Title:  Reversibly cross-linked hydrogels

United States Patent:  6,790,840

Issued:  September 14, 2004

Inventors:  Lee; Kuen Yong (Ann Arbor, MI); Mooney; David J. (Scio. Township, MI)

Assignee:  The Regents of the University of Michigan (Ann Arbor, MI)

Appl. No.:  722010

Filed:  November 27, 2000

Abstract

A hydrogel composition and methods of preparing and using the same are disclosed. The hydrogel composition comprises an oxidized polysaccharide, and a cross-linker having at least two functional cross-linking groups. The cross-linker reversibly cross-links the polysaccharide and is provided in an amount to provide dangling cross-linkers. The method of preparing a hydrogel comprises the steps of providing an oxidized polysaccharide, and providing a cross-linker having at least two functional cross-linking groups. The cross-linker is mixed with the polysaccharide at a concentration of the cross-linker sufficient to form a hydrogel wherein the cross-linker reversibly cross-links the polysaccharide and has dangling cross-linkers. The hydrogels are useful, for example, for tissue engineering, cell transplantation and drug delivery applications.

FIELD OF THE INVENTION

The present invention includes hydrogel compositions and methods for the fabrication and use thereof.

BACKGROUND OF THE INVENTION

Water-containing hydrogels have numerous applications including as food additives, blood contact materials, bioadhesives, contact lenses, wound dressings, artificial organs, drug delivery, controlled release formulations, membranes, superabsorbents, cell encapsulation and immunoisolation materials, and delivery carriers for bioactive agents, including drugs. Their biocompatibility is likely related to their high water content and low interfacial tension with surrounding biological environment. One of the most recent applications of hydrogels is as delivery vehicles of cells for tissue engineering approaches. The aim of this approach is the reconstruction of tissues and organs using three-dimensionally designed synthetic matrices which mimic the function of the extracellular matrix, and offers an alternative to the patient who needs new tissues or organs. Hydrogels may be potent materials for soft tissue engineering applications due to their similarity to the highly hydrated macromolecular-based materials in the body. Critical properties of hydrogels utilized in these applications include their degradation time and mechanical properties. One typically desires to time the rate of hydrogel degradation to the rate of new tissue formation, and this time may vary significantly for different tissues. The mechanical properties of these materials are critical to their ability to create and maintain a space for new tissue formation in vivo, and the mechanical properties of the materials to which cells adhere can also regulate the gene expression of the cells. A number of synthetic and naturally derived materials may be used in the formation of hydrogels. One widely used material in hydrogel formation is alginate, a hydrophilic polysacchoride derived from seaweeds. Alginate comprises a family of natural copolymers of .beta.-D-mannronic acid and .alpha.-L-guluronic acid. See Martinsen et al., Biotechnology and Bioengineering, 33: p. 79-89 (1989); Draget et al., Carbohydrate Polymers, 14: p. 159-178 (1991).

One particularly promising application of hydrogels is in tissue engineering. Tissue engineering is directed towards creating biological tissue rather than rely on scarce transplantable organs. An extracellular matrix (ECM) of noncellular material has been identified in many multi-cellular organisms, including human beings. ECM molecules include specialized glycoproteins, proteoglycans, and complex carbohydrates. A wide variety of ECM structures have been identified, and ECM has been implicated in tissue formation. Simply put, the method of tissue engineering is tissue and organ reconstruction using synthetic (e.g., polymeric), three-dimensional matrices, also referred to as "scaffolds" which mimic a body's ECM to provide a space for new tissue formation in vivo. Because alginate exhibits a high degree of biocompatability, is abundant, and inexpensive, it is well suited to application in tissue engineering as well as other applications.

Use of hydrogels in tissue engineering applications particularly is dependent upon hydrogel degradation time and mechanical properties. While alginate is widely employed to fabricate hydrogels for various biomedical applications, ionically cross-linked alginate hydrogels have uncontrollable mechanical properties and disintegration behavior. Preferably, however, hydrogels used in tissue engineering, for example, persist as a tissue generation "scaffold" at least as long as required for new tissue formation. Additionally, the molecular weight of alginate as commonly used in such hydrogels is greater than the limit of renal clearance in humans, such that the disintegrated hydrogel cannot be processed by human kidneys.

Use of hydrogels in injectable form for the delivery of drugs and/or cells has also been of advantageous use. The ability to inject these materials minimizes the pain and cost of delivery to the patient.

Because it is conventionally considered that hydrogel degradation is a function of cross-linking density, one solution to the problem of rapid bydrogel degradation has been the creation of hydrogels characterized by high cross-linking density. However, highly cross-linked hydrogels display mechanical stiffness, an undesirable characteristic particularly in biomedical applications.

What is needed is a hydrogel composition with both desirable mechanical properties and degradation characteristics.

SUMMARY OF THE INVENTION

The hydrogel compositions of the invention are provided with excess reversible cross-linking agent(s) such that some binding sites on the cross-linking agent(s) are initially unbound to the polymer, but are capable of binding to other sites on the polymer as those sites become available through degradation of other cross-links. The cross-linkers which have at least one site bonded to the polymer and at least one site open for reversible bonding will be referred to as dangling cross-linkers or danglers. The conventional view in the art was that such dangling cross-linkers were disadvantageous and to be avoided because the danglers block the site of the polymer to which they are attached. The inventors have discovered, however, how to put this supposed disadvantage to advantageous use according to their invention. For the hydrogels of the invention, the provision of dangling cross-linkers advantageously results in a hydrogel with less mechanical stiffness because not all of the potentially cross-linked sites can be cross-linked due to blocking by the danglers.

It has surprisingly been discovered that the lower mechanical stiffness is not coupled with a corresponding loss of stability to degradation. As cross-linking sites degrade, the presence of the dangling cross-linkers allows formation of new cross-links, thus, compensating for and slowing the degradation rate. The invention therefore results in hydrogels where the mechanical stiffness properties do not have to correspond or be coupled with the degradation properties. In a particular embodiment, hydrogels with desired slow degradation but not with undesired high mechanical stiffness are provided. These hydrogels are particularly useful in drug delivery and tissue engineering applications where it is desirable that the hydrogel not be too stiff to manipulate, administer and/or implant, but which still is resistant to degradation until its function has been served.

The present invention thus relates to an improved polymeric hydrogel composition and method of making the same, and in particular to such a hydrogel composition comprising a hydrogel polymer, preferably an oxidized polysaccharide, and at least one cross-linker having two or more functional groups capable of reversibly cross-linking the polysaccharide in the hydrogel system. The cross-linker is provided as described above to have dangling cross-linkers. In an exemplary hydrogel, the hydrogel polymer is a polysaccharide comprising a synthetic or naturally derived alginate polymer having aldehyde groups, and the cross-linking agent is one having at least two hydrazide groups, such as adipic acid dihydrazide (AAD). Because the hydrogel has preferably many dangling cross-linkers capable of reversibly cross-linking the polymer, the inventive hydrogel compositions display surprisingly improved degradation characteristics and improved mechanical properties as compared with hydrogels having higher cross-linking densities, and/or no dangling cross-linkers.

As indicated, the hydrogel polymer is preferably an oxidized polysaccharide, particularly an alginate. Preferably, such alginate polymer comprises any of several derivatives of alginic acid, including calcium, sodium, or potassium salts or propylene glycol alginate, and most preferably comprises an alginate salt of high guluronate content. The cross-linking agent preferably comprises at least two functional groups which are capable of reversibly cross-linking the polymer, preferably at least two hydrazide groups, and most preferably the cross-linker comprises AAD. Further exemplification of useful polymers and cross-linkers for the hydrogel is provided by reference to WO 98/12228 published Mar. 26, 1998.

The hydrogel polymer and cross-linking agent are admixed in amounts providing an excess of cross-linker so that dangling cross-linkers result and block a high-density extent of cross-linking.

It is preferred that the hydrogels have a cross-linking efficiency for single-end dangling cross-linkers of from 20-90%, more preferably in the range of 20-80%, 20-70% or 30-50%. The creation of significant dangling cross-linkers is facilitated by the use of an excess amount of cross-linker. Also, it is preferred that hydrogel formation be conducted in a salt solution. Such solution preferably contains 0.01-20 g/l (more preferably 2.0-10.0 g/l) of NaCl and may optionally additionally contain one or more of:

0.01-1.0 (pref. 0.1-0.5) g/L of CaCl₂ ;

0.01-2.0 (pref. 0.2-1.0) g/L of KCl;

0.01-1.0 (pref. 0.05-0.5) g/L of NaH₂ PO₄.H₂ O;

or

0.01-1.0 (pref. 0.05-0.5) g/L of MgSO₄.

The hydrogel polymer is preferably of low molecular weight (Mw) so as to be suited for biomedical applications. However, applications using hydrogels with molecular weight up to 50,000 Daltons are possible. Hydrogels with molecular weight (Mw) from 1,000 to 30,000 or 1,000 to 10,000 are more preferred. Molecular weight can be modified by means such as acid hydrolysis and oxidation, as necessary. According to the illustrated example, an alginate material is hydrolyzed under acidic conditions to yield sodium poly(guluronate) (PG) of relatively low molecular weight (e.g. Mw about 7,000). The PG precipitate is then oxidized by sodium periodate to form the alginate polymer, PAG (e.g. Mw about 5,700). This PAG intermediate is subsequently cross-linked with a suitable cross-linker, such as AAD, in the manner discussed above to form hydrogels with dangling cross-linkers.

The resultant PAG hydrogels exhibited a higher degree of swelling (Q) and lower shear modulus (G) than PAG hydrogels with a higher cross-linking density (e.g., those on the order of 16.0x10⁵ mol/cm³ or higher). The preferred degree of swelling (Q) is from 1 to 200, more preferably 5 to 100. The preferred shear modulus (G) is from 0.005 to 200 kPa, more preferably 0.05 to 100 kPa.

The hydrogels are further characterized by increased stability over time; that is, slower degradation. Hydrogels having this characteristic retarded degradation imparted by reversibly cross-linking dangling cross-linkers are well suited to numerous applications, including biomedical applications such as tissue engineering cell transplantation and drug delivery. Further discussion of useful applications is provided by reference to WO 98/12228 published Mar. 26, 1998.

The present invention relates to polymer hydrogel compositions and methods of making and using the same, and particularly to hydrogels characterized by a cross-linker having at least two functional groups able to reversibly cross-link the polymer. The hydrogels are further characterized by an extent of cross-linking such that some potentially cross-linkable sites are not cross-linked because two dangling cross-linkers are occupying sites which are cross-linkable by a single cross-linker. Such hydrogels display improved mechanical properties and retarded degradation as compared to conventional hydrogel systems.

EXAMPLES

In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and, unless otherwise indicated, all parts and percentages are by weight.

Hydrogel Preparation

In brief, the exemplary hydrogel of the present invention is prepared by cross-linking a hydrogel polymer, preferably an oxidized polysacharide such as an alginate polymer, with a cross-linking agent having at least two functional groups, the cross-linking agent capable of reversibly cross-linking the polymer and provided in an amount to result in the discussed dangling cross-linkers. In the exemplary embodiment, the hydrogel polymer is an alginate cross-linked with a cross-linker having two hydrazide functional groups, the polymer and cross-linker admixed in relative amounts such that the resultant hydrogel is characterized by the presence of a significant amount of dangling cross-linkers. The hydrogel preferably has a relatively low cross-linking density, as determined by the Flory-Rehner equation. In an exemplary hydrogel system comprising poly(aldehyde guluronate) (PAG) cross-linked with AAD, the preferred cross-linking density (Ve) is less than 16.0x10⁵ mol/cm³, and most preferably less than approximately 12.3x10⁵ mol/cm³.

According to the illustrated examples, polymer bydrogels were generally prepared by hydrolyzing an alginate under acidic conditions, isolating and subsequently oxidizing polyguluronate (PG) therefrom to prepare poly(aldehyde guluronate) (PAG), and cross-linking the PAG (20% wt solution) with a cross-linker having at least two hydrazide groups, for example adipic acid dihydrazide (AAD), to form hydrogels. The concentration of cross-linker was varied from 50 mM to 250 mM.

The hydrogel polymer preferably comprises an oxidized polysaccharide, for example an alginate such as the commercially available sodium alginate (PROTANAL LF 20/60) obtained from PRONOVA (Drammen, Normay). Preferably, such alginate is characterized by a high guluronate content, since the guluronate units provide sites for ionic cross-linking through divalent cations to gel the polymer. However, it will be understood that a variety of alginates may serve for the present invention. "Alginate", as that term is used herein, refers to any of a number of derivatives of alginic acid (e.g., calcium, sodium, or potassium salts or propylene glycol alginate). These compounds may be synthetic or naturally derived. Both natural and synthetic alginates are commercially available or may be prepared, and may be substituted in preparation of the present inventive hydrogel according to this disclosure. Natural source alginates may be derived from seaweed or bacteria according to conventional methods. See Biomaterials: Novel Materials from Biolopical Sources, ed. Byrum, Alginates chapter (ed. Sutherland), p. 309-331 (1991). Both naturally derived and synthetically prepared alginates can be fabricated, according to known methods, to provide side chains with a desired mannuronate and guluronate proportion. It is not intended that the present invention be limited to alginate or any particular polymer, or a particular method of making the same. For instance, any of a number of polysaccharides, both synthetic and naturally derived, including cellulose, agarose, dextran, pullulan, starch, hyaluronate, etc., may be substituted for the alginate of the exemplary disclosure. Additionally, other polymers may be used which are biocompatible, can provide hydrogels and are cross-linkable according to the invention. Many synthetic polymers and proteins, optionally modified to facilitate gelling and/or cross-linking.

PAG was prepared for the exemplary hydrogels according to the method of Haug et al., reported in Acta. Chem. Scand., 20: p. 183-190 (1966), which disclosure is incorporated herein in its entirety. According to this method, the alginate material underwent acid hydrolysis to break down the .beta.-glycosidic linkages between mannuronate and guluronate residues, thereby providing a lowered molecular weight PG which is essentially lacking mannuronic acid units. The PG was then isolated at pH 2.85, and determined by size-exclusion chromatography (SEC) to have a molecular weight (Mw) of 7,000 (Mw/Mn=1.60). SEC was performed on a triple detector system including a laser refractometer (LR40, VISCOTEK), a differential viscometer, and RALLS (T60, VISCOTEK), 0.1 M NaNO₃ buffer solution (pH 6.3) was used as a mobile phase with a flow rate of 0.7 ml/min. Two TSK-gel columns (G4000PW_XL and G3000PW_XL) were used for separation.

Further purification of the PG precipitate was carried out by dissolving the PG in double distilled water at neutral pH, to which activated carbon was added. The resultant solution was thoroughly stirred, the activated carbon removed by filtration, and PG precipitated by ethanol and lyophilized.

The isolated PG was oxidized at room temperature with 0.25 M sodium periodate (ALDRICH, Milwaukee, Wis.) to prepare PAG. The ratio between guluronate units and periodate was 1:1. After 19 hours of oxidation, an equimolar amount of ethylene glycol was added to arrest the reaction. The resultant solution was filtered and precipitated by ethanol. The collected precipitate was redissolved in double distilled water and dialyzed (MWCO 1000, SPECTRA/POR) for 3 days, following which the solution was concentrated under reduced pressure and lyophilized. The Mw of the PAG was 5,700 (Mw/Mn=1.64) as determined by SEC, and the degree of oxidation was determined to be 66.5% (defined by the number of oxidized guluronate residues per 100 guluronate units) by measuring the number of aldehyde groups in the PAG. This measurement was taken by adding an excess amount of t-butyl carbazate to PAG solution, and measuring the amount of unreacted t-butyl carbazates through the addition of trinitrobenzene sulfonic acid (TNBS) solution (the colored complex of t-butyl carbazates and TNBS was quantified spectrophotometrically at 334 nm).

Preferably, the molecular weight of the hydrogel polymer material so prepared is at or below the renal threshold for clearance by the host, whether human or other, which reduction in molecular weight can be effected by the oxidation reaction described above with reference to the exemplary alginate polymer.

Those of ordinary skill will understand that the method of preparing the hydrogel polymer for the exemplary hydrogel of this disclosure is not intended to be limiting of the present invention, according to which a variety of polymers, including the preferred oxidized polysaccharides, prepared according to the above or other known methods, may be substituted in the hydrogel system.

The PAG so obtained was cross-linked with varying amounts of AAD (ALDRICH, Milwaukee, Wis.), a bi-functional cross-linker. Cross-linking of the PAG and AAD occurs in the absence of a catalyst or additive, as aldehyde groups are known to be much more reactive towards hydrazide groups as compared to carboxyl groups. While AAD is a preferred cross-linker for the exemplary hydrogel, other preferred cross-linking agents include compounds with at least two functional groups capable of reversibly cross-linking the hydrogel polymer. Preferred functional groups are hydrazide groups, and any multi-hydrazide cross-linkers will be suited to this invention. However, it will be understood that the functional groups may vary according to such factors as the hydrogel polymer employed. The cross-linker may comprise amine groups, for example. In a hydrogel system comprising aldehyde and multi-hydrazide functional groups, it will also be understood that the functional groups of the polymer and cross-linker may be reversed from the exemplary hydrogel; that is, the cross-linker may comprise aldehyde functional groups, while the hydrogel polymer comprises the hydrazide groups.

In formation of the exemplary hydrogels, a 20 wt % solution of PAG was admixed with AAD in concentrations of from 50 mM to 250 mM. All such solutions were prepared in Dulbecco's Modified Eagle's Medium (DMEM) (LIFE TECHNOLOGIES, Grand Island, N.Y.) having an adjusted pH of 7.4 prior to mixing. The final concentration of PAG in the hydrogel was fixed at 6 wt %. The hydrogel solution was plated in tissue culture plates and incubated at room temperature for 4 hours to permit hydrogel formation. Coupling of aldehyde and hydrazide groups was confirmed by FP-ir spectra on an AVATAR 360 spectrophotometer (NICOLET, Wisconsin) using the KBr pellet method (resolution 2 cm^-1 with 32 scan repetition). Upon coupling, a characteristic symmetric vibrational band of the aldehyde group at 1735 cm^-1 disappears, and a hydrazone band at 1658 cm^-1 appears.

Claim 1 of 14 Claims

What we claim is:

1. A hydrogel composition comprising a hydrogel polymer, which polymer is a natural or synthetic alginate, optionally hydrolyzed and/or oxidized, and which polymer is prepared using an excess amount of cross-linker having two or more functional groups capable of cross-linking the polymer such that the polymer has cross-links to other hydrogel polymer molecules and also has dangling cross-linkers with at least one functional group bound to a hydrogel polymer and at least one unbound functional group capable of reversibly cross-linking the polymer, wherein the amount of dangling cross-linkers, based on the total amount of cross-linkers bound to the polymer by at least one functional group, is from 20% to 90%.

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