Photoresponsive protein hydrogels and methods and uses thereof
The present disclosure provides light-sensitive protein hydrogels, and methods of their use thereof. The hydrogels can be used for cell encapsulation, culturing, and selective release under appropriate light conditions.
1. A light-responsive hydrogel biopolymer matrix comprising:
(a) one or a plurality of light-responsive gelation initiator complexes comprising the protein photoreceptor CarH c and the multi-dentate ligand adenosylcobalamin, connected to one or a plurality of a first extracellular matrix protein fragments, and
(b) two or more cross-linkable proteins connected to one or a plurality of a second extracellular matrix protein fragments,
wherein the first extracellular matrix protein fragments and second extracellular matrix protein fragments are independently selected from elastin fragments which comprise a repeating polypeptide having the sequence of (VPGXG) n (SEQ ID NO: 23), wherein X represents valine or glutamate, the ratio of valine to glutamate ranges from 10:1 to 1:10, n is an integer selected from 2 to 25, the two or more cross-linkable proteins are SpyTag and SpyCatcher, and
the matrix is responsive to light comprising a wavelength from about 300 nm to about 600 nm.
2. The light-responsive hydrogel biopolymer matrix of claim 1 ,
wherein upon exposure to light with a wavelength from about 300 nm to about 600 nm, the matrix exhibits a phase transition from gel to solution.
3. The light-responsive hydrogel biopolymer matrix of claim 1 further comprising a mammalian cell.
4. The light-responsive hydrogel biopolymer matrix of claim 3 , wherein the mammalian cell is a fibroblast or a stem cell.
5. The light-responsive hydrogel biopolymer matrix of claim 4 , wherein the stem cell is a mesenchymal stem cell (hMSC).
6. The light-responsive hydrogel biopolymer matrix of claim 1 further comprising a non-covalently bound protein selected from a therapeutic protein, cytokine, or fluorescent protein.
7. The light-responsive hydrogel biopolymer matrix of claim 6 , wherein the fluorescent protein is mCherry.
8. The light-responsive hydrogel biopolymer matrix of claim 1 , further comprising water.
9. The light-responsive hydrogel biopolymer matrix of claim 8 , wherein the water content of the hydrogel is in the range from 70% to 99.5% (w/w).
10. The light-responsive hydrogel biopolymer matrix of claim 1 , wherein the protein photoreceptor content of the hydrogel is in the range from 0.001% to 0.5% (w/w).
11. A light-responsive hydrogel biopolymer matrix comprising:
a first telechelic biopolymer which comprises a first light-responsive gelation initiator complex covalently bound to the first extracellular matrix protein fragments which are covalently bound to a first two or more cross-linkable proteins, and
a second telechelic biopolymer which comprises a second light-responsive gelation initiator complex covalently bound to the second extracellular matrix protein fragments which are covalently bound to a second two or more cross-linkable proteins.
12. The light-responsive hydrogel biopolymer matrix of claim 11 ,
wherein said first telechelic biopolymer is a recombinant protein encoding for the first light-responsive gelation initiator complex covalently bound to the first extracellular matrix protein fragments which are covalently bound to a first two or more cross-linkable proteins, and said second telechelic biopolymer is a recombinant protein encoding for the second light-responsive gelation initiator complex covalently bound to the second extracellular matrix protein fragments which are covalently bound to a second two or more cross-linkable proteins.
13. The light-responsive hydrogel biopolymer matrix of claim 12 ,
wherein the first telechelic biopolymer and second telechelic biopolymer independently have the linkage structure selected from: SpyTag-CarH c -SpyTag, SpyCatcher-CarH c -SpyCatcher, SpyTag-CarH c -SpyCatcher, SpyTag-CarH c , SpyCatcher-CarH c , and CarH c -CL7.
14. The light-responsive hydrogel biopolymer matrix of claim 12 , wherein the first telechelic biopolymer and second telechelic biopolymer independently have the linkage structure selected from: SpyTag-elastin-CarH c -elastin-SpyTag, SpyCatcher-elastin-CarH c -elastin-SpyCatcher, SpyTag-elastin-CarH c -elastin-SpyCatcher, SpyTag-elastin-CarH c , SpyCatcher-elastin-CarH c , and CarH c -elastin-CL7,
wherein the elastin segments comprise a repeating polypeptide having the sequence of (VPGXG) n (SEQ ID NO: 23), wherein X represents valine or glutamate, the ratio of valine to glutamate ranges from 10:1 to 1:10, and n is an integer selected from 2 to 25.
15. A polypeptide corresponding to the linkage structure of claim 13 , comprising the sequence of SEQ ID NO:2.
16. An oligonucleotide encoding the linkage structure of claim 14 , comprising the sequence of SEQ ID NO:1.
17. A method for producing a light-responsive hydrogel biopolymer matrix, the method comprising dissolving one or a plurality of light-responsive gelation initiator complexes comprising the protein photoreceptor CarH c and the multi-dentate ligand adenosylcobalamin connected to one or a plurality of a first extracellular matrix protein fragments and two or more cross-linkable proteins connected to one or a plurality of a second extracellular matrix protein fragments in an aqueous solution to form a gelation mixture under light which does not comprise a wavelength of less than about 600 nm,
wherein the first extracellular matrix protein fragments and second extracellular matrix protein fragments are independently selected from elastin fragments which comprise a repeating polypeptide having the sequence of (VPGXG) n (SEQ ID NO: 23), wherein X represents valine or glutamate, the ratio of valine to glutamate ranges from 10:1 to 1:10, n is an integer selected from 2 to 25, and
the two or more cross-linkable proteins are SpyTag and SpyCatcher.
18. A method of culturing cells in a photoresponsive hydrogel matrix, the method comprising the steps of:
(a) dissolving one or more cells in an aqueous solution,
(b) dissolving one or a plurality of light-responsive gelation initiator complexes comprising the protein photoreceptor CarH c and the multi-dentate ligand adenosylcobalamin connected to one or a plurality of a first extracellular matrix protein fragments and two or more cross-linkable proteins connected to one or a plurality of a second extracellular matrix protein fragments in an aqueous solution comprising one or more cells to form a gelation mixture under light which does not comprise a wavelength of less than about 600 nm,
wherein the first extracellular matrix protein fragments and second extracellular matrix protein fragments are independently selected from elastin fragments which comprise a repeating polypeptide having the sequence of (VPGXG) n (SEQ ID NO: 24), wherein X represents valine or glutamate, the ratio of valine to glutamate ranges from 10:1 to 1:10, n is an integer selected from 5 to 25, and
the two or more cross-linkable proteins are SpyTag and SpyCatcher.
19. The method of claim 18 , further comprising the steps of:
(c) contacting the gelation mixture with cell growth media to form a gelation growth media, and
(d) incubating the gelation growth media to form incubated gelation growth media.
20. The method of claim 19 , further comprising the step of:
(e) irradiating the incubated gelation growth media with light comprising a wavelength between about 300 nm and about 600 nm to transform the hydrogel into a liquid resulting in release of the cells from the gelation mixture.