Silk Fibroin Hydrogels and Uses Thereof
The present specification provides for methods for purifying fibroins, purified fibroins, methods of conjugating biological and synthetic molecules to fibroins, fibroins conjugated to such molecules, methods of making fibroin hydrogels, fibroin hydrogels and fibroin hydrogel formulations useful for a variety of medical uses, including, without limitation uses as bulking agents, tissue space fillers, templates for tissue reconstruction or regeneration, cell culture scaffolds for tissue engineering and for disease models, surface coating to improve medical device function, or drug delivery devices.
1 . A hydrogel comprising a substantially sericin-depleted silk fibroin and an amphiphilic peptide.
2 . The hydrogel of claim 1 , wherein the hydrogel comprises about 1% (w/v) to about 10% (w/v) of silk fibroin.
3 . The hydrogel of claim 1 , wherein the amphiphilic peptide comprises a RGD motif.
4 . The hydrogel of claim 3 , wherein the amphiphilic peptide is 23 RGD.
5 . The hydrogel of claim 1 , wherein the amphiphilic peptide comprises of a tail region, followed by a spacer region and finally a RGD motif.
6 . The hydrogel of claim 1 , wherein the hydrogel comprises a molar ratio of 1:10 to 10:1 moles of the amphiphilic peptide per mole of the silk fibroin.
7 . The hydrogel of claim 6 , wherein the hydrogel comprises a molar ratio of 3:1 moles of the amphiphilic peptide per mole of the silk fibroin.
8 . The hydrogel of claim 1 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 80%.
9 . The hydrogel of claim 1 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 50%.
10 . The hydrogel of claim 1 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 20%.
11 . The hydrogel of claim 1 , wherein the hydrogel comprises a protein structure having an α-helical and random coil conformation of at most 20%.
12 . The hydrogel of claim 1 , wherein a synthetic molecule having the formula: (molecule X) n -(spacer peptide) 0-300 -(five-amino-acid peptide tail) is conjugated to the silk fibroin.
13 . A method of making a hydrogel, the method comprising the steps of:
a) obtaining a solution comprising a substantially sericin-free, depolymerized silk fibroin; and
b) adding an enhancer solution comprising an alcohol and an amphiphilic peptide to the silk fibroin solution; wherein addition of the enhancer solution causes polymerization of the depolymerized silk fibroin, thereby generating a hydrogel.
14 . The method of claim 13 , wherein the alcohol is ethanol.
15 . The method of claim 13 , wherein the method comprises a further step (c) of rinsing the hydrogel in a solvent to remove residual enhancer solution.
16 . The method of claim 13 wherein the concentration of the depolymerized silk fibroin is 1% (w/v) to 10% (w/v).
17 . The method of claim 13 , wherein addition of the enhancer solution results in a molar ratio of 1:10 to 10:1 moles of the amphiphilic peptide per mole of the depolymerized silk fibroin.
18 . A method of conjugating a molecule X to a silk fibroin, the method comprising the step of mixing a synthetic molecule having the formula: (molecule X) n -(spacer peptide) 0-300 -(five-amino-acid peptide tail) with a silk fibroin or silk solution.
19 . The method of claim 18 , wherein the five-amino-acid peptide “tail” comprises hydrophobic and/or apolar (non polar) amino acid residues.
18 . The method of claims 18 , wherein the five-amino-acid peptide “tail” comprises arginine or lysine.
20 . The method of claim 18 , wherein the five-amino-acid peptide “tail” is flanked by arginine or lysine on either side of the peptide.
21 . The method of claim 18 , wherein the five-amino-acid peptide “tail” is SEQ ID NO: 15.
22 . A hydrogel comprising a substantially sericin-depleted silk fibroin and an amphiphilic peptide made from a method comprising the steps of:
a) obtaining a solution comprising a substantially sericin-free, depolymerized silk fibroin; and
b) adding an enhancer solution comprising an alcohol and an amphiphilic peptide to the silk fibroin solution;
wherein addition of the enhancer solution causes polymerization of the depolymerized silk fibroin, thereby generating a hydrogel.
23 . The hydrogel of claim 22 , wherein the hydrogel comprises about 1% (w/v) to about 10% (w/v) of silk fibroin.
24 . The hydrogel of claim 22 , wherein the amphiphilic peptide comprises a RGD motif.
25 . The hydrogel of claim 24 , wherein the amphiphilic peptide is 23 RGD.
26 . The hydrogel of claim 22 , wherein the amphiphilic peptide comprises of a tail region, followed by a spacer region and finally a RGD motif.
27 . The hydrogel of claim 22 , wherein the hydrogel comprises a molar ratio of 1:10 to 10:1 moles of the amphiphilic peptide per mole of the silk fibroin.
28 . The hydrogel of claim 27 , wherein the hydrogel comprises a molar ratio of 3:1 moles of the amphiphilic peptide per mole of the silk fibroin.
29 . The hydrogel of claim 22 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 80%.
30 . The hydrogel of claim 22 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 50%.
31 . The hydrogel of claim 22 , wherein the hydrogel comprises a protein structure having a β-sheet conformation of at least 20%.
32 . The hydrogel of claim 22 , wherein the hydrogel comprises a protein structure having an α-helical and random coil conformation of at most 20%.
33 . The hydrogel of claim 22 , wherein a synthetic molecule having the formula: (molecule X) n -(spacer peptide) 0-300 -(five-amino-acid peptide tail) is conjugated to the silk fibroin.