# US 9,434,926 B1 · Graphene hydrogel and methods of using the same

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- Number: US 9,434,926 B1
- Document ID: US9434926B1
- Status: Granted
- Kind: B1
- Application number: 14/627,671
- Filed: 2015-02-20
- Granted: 2016-09-06
- Earliest claimed: 2012-02-23
- Assignees: University of South Florida
- Applicants: Subhra Mohapatra; Chunyan Wang
- Inventors: Subhra Mohapatra; Chunyan Wang
- Web page: https://patentgrep.com/patent/US9434926B1

## CPC

- C12N 5/0663: {Bone marrow mesenchymal stem cells (BM-MSC)}
- A61K 9/06: Ointments; Bases therefor; {Other semi-solid forms, e.g. creams, sticks, gels (composition of ointments, creams or gels A61K47/00)}
- A61K 35/28: Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
- A61K 47/02: Inorganic compounds
- A61K 47/14: Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
- A61K 47/36: Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61L 27/20: Polysaccharides
- A61L 27/3821: {Bone-forming cells, e.g. osteoblasts, osteocytes, osteoprogenitor cells}
- A61L 27/52: Hydrogels or hydrocolloids
- C08B 37/003: {Chitin, i.e. 2-acetamido-2-deoxy-(beta-1,4)-D-glucan or N-acetyl-beta-1,4-D-glucosamine; Chitosan, i.e. deacetylated product of chitin or (beta-1,4)-D-glucosamine; Derivatives thereof}
- C08F 251/00: Macromolecular compounds obtained by polymerising monomers on to polysaccharides or derivatives thereof
- C08F 290/062: {Polyethers}
- C08F 292/00: Macromolecular compounds obtained by polymerising monomers on to inorganic materials
- C08G 65/3322: {acyclic}
- C08J 3/075: Macromolecular gels
- C08L 5/08: Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
- C08L 71/02: Polyalkylene oxides
- C12N 5/0068: {General culture methods using substrates (for specific animal cell type C12N5/06)}
- C08F 220/54: Amides {, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide}
- C08F 222/102: {of dialcohols, e.g. ethylene glycol di(meth)acrylate or 1,4-butanediol dimethacrylate}
- C08G 83/001: {Macromolecular compounds containing organic and inorganic sequences, e.g. organic polymers grafted onto silica}
- C08J 2387/00: Characterised by the use of unspecified macromolecular compounds, obtained otherwise than by polymerisation reactions only involving unsaturated carbon-to-carbon bonds
- C12N 2501/00: Active agents used in cell culture processes, e.g. differentation
- C12N 2533/20: Small organic molecules
- C12N 2533/30: Synthetic polymers
- C12N 2533/72: Chitin, chitosan
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)
- 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)
- C08B: POLYSACCHARIDES; DERIVATIVES THEREOF (polysaccharides containing less than six saccharide radicals attached to each other by glycosidic linkages C07H; fermentation or enzyme-using processes C12P19/00; sugar industry C13; production of cellulose D21)
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08G: MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- 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)
- C08L: COMPOSITIONS OF MACROMOLECULAR COMPOUNDS (compositions based on polymerisable monomers C08F, C08G; artificial filaments or fibres D01F; textile treating compositions D06)

## Abstract

Provided herein are hydrogel compositions containing graphene, chitosan, a polyethylene (glycol) diacrylate (PEGDA). The hydrogel can optionally contain an N-isopropylacrylamide (NIPAM) (TPCG hydrogel). Also provided are methods for differentiating a cell on the hydrogels described herein. Further provided herein are methods for delivering a compound via the hydrogels described herein.

## Claims

### Claim 1

A method comprising:
contacting a cell with a hydrogel for a period of time, where the hydrogel comprises:
at least two acrylated chitosan-graphene monomers, where each chitosan-graphene monomer comprises:
carboxylic acid functionalized graphene, where the chitosan component of the chitosan-graphene monomer is covalently bonded to the carboxylic acid functionalized graphene component of the chitosan-graphene monomer; and
polyethylene (glycol) diacrylate (PEGDA), where the (PEGDA) covalently crosslinks the at least two acrylated chitosan-graphene monomers.

### Claim 2 (depends on 1)

The method of claim 1, further comprising the step of culturing the cell and the hydrogel in cell culture media.

### Claim 3 (depends on 2)

The method of claim 2, where the cell culture media is a differentiation cell culture media.

### Claim 4 (depends on 3)

The method of claim 3, wherein the differentiation cell culture media is an adipogenic cell culture media, osteogenic cell culture media, chondrogenic cell culture media, or combination thereof.

### Claim 5 (depends on 3)

The method of claim 3, wherein the cell and hydrogel are cultured in the differentiation cell culture media for about 7 days.

### Claim 6 (depends on 1)

The method of claim 1, wherein the cell is a stem cell.

### Claim 7 (depends on 6)

The method of claim 6, wherein the stem cell is selected from the group consisting of: a pluripotent stem cell, a totipotent stem cell, a multipotent stem cell, an adult stem cell, and an induced pluripotent stem cell.

### Claim 8 (depends on 1)

The method of claim 1, wherein the cell is a bone-marrow mesenchymal stem cell.

### Claim 9 (depends on 1)

The method of claim 1, where the period of time ranges from about 1 hour to about 14 days.

### Claim 10 (depends on 1)

The method of claim 1, wherein the cell proliferates to form a population of cells after the period of time.

### Claim 11 (depends on 10)

The method of claim 10, wherein the population of cells is harvested by separating the population of cells from the hydrogel to form harvested cells.

### Claim 12 (depends on 11)

The method of claim 11, wherein the harvested cells are delivered to a subject in need thereof.

### Claim 13 (depends on 12)

The method of claim 12, wherein the cell is autologous, allogenic, xenogeneic, or syngeneic.

### Claim 14 (depends on 1)

The method claim 1, wherein after the period of time the cell differentiates to form a differentiated population of cells.

### Claim 15 (depends on 14)

The method of claim 14, wherein the differentiated population of cells contains at least one cell selected from the group consisting of: a chondrocyte, an adipocyte, and an osteocyte.

### Claim 16 (depends on 14)

The method of claim 14, wherein the differentiated population of cells is harvested by separating the differentiated population of cells from the hydrogel.

### Claim 17 (depends on 16)

The method of claim 16, wherein the cell is autologous, allogenic, xenogeneic, or syngeneic.

### Claim 18 (depends on 14)

The method of claim 14, wherein the graphene enhances differentiation of the cell.

### Claim 19 (depends on 10)

The method of claim 10, further comprising the step of delivering the hydrogel and the population of cells to a subject in need thereof.

### Claim 20 (depends on 14)

The method of claim 14, further comprising the step of delivering the hydrogel and the differentiated population of cells to a subject in need thereof.

### Claim 21 (depends on 1)

The method of claim 1, wherein the hydrogel further comprises N-Isopropylacrylamide (NIPAM), where the NIPAM is crosslinked at least one of the acrylated chitosan-graphenemonomers.