# US 11,298,424 B2 · Non-ionic and thermoresponsive diblock copolypeptide hydrogels for delivery of molecules and cells

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- Number: US 11,298,424 B2
- Document ID: US11298424B2
- Status: Granted
- Kind: B2
- Application number: 15/516,211
- Filed: 2015-10-01
- Published: 2017-10-19
- Granted: 2022-04-12
- Earliest claimed: 2014-10-01
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Timothy J. Deming; Michael V. Sofroniew; Shanshan Zhang
- Web page: https://patentgrep.com/patent/US11298424B2

## CPC

- 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 9/06: Ointments; Bases therefor; {Other semi-solid forms, e.g. creams, sticks, gels (composition of ointments, creams or gels A61K47/00)}
- A61K 35/12: Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells (vaccines or medicinal preparations containing antigens or antibodies A61K39/00)
- A61K 35/30: Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
- A61P 25/00: Drugs for disorders of the nervous system
- C12N 11/02: Enzymes or microbial cells immobilised on or in an organic carrier
- C12N 11/04: entrapped within the carrier, e.g. gel or hollow fibres
- G01R 33/46: NMR spectroscopy
- G02B 21/0076: {arrangements using fluorescence or luminescence}
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01R: MEASURING ELECTRIC VARIABLES; MEASURING MAGNETIC VARIABLES (indicating correct tuning of resonant circuits H03J3/12)
- G02B: OPTICAL ELEMENTS, SYSTEMS OR APPARATUS

## Abstract

The present invention is directed to copolypeptide hydrogels (DCH) containing non-ionic hydrophilic residues (DCHEO), incorporation of thermoresponsive elements into DCHEO, to generate thermoresponsive DCH (DCHT), and hydrogels that include a combination of DCHEO and DCHT. The invention includes preparation, uses, compositions containing the hydrogels and methods of tuning the hydrogels. The hydrogels can be used to deliver an agent or cells to an organism.

## Claims

### Claim 1

A composition comprising:
an aqueous medium; and
a copolypeptide hydrogel forming composition,
wherein said copolypeptide composition comprises at least one hydrophilic polypeptide segment or hydrophilic copolypeptide segment and at least one hydrophobic polypeptide segment or hydrophobic copolypeptide segment,
wherein the hydrophilic polypeptide segment or hydrophilic copolypeptide segment consists of residues selected from lysine, glutamate, aspartate, arginine, ornithine, homoarginine, a residue of Formula I, a residue of Formula II, a residue of Formula III, a residue of Formula IV, a residue of Formula V, a residue of Formula VI, and combinations thereof; and
wherein the hydrophilic polypeptide segment or hydrophilic copolypeptide segment contains less than 50 mol % ionic amino acid residues, wherein an ionic amino acid residue is an amino acid residue having a charged side-chain at pH=7 in water;
wherein:
R1 is, independently at each occurrence, —(CH2CH2O)nCH3 or
R1a is —(CH2CH2O)nCH3;
R2 is, independently at each occurrence, —(CH2CH2O)nCH3 or
R2a is —(CH2CH2O)nCH3;
X1 is O;
Y1 is, independently at each occurrence, absent or O;
R3 is, independently at each occurrence, selected from —(CH2CH2O)mCH3, —CH2CH2CH2(sugar), and -sugar;
X2 is, independently at each occurrence, absent or O;
Y2 is, independently at each occurrence, absent or O;
R4 is, independently at each occurrence, selected from —(CH2CH2O)nCH3, —CH2CH2CH2(sugar), —CH2CHR4aC(O)OR4b, and —CH2CH2SO2CH2CH2SR4c;
R4a is, independently at each occurrence, —H or —CH3;
R4b is —(CH2CH2O)CH3;
R4c is —(CH2CH2O)nCH3;
X3 is, independently at each occurrence, absent or O;
Y3 is, independently at each occurrence, absent or O;
R5 is, independently at each occurrence, —(CH2CH2O)nCH3 or -sugar;
n is an integer from 1-4;
m is an integer from 1-6; and
p is an integer from 1-9.

### Claim 2 (depends on 1)

The composition of claim 1, further comprising an agent or a cell.

### Claim 3 (depends on 1)

The composition of claim 1, further comprising:
a second copolypeptide hydrogel forming composition,
wherein said second copolypeptide composition comprises at least one hydrophilic polypeptide segment or hydrophilic copolypeptide segment and at least one thermoresponsive polypeptide segment or thermoresponsive copolypeptide segment,
wherein said second copolypeptide composition undergoes a temperature induced transition between a liquid and a transparent hydrogel in said aqueous medium.

### Claim 4 (depends on 3)

The composition of claim 3, wherein the at least one thermoresponsive copolypeptide segment comprises at least one thermoresponsive residue and at least one non-ionic residue.

### Claim 5 (depends on 3)

The composition of claim 3, further comprising an agent or a cell.

### Claim 6

A method of delivering an agent or a cell into an organism comprising:
a) creating a mixture comprising:
the agent or the cell,
an aqueous medium, and
a copolypeptide hydrogel forming composition, wherein said copolypeptide composition comprises at least one hydrophilic polypeptide segment or hydrophilic copolypeptide segment and at least one hydrophobic polypeptide segment or hydrophobic copolypeptide segment; wherein the hydrophilic polypeptide segment or hydrophilic copolypeptide segment consists of residues selected from lysine, glutamate, aspartate, arginine, ornithine, homoarginine, a residue of Formula I, a residue of Formula II, a residue of Formula III, a residue of Formula IV, a residue of Formula V, a residue of Formula VI, and combinations thereof; and wherein the hydrophilic polypeptide segment or hydrophilic copolypeptide segment contains less than 50 mol % ionic amino acid residues, wherein an ionic amino acid residue is an amino acid residue having a charged side-chain at pH=7 in water; and
b) introducing the mixture into the organism;
wherein:
R1 is, independently at each occurrence, —(CH2CH2O)nCH3 or
R1a is —(CH2CH2O)nCH3;
R2 is, independently at each occurrence, —(CH2CH2O)nCH3 or
R2a is —(CH2CH2O)nCH3;
X1 is O;
Y1 is, independently at each occurrence, absent or O;
R3 is, independently at each occurrence, selected from —(CH2CH2O)mCH3, —CH2CH2CH2(sugar), and -sugar;
X2 is, independently at each occurrence, absent or O;
Y2 is, independently at each occurrence, absent or O;
R4 is, independently at each occurrence, selected from —(CH2CH2O)pCH3, —CH2CH2CH2(sugar), —CH2CHR4aC(O)OR4b, and —CH2CH2SO2CH2CH2SR4c;
R4a is, independently at each occurrence, —H or —CH3;
R4b is —(CH2CH2O)pCH3;
R4c is —(CH2CH2O)pCH3:
X3 is, independently at each occurrence, absent or O;
Y3 is, independently at each occurrence, absent or O;
R5 is, independently at each occurrence, —(CH2CH2O)nCH3 or -sugar;
n is an integer from 1-4;
m is an integer from 1-6; and
p is an integer from 1-9.

### Claim 7 (depends on 6)

The method of delivering an agent or a cell into an organism according to claim 6, wherein the mixture further comprises:
a second copolypeptide hydrogel forming composition,
wherein said second copolypeptide composition comprises at least one hydrophilic polypeptide segment or hydrophilic copolypeptide segment and at least one thermoresponsive polypeptide segment or thermoresponsive copolypeptide segment, and
wherein said second copolypeptide composition undergoes a temperature induced transition between a liquid and a transparent hydrogel in said aqueous medium.

### Claim 8 (depends on 1)

The composition of claim 1, wherein a plurality of residues in the hydrophilic polypeptide segment or hydrophilic copolypeptide segment are selected from a residue of Formula I, a residue of Formula II, a residue of Formula III, a residue of Formula IV, a residue of Formula V, and a residue of Formula VI.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the hydrophilic polypeptide segment or hydrophilic copolypeptide segment consists of residues selected from a residue of Formula I, a residue of Formula II, a residue of Formula III, a residue of Formula IV, a residue of Formula V, and a residue of Formula VI.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the hydrophobic polypeptide segment or hydrophobic copolypeptide segment comprises residues selected from leucine, alanine, phenylalanine, methionine, tyrosine, tryptophan, valine, isoleucine, serine, cysteine, glutamine, asparagine, a γ-alkyl glutamate ester, a β-alkyl aspartate ester, and a ε-modified lysine.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the copolypeptide composition is selected from:

### Claim 12 (depends on 1)

The composition of claim 1, wherein the copolypeptide contains less than 50 mol % ionic amino acid residues.

### Claim 13 (depends on 1)

The composition of claim 1, wherein, after exposure of a suspension of HeLa cells to the composition at a concentration of 2% for 24 hours, greater than 71% of the HeLa cells are viable.

### Claim 14 (depends on 1)

The composition of claim 1, wherein sugar is selected from galactose, glucose, and mannose.

### Claim 15 (depends on 6)

The method of claim 6, wherein sugar is selected from galactose, glucose, and mannose.