The invention is related to biomimetic gels that are prepared enzymatically, using a transglutaminase to cross-link polymer-peptide conjugates of rational design.

BACKGROUND OF THE INVENTION

Polymer hydrogels have structural similarities to numerous macromolecular components in the human body, are generally considered biocompatible, and have been investigated extensively as materials useful for drug delivery, tissue repair and tissue engineering, as well as for use as surgical sealants and adhesives. With increasing frequency, polymer hydrogels are designed for in situ gelation from a liquid precursor, thereby allowing minimally invasive administration via syringe or needle.

Existing hydrogel systems, formed chemically or physically, are subject to several limitations. Chemically cross-linked hydrogels often employ toxic cross-linking agents and/or radicals, and the resulting hydrogels are often non-biodegradable. On the other hand, physical hydrogels--formed through ionic interactions, hydrophobic interactions, hydrogen bonding, or phase transition--are relatively weak and can be prone to unwanted or uncontrollable degradation through ion exchange, ion diffusion, or monomer dissolution. An alternate approach--solidification by enzymatic cross-linking--has two principal advantages, compared to other hydrogel systems. First, an enzyme has substrate specificity to allow controllable gel formation. Second, an enzymatic method can be applied to the in vivo utilization of cross-linked hydrogels, under appropriate physiological conditions.

Transglutaminases (e.g. protein-glutamine:amine .gamma.-glutamyltransferase, EC 2.3.2.13) catalyze a post-translational acyl-transfer reaction between the .gamma.-carboxamide groups of peptide-bound glutamine residues and the .epsilon.-amino groups of lysine residues in proteins, or certain primary amino groups, resulting in the cross-linking of proteins through the formation of .epsilon.-(.gamma.-glutamyl)lysine isopeptide side-chain bridges. Although several biological fluids are known to undergo rapid transglutaminase (TGase) catalyzed hydrogel formation, previous attempts to use TGase with peptide modified synthetic polymers have resulted in slowly gelling systems. Although proteins are typical TGase substrates, the prior art has demonstrated that synthetic poly(amino acids) and peptide-modified poly(ethylene glycol) can be cross-linked with hydrogel formation. Recently, factor XIII (plasma transglutaminase) was reported to catalyze a hydrogel formation. However, due to the stringent substrate specificity of factor XIII, a 20 amino acid-long peptide from the cross-linking .gamma.-chain of fibrinogen was first, necessarily, synthesized and conjugated to a branched polyethylene glycol (PEG) polymer. The complex preparation of the initial conjugate made a large-scale hydrogel preparation difficult. In both this instance and using synthetic peptides, the time required for gelation was considerably longer than useful for many clinical applications.

SUMMARY OF THE INVENTION

In light of the foregoing, it is an object of the present invention to provide hydrogel systems and/or method(s) for their preparation or formation, thereby overcoming various limitations of the prior art, including those outlined above. It will be understood by those skilled in the art that one or more aspects of this invention can meet certain objectives, while one or more other aspects can meet certain other objectives. Each objective may not apply equally, in all its respects, to every aspect of this invention. As such, the following objects can be viewed in the alternative with respect to any one aspect of this invention.

It is an object of the present invention to provide a range of polymeric components which undergo rapid gelation under physiological conditions.

It is another object of the present invention to identify suitable TGase substrates, short amino acid sequences readily available through known synthetic techniques, having enhanced specificities for TGase activity.

It is another object of the present invention to provide for rational peptide design and conjugation with a range of biocompatible polymeric components, for enzymatic gelation within the minute-timeframe necessary for many medical or political applications.

Other objects, features, benefits and advantages of the present invention will be apparent from this summary and its descriptions of various embodiments, and will be readily apparent to those skilled in the art having knowledge of various polymeric components and enzymatic cross-linking techniques. Such objects, features, benefits and advantages will be apparent from the above as taken into conjunction with the accompanying examples, data, figures and all reasonable inferences to be drawn therefrom.

This invention relates, generally, to a system in which a fluid solution of polymer-peptide conjugates forms covalently cross-linked hydrogels in the presence of an enzyme under physiological or comparable reaction conditions. More specifically, this invention includes the design and preparation of peptide substrates for use in transglutaminase-catalyzed gelation of polymer-peptide conjugates.

In part, the present invention comprises a component system and/or method of using a transglutaminase for polymeric gelation. Such a method comprises (1) providing a first polymeric component coupled to at least one peptide comprising at least one .epsilon.-amino moiety; (2) providing a second polymeric component coupled to at least one peptide comprising a .gamma.-carboxamide moiety; or, optionally, providing a polymeric component coupled to at least one peptide comprising at least one .epsilon.-amino moiety and at least one peptide comprising at least one .gamma.-carboxamide moiety; and (3) introducing a transglutaminase to the first and second polymeric components, or to a polymeric component comprising both moieties. Generally, such peptides exhibit substrate (e.g., acyl acceptor or acyl donor) specificity with respect to transglutaminase activity, as do the coupled peptide-polymer conjugates. A transglutaminase enzyme is available, as known to those in the art, from a variety of biological, bacterial and/or recombinant sources. A gelation system comprising such polymer and peptide components can be used in conjunction with such a biomimetic method.

As demonstrated herein, illustrated by several of the examples provided below, an .epsilon.-amino moiety can be provided with a lysine residue of such a peptide. Alternatively, an amino moiety can be provided by a component chemically or structurally similar to such a lysine residue, providing comparable functional effect and/or substrate specificity with regard to the enzymatic activity utilized in this invention. Likewise, a .gamma.-carboxamide moiety can be provided with a glutaminyl residue of such a peptide. In certain embodiments, such a peptide comprises at least two glutamine or glutamic acid residues, or optionally, two to about thirty glutamine of and/or glutamic acid residues. Alternatively, a carboxamide moiety can be incorporated into such a peptide using a chemically or structurally-similar component providing functional effect or substrate specificity comparable to a reactive glutaminyl residue(s).

The peptides of this invention are rationally designed in consideration of the foregoing. Without limitation, such peptides, alone or as coupled or conjugated with a suitable polymeric component, provide an amino moiety, a lysine residue and/or a functionally-comparable acyl acceptor component at between about the second residue position and about the fourth residue position from the peptide N-terminal. Conversely, other such peptides useful with this invention--again, either alone or as coupled or conjugated with a suitable polymeric component--provide at least two contiguous carboxamide moieties, glutaminyl residues and/or functionally comparable acyl donor components of such a peptide.

A range of polymeric components can be utilized in conjunction with the methods of this invention. Without limitation and for purposes of illustration, alginic acid (or a salt thereof) and/or a poly(ethylene oxide), one of numerous linear or branched compounds commercially available, can be used with good effect, synthetically modified as may be required for coupling to an enzyme specific peptide(s) of this invention. Other polymers or polymeric components, including but not limited to those described herein, synthetically modified as desired, can be used with comparable effect in conjunction with the aforementioned biomimetic peptides. Polymer molecular weights and other such physical or chemical parameters can be varied, limitations on which are imposed only by way of enzyme activity and/or resultant gelation. Accordingly, the present invention can also comprise peptides and polymer-peptide conjugates, their corresponding gels or matrix materials and/or systems of gels/matrices having cellular, chemical or therapeutic agents incorporated therein.

In part, the present invention may also be a method of using a biomimetic peptide for polymer gelation. Such a method includes (1) providing a first polymeric component and coupling thereto at least one acyl acceptor peptide comprising at least one lysine residue; (2) providing a second polymeric component and coupling thereto at least one acyl donor peptide comprising at least two contiguous glutaminyl residues; or optionally, providing a polymeric component coupled both to at least one peptide comprising at least one .epsilon.-amino moiety and at least one peptide comprising at least one .gamma.-carboxamide moiety, such a polymeric component providing both acyl donor and acceptor substrates sufficient for transglutaminase activity, and (3) introducing a transglutaminase enzyme to the aforementioned polymeric component(s) in an amount and for a time at least partially sufficient for gelation.

For the first time, this invention provides new peptide substrates for tissue transglutaminase, polymer-peptide conjugates, and bioadhesive hydrogels formed by tissue transglutaminase cross-linking. As mentioned above, the new peptide substrates are rationally designed and synthesized. In designing short peptide substrates of TGase, it was recognized that primary amino acid sequence is a major factor in determining the suitability of a peptide as a TGase substrate, and that not all peptides containing Gln or Lys residues are useful good substrates. In acyl donor substrates, primary peptide/protein sequence near the Gln residue is known to play a role in substrate properties. For instance, short peptides containing Gln repeats, as found in involucrin and other proteins, are known to be excellent acyl donors. Regarding an acyl acceptor, it has been shown in the art that the composition and sequence of the residues adjacent to Lys in peptide and protein substrates can have an effect on the amine specificity.

The substrate specificity of these peptides toward transglutaminase was measured by enzymatic kinetic studies. Peptide substrates with high substrate specificity were coupled to polymer molecules, and the polymer-peptide conjugates were purified. These polymer-peptide conjugates were then cross-linked to rapidly form hydrogels by transglutaminase, as may be achieved through a biomimetic approach under physiological conditions. Such covalently cross-linked hydrogels are injectable, may be formed in-situ, and are biodegradable--and are equal to or better than fibrin glue in adhesion to skin tissue or extracellular matrix protein (collagen) membranes.

For purposes of illustration (Table 1 -- see Original Patent), eight acyl acceptor lysine-peptide substrates and three acyl donor glutaminyl-peptide substrates were rationally designed, in light of the preceding, and synthesized by solid phase peptide synthesis using known literature techniques. As shown in certain embodiments, these peptides may be relatively short (usually about four to about six amino acid residues), such that their preparation can be easily and economically scaled up by standard solution synthetic methods.

For acyl donor attention was focused on peptides containing two to about 5 contiguous glutaminyl (e.g., Gln residues), whereas for acyl acceptor a variety of residues were placed near or adjacent Lys. The peptides were assayed as TGase substrates by monitoring the rate of cross-linking reaction with a known dansyl labeled substrate. The use of fluorescently labeled compounds and RP-HPLC analysis with the aid of LC-ESI/MS, allowed for identification of products and comparison of the kinetic constants of the candidate peptides. The specificity (k.sub.cat/K.sub.m, app) values determined under identical experimental conditions reflect the relative specificity of the enzyme toward the substrates; peptides with higher specificities are better substrates for TGase enzyme, indicating the enzyme-catalyzed cross-linking reaction of that peptide will proceed more rapidly.

Under experimental conditions, the specificities of the Lys peptides varied by several orders of magnitude, whereas the specificities of the Gln peptides varied only modestly (Table 1). The acetylated dipeptide Ac--KG-NH.sub.2 had a specificity of 10.6 (min.sup.-1 mM.sup.-1) for TGase. Addition of a hydrophobic residue (F or L) to the N-terminus of the dipeptide resulted in an approximately 5-fold increase in specificity, and acetylation of the N-terminus of the F/LKG-NH.sub.2 tripeptide further increased the specificity ca. 10-fold.

Incorporation of a DOPA residue directly preceding the Lys residue of the peptide KG-NH.sub.2 caused the side-chain primary amine of the Lys to entirely lose its ability to serve as an acyl acceptor. However, addition of an N-terminal DOPA residue to the tripeptides Ac--F/LKG-NH.sub.2 resulted in ca. 2.4-fold increases in specificity. Remarkably, the specificities of tetrapeptides DOPA-F/LKG-NH.sub.2 were enhanced ca. 100-fold compared to the dipeptide Ac--KG-NH.sub.2: representing the first successful incorporation of DOPA into TGase substrate.

More subtle differences were noted in the specificities of the acyl donor peptides, with all three designed Gln peptides exhibiting good substrate properties. It is interesting to note that the specificities of representative short peptides of this invention, Ac-(SEQ ID NO:2)GQQQLG-NH.sub.2 and DOPA-(SEQ ID NO:2)GQQQLG-NH.sub.2, compared favorably to the specificity of NH.sub.2-(SEQ ID NO:3)GQLKHLEQQEG-NH.sub.2, a peptide derived from the repeat motif found in the keratinocyte protein involucrin, which is known to be an excellent substrate for TGase.

The acyl donor and acyl acceptor peptides of this invention can be separately conjugated or coupled with, for example, PEG. Solutions of such polymer-peptide conjugates rapidly form hydrogels in the presence of transglutaminase under physiological and/or appropriate reaction or end-use conditions. The hydrogels of this invention are adhesive, for example, comparable to type I collagen and guinea pig skin. For example, based on the results of substrate specificity studies, DOPA-FKG (acyl acceptor) and Ac-(SEQ ID NO:2)GQQQLG (acyl donor) were selected and separately coupled to a PEG to form PEG-peptide conjugates 1 and 2 shown in FIGS. 7 8. The PEG-peptide conjugates were analyzed and purified by RP-HPLC, and their structures confirmed by MALDI TOF-MS analysis.

In the presence of TGase, an aqueous fluid containing equimolar amounts of PEG-peptide conjugates 1 and 2 formed a hydrogel within minutes under physiologic conditions. Rheological studies indicated that the hydrogel formed in less than two minutes, as indicated by the crossover of the storage (G') and loss (G'') moduli (FIG. 1). The elastic nature of the cross-linked gel was demonstrated by constant values of G' obtained over several decades of frequency in an oscillatory frequency sweep experiment, and the resulting hydrogel was found to be highly elastic, as indicated by constant values of G' at up to 100% strain during a strain sweep experiment (see examples 8 9).

The conjugated peptides clearly retained their TGase substrate characteristics after coupling to PEG, as evidenced by the rapid gelation time observed. The multifunctionality of the PEG-peptide conjugates result in gel network formation. It should be noted, however, that formulations in which Lys-PEG-Lys was used in place of 1 did not form gels under identical conditions (data not shown), demonstrating that at least under the conditions and acceptor conjugate employed, it may not be sufficient to have Lys residues present in one of the PEG polymers, and thereby emphasizing the benefit of using Lys and Gln peptides with high TGase reactivity.

Although the enzyme concentration necessary for rapid hydrogel formation was considerably higher than for TG-induced cross-linking of free peptide, this may be explained in part by the effect of gel network formation on the mobility of TGase enzyme. During incipient network formation resulting from partial cross-linking of the PEG-peptide conjugates, the solution viscosity rapidly increases while the mobility of the enzyme rapidly decreases. Higher enzyme concentration is therefore necessary to ensure sufficient enzyme available to the cross-linking sites during the later stages of gelation, when enzyme mobility is increasingly restricted. Nevertheless, the gelation time demonstrated here approaches that desired for tissue engineering and surgical adhesive applications, and the presence of DOPA suggests that these new hydrogel materials may prove useful in applications where adhesion to tissues is paramount.

As mentioned above, a range of available transglutaminases catalyze a cross-linking reaction between the .gamma.-carboxamide groups of peptide-bound glutamine residues and the .epsilon.-amino groups of lysine residues in proteins. Substrate specificity may be optimized through this invention using, in accordance herewith, short peptides by rational design: typically less than about 20 residues and, in certain embodiments, less than about 10 residues, for easier, quicker and more cost effective peptide synthesis. Optimized peptides can be covalently conjugated to linear or branched polymers such as PEG (e.g., linear PEG, and branched PEG--4-arm, 6-arm or 20-arm), hyper-branched polymers such as dendrimers, or linear polymers with multi-functional groups such as but not limited to chitosan, gelatin, soluble collagens, hyaluronic acid, alginates, and albumins. Solutions of such polymer-peptide conjugates can be mixed with therapeutic agents or cells, which can be injected and triggered to hydrogel and entrap the therapeutics or cells in the presence of tissue or an otherwise suitable transglutaminase under physiological conditions in vivo. Alternatively, solutions of these polymer-peptide conjugates can also be applied onto a recipient surface or an anatomical surface for use as a surgical sealant and/or medical adhesive.

In various embodiments, polymer-peptide conjugates in solution or liquid form can be positioned or injected then triggered to rapidly form hydrogels in the presence of tissue transglutaminase in vivo. Such injectable in-situ formed and cross-linked hydrogels may be useful in sustained drug delivery applications as biodegradable reservoirs, tissue repair and engineering as polymeric scaffolds, gene therapy, soft tissue augmentation and cosmetic tissue augmentation in a minimally invasive manner, and as surgical sealant, medical and dental adhesives, and wound dressings.

As demonstrated below, for purposes of comparison, a Fibrin sealant of the prior art (available under the Tisseel trademark) is currently approved for clinic use as a surgical sealant and medical adhesive. However, it contains human and bovine derived proteins, which pose a risk of contamination with viruses or the prions that cause mad cow disease, or bovine thrombin sensitization. The present hydrogel systems have two primary advantages over fibrin sealant: First, the polymer-peptide conjugates are chemically synthesized, and the enzyme could be prepared by recombinant DNA technology. Second, the hydrogel systems of this invention provide better adhesive qualities to type I collagen.
 

Claim 1 of 3 Claims

1. A biomimetic gelation system comprising: a) a first polymeric component conjugated to at least one acyl donor peptide, wherein the at least one donor peptide comprises at least two contiguous glutaminyl residues; b) a second polymeric component conjugated to at least one acyl acceptor peptide, wherein the at least one acceptor peptide comprises a lysine residue; and c) a transglutaminase; wherein at least one of said first and second polymeric components comprises poly(ethylene glycol).

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