Title:  Polysaccharide-based hydrogels and pre-gel blends for the same

United States Patent:  6,586,493

Issued:  July 1, 2003

Inventors:  Massia; Stephen P. (Apache Junction, AZ); Trudel; Julie (Sunnyvale, CA)

Assignee:  Arizona Board of Regents Arizona State University (Tempe, AZ)

Appl. No.:  801373

Filed:  March 7, 2001

Abstract

Disclosed are hyaluronate-containing hydrogels having angiogenic and vascularizing activity and pre-gel blends for preparing the hydrogels. The hydrogels contain a cross-linked matrix of a non-angiogenic hyaluronate and a derivatized polysaccharide material, in which cross-linking is effected by free-radical polymerization. Kits for preparing the hydrogels are also disclosed.

SUMMARY OF THE INVENTION

The present invention provides hydrogels having angiogenic activity and pre-gel blends for synthesizing the hydrogels. The pre-gel blend includes a mixture of a polysaccharide material at least partially substituted with an unsaturated, cross-linking moiety, and a non-angiogenic hyaluronic acid or salt thereof. Preferably, the polysaccharide material has an average molecular weight of at least about 10,000 Daltons, with at least about 40,000 Daltons being more preferred. Classes of polysaccharide material to be used are starch or starch derivatives, water-soluble gums, or mixtures thereof. Examples of starch or starch derivatives are dextrans, curdlans, succinoglycans, pullulans, cellulose derivatives, cyclodextrins, or mixtures thereof Examples of water-soluble gum are alginates, carageenens, xanthans, galactomannans, or mixtures thereof. In a particular embodiment, the starch or starch derivative is dextran. Preferably, the unsaturated, cross-linking moiety is selected from the group consisting of acrylates, esters, ethers, thioethers, amides, enamides, sulfonyl esters or mixtures thereof, with acrylate being particularly preferred. The non-angiogenic hyaluronic acid or salt preferably has an average molecular weight of at least about 500,000 Daltons, with at least about 1,000,000 Daltons being more preferable.

The hydrogels of the present invention are a reaction product prepared by a process which includes: admixing the polysaccharide material that is at least partially substituted with the unsaturated, cross-linking moiety, and the non-angiogenic hyaluronic acid or salt thereof, with a free-radical initiator in a solvent; and cross-linking the mixture to form the hydrogel. The free-radical initiator is either a chemical initiator and a non-chemical initiator, with a non-chemical initiator (e.g., UV initiator) being preferred. A preferred class of UV initiators are acetophenone derivatives. The solvent is preferably water, a water-based solvent, a water-miscible organic solvent, or mixtures thereof. The other components are as described above.

The present invention also provides kits for preparing the hydrogels. In one embodiment the kit includes: a first container containing a mixture of the partially substituted, polysaccharide material and the non-angiogenic hyaluronic acid or salt thereof; and a second container containing a free-radical initiator. In another embodiment the kit includes: a first container containing the partially substituted, polysaccharide material; a second container containing the non-angiogenic hyaluronic acid or salt thereof; and a third container containing the free-radical initiator. Preferably, the kits further include an instruction pamphlet for preparing the hydrogel.

Advantageously, the pre-gel blends and hydrogels of the present invention omit the use of angiogenic hyaluronate fragments as a starting material while providing angiogenic hyaluronate-containing hydrogels. These and other advantages of the invention will become more readily apparent from the detailed description set forth below.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides hydrogels having angiogenic activity and pre-gel blends for synthesizing the hydrogels. The hydrogels of the invention advantageously avoid the use of small hyaluronate fragments while at the same time providing angiogenic and vascularizing activity.

The pre-gel blend of the invention includes a mixture of a polysaccharide material at least partially substituted with an unsaturated, cross-linking moiety, and a non-angiogenic hyaluronic acid or a salt thereof. In accordance with the present invention, the polysaccharide material is any polysaccharide material with a polysaccharide material other than hyaluronic acid or its salt being preferred. In another embodiment, the polysaccharide material is preferably a water-soluble polysaccharide. Two preferred classes of polysaccharides to be used are starch or starch derivatives and water-soluble gums. Representative examples of starch or starch derivatives to be used include, but are not limited to, dextrans, curdlans, succinoglycans, pullulans, cellulose derivatives, cyclodextrins and mixtures thereof. One particularly preferred starch derivative to be used is dextran. Representative examples of water-soluble gums to be used include, but are not limited to, alginates, carrageenans, xanthans, galactomannans and mixtures thereof.

In accordance with the invention, the polysaccharide material can have varying molecular weights. In a preferred embodiment a polysaccharide material with an average molecular weight of at least about 10,000 Daltons is used, with at least about 40,000 Daltons being more preferred. Likewise, the polysaccharide material should preferably have an average molecular weight less than about 2,000,000 Daltons for ease of preparation. Polysaccharide materials with higher molecular weights can be used however decreased water solubility becomes a factor.

The polysaccharide material has at least a portion of its substituents derivatized with an unsaturated, cross-linking moiety. The cross-linking moiety is derived from any heterofunctional cross-linking agent that can bonded to the saccharide residues while providing at least one vinyl group to effect cross-linking via free-radical polymerization. The polysaccharide material is substituted with the unsaturated, cross-linking moiety using any technique known in the art. As will apparent to the skilled artisan, the specific technique utilized will depend on the functional groups available on the saccharide residues and the cross-linking agent. In most instances, the saccharide residue will have free hydroxyl groups that provide available sites for attachment of the cross-linking agent. In a preferred embodiment, the cross-linking moiety is non-photoreactive and contains a hydrophilic functional group. "Non-photoreactive" means that the cross-linking moiety is substantially light insensitive (i.e., does not undergo free-radical polymerization without a free-radical initiator). Examples of functional groups in which available hydroxyl groups of the polysaccharide material can be derivatized to include, but are not limited to, acrylates, esters, ethers, thioethers, amides, enamides, sulfonyl esters and mixtures thereof.

In a particularly preferred embodiment, the unsaturated cross-linking moiety is an acrylate (i.e., an acryloyl) group. An example of a cross-linking agent to be used to derivatize the polysaccharide material is a glycidyl acrylate such as glycidyl methacrylate. Other cross-linking agents to be used are epichlorohydrin, borate, glyoxal, and glutaraldehyde. The derivatization of free-hydroxyl groups of a polysaccharide materials is achieved following conventional techniques known in the art. One technique is described in van Dijk-Wolthuis, et al., Synthesis, Characterization, and Polymerization of Glycidyl Methacrylate Derivatized Dextran. Macromolecules, 28:6317-6322 (1995), which is incorporated herein by reference. Following synthesis, the derivatized polysaccharide material can be lyophilized (i.e., freeze-dried) for storage.

In accordance with the invention, the polysaccharide material is partially derivatized with the unsaturated cross-linking moiety. The level of derivatization is variable and is dependent on the desired physical properties (e.g., viscosity) for the hydrogel end product. In a preferred embodiment, the polysaccharide material has a degree of substitution (DS) of at least about 4, with about 7 being more preferred and about 15 being even more preferred. If desired, DS levels of 22, 35 or greater can also be utilized. As will be apparent to those skilled in the art, degree of substitution refers to the molar ratio of cross-linking moiety per saccharide residue. The advantage of greater DS levels is reduced gelation time. The DS level of the derivatized material can be ascertained following conventional techniques, such a Nuclear Magnetic Resonance (NMR). These and other parameters can be easily ascertained by those skilled in the art following the teachings of the invention.

As previously described, the second component for the pre-gel blends and hydrogels of the invention is a non-angiogenic hyaluronic acid or salt thereof (i.e., a hyaluronate). In the context of the invention, a non-angiogenic hyaluronate is a hyaluronate that contains greater than 25 disaccharide units which corresponds to an average molecular weight of greater than 10,000 Daltons. As known from the prior art, hyaluronate fragments greater than 25 disaccharide units do not exhibit angiogenic activity. In a preferred embodiment, the hyaluronate has an average molecular weight of at least about 500,000 Daltons, with an average molecular weight of at least about 1,000,000 Daltons being preferred. A particular advantage of the invention is that unfractionated (i.e., native) hyaluronates are used as a starting material rather than fractionated hyaluronate fragments, which significantly reduces costs and time constraints for producing angiogenic hyaluronate-containing hydrogels.

To form the hydrogel, the derivatized polysaccharide material and the hyaluronate are mixed in the presence of a suitable solvent. Suitable solvents to be used are water or water-based solvents, water-miscible organic solvents, or combinations thereof. Example of water-miscible organic solvents include, but are not limited to, methanol, ethanol, acetone, dioxane, dimethylformamide, and tetrahydrofuran. In a preferred embodiment water or a water-based solvent (e.g., buffered saline) is used to induce sufficient swelling of the polysaccharide material to provide a viscous solution (a hydrogel precursor). The desired viscosity of the solution and resulting hydrogel end product is variable and can be tailored for the particular end use for contemplated for the hydrogel. For example, to impregnate an arterial prostheses, a relatively low viscosity solution is preferable for ease of use.

The hyaluronate and the derivatized polysaccharide material are mixed using any conventional technique known in the art. The ratio of hyaluronate to derivatized polysaccharide material is any ratio that exhibits increased angiogenesis or vascularization over either component alone. Preferably, the resulting mixture of the two components contains at least about 0.1 percent by weight of hyaluronate, with at least about 0.4 weight percent being more preferred, and about 4 weight percent being even more preferred. Greater concentrations of hyaluronate can also be used but may require increased preparation times due to increasing solution viscosities.

Cross-linking of the mixture is effected by the addition of a free-radical polymerization initiator to the solution to induce cross-linking via the vinyl groups of the cross-linking moieties. Examples of free radical initiators are well-known in the art with a description of free-radical initiators being found in Kirk-Othmer, Encyclopedia of Chemical Technology, 14:431-460 (1995), which is incorporated herein by reference. In accordance with the invention, any free-radical initiator is used to effect cross-linking. In a preferred embodiment, non-chemical initiators are used to allow for subsequent handling of the mixture before cross-linking. Chemical initiators (e.g., peroxide initiators) are used if handling of the mixture after addition of the initiator is not required or limited. One particularly preferred class of non-chemical initiators are photoinitiators (i.e., UV-initiators) such as acetophenone derivatives. An example of an acetophenone derivative is 2,2-dimethoxy-2-phenylacetophenone.

In another embodiment, the present invention provides a kit for preparing a hydrogel having angiogenic and vascularizing activity. The kit includes a first container which contains the pre-gel blend and a second container which contains the free-radical polymerization initiator. In accordance with the invention, the first and second containers can also be the first and second compartments of a single container structure. The kit can also include an instruction pamphlet providing instructions on using the kit to synthesize the hydrogel. In an alternative embodiment the kit includes a first container which contains the derivatized polysaccharide material, a second container which contains the non-angiogenic hyaluronic acid or salt thereof, and a third container which contains the free-radical polymerization initiator.

The hydrogels of the present invention due to their angiogenic and vascularizing activity are useful for a variety of medical applications. In particular, the hydrogels are particularly suitable for wound healing applications such as wound dressings and porous prostheses.

Claim 1 of 14 Claims

We claim:

1. A hydrogel having angiogenic and vascularizing activity prepared by a process which comprises:

admixing a polysaccharide material at least partially substituted with an unsaturated, cross-linking moiety, and a non-angiogenic hyaluronic acid or a salt thereof, with a free-radical initiator in a solvent; and

cross-linking the mixture to form the hydrogel having angiogenic and vascularizing activity.

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