaporation resulting in a solid polymer containing aqueous droplets. This type of system suffers from the need to use organic solvents which would be undesirable for protein delivery since the solvent may denature the protein. Therefore it is envisioned that hydrophilic biodegradable network polymers will improve the range of drugs delivered from this general glass of polymers. Biodegradable Nanoparticles The use of nanoparticles for colloidal drug delivery has been a goal of formulation scientists for the last 20 years. Nanoparticles are defined as any solid particle between 10 and 1000 nm and are composed of natural or more commonly synthetic polymers. The most useful method of production for the lower end of this size range is emulsion polymerization, where micelles act as a reaction template for the formation of a growing polymer particle. For passive delivery of anticancer agents to tumors, nanometer size particles (50-200 nm) are required. The small size is required for extravasation of the nanoparticles through the permeable tumor vasculature in a process termed the EPR effect (enhanced permeability and retention) (Duncan). Another important feature of any nanocarrier is the biocompatibility of the particle. This requires that the polymer particle degrades after some period so that it may be excreted. These criteria require polymer compositions that are well tolerated. To date there are no reports in the literature of degradable nanogels composed of well-tolerated parenternal polymers. Hydrogel particles can be made in several sizes according to the performance requirements of the drug delivery system being engineered. Gel particles in the nanometer size range that are capable of being retained in tumor tissue are preferred for delivery of anticancer agents. Methods for the creation of approximately 100 nm in diameter hydrogel particles involve the use of surfactant-based emulsion polymerizations in water. To make ionomeric nanogels by this method it is necessary to include a hydrophobic component in the monomer mixture, thus allowing partitioning of the monomers into the micellar phase followed by particle nucleation and further monomer adsorption (normally emulsion polymerizations are used to make hydrophobic latexes). Another important consideration is the means by which the carrier will load the drug substance to be delivered. The loading capacity of non-ionic hydrogels is generally limited by the aqueous solubility of the drug. However if the drug is charged, groups of opposite charge to the drug can be incorporated into the polymer to allow high drug loading through ion exchange. An interesting and perhaps useful property resulting from inclusion of charged monomers in the polymer network is a pH induced volume response of the polymer. Current State of the Art To date most biodegradable polymers have been synthesized using stepwise condensation of monomer resulting in a polydispersed molecular architecture. Since the rate of degradation is in part directly related to this architecture, this method results in the undesirable property that the material will contain cross-links with a variety of degradation rates. Secondly, since synthetic biodegradable polymers are generally water insoluble, there is a need for degradable moieties that are readily incorporated into water soluble monomers or polymers. Biodegradable moieties based on the non-soluble degradable units can be combined with water soluble oligomeric regions or polymers, resulting in a biodegradable structure. Therefore as an object of the present invention the new material would have the preferred characteristics that it was easily synthesized, composed of biocompatible components, and have a well defined molecular structure leading to defined biodegradation rates. It is a further object of the present invention that it be easily incorporated in many different polymer processing options such as polymer microparticles, nanoparticles and slab gels. Therefore, the use of organic synthesis methodology to incorporate monodispersed degradable sequences into the monomer structure before polymer formation permits control of overall degradation as well as the release rate of entrapped substances. Previous work in the area of creating biodegradable cross-linkers by Hubbel teaches a method to create degradable sequences using ring opening polymerization of lactide or glycolide. This method creates a mixture of degradable units with varying molecular weights or chain lengths in the end product. The present invention described herein teaches a method of stepwise synthesis of the degradable region which creates a pure compound at the end of the synthesis. Therefore, since the length of the degradable region will be the major structural determinant of the degradation rate, the present invention provides for a more controlled degradation rate than the Hubbel invention. Our invention also provides compounds which will be easier to purify than the Hubbel invention owing to stepwise syntheses of the degradable region and the resulting purity of the reaction product. Other advantages of our invention over Hubbel's invention are that the invention described herein is applicable to hydrophobic networks as well as hydrophilic networks whereas Hubbel is restricted to hydrophilic networks, and the invention herein can generate all useful properties such as rapid degradation rate and water solubility through the syntheses of oligomeric cross-linking compounds without resorting to polymeric cross-linking compounds. The present preferred embodiment of this invention is as cross-linkers which are composed of a symmetrical diacid attached to at least one biodegradable region. These regions may consist of alpha hydroxy acids such as glycolic or lactic acid. These degradable portions are then terminated directly or indirectly by a functional group which may be polymerized. Moreover component pieces of the degradable gel such as lactic, glycolic and succinic acids are members of the Krebs cycle and therefore readily metabolized in vivo, while the end groups become incorporated into water-soluble polymer, which is eliminated by renal excretion. SUMMARY OF THE INVENTION In one important aspect the present invention concerns a monomeric or oligomeric cross-linker comprising a polyacid core with at least two acidic groups directly or indirectly connected to a reactive group usuable to cross-link polymer filaments, with at least one acidic group being connected to a region degradable under aqueous conditions and the degradable regions or (in the case of a single degradable region), the degradable region at at least one other acidic group directly or indirectly having a covalently attached reactive group usable to cross-link polymer filaments interceding between the acidic group and a reactive group. Thus the at least two reactive groups are always interspaced by at least one degradable region. In many preferred applications, the cross-linker is utilized to cross-link water soluble polymeric filaments. The polyacid core may be attached to a water soluble region that is in turn attached to a degradable region (or vice versa) having an attached reactive group. A polycarboxylic acid is the preferred polyacid. The polyacid core is preferably a diacid, triacid, tetraacid or pentaacid. The most preferred polyacid core is a diacid. Preferred polyacids or polycarboxylic acids. Alkyl-based diacids such as malonic, succinic, adipic, fumaric, maleic, sebacic and tartaric are preferred. Diacids such as succinic, adipic or malonic acid are particularly preferred. A triacid such as citric acid, for example, is usable. Tetra-and penta-acids such as ethylenediamine tetraacidic acid (EDTA) or diethylenetramine pentaacetic acid (DTPA) are usable, for example. When cross-linked polymer filaments are formed according to the present invention, they are cross-linked by a component having at least one degradable region. Preferred degradable regions include poly(alpha-hydroxy acids), although other hydroxy alkyl acids that may form polyesters can be used to form biodegradable regions. Preferred polyesters include those of glycolic acid, DL lactic acid, L lactic acid, oligomers, monomers or combinations thereof. Cross-linkers of the present invention may also include a degradable region containing one or more groups such as anhydride, a orthoester and/or a phosphoester. In certain cases the biodegradable region may contain at least one amide functionality. The cross-linker of the present cross-linker may also include an ethylene glycol oligomer, oligo(ethylene glycol), poly(ethylene oxide), poly(vinyl pyrolidone), poly(propylene oxide), poly(ethyloxazoline), or combinations of these substances. Preferred reactive groups are those that contain a carbon-carbon double bond, a carbonate, a carbamate, a hydrazone, a hydrazino, a cyclic ether, acid halide, a acylazide, succinimidyl ester, imidazolide or amino functionality. Other reactive groups may be used that are known to those skilled in the art to be precursors to polymers. Utilizing the cross-linkers of the present invention, networks of polymer filaments may be formed by thermal, catalytic or photochemical initiation. Networks of polymer filaments may likewise be formed by pH changes. Networks of polymer filaments may also be formed for example by free radical addition or Michael addition. The present invention comprises a network of polymer filaments formed by precipitation or emulsion polymerization and cross-linked by a monomeric or oligomeric cross-linker comprising a poly acid core with at least one acidic group connected to a region degradable under in vivo conditions and having at least two covalently attached reactive groups usable to cross-link polymer filaments. Polymeric filaments to be cross-linked include preformed polymer filaments such as polynucleic acids, polypeptides, proteins or carbohydrates. Such cross-linked polymeric filaments may be utilized to contain biologically active molecules. The biologically active molecules may be organic molecules, proteins, carbohydrates, polynucleic acids, whole cells, tissues or tissue aggregates. The preferred monomeric or oligomeric cross-linker of the present invention has a polyacid core with a molecular weight between about 60 and about 400 Daltons. The degradable region(s) has a preferable molecular weight range of about 70 to about 500 Daltons. The reactive groups of the cross-linker of the present invention may be end groups and have preferred molecular weights between about 10 and 300 Daltons. An important aspect of the present invention is a monomeric or oligomeric cross-linker comprising a polyacid core with at least two esterified groups being connected (directly or indirectly) to reactive groups usable to cross-link polymer filaments. Between at least one reactive group and polyacid core is a region degradable under aqueous conditions. Thus the cross-linker is usable to form cross-linked polymer filaments. In a preferred embodiment, the polyacid core has two acidic groups connected to a region degradable under aqueous conditions, each having a covalently attached reactive group usable to form cross-linked polymer filaments. In certain cases the cross-linkers of the present invention may contain a water soluble region located between at least one carboxyl group and its associated reactive group. A preferred polymer filament for cross-linking is a hydrogel. In certain cases the polymer filament being cross-linked may be hydrophobic. In many cases the polyacid core of the present inventive cross-linker is a diacid, such as for example succinic acid, adipic acid, fumaric acid, maleic acid, sebacic acid or malonic acid. Triacids such as citric acid are also usable. Other triacids will be apparent to those of skill in the art. Tetraacids and pentaacids may also be used. A preferred tetraacid is ethylene diamine tetraacetic acid (EDTA) and a preferred pentaacid is diethylenetriamine pentaaceticic acid (DTPA). Acids that may be used as a polyacid core include citric acid, tartaric acid and the like. A preferred biodegradable region for use in the cross-linkers of the present invention is one that comprises a hydroxy alkyl acid ester. A preferred hydroxy acid ester is an alpha hydroxy acid ester. Under some circumstances the degradable region may be a peptide. Preferred degradable polyesters include glycolic polyester, DL lactic acid polyester and L lactic acid ester or combinations thereof. In certain cases the degradable region of the cross-linker of the present invention may comprise an anhydride, orthoester or phosphoester linkages. In certain cases the reactive group of the present inventive cross-linker contains a carbon-carbon double bond. In some cases the reactive group is an end group, e.g. at the end of a degradable region. The reactive group may also contain a carbonate, carbamate hydrazone, hydrazino, cyclic ether, acid halide, acyl azide, succinimidyl ester, imidazolide or amino functionality. The cross-linker of the present invention may be utilized to form networks of polymer films formed by thermal catalytic or photochemical initiation. In certain cases networks of polymer films may be formed as induced by a pH change and then cross-linked. In other cases, networks of polymer films may be formed through reactions involving free radical addition or Michael addition. The aqueous conditions under which the cross-linkers of the present invention are degradable are most frequently physiological conditions. In an important aspect, the present invention comprises a network of polymer filaments formed by precipitation, dispersion or emulsion polymerization and cross-linked by a monomeric or oligomeric cross-linker having a polyacid core with at least two esterified groups connected to a covalently attached reactive group used to cross-link polymer filaments and at least one acidic group having a region degradable under aqueous conditions between the acidic group and the reactive group. Also included in the present invention are networks of polymer filaments of polynucleic acids, polypeptides, proteins or carbohydrates and cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid core with at least two esterified groups connected to at least one region degradable under in vivo conditions, and having a covalently attached reactive group cross-linking the polymer filaments. In both cases of networked polymer filaments, these networks may contain biologically active molecules. Because the cross-links are degradable, these biological molecules will be expected to be released. In one important aspect, the present invention comprises a network of polymer filaments cross-linked by a monomeric or oligomeric cross-linker comprising a polyacid core with at least two acidic groups connected to at least one region degradable under in vivo conditions, and both acidic groups connected to a covalently attached reactive group and defined further as comprising an organic molecule, inorganic molecule, protein, carbohydrate, poly(nucleic acid), cell, tissue or tissue aggregate. Additionally, the invention includes a network of polymer filaments cross-linked by monomeric or oligomeric cross-linker comprising a central polyacid core with at least two acidic groups connected to at least one region degradable under in vivo conditions, and terminated by a covalently attached reactive end group usable to cross-link polymer filaments, the network comprising an organic radioisotope, inorganic radioisotope or nuclear magnetic resonance relaxation reagent. According to the present invention the polyacid core has a preferred molecular weight between about 60 and about 400 daltons. The degradable region of the cross-linker has a preferred molecular weight between about 70 and about 500 daltons. The reactive groups of the present invention generally have molecular weights between about 10 and about 300 daltons. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 schematically illustrates a representative lactate-based cross-linking agent of the present invention. FIG. 2 schematically displays a synthetic method for symmetrical biodegradable cross-linkers such as HPMALacSuc 5a, HPMAGlySuc 5b, HPMALacLacSuc 7a, and HPMAGlyGlySuc 7b. Conditions: (a) CH2Cl2, pyridine 0.degree. C.; (b) Pd/C 50 psi H2, i-PrOH; 0.degree. C.; (c) carbonyldiimidazole CDI, DMF, 0.degree. C.; HPMA, rt.; (e) (CDI), DMF, 0.degree. C.; benzyl lactate (6a); benzyl glycolate (6b); (f) Pd/C 50 psi H2, i-PrOH. FIG. 3 displays a photograph of biodegradable gels of the same composition with 1.5 mole % cross-linker after incubation in pH 7 phosphate buffer at 37.degree. C. for varying amounts of time. (a) control gel made up of compound 2 after 15 days (b-d) compound 5b after 2, 5 and 15 days, respectively. FIG. 4 displays the degradative swelling of HPMA-co-XL gels made from 4 different cross-linkers in pH 7.3 buffer; 100 mM phosphate buffer; 1=200 mM at 37.degree. C. The cross-linker labeled HPMASuc is non-degradable. FIG. 5 displays a plot of the half-life to dissolution versus pH for three different degradable cross-linkers studied at 37.degree. C. FIG. 6 displays a photograph of p (HPMA) degradable gels with 1.5 mole % cross-linker and containing a deep red fluorescent dye--thus the dark color--after incubation in pH 7 phosphate buffer at 37.degree. C. for varying amounts of time. (a) control gel made from compound 2 after 15 days (b and c) compound 7b after 4, 8 days respectively. FIG. 7 displays a plot comparing the swelling response and the release of tetramethyl rhodamine labeled albumin from the degradable gel network for HPMAGlyGlySuc 7b at pH 7.3 at 37.degree. C. DESCRIPTION OF PREFERRED EMBODIMENTS This invention discloses a representative synthesis and application of symmetrical biodegradable cross-linking agents for use in cross-linked polymer matrices formed into particles or slabs that may be used e.g., in drug delivery. The cross-linking agents will be monomers or oligomers of biocompatible units in the preferred biological applications. In the preferred practice of this invention the cross-linker is composed of a central diacid (such as succinic); to this diacid is attached one or more biodegradable regions, which are then terminated by reactive moieties which are used for incorporation into the polymer network. This invention requires there be at least two reactive moieties (two representative cross-linkers are depicted in FIG. 1). The cross-linkers may be incorporated into matrices of various sizes ranging from hundreds of cm's to 10 nm so as to control the diffusion of substance such as drugs e.g., from the matrix by biodegradation of the cross-linkers under physiological conditions. Ultimately the cross-linkers described above may be included in all variety of hydrophilic and hydrophobic polymer networks to which the desirable property of degradation is required. Design of Centro-symmetric Degradable Cross-linkers Based on the Alpha-hydroxy Acids Of importance in hydrogel engineering is the control the structural properties of a random polymeric network. In standard stepwise growth of polymers there is heterogeneity in copolymer composition and dispersity in the molecular weight of the polymer filaments thus making it difficult to precisely control bulk material properties of the polymer network such as crystallinity and mesh size. By engineering homogeneous structures into the polymer structure, usefully tuned macromolecular properties such as biodegradability can be obtained. Hydrogel networks in the form of colloidal particles which are being explored for use in drug delivery (Kiser et al.) are not biodegradable owing to their carbon-carbon bond containing backbone and their methylene-bis-acrylamide cross-links. This fact initiated the design of a new class of centro-symmetric cross-linking monomers. One of the preferred characteristics of the new material was that it must be easily synthesized. A second preferred characteristic is that the cross-linkers be composed of biocompatible components. The third characteristic which separates this work from all other work in this area is that the biodegradable cross-linker be synthesized to be a single pure molecule and not a mixture. This characteristic should lead to defined biodegradation rates versus the use of a cross-linker mixture as in previous work (Pathak et al.). Therefore by utilizing classical organic synthesis methodology to synthesize monodispersed degradable sequences into the monomer structure before polymer formation presents an opportunity to carefully control the overall degradation as well as possibly the release rate of entrapped substances. One of the particularly preferred embodiments of these cross-linkers is that they are composed of a symmetrical diacid each acid attached to a biodegradable regions consisting of acids, such as the alpha-hydroxy acids glycolic or lactic acid for example. These portions are then preferably terminated by the monomer methacrylate. The Monomers The monomers are composed of a central. polyacid as in FIG. 1 and are attached to the degradable region through oxygen, nitrogen, or phosphorous atoms. Structure A shows a monomer having a central diacid region {character pullout}, and a degradable region {character pullout} which is then terminated by a reactive polymerizable region {character pullout}. Structure B is similar and uses the same symbols except that the central core is a triacid symbolized by a T structure. FIG. 2 displays a more specific embodiments of this invention. In structure C, a symmetrical centerpiece (succinic acid) is attached to two degradable regions containing alpha-hydroxy esters. These are then attached to a moiety (R.sub.2) which may or may not impart water solubility through the connecting portion labeled Y. Finally, the cross-linker is terminated with vinyl groups. Structure D is again similar to structure C except in this case the monomer is terminated with two nucleophilic moieties which could be used to cross-link preformed polymer chains. These structures are exemplary only. Many more are conceivable by those skilled in the art. In a preferred embodiment the network begins with a cross-linker containing two equal degradable regions attached to a central diacid and each containing a terminal reactive group. In a particularly preferred embodiment, the core is made of succinic acid, each degradable region is composed of either symmetrical units of glycolic or lactic acid where n in FIG. 1 is between 1 and 5 and the terminal reactive group is a acrylate type moiety where R2 in FIG. 1 is CH(CH3)CH2CO and Y is equal to oxygen. Central Component In preferred embodiments the central piece can consist of esters of dicarboxylic acids such as malonic succinic, adipic, sebacic, maleic fumaric acids or even possibly (alpha, omega-(oligo(ethylene glycol)) dicarboxylic acid (alpha, omega-(oligo(propylene glycol)) dicarboxylic acid. Other diacids such as aromatic polycarboxylic acids may also be used. In another embodiment tri-acids such as citric acid or tetra and penta acids such as EDTA and DTPA (possibly as protected derivatives) could also be utilized. Also protected versions of tartaric, citric, aspartic or glutamic acid may be used in certain embodiments. Biodegradable Component The biodegradable region is preferred to be hydrolyzable under environmental or in vivo conditions. In the most preferred embodiment the degradable regions will be composed of glycolic or lactic acid domains containing anywhere from one to six members in each oligomeric region attached to the central piece. Other hydroxy esters that may be embodied include: (3-hydroxy butyric acid, 2-hydroxy propanoic acid, and 5-hydroxy caproic acid. Other useful biodegradable regions include amino acids, ortho-esters, anhydrides, phosphazines, phosphoesters and their oligomers and polymers. Reactive Cross-linking Polymerizable Region This region is necessary for the invention because it is the chemical functionality terminating the two or more ends of the cross-linker which will chemically bind polymer filaments together. The preferred method of achieving this end is through an acrylate moiety, with polymerization through free radical generation. Free radical generation can be accomplished via thermal, photochemical or redox catalysis initiation systems (Odian). The preferred polymerizable regions for free radical generation are acrylates, vinyl ethers, diacrylates, oligoacrylates, methacrylates, dimethacrylates, and oligomethacrylates. Alternatively another preferred method of cross-linking preformed chains in solution is to attach two or more nucleophiles to the end of the chains which would be reactive with an electrophile attached to the polymer chain. The preferred chemical reactive moieties for this method are carbonate, carbamate, hydrazone, hydrazino, cyclic ether, acid halide, acyl azide, alkylazide, succinimidyl ester, imidazolide, amino groups, alcohol, carbonyl, carboxylic acid, carboxylic ester, alkyl halide, aziridino, nitrile, isocyanate, isothiocyanate, phosphine, phosphonodihalide, sulfide, sulfonate, sulfonamide, sulfate, silane, or silyloxy groups. Initiators Several initiation systems for the formation of polymer networks are useful with these compounds, depending on the application and the conditions used. For generation of polymer slabs either irradiation of vinyl groups with high energy light such as in the UV is a suitable method for initiation. Other preferred methods include the use of thermally activated initiators such as azobisisobutyronitrile or benzoyl peroxide for initiation in water or mixed water/organic solvents, other water soluble alkyl diazo compounds, ammonium persulphate with or without N,N,N',N'-tetramethyethylene diamine. For generation of particles by emulsion polymerization generation of radicals by thermal initiation is convenient. Generally this is accomplished with water soluble initiators such as ammonium persulphate. Other initiators include the water soluble alkyl diazo compounds. For generation of polymer networks in vivo the most useful initiation system is photochemical. Photochemical initiation of free radical polymerization involves light activation of a light absorbing compound (a dye), radical abstraction of a hydrogen to generate the initiation radical (usually an amine), and attack of this radical on a vinylic moiety beginning the polymerization. This system preferably requires free radicals to be generated locally and within a short time period, preferably in seconds. Initiation in this system begins with irradiation of light at the appropriate wavelength. The wavelength is chosen to be as close to the absorption maximum of the dye as possible. The preferred light absorbing compounds which will begin the radical generation process are eosin dyes, 2,2'-dimethoxy-2-phenyl acetophenone and other acetophenone derivatives. Other photo redox active dyes include acridine dyes, xanthene dyes and phenazine dyes, for example, acriblarine, rose bengal and methylene blue, respectively. These dyes when photoactivated assume a triplet excited state which can abstract a proton from an amine and thus generate a radical which begins the polymerization. Compounds which act as the initiating radical are amines such as triethanolamine, sulfur containing compounds such as ammonium persulphate, and nitrogen containing-heterocycles such as imidazoles. Applications for the Cross-linkers Nature of the Polymer In the preferred embodiment of this invention, these cross-linkers can be incorporated in biodegradable network polymers that are either hydrophilic or are hydrophobic. Hydrophobic networks will contain less than 5% of the total mass of the polymer network as water. Whereas hydrophilic networks can contain as great as 99% water as the total mass. Hydrophilic network polymers are known as hydrogels to those skilled in the art. Those skilled in the art will generally recognize the polymer structures which are generally considered to be hydrophilic or hydrophobic. In Vivo Drug Delivery One preferred application of these materials is in the use of controlled delivery of bioactive compounds. In this method the cross-linker is homopolymerized or copolymerized with other monomer or polymers which may be charged or uncharged. The drug is placed in the polymer network by polymerizing the network around the drug (i.e., by co-dissolving or dispersing the drug with the monomer solution) or by incubating the resulting polymer with a solution of the drug whereby it diffuses into the polymer network. In this embodiment the drug may be anywhere from 1 to 90% by weight of the device. The biologically active compounds can be (but are not limited to) proteins, peptides, carbohydrates, polysaccharides, antineoplastic agents, water soluble linear and branched polymeric prodrugs, particles containing drug, antibiotics, antibodies, neurotransmitters, psychoactive substances, local anesthetics, anti-inflammatory agents, spermicidal agents, imaging agents, phototherapeutic agents, DNA, oligonucleotides and anti-sense oligonucleotides. An alternative method of producing a biodegradable drug delivery system is through the production of particles. The preferred size range is between 10 nm and 10 .mu.m. These particles can be produced by emulsion polymerization in water containing a surfactant such as sodium dodecyl sulfate, an initiator such as ammonium persulphate, and cross-linking monomer and co-monomer(s) such as 2-hydroxypropyl methacrylamide, 2-hydroxyethylmethacrylate, acrylic acid, methacrylic acid, methyl methacrylate, methyl acrylate, or other suitable monomers by themselves or in mixtures. Alternatively the particles can be synthesized by precipitation polymerization in organic solvent containing organic soluble initiator such as azobisisobutronitrile and co-monomer(s) such as acrylamide, as 2-hydroxypropyl methacrylamide, 2-hydroxyethyl methacrylate, acrylic acid, methacrylic acid, methyl methacrylate or methyl acrylate by themselves or in mixtures. In this method the preferred route of incorporating drug in the particles is by first synthesizing the particle, followed by purification through washing. The particle is then incubated with drug which is bound to the polymer network by either hydrophilic or ionic forces or by entrapment within the network. Another method which is well known to those skilled in the art of producing polymer particles includes dissolving the cross-linking monomer, co-monomer, initiator with or with our drug in water and then dispersing this solution in oil. The resulting oil droplets then act as templates for the formation of the gel network. Polymerization is initiated either thermally, chemically or photochemically depending on the monomer system and initiator system. Which combination of systems to use will be obvious to those skilled in the art. The resulting particles can then be sedimented and isolated and purified. This technique is particularly useful for producing larger particles in the 5- to 1000 micron in diameter size range. Another preferred method for the creation of a drug delivery device is to create a homopolymer network of the cross-linker in organic solvent in the presence of a organic soluble drug. The network is then dried and contains drug dispersed within it. The highly cross-linked network will begin to erode when hydrated and release drug. Water Absorbents In this application an important consideration is to copolymerize the biodegradable cross-linker with charged monomers (either negative charges or positive charges or mixtures thereof). Very high charge densities in the polymer network can be obtained by copolymerization of charge monomers into networks (>5 M). The presence of charges in the polymer network require counterions for electroneutrality. These counterions bind water to a lesser or greater extent, depending on their size and polarizabilities. Since the volume of the hydrated gel is equal to the volume of polymer, the volume of water bound to the polymer and the volume of the hydrated ions bound to the polymer, the presence of a large amount of hydrated ions can create a super-water adsorbent hydrogel. The molar ratio of cross-linker to other monomers should be kept as low as possible so as to not inhibit the swellability of the network, preferably in the range of 5 mol % or less. The preferred copolymers include methacrylic acid, acrylic acid, acrylic and methacrylic monomers containing sulfate, alkyl carboxylate, phosphate, amino, quaternary amino and other charged groups and their salts. In this application large batches of the degradable network will be synthesized either by dispersion polymerization or in bulk. The material could be synthesized in the presence of a suitable counterion such as sodium for negatively charged filaments or chloride for positively charged filaments. Alternatively the polymer may be formed in its neutral state and then incubated with a suitable acid or base such as hydrochloride in the case of nitrogen containing co-monomers, and soluble metal hydroxides in the case of acidic co-monomers. The most preferred method is to polymerize the cross-linker with the salt form of the co-monomer. Adhesives Another use of the monomer is in temporarily binding two surfaces together. The biodegradable cross-linking monomer and co-monomer or just the biodegradable cross-linking monomer itself are mixed together with a solvent and an initiator by itself or with a co-catalyst. The mixture is then spread on the surfaces which are to be adhered, then polymerization is initiated by addition of heat or by light. In the case of light initiation at least one of the surfaces to be adhered must be transparent to the light beam in order for the polymer network to form. The initiation systems described above can be used to this end. Such biodegradable adhesives should have many uses. Tissue Supports There is a need for degradable polymers as cell scaffolds in tissue engineering. In this application the tissue scaffold would be synthesized under sterile conditions in a suitable biocompatible buffer. The cross-linking density should be controlled so as to obtain a pore size large enough to allow cell migration. Pore size may be determined by scanning electron microscopy and by using macromolecular probes. A cell suspension containing cells such as, but not limited to, keratinocytes, chrondocytes and osteoblasts, would be injected into the polymer network along with suitable growth factors. The cells would then be allowed to grow within the network. As the cells grow the network around them would degrade. Bioadhesive moieties such as RGD peptide sequence (Arg-Gly-Asp) could be connected to matrix and thereby provide adhesive domains for the growing cells. The timing of the network degradation should coincide with the cells forming their own network (artificial tissue) through inter-cell contacts. The following examples are presented to describe preferred embodiments and utilities of this invention but are not intended to limit the use or scope of the methods, compositions or compounds claimed in this invention unless otherwise stated in the claims. Taken together, these examples describe the best currently understood mode of synthesizing and incorporating these materials into polymer networks. The synthesis of the four members of the preferred class of molecules claimed herein are given in FIG. 2. This invention has several advantages over related inventions in this area, including: (1) the cross-linking agents are biodegradable to biocompatible substances, (2) the syntheses are both general and flexible, allowing for a variety of monomeric units to be incorporated, (3) the end groups (e.g., acrylate or hydrazide) can be readily modified to accommodate either condensation or radical-type polymerizations. |
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