# US 2016/0256604 A1 · Tunable covalently crosslinked hydrogels and methods of making the same

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- Number: US 2016/0256604 A1
- Document ID: US20160256604A1
- Status: Published application
- Kind: A1
- Application number: 15/027,468
- Filed: 2014-10-06
- Published: 2016-09-08
- Earliest claimed: 2013-10-08
- Assignees: Trustees of Tufts College
- Applicants: Trustees of Tufts College
- Inventors: Craig W. Hanna; Benjamin P. Partlow; David L. Kaplan
- Web page: https://patentgrep.com/patent/US20160256604A1

## CPC

- A61L 27/227: {Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24}
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 9/06: Ointments; Bases therefor; {Other semi-solid forms, e.g. creams, sticks, gels (composition of ointments, creams or gels A61K47/00)}
- A61K 35/00: Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K 47/42: Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein (oligopeptides having up to five amino acids {A61K47/183}; polyamino acids A61K47/34)
- A61K 47/46: Ingredients of undetermined constitution or reaction products thereof, e.g. skin, bone, milk, cotton fibre, eggshell, oxgall or plant extracts
- A61L 27/18: obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
- A61L 27/20: Polysaccharides
- A61L 27/22: Polypeptides or derivatives thereof {, e.g. degradation products}
- A61L 27/3604: {characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel}
- A61L 27/3834: {Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells}
- A61L 27/50: Materials characterised by their function or physical properties {, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials}
- A61L 27/52: Hydrogels or hydrocolloids
- A61L 27/54: Biologically active materials, e.g. therapeutic substances {(A61L27/227 takes precedence)}
- A61L 27/58: Materials at least partially resorbable by the body
- C07K 14/43586: {from silkworms}
- C08J 3/075: Macromolecular gels
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- A61L 2300/64: Animal cells
- A61L 2400/06: Flowable or injectable implant compositions
- C08J 2389/00: Characterised by the use of proteins; Derivatives thereof
- A61L: METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES (preservation of bodies or disinfecting characterised by the agents employed A01N; preserving, e.g. sterilising, food or foodstuffs A23; preparations for medical, dental or toiletry purposes A61K)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C08J: WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H (working, e.g. shaping, of plastics B29)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

The present invention provides, among other things, a elastomeric biomaterial having enzymatically cross-linked amino acid phenolic side chains to generate highly elastic hydrogels. Materials are characterized by tunable mechanical properties, gelation kinetics and swelling properties of these new protein polymers. Provided materials are support encapsulation of cells. Methods of making and using of provided particles are also disclosed.

## Claims

### Claim 1

A covalently crosslinked hydrogel, comprising:
a protein polymer having phenolic side chains, and
covalent crosslinks between at least some such phenolic side chains,
so that the hydrogel is characterized by a storage modulus value that is between about 50 Pa and about 100 kPa without an indication of a plastic deformation.

### Claim 2 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the protein polymer is silk fibroin, elastin, chitosan, collagen, gelatin, agarose, alginate, chitin, polyhydroxyalkanoates, pullan, starch, cellulose, hyaluronic acid, polydimethylsiloxane, poly(lactide-co-glycolide), resilin, poly(ethylene glycol) or combinations thereof.

### Claim 3 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the covalent crosslinks comprise covalent dityrosine bonds.

### Claim 4 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is formed from a solution having a protein polymer to solvent concentration between about 0.1 wt % to about 30 wt %.

### Claim 5 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the protein polymer has an average molecular weight between about 10 kDa and about 400 kDa.

### Claim 6 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is further characterized by swelling up to 400% when exposed to solvent.

### Claim 7 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is configured to support encapsulation of cells.

### Claim 8 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is configured to support incorporation of functional moieties.

### Claim 9 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is configured to support incorporation of at least one agent.

### Claim 10 (depends on 9)

The covalently crosslinked hydrogel of claim 9, wherein the hydrogel degrades releasing the at least one agent.

### Claim 11 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel recovers from a shear strain of at least 100% without showing an indication of a plastic deformation.

### Claim 12 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel recovers from a compressive strain of at least 75% without showing an indication of a plastic deformation.

### Claim 13 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is further characterized by a tangent modulus between about 200 Pa to about 400 kPa.

### Claim 14 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel is configured to be injectable.

### Claim 15 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel exhibits negligible absorbance above 290 nm so that the biomaterial is optically clear.

### Claim 16 (depends on 1)

The covalently crosslinked hydrogel of claim 1, wherein the hydrogel further includes a crystalline structure.

### Claim 17 (depends on 16)

The covalently crosslinked hydrogel of claim 16, wherein the crystalline structure comprises beta sheet structures and hydrogen bonding.

### Claim 18

A method of manufacturing a covalently crosslinked hydrogel, comprising steps of:
providing a protein polymer having phenolic side chains, wherein the protein polymer has an average molecular weight in a range of about 10 kDa to about 400 kDa;
solubilizing the protein polymer to form an aqueous protein polymer solution, wherein a concentration of the aqueous protein polymer solution is in a range of about 0.1 wt % to about 30 wt %;
providing a solution of peroxidase;
combining the aqueous protein polymer solution with the solution of peroxidase;
inducing a gelation reaction in the combined solution when a peroxide is added to the combined solution; and
covalently crosslinking at least some of the phenolic side chains of the protein polymer thereby forming a hydrogel, wherein the hydrogel is characterized by a storage modulus value that is between about 50 Pa and about 100 kPa without an indication of a plastic deformation.

### Claim 19 (depends on 18)

The method of claim 18, the step of providing the protein polymer having phenolic side chains, wherein the protein polymer is silk, elastin, chitosan, collagen, gelatin, agarose, chitin, polyhydroxyalkanoates, pullan, starch, cellulose, hyaluronic acid, polydimethylsiloxane, poly(lactide-co-glycolide), resilin, poly(ethylene glycol) or combinations thereof.

### Claim 20 (depends on 18)

The method of claim 18, the step of solubilizing the protein polymer to form the aqueous protein polymer solution, wherein a solvent is water, DMEM, PBS, organic solvents or combinations thereof.

### Claim 21 (depends on 18)

The method of claim 18, the step of providing the polymer solution having phenolic side chains, wherein the phenolic side chains comprise tyrosine.

### Claim 22 (depends on 18)

The method of claim 18, the step of providing the solution of peroxidase, wherein the peroxidase comprises horseradish peroxidase, glutathione peroxidase, thyroid peroxidase, haloperoxidase, myeloperoxidase, animal heme-dependent peroxidase, vanadium bromoperoxidase, lactoperoxidase or combinations thereof.

### Claim 23 (depends on 18)

The method of claim 18, the step of providing the solution of peroxidase, wherein a peroxidase concentration is between about 0.001 mg/mL and about 10 mg/mL.

### Claim 24 (depends on 18)

The method of claim 18, the step of inducing the gelation reaction in the combined solution when the peroxide is added to the combined solution, wherein the peroxide comprises hydrogen peroxide, barium peroxide, calcium peroxide, sodium peroxide, organic peroxides or combinations thereof.

### Claim 25 (depends on 18)

The method of claim 18, the step of inducing the gelation reaction in the combined solution when the peroxide is added to the combined solution, wherein the peroxide concentration is between about 0.01 wt % and about 30 wt %.

### Claim 26 (depends on 18)

The method of claim 18, the step of inducing the gelation reaction in the combined solution when the peroxide is added to the combined solution, wherein the gelation reaction forms the hydrogel and the gelation reaction is complete between about 200 seconds and about 5000 seconds after the gelation reaction was induced.

### Claim 27 (depends on 18)

The method of claim 18, further comprising incorporating functional moieties.

### Claim 28 (depends on 18)

The method of claim 18, further comprising incorporating at least one agent.

### Claim 29 (depends on 28)

The method of claim 28, further comprising releasing the at least one agent when the hydrogel degrades.

### Claim 30 (depends on 18)

The method of claim 18, further comprising tuning the storage modulus value.

### Claim 31 (depends on 18)

The method of claim 18, further comprising encapsulating a plurality of cells within the hydrogel, wherein the hydrogel is a matrix.

### Claim 32 (depends on 30)

The method of claim 30, the step of tuning the storage modulus value comprises varying an average molecular weight of the protein polymer between about 10 kDa and about 400 kDa.

### Claim 33 (depends on 30)

The method of claim 30, the step of tuning the storage modulus value comprises varying the concentration of the aqueous protein polymer solution between about 0.5 wt % and about 30 wt %.

### Claim 34 (depends on 1)

The method of claim 1, further comprising molding channels into the hydrogel, whereby the channels enhance diffusion of oxygen and nutrients and promote vascularization.

### Claim 35 (depends on 1)

A pharmaceutical composition comprising the covalently crosslinked hydrogel of claim 1, further comprising: a plurality of cells, wherein a cell shape is influenced when the storage modulus value is tuned, thereby effecting an elasticity of the hydrogel.

### Claim 36 (depends on 35)

The pharmaceutical composition comprising covalently crosslinked hydrogel of claim 35, wherein the hydrogel is configured to be in an injectable dosage form.

### Claim 37

A covalently crosslinked hydrogel prepared by a process comprising steps of:
providing a solution of a protein polymer having phenolic side chains;
providing a solution of peroxidase;
combining the polymer solution with the solution of peroxidase;
inducing a gelation reaction in the combined solution when a peroxide is added to the combined solution; and
inducing a gelation reaction in the combined solution when a peroxide is added to the combined solution; and
covalently crosslinking at least some of the phenolic side chains of the protein polymer forming a hydrogel, wherein the hydrogel is characterized by a storage modulus value that is between about 50 Pa and about 100 kPa and/or a tangent modulus between about 200 Pa to about 400 kPa without deforming.

### Claim 38 (depends on 37)

The covalently crosslinked hydrogel of claim 37, prepared by a process, wherein the step of providing the solution of the protein polymer having phenolic side chains further comprises selecting a molecular weight of the polymer for matching, tuning, adjusting, and/or manipulating mechanical properties of the covalently crosslinked hydrogel.

### Claim 39 (depends on 38)

The covalently crosslinked hydrogel of claim 38, prepared by a process, wherein the molecular weight of the polymer is an average molecular weight between about 10 kDa and about 400 kDa.

### Claim 40 (depends on 37)

The covalently crosslinked hydrogel of claim 37, prepared by a process, wherein the step of providing a solution of the protein polymer having phenolic side chains further comprises selecting a specific polymer, for matching, tuning, adjusting, and/or manipulating mechanical properties of the covalently crosslinked hydrogel.

### Claim 41 (depends on 37)

The covalently crosslinked hydrogel of claim 37, prepared by a process, wherein the step of providing the solution of the protein polymer having phenolic side chains further comprises selecting a concentration of a polymer solution, for matching, tuning, adjusting, and/or manipulating mechanical properties of the covalently crosslinked hydrogel.

### Claim 42 (depends on 41)

The covalently crosslinked hydrogel of claim 41, prepared by a process, wherein the polymer solution concentration is between about 0.5 wt % and about 30 wt %.

### Claim 43 (depends on 37)

The covalently crosslinked hydrogel of claim 37, prepared by a process, wherein the step of providing the solution of peroxidase further comprises selecting a specific peroxidase, for matching, tuning, adjusting, and/or manipulating mechanical properties of the covalently crosslinked hydrogel.

### Claim 44 (depends on 37)

The covalently crosslinked hydrogel of claim 37, prepared by a process, wherein the step of inducing the gelation reaction in the combined solution when the peroxide is added to the combined solution, further comprises selecting a specific peroxide, for matching, tuning, adjusting, and/or manipulating mechanical properties of the covalently crosslinked hydrogel.