Patent Application
20090238788
Hydrogels are a class of materials that have significant promise for use in soft tissue and bone engineering and wound sealing, in part because of their well-hydrated, porous structure. For use in tissue regeneration, however, a material must be cyto-compatible, i.e., not toxic to the target cells, biocompatible, i.e., does not cause a significant immunological and inflammatory response in vivo and is preferably biodegradable, and have some rigidity.
To these ends, β-sheet peptide-based hydrogels that are capable of self-assembly in vivo or in vitro in response to environmental stimuli, such as pH, temperature, salt concentration, or specific ion concentrations, have been developed. In solution, the peptides are unfolded. Upon stimulation, the peptides first fold to form β-hairpins. The β-hairpin peptides then self-assemble to form β-sheet hydrogels. These peptide-based hydrogelation systems have been described in J. P. Schneider, et al., J Am Chem Soc 124: 15030-15037, 2002; D. J. Pochan, et al., J Am Chem Soc 125: 11802-11803, 2003; B. Ozbas, et al., Macromolecules 37: 7331-7337, 2004; K. Jajagopal and J. P. Schneider, Curr. Opin. Structural Biol. 14: 480-486, 2004; L. Haines-Butterick, et al., PNAS 104: 7791-7796, 2007. They are additionally described in US 2006/0025524 and US 2007/0128175, which are incorporated herein, in entirety, by reference.
When hydrogels are applied in vivo, the gel materials or precursor liquids do not remain localized to the site of application unless hydrogelation is rapid or the material is applied to a well-defined cavity. Therefore, a method is needed to apply hydrogels to tissue in such a way that the hydrogels remain localized to the site of application. In addition, methods for depositing thin hydrogel films on cell and tissue culture substrates and for depositing hydrogels over a broad surface, e.g., in wound sealing, are required.
A method of delivering a preformed β-sheet hydrogel to a target site is provided, the method comprising shearing the hydrogel and spraying the sheared hydrogel onto the target site. Embodiments of the method include, but are not limited to, methods of spraying hydrogels comprising peptides selected from the group consisting of SEQ ID NOs: 1-69 to a target site. In a further embodiment, the hydrogel comprises a therapeutic agent.
As described below, preformed β-sheet peptide hydrogels can be shear-thin delivered as a spray to provide broad surface coverage at target sites, such as tissue surfaces and tissue or cell-culture surfaces. Shear-thin delivery is achieved by using mechanical shear forces, for example, educting with gas or spraying through an aperture at high pressure, to thin the gel material allowing it to flow. Any spraying apparatus appropriate for delivering the hydrogel to a target surface in the desired amount and thickness may be used. In a preferred embodiment, the shearing and spraying of the hydrogel are substantially simultaneous. After delivery as a spray, the hydrogel remains localized at the site of application and retains its β-sheet structure. These peptide hydrogels can be deposited over a range of thickness from 1 μm to 1 mm, and can be used to pattern surfaces for site-selective cell attachment. Shear-thin delivery of the hydrogel as a spray allows broad surface coverage.
The hydrogel can also comprise a therapeutic agent and be utilized to deliver the therapeutic agent to a target site, such as to a tissue in vivo or in vitro. For example, the hydrogel may contain agents that stimulate cell proliferation or differentiation, stimulate wound healing, or inhibit bacterial growth. Such agents may include, but are not limited to analgesics, antibiotics, antineoplastics, hemostatic agents, anticoagulants, cytokines, growth factors, anti-inflammatories, small molecules, proteins, peptides, nucleotides, or cells. Spray delivery for β-sheet peptide hydrogels has broad medical application in, for example, tissue and bone engineering, regenerative and cosmetic treatment for hair and skin, cell-based diagnostics, surgery, wound-healing and wound-sealing. Spray delivery of β-sheet peptide hydrogels can also be used to apply gels to plastic or glass substrates for cell culture. The sprayed hydrogels may also be used in applying protective anti-bacterial coatings to a surface.
Examples of peptides that may be used in the practice of one or more aspect of the invention include, but are not limited to, the following:
In addition to the amino acids specifically recited above, at any position of any of the above peptides indicated with X, each X can independently be any natural or non-natural amino acid (L or D stereochemistry) or any analog of an amino acid known to those skilled in the art. In this application, D stereochemistry will be indicated by a superscript before the D amino acid, thus DP is D-proline.
In some embodiments of the invention, peptides may fit the general formula VKVKVKVK(XXXX)aKVKVKV(XXXX)bKVKVKVKV (SEQ ID NO:5). Each of these peptides adopts a 3-stranded β-sheet conformation. (Rughani, et al., Biomacromolecules, accepted Mar. 4, 2009). Specific examples of 3-stranded β-sheet forming peptides include, but are not limited to,
In addition to the amino acids specifically recited above, at any position of any of the above peptides indicated with X, each X can independently be any natural or non-natural amino acid (L or D stereochemistry) or any analog of an amino acid known to those skilled in the art. Preferably, each (XXXX)a and (XXXX)b may comprise a sequence capable of forming a turn (e.g., a β-turn).
In some embodiments of the invention, peptides may fit the following general formulas:
wherein each m and n may independently be from 1-100 and m may or may not equal n;
wherein Na-butylated lysine residues are present at the bold positions;
where m=1-20 and n=1-20 and m may be the same or different as n in any given peptide.
In some embodiments, one or more amino acids of the turn region may be substituted and/or modified as compared to the turn region of MAX1. In some embodiments, turn sequences may be incorporated that not only play a structural role but also play a biofunctional role. For example, RGD (SEQ ID NO:66) binding epitopes are normally found within turn regions of proteins known to be important in cell adhesion events, and residues that flank RGD provide additional specificity to the binding event. Incorporating these epitopes into the turn regions of self-assembling hairpins may lead to hydrogel scaffolds having enhanced cell adhesion properties.
The peptide, HPL8 (SEQ ID NO: 21), in the folded state, is a 20 amino acid β-hairpin comprised of β-strands of alternating valine and lysine residues flanking a type II′ turn (
An HPL8 hydrogel was prepared by adding 500 μL of 25 mM HEPES, pH 7.4 to a vial containing 10 mg of HPL8 peptide, giving rise to a soluble 2 wt % HPL8 peptide solution. An equal volume of DMEM supplemented with 25 mM HEPES, pH 7.4 was added to the soluble 2 wt % HPL8 peptide solution and the mixture was immediately transferred to a 15 ml gravity-feed cup on top of an Iwata Revolution CR airbrush equipped with a 0.5 mm screw-in nozzle (
The airbrush described in Example 1 was connected to a nitrogen tank equipped with a regulator set to a range of approximately 10 psi to approximately 20 psi. Nitrogen (N2) was passed through the airbrush to provide a shear force that disrupted the non-covalently cross-linked network of the hydrogel in the gravity-feed cup. Pulling back on the airbrush lever exposed the gel in the gravity feed cup to the nitrogen gas below, which flows through the brush. The flowing gas provided suction to pull the gel into the flowing gas and consequently shear-thinned the gel into particles that were sprayed through the brush nozzle with the exiting gas. Thus, the shear-thinning procedure produces gel particles that can be sprayed through the airbrush and onto a surface. Using this method, the shearing and spraying of the hydrogel are substantially simultaneous. The mist of hydrogel produced immediately recovered rigidity upon contact with the surface and remained fixed on the surface at the site of application. Hydrogels remained fixed at the site of application, even when submerged in water and agitated. The shear-thin and spraying protocol is shown in
CD wavelength spectra were collected on a Jasco J-810 spectropolarimeter employing a 0.01 mm detachable quartz cell. HPL8 gels (1 wt %) were prepared as described in Example 1 and shear-thin delivered via the airbrush to the detachable quartz cell. Measurements were taken immediately after shear-thin delivery. Ellipticity in millidegrees was monitored from 260 nm to 200 nm at 37° C. using a step size of 2 nm. The CD wavelength spectrum, shown in
An LSCM (laser scan confocal microscope) z-stack image of a 1 wt % HPL8 hydrogel sprayed onto the surface of one well of an 8-well confocal plate and stained with calcein for visualization is shown in
The height of the sprayed hydrogel on the surface can be as thin as approximately 1 μm. The height of the hydrogel can be adjusted to any desired height by manipulating the rate of gel delivery within the spray brush and by using multiple, consecutive sprays.
4. Patterning with the Sprayed Hydrogel.
Hydrogels can be sprayed in patterns by laying a mask on the surface to be sprayed.
Hydrogel sprays can also be used to create cell patterns on surfaces. Patterned surfaces were generated by first spin-coating microscope slides with a triblock comb polymer (61 wt % (methyl methacrylate (MMA), 21 wt % hydroxyl poly(oxyethylene) methacrylate (HPOEM), and adding 18 wt % poly(ethylene glycol) methyl ether methacrylate (POEM)) to the slides. The slides were centrifuged at about 2500 rpm for 20 seconds then cured at 60° C. under vacuum overnight. Cells are unable to attach to the polymer-coated slides. A mask containing small holes of about 2 mm in diameter was placed on top of the polymer-coated slides, and HPL8 hydrogel was then sprayed through the mask. The mask was removed leaving behind a pattern of small islands of hydrogel, each about 2 mm in diameter, surrounded the by polymer.
A solution of 5×106 C3H10t1/2 cells/mL, suspended in DMEM cell culture medium, was prepared and 2 ml of this solution was added to the surface of the patterned slide and incubated for 5 minutes. The slide was then washed in DMEM to remove unattached cells. A live/dead assay (Molecular Probes) was performed to determine cellular attachment and viability and to visualize the location of cells. With this assay, live cells fluoresce green and dead cells fluoresce red.
Although the invention is illustrated and described herein with reference to specific embodiments, the invention is not intended to be limited to the details shown. Rather, various modifications may be made in the details within the scope and range of equivalents of the claims and without departing from the invention.
This application claims benefit of priority to U.S. Provisional Application Ser. No. 61/037,996, filed Mar. 19, 2008, and which is incorporated herein, in its entirety, by reference.
This invention was made with government support under DE0163861, awarded by the National Institutes of Health. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61037996 | Mar 2008 | US |
