# US 11,998,658 B2 · Injectable porous hydrogels

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- Number: US 11,998,658 B2
- Document ID: US11998658B2
- Status: Granted
- Kind: B2
- Application number: 16/786,312
- Filed: 2020-02-10
- Published: 2020-08-13
- Granted: 2024-06-04
- Earliest claimed: 2019-02-08
- Assignees: University of New Hampshire
- Applicants: University of New Hampshire
- Inventors: Kyung Jae Jeong
- Web page: https://patentgrep.com/patent/US11998658B2

## CPC

- A61L 27/222: {Gelatin}
- A61L 27/56: Porous materials, {e.g. foams or sponges}
- A61L 27/3687: {characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents}
- A61L 27/38: {containing added animal cells (organs or tissue containing native cells A61L27/36)}
- 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 2400/06: Flowable or injectable implant compositions
- 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)

## Abstract

Injectable macroporous hydrogels, and methods of producing same, are described. The injectable macroporous hydrogels may be formed by mixing gelatin microgels with an enzyme. In at least some embodiments, the enzyme may be transglutaminase, and more specifically microbial transglutaminase (mTG).

## Claims

### Claim 1

A method for producing a porous hydrogel, the method comprising:
preparing gelatin/gelatin methacrylate (GelMA) microgels comprising gelatin and GelMA, wherein the gelatin/GelMA microgels have an average diameter between 130 μm and 250 μm when in a hydrated state;
preparing a microgel solution comprising the gelatin/GelMA microgels, cross-linking enzyme comprising transglutaminase, and photoinitiator present at a concentration range of 0.001% to 0.1% (w/v) to minimize cytotoxic effects on live cells; and
curing the microgel solution by contacting the gelatin/GelMA microgels with ultraviolet (UV) light for a time effective for the photoinitiator to activate cross-linking of the gelatin/GelMA microgels, to produce a porous hydrogel having a porosity sufficient for cell migration and proliferation in the interstitial space of the porous hydrogel.

### Claim 2 (depends on 1)

The method of claim 1, wherein the transglutaminase is microbial transglutaminase (mTG).

### Claim 3 (depends on 1)

The method of claim 1, wherein curing the microgel solution comprises incubating the microgel solution at about 37° C.

### Claim 4 (depends on 1)

The method of claim 1, wherein the photoinitiator comprises at least one of Irgacure 2959, vitamin B12, and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

### Claim 5 (depends on 1)

The method of claim 1, further comprising: preparing the microgel solution to further comprise one or more live cells.

### Claim 6 (depends on 5)

The method of claim 5, wherein the one or more live cells comprise one or more stem cells.

### Claim 7

A microgel solution, comprising:
(a) gelatin/gelatin methacrylate (GelMA) microgels comprising gelatin and GelMA, wherein the gelatin/GelMA microgels have an average diameter between 130 μm and 250 μm, the average diameter of the microgels being sufficient to create an interstitial space in a resulting hydrogel of sufficient porosity for cell migration and proliferation;
(b) cross-linking enzyme comprising transglutaminase; and
(c) photoinitiator present at a concentration range of 0.001% to 0.1% (w/v) to minimize cytotoxic effects on live cells.

### Claim 8 (depends on 7)

The microgel solution of claim 7, wherein the cross-linking enzyme further comprises one or more of genipin, factor XIIIa and horse radish peroxidase (HRP).

### Claim 9 (depends on 7)

The microgel solution of claim 7, further comprising an antioxidant agent.

### Claim 10 (depends on 7)

The microgel solution of claim 7, further comprising one or more live cells.

### Claim 11

A method of delivering one or more live cells into a subject, the method comprising:
(a) preparing a microgel solution comprising:
gelatin/gelatin methacrylate (GelMA) microgels comprising gelatin and GelMA, wherein the gelatin/GelMA microgels have an average diameter between 130 μm and 250 μm,
(ii) cross-linking enzyme comprising transglutaminase,
(iii) photoinitiator present at a concentration range of 0.001% to 0.1% (w/v) to minimize cytotoxic effects on live cells, and
(iv) one or more live cells;
(b) administering the microgel solution to a target in a subject where the one or more live cells are intended to be delivered to the subject; and
(c) curing the microgel solution by contacting the gelatin/GelMA microgels with ultraviolet (UV) light for a time effective for the photoinitiator to activate cross-linking of the gelatin/GelMA microgels, to produce a porous hydrogel that is adhered by transglutaminase cross-linking to the target and having a porosity sufficient for cell migration and proliferation of the one or more live cells in the interstitial space of the porous hydrogel.

### Claim 12 (depends on 11)

The method of claim 11, wherein the one or more live cells comprise one or more stem cells.

### Claim 13 (depends on 7)

The microgel solution of claim 7, wherein the microgel solution comprises 0.05% (w/v) concentration of the photoinitiator.

### Claim 14 (depends on 7)

The microgel solution of claim 7, where the transglutaminase is microbial transglutaminase (mTG).

### Claim 15 (depends on 11)

The method of claim 11, where the transglutaminase is microbial transglutaminase (mTG).

### Claim 16 (depends on 1)

The method of claim 1, wherein the gelatin/GelMA microgels are prepared from lyophilized gelatin/GelMA microgels.

### Claim 17 (depends on 7)

The microgel solution of claim 7, wherein the gelatin/GelMA microgels are hydrated from lyophilized gelatin/GelMA microgels.

### Claim 18 (depends on 11)

The method of claim 11, wherein, the gelatin/GelMA microgels are prepared from lyophilized gelatin/GelMA microgels.

### Claim 19 (depends on 7)

The microgel solution of claim 7, wherein the microgel solution comprises 0.02% to 0.08% (w/v) concentration of the photoinitiator.

### Claim 20 (depends on 7)

The method of claim 7, wherein the microgel solution comprises 0.02% to 0.08% (w/v) concentration of the photoinitiator.