Silk fibroin glycerol membranes

1. A formic acid treated silk fibroin glycerol membrane matrix prepared in the presence of formic acid, wherein the membrane:

(a) is fabricated from a glycerol and silk protein complex solution prepared in the presence of formic acid, comprising silk fibroin in an amount ranging from about 0.1% to about 20% (w/v);

(b) the glycerol and silk protein complex solution when dried forms the membrane, the membrane comprises about 5% (w/w) to 60% (w/w) glycerol; and

(c) transmits sound waves between 20 Hz and 20 KHz to the middle ear in vivo;

(d) has a tensile strength between 10 MPa to 100 MPa; and

(e) has at least one of:

i. a secondary structure dominated by β-sheet motifs, as determined by the analysis of deconvoluted fourier transform infrared (FTIR) peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor;

ii. at least 45% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, or solvent/glycerol or solvent vapor;

iii. a % β-sheet motif content, that is greater than the sum of the % content of side chain, random coil, α-helix and β-turn motifs, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent or solvent/glycerol or solvent vapor; or

iv. 5% to 64% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent or solvent/glycerol or solvent vapor.

2. A method of fabricating a formic acid treated silk fibroin glycerol membrane matrix comprising the steps of:

(a) preparing silk protein or a silk protein complex after removal of sericin from a cocoon or fibre;

(b) dissolving glycerol and the silk protein or a silk protein complex using formic acid to form a silk protein or a silk protein complex solution, comprising silk fibroin in an amount ranging from about 0.1% to about 20% (w/v); and

(c) drying the silk protein or silk protein complex solution to fabricate the formic acid treated silk fibroin glycerol membrane matrix;

wherein the dried formic acid treated silk fibroin glycerol membrane matrix of step (c):

1. comprises about 5% (w/w) to 60% (w/w) glycerol;

2. is capable of transmitting sound waves between 20 Hz and 20 KHz to the middle ear in vivo;

3. has a tensile strength between 10 MPa to 100 MP; and

4. has at least one of:

(i). a secondary structure, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , dominated by β-sheet motifs without an annealing treatment by solvent solvent/glycerol or solvent vapor;

(ii). at least 45% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor;

(iii). a % β-sheet motif content, that is greater than the sum of the % content of side chain, random coil, α-helix and β-turn motifs, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor; or

(iv). 5% to 64% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor.

3. The method according to claim 2 , wherein after step (c), the formic acid treated silk fibroin glycerol membrane matrix is recrystallized by heat, a solvent, solvent/glycerol, or solvent vapor treatment to reduce solubility to water.

4. The method of claim 3 wherein the formic acid treated silk fibroin glycerol membrane matrix is exposed to ethanol or another C 1 to C 3 alcohol, or a combination thereof to induce protein conformational transition to β-sheet structure and to secure insolubility in phosphate-buffered saline (PBS) or water.

5. The formic acid treated silk fibroin glycerol membrane matrix prepared according to a method of claim 2 .

6. The formic acid treated silk fibroin glycerol membrane matrix according to claim 5 comprising at least one active agent.

7. The formic acid treated silk fibroin glycerol membrane matrix according to claim 6 wherein the active agent is selected from the group of cells, proteins, peptides, nucleic acid analogues, nucleotides oligonucleotides, peptide nucleic acids, aptamers, antibodies fragments or portions thereof, hormones, hormone antagonists, growth factors recombinant growth factors and fragments of recombinant growth factors and variants of recombinant growth factors, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, antivirals, toxins, prodrugs, chemotherapeutic agents, small molecules, drugs, and combinations thereof.

8. The formic acid treated silk fibroin glycerol membrane matrix according to claim 5 wherein the membrane supports growth of keratinocytes, fibroblasts, mucosal epithelium, endothelial cells, chondrocytes, induced pluripotent stem cells, adult stem cells, embryonic stem cells, and combinations thereof.

9. The formic acid treated silk fibroin glycerol membrane matrix prepared according to a method of claim 3 .

10. The formic acid treated silk fibroin glycerol membrane matrix according to claim 9 comprises at least one active agent.

11. The formic acid treated silk fibroin glycerol membrane matrix according to claim 10 wherein the active agent is selected from the group of cells, proteins, peptides, nucleic acid analogues, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments of recombinant growth factors and variants of recombinant growth factors, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, antivirals, toxins, prodrugs, chemotherapeutic agents, small molecules, drugs, and combinations thereof.

12. The formic acid treated silk fibroin glycerol membrane matrix according to claim 9 wherein the membrane supports growth of keratinocytes, fibroblasts, mucosal epithelium, endothelial cells, chondrocytes, induced pluripotent stem cells, adult stem cells, embryonic stem cells, and combinations thereof.

13. The formic acid treated silk fibroin glycerol membrane matrix prepared according to a method of claim 4 .

14. The formic acid treated silk fibroin glycerol membrane matrix according to claim 13 , comprising at least one active agent.

15. The formic acid treated silk fibroin glycerol membrane matrix according to claim 14 , wherein the active agent is selected from the group of cells, proteins, peptides, nucleic acid analogues, nucleotides or oligonucleotides, peptide nucleic acids, aptamers, antibodies or fragments or portions thereof, hormones, hormone antagonists, growth factors or recombinant growth factors and fragments of recombinant growth factors and variants of recombinant growth factors, cytokines, enzymes, antibiotics or antimicrobial compounds, viruses, antivirals, toxins, prodrugs, chemotherapeutic agents, small molecules, drugs, and combinations thereof.

16. The formic acid treated silk fibroin glycerol membrane matrix according to claim 13 , wherein the membrane supports growth of keratinocytes, fibroblasts, mucosal epithelium, endothelial cells, chondrocytes, induced pluripotent stem cells, adult stem cells, embryonic stem cells, and combinations thereof.

17. A method of fabricating a silk fibroin membrane comprising the steps of:

(a) dissolving silk fibroin and glycerol in formic acid to form a silk fibroin solution; and

(b) drying the silk fibroin solution to fabricate the silk fibroin membrane wherein the dried silk fibroin membrane of step (b):

(1) is capable of transmitting sound waves between 20 Hz and 20 KHz to the middle ear in vivo;

(2) has a tensile strength between 10 MPa to 100 MP; and

(3) has at least one of:

(i). a secondary structure dominated by β-sheet motifs, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor;

(ii). at least 45% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor;

(iii). a % β-sheet motif content, that is greater than the sum of the % content of side chain, random coil, α-helix and β-turn motifs, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor; or

(iv). 51% to 75% β-sheet motif content, as determined by the analysis of deconvoluted FTIR peaks in the amide I region of 1705 to 1595 cm −1 , without an annealing treatment by solvent, solvent/glycerol or solvent vapor.

18. The method of claim 17 , wherein the silk fibroin membrane comprises 5% to 60 (w/w) glycerol.

19. The method of claim 17 , further comprising recrystallizing the membrane by heat or a solvent or solvent/glycerol or solvent vapor treatment.

20. The method of claim 17 , further comprising annealing with methanol or ethanol vapor.