Freeze-dried and sprayable silk fibroin and modified silk fibroin forms

Abstract

The present disclosure relates to lyophilized or sprayable peptide compositions, for example, lyophilized or sprayable silk fibroin-derived peptide compositions.

Description

[Technical Field]

[0001] The present disclosure relates to peptide compositions, for example, silk fibroin-derived peptide compositions. [Background technology]

[0002] Silk is a natural polymer produced by a variety of insects and spiders and comprises a filament core protein, silk fibroin, and a colloidal coating consisting of the non-filamentous protein, sericin. Summary of the Invention

[0003] Embodiments of the present disclosure provide a plurality of substantially solid silk fibroin particles comprising silk fibroin fragments, the particles characterized by at least one of bulk density, surface area, pore size, pore volume, aspect ratio, and / or Hausner ratio. In some embodiments, the substantially solid silk fibroin particles are annealed. In some embodiments, the substantially solid silk fibroin particles are milled. In some embodiments, the substantially solid silk fibroin particles have a viscosity of less than 0.03 g / ml, less than 0.04 g / ml, less than 0.05 g / ml, less than 0.06 g / ml, less than 0.07 g / ml, less than 0.08 g / ml, less than 0.09 g / ml, less than 0.10 g / ml, less than 0.11 g / ml, less than 0.12 g / ml, less than 0.13 g / ml, less than 0.14 g / ml, less than 0.15 g / ml, less than 0.16 g / ml, less than 0.17 g / ml, less than 0.18 g / ml or less than 0.25 g / ml, less than 0.26 g / ml, less than 0.27 g / ml, less than 0.28 g / ml, less than 0.29 g / ml, less than 0.30 g / ml, less than 0.31 g / ml, less than 0.32 g / ml, less than 0.33 g / ml, less than 0.34 g / ml, or less than 0.35 g / ml. In some embodiments, the substantially solid silk fibroin particles have a viscosity of about 0.03 g / ml, about 0.04 g / ml, or about 0.05 g / ml, about 0.03 g / ml, about 0.04 g / ml, about 0.05 g / ml, about 0.06 g / ml, about 0.07 g / ml, about 0.08 g / ml, about 0.09 g / ml, about 0.10 g / ml, about 0.11 g / ml, about 0.12 g / ml, about 0.13 g / ml, about 0.14 g / ml, about 0.15 g / ml, about 0.16 g / ml, and having an average bulk density of about 0.17 g / ml, about 0.18 g / ml, about 0.19 g / ml, about 0.20 g / ml, about 0.21 g / ml, about 0.22 g / ml, about 0.23 g / ml, about 0.24 g / ml, about 0.25 g / ml, about 0.26 g / ml, about 0.27 g / ml, about 0.28 g / ml, about 0.29 g / ml, about 0.30 g / ml, about 0.31 g / ml, about 0.32 g / ml, about 0.33 g / ml, about 0.34 g / ml, or about 0.35 g / ml.In some embodiments, the substantially solid silk fibroin particles have a Hausner ratio of 1.00 to 1.11, 1.12 to 1.18, 1.19 to 1.25, 1.26 to 1.34, or 1.35 to 1.45. In some embodiments, the substantially solid silk fibroin particles have an average diameter of about 3 mm to about 10 mm. In some embodiments, the average diameter of the substantially solid silk fibroin particles is about 1 mm, about 2 mm, about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, about 10 mm, about 11 mm, about 12 mm, about 13 mm, about 14 mm, about 15 mm, about 16 mm, about 17 mm, about 18 mm, about 19 mm, or about 20 mm. In some embodiments, the substantially solid silk fibroin particles have an aspect ratio of 1 to about 1.45. In some embodiments, the substantially solid silk fibroin particles have an aspect ratio of 1, about 1.10, about 1.15, about 1.20, about 1.25, about 1.30, about 1.35, about 1.40, or about 1.45. In some embodiments, the substantially solid silk fibroin particles have an aspect ratio of 1, about 1.10, about 1.11, about 1.12, about 1.13, about 1.14, about 1.15, about 1.16, about 1.17, about 1.18, about 1.19, about 1.20, about 1.21, about 1.22, about 1.23, about 1.24, about 1.25, about 1.26, about 1.27, about 1.28 , about 1.29, about 1.30, about 1.31, about 1.32, about 1.33, about 1.34, about 1.35, about 1.36, about 1.37, about 1.38, about 1.39, about 1.40, about 1.41, about 1.42, about 1.43, about 1.44, about 1.45, about 1.46, about 1.47, about 1.48, about 1.49, or about 1.50. In some embodiments, the substantially solid silk fibroin particles are substantially spherical. In some embodiments, the substantially solid silk fibroin particles are mesoporous. In some embodiments, the substantially solid silk fibroin particles are about 2.50 m. 2 / g ~ approx. 6.50m 2 In some embodiments, the substantially solid silk fibroin particles have a BET (Brunauer-Emmett-Teller) surface area of ​​about 2.50 m / g. 2 / g~approx.3.00m 2 / g, approx. 3.00m2 / g ~ approx. 3.50m 2 / g, approx. 3.50m 2 / g ~ approx. 4.00m 2 / g, approx. 4.00m 2 / g ~ approx. 4.50m 2 / g, approx. 4.50m 2 / g ~ approx. 5.00m 2 / g, approx. 5.00m 2 / g~approx.5.50m 2 / g, approx. 5.50m 2 / g~about 6.00m 2 / g, or approximately 6.00 m 2 / g ~ approx. 6.50m 2 / g BET (Brunauer-Emmett-Teller) surface area. In some embodiments, the substantially solid silk fibroin particles have an average pore size of about 25 Å to about 500 Å. In some embodiments, the substantially solid silk fibroin particles have an average pore size of about 25 Å to about 30 Å, about 30 Å to about 35 Å, about 35 Å to about 40 Å, about 40 Å to about 45 Å, about 45 Å to about 50 Å, about 50 Å to about 55 Å, about 55 Å to about 60 Å, about 60 Å to about 65 Å, about 65 Å to about 70 Å, about 70 Å to about 75 Å, about 75 Å to about 80 Å, about 80 Å to about 85 Å, or about 85 Å to about The substantially solid silk fibroin particles have an average pore diameter of 90 Å, about 90 Å to about 95 Å, about 95 Å to about 100 Å, about 100 Å to about 105 Å, about 105 Å to about 110 Å, about 110 Å to about 115 Å, about 115 Å to about 120 Å, about 120 Å to about 125 Å, about 125 Å to about 130 Å, about 130 Å to about 135 Å, about 135 Å to about 140 Å, about 140 Å to about 145 Å, or about 145 Å to about 150 Å. In some embodiments, the substantially solid silk fibroin particles comprise a plurality of radially oriented microchannels. In some embodiments, the substantially solid silk fibroin particles comprise an emulsifier, a surfactant, a buffer, an amino acid, or a sugar. In some embodiments, the substantially solid silk fibroin particles comprise a polysaccharide, polysorbate, glycoside, PBS, arginine, trehalose, glucose, or sucrose. In some embodiments, the substantially solid silk fibroin particles comprise a surfactant selected from sucrose esters, cetearyl glucoside, caprylyl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof. In some embodiments, the substantially solid silk fibroin particles comprise an additional protein or peptide, a C12-C24 fatty alcohol, a glycolipid, or a lipid. In some embodiments, the substantially solid silk fibroin particles comprise a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination. In some embodiments, the substantially solid silk fibroin particles comprise a sugar fatty acid ester, a sugar fatty acid monoester, a sugar fatty diester, a sugar fatty triester, or a sugar fatty and polyester.In some embodiments, the substantially solid silk fibroin particles comprise sucrose fatty acid esters, sorbitan or sorbitol fatty acid esters, alkyl glucosides, alkyl polyglucosides, or combinations thereof. In some embodiments, the substantially solid silk fibroin particles comprise KCl, NaCl, MgCl, CaCl, PBS, Tris, polysorbate 20, polysorbate 80, capryl glucoside, sucrose, histidine, glycine, or arginine. In some embodiments, the stabilizer is selected from polysaccharides, such as, but not limited to, maltodextrin, dextran, surfactants, such as, but not limited to, polysorbate (20, 60, 80), capryl glucoside, buffers, such as, but not limited to, PBS, Tris acetate, sodium phosphate, amino acids, such as, but not limited to, arginine, glycine, cysteine, histidine, lysine, serine, and / or sugars, such as, but not limited to, trehalose, glucose, sucrose, maltose, fucose.

[0004] Embodiments of the present disclosure provide silk fibroin nanoclay composites or films comprising silk fibroin fragments and clay, wherein the % (w / w) of clay in the composite is from about 1% to about 99%. In another embodiment, the clay is bentonite. In some embodiments, the concentration of clay in the composite or film is from about 20% (w / w) to about 33% (w / w), from about 33% (w / w) to about 50% (w / w), or from about 50% (w / w) to about 67% (w / w). In some embodiments, the water vapor transmission rate (WVP) of the composite is inversely proportional to the concentration of clay in the composite. In some embodiments, the water vapor transmission rate (WVP, g / m) of the composite is 2 × Pa × 24 hours) is about 0.20 to about 0.30, about 0.30 to about 0.35, about 0.35 to about 0.40, about 0.40 to about 0.45, about 0.45 to about 0.50, about 0.50 to about 0.55, about 0.55 to about 0.60, about 0.60 to about 0.65, about 0.65 to about 0.70, about 0.70 to about 0.75, about 0.75 to about 0.80, or about 0.80 to about 0.85.

[0005] Embodiments of the present disclosure provide a stabilized silk fibroin solution comprising silk fibroin fragments and a stabilizer, wherein the solution has a lower z-average value than a substantially similar silk fibroin solution comprising the silk fibroin fragments but excluding the stabilizer, and / or the solution has a lower z-average plateau value than a substantially similar silk fibroin solution comprising the silk fibroin fragments but excluding the stabilizer. In some embodiments, the z-average is measured after a period of time following co-combination of the silk fibroin fragments and the stabilizer, the period of time being in the range of 1 hour to 250 hours, 1 hour to 350 hours, 1 hour to 450 hours, 1 hour to 550 hours, 1 hour to 650 hours, or 1 hour to 1000 hours. In some embodiments, the z-average is measured after a period of time after co-combining the silk fibroin fragments with the stabilizer, the period of time being in the range of 1 minute to 10 minutes, 1 minute to 20 minutes, 1 minute to 30 minutes, 1 minute to 40 minutes, 1 minute to 50 minutes, 1 minute to 60 minutes, 1 minute to 70 minutes, or 1 minute to 80 minutes. In some embodiments, the stabilizer is an emulsifier, surfactant, buffer, amino acid, or sugar. In some embodiments, the stabilizer is a polysaccharide, polysorbate, glycoside, PBS, arginine, trehalose, glucose, or sucrose. In some embodiments, the stabilizer is a surfactant selected from sucrose esters, cetearyl glucoside, caprylyl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof. In some embodiments, the stabilizer is an additional protein or peptide, a C12-C24 fatty alcohol, a glycolipid, or a lipid. In some embodiments, the stabilizer is a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination. In some embodiments, the stabilizer is a sugar fatty acid ester, a sugar fatty acid monoester, a sugar fatty diester, a sugar fatty triester, or a sugar fatty and polyester. In some embodiments, the stabilizer is a sucrose fatty acid ester, a sorbitan or sorbitol fatty acid ester, an alkyl glucoside, an alkyl polyglucoside, or a combination thereof.In some embodiments, the stabilizer is KCl, NaCl, MgCl, CaCl, PBS, Tris, polysorbate 20, polysorbate 80, caprylglucoside, sucrose, histidine, glycine, or arginine. In some embodiments, the stabilizer is selected from polysaccharides such as, but not limited to, maltodextrin, dextran, surfactants such as, but not limited to, polysorbate (20, 60, 80), caprylglucoside, buffers such as, but not limited to, PBS, Tris acetate, sodium phosphate, amino acids such as, but not limited to, arginine, glycine, cysteine, histidine, lysine, serine, and / or sugars such as, but not limited to, trehalose, glucose, sucrose, maltose, and fucose.

[0006] In some embodiments, the solution is sprayable. Embodiments of the present disclosure provide a liquid suspension in air comprising a plurality of droplets comprising the stabilized silk fibroin solution described above, wherein the droplets are sufficiently stable after being sprayed for the period of time required to reach a surface. In some embodiments, the droplets or liquid droplets are sufficiently stable after being formed for the period of time required to reach a surface.

[0007] In some embodiments, the silk fibroin fragments in any of the embodiments of the plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stabilized silk fibroin solutions, liquid-in-air suspensions, or plurality of droplets or droplets described herein are between about 1 kDa and about 5 kDa, between about 5 kDa and about 10 kDa, between about 6 kDa and about 17 kDa, between about 10 kDa and about 15 kDa, between about 14 kDa and about 30 kDa, between about 15 kDa and about 20 kDa, between about 17 kDa and about 39 kDa, between about 20 kDa and about 40 kDa, between about 40 kDa and about 50 kDa, between about 50 kDa and about 60 kDa, between about 50 kDa and about 70 kDa, between about 50 kDa and about 80 kDa, between about 50 kDa and about 90 kDa, between about 60 kDa and about 100 kDa, between about 60 kDa and about 17 kDa, between about 10 kDa and about 15 kDa, between about 14 kDa and about 30 kDa, between about 15 kDa and about 20 kDa, between about 17 kDa and about 39 kDa, between about 20 kDa and about 40 kDa, between about 40 kDa and about 50 kDa, between about 50 kDa and about 60 kDa, between about 50 kDa and about 70 kDa, between about 50 kDa and about 80 kDa, between about 50 kDa and about 90 kDa, between about 50 kDa and about 90 kDa, between about 50 The polysaccharide has a weight average molecular weight selected from about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, about 80 kDa to about 144 kDa, about 144 kDa to about 250 kDa, or about 250 kDa to about 350 kDa, and a polydispersity of 1 to about 5. In some embodiments, the polydispersity of any of the above-described embodiments is 1 to about 1.5, about 1.5 to about 2.0, about 2.0 to about 2.5, about 2.5 to about 3.0, about 3.0 to about 3.5, about 3.5 to about 4.0, about 4.0 to about 4.5, or about 4.5 to about 5.0. In any of the above-described embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin fragments. In any of the above-described embodiments, the silk fibroin fragments do not spontaneously or gradually gel or show a visible change in color or turbidity when in aqueous solution for at least 10 days prior to being incorporated into the substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, or stabilized silk fibroin solution.

[0008] In any embodiment of the plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets described herein, the silk fibroin fragment may comprise a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W, wherein at least one of the amino acids is modified, substituted, or replaced. In any of the above-described embodiments, the fibroin is a fibroin heavy chain, a fibroin light chain, or fibrohexamerin. In any of the above-described embodiments, the silk fibroin fragment comprises from about 2 to about 100 amino acids. In any of the above-described embodiments, the silk fibroin fragment comprises from 1 to 5 modifications, substitutions, and / or replacements. In any of the above-described embodiments, the modification, substitution, and / or substitution is selected from an asparagine to aspartic acid modification, substitution, and / or substitution, a glutamine to glutamic acid modification, substitution, and / or substitution, and a methionine to methionine oxide modification, substitution, and / or substitution. In any of the above-described embodiments, the fibroin is a fibroin heavy chain, and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-5263 of the fibroin heavy chain. In any of the above-described embodiments, the modification, substitution, and / or substitution is at Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, and / or N5262. In any of the above-described embodiments, the fibroin is a fibroin light chain and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-262 of the fibroin light chain. In any of the above-described embodiments, the modification, substitution, and / or substitution is at N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, and / or Q255. In any of the above-described embodiments, the fibroin is fibrohexamerin (p25) and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-220 of fibrohexamerin (p25).In any of the above-described embodiments, the modification, substitution, and / or substitution is at Q62, N93, M120, N149, N172, N174, and / or N202. In any of the above-described embodiments, each modification, substitution, and / or substitution independently ranges from about 1% to about 99% of the silk fibroin fragment portion of the substantially solid silk fibroin particle, silk fibroin nanoclay composite or film, or stabilized silk fibroin solution composition. In any of the above-described embodiments, as used herein, % modification, substitution, and / or substitution is defined as (the number of peptides or protein fragments containing the modification, substitution, and / or substitution at a particular position divided by the total number of peptides or protein fragments containing the particular position, regardless of whether they contain the modification, substitution, and / or substitution) × 100.

[0009] In any of the plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composites or films, stabilized silk fibroin solutions, liquid-in-air suspensions, or plurality of droplets or droplets described herein, the silk fibroin fragments are contained in one or more fractions, each fraction independently comprising a plurality of fibroin heavy chain fragments, a plurality of fibroin light chain fragments, and / or a plurality of fibrohexamerin (p25) fragments. In any embodiment, the silk fibroin fragment has a weight average molecular weight (M) selected from about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 250 kDa. w In any of the above-described embodiments, the silk fibroin fragments have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. w), and a polydispersity of 1 to about 1.1, or 1 to about 1.2. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ), and a polydispersity of 1 to about 1.1, or 1 to about 1.2. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. w In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w ), and a polydispersity of 1 to about 1.1, or 1 to about 1.2. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. w ), and a polydispersity of 1 to about 1.1. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 20 kDa to about 40 kDa, or about 40 kDa to about 60 kDa. w), and a polydispersity of 1 to about 1.1, or 1 to about 1.2. In any of the above-mentioned embodiments, one or more fractions are selected from AS77, AS78, AS79, AS80, and AS81. In any of the above-mentioned embodiments, one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. In any of the above-mentioned embodiments, one or more fractions are selected from AS90, AS91, AS92, AS93, and AS94. In any of the above-mentioned embodiments, one or more fractions are selected from AS95, AS96, AS97, AS98, AS99, and ASIOO. In any of the above-described embodiments, the silk fibroin fragments have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. w In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w ), and a polydispersity of 1 to about 1.2, or 1 to about 1.3. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. w), and a polydispersity of 1 to about 1.1, or 1 to about 1.2. In any of the above-described embodiments, the silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. w), and a polydispersity of 1 to about 1.2, or 1 to about 1.3. In any of the above-mentioned embodiments, the one or more fractions are selected from AS101, AS102, AS103, AS104, and AS105. In any of the above-mentioned embodiments, the one or more fractions are selected from AS106, AS107, AS108, AS109, AS110, and AS111. In any of the above-mentioned embodiments, the silk fibroin fragments comprise one or more amino acid modifications, substitutions, or replacements of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W. In any of the above-mentioned embodiments, the silk fibroin fragments comprise from about 2 to about 100 amino acids. In any of the above-mentioned embodiments, the silk fibroin fragments comprise from 1 to 5 modifications, substitutions, and / or replacements. In any of the above-described embodiments, the fibroin is a fibroin heavy chain and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-5263 of the fibroin heavy chain. In any of the above-described embodiments, the fibroin is a fibroin light chain and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-262 of the fibroin light chain. In any of the above-described embodiments, the fibroin is a fibrohexamerin (p25) chain and the modification, substitution, and / or substitution is at a position corresponding to any one of positions 1-220 of the fibrohexamerin (p25) chain. In any of the above-described embodiments, the modification, substitution, and / or substitution is selected from an asparagine to aspartic acid modification, substitution, and / or substitution, a glutamine to glutamic acid modification, substitution, and / or substitution, and a methionine to methionine oxide modification, substitution, and / or substitution. In any of the above-described embodiments, the modification, substitution, and / or substitution is at a fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, and / or N5262. In any of the above-described embodiments, the modification, substitution, and / or substitution is at a fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, and / or Q255.In any of the above-described embodiments, the modification, substitution, and / or substitution is at a fibrohexamerin (p25) position selected from Q62, N93, M120, N149, N172, N174, and / or N202. In any of the above-described embodiments, each modification, substitution, and / or substitution independently ranges from about 1% to about 99% of the composition. In any of the above-described embodiments, as used herein, % modification, substitution, and / or substitution is defined as (the number of peptides or protein fragments containing the modification, substitution, and / or substitution at a particular position divided by the total number of peptides or protein fragments containing the particular position, regardless of whether they contain the modification, substitution, and / or substitution) x 100.

[0010] In any of the above embodiments, the molecular weight is determined by MALS.

[0011]

[0010] Embodiments of the present disclosure provide a pouch or laundry pod comprising a plurality of substantially solid silk fibroin particles of any of the above-described embodiments. In some embodiments, the plurality of substantially solid silk fibroin particles are compressed into a multi-particle puck. In some embodiments, the laundry pod further comprises a dissolvable enclosure comprising polyvinyl alcohol (PVA) or a derivative of PVA. In some embodiments, the laundry pod further comprises an enclosure comprising one or more of nylon, polyglycolide (PGA), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), poly(butylene succinate) (PBS), polybutylene succinate adipate, poly(p-dioxanone) (PPDO), poly(butylene adipate-co-terephthalate) (PBAT), copolyester of terephthalic acid and lactic acid, copolyester of terephthalic acid and glycolic acid, copolyester of terephthalic acid and succinic acid, poly(hydroxybutyrate), poly(hydroxyvalerate), polyhydroxyhexanoate, poly(hydroxyalkanoate) (PHA), polymethylene adipate / terephthalate.

[0012] Embodiments of the present disclosure provide methods for making a plurality of substantially solid silk fibroin particles of any of the above-described embodiments, the method comprising: dripping a solution containing a plurality of silk fibroin fragments into liquid nitrogen. In some embodiments, the method further comprises a freeze-drying step. In some embodiments, the concentration of the silk fibroin fragments in the solution is between about 3% (w / w) and about 50% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is between about 6% (w / w) and about 25% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is between about 6% (w / w) and about 20% (w / w). In some embodiments, the concentration of the silk fibroin fragments in the solution is about 3% (w / w), about 4% (w / w), about 5% (w / w), about 6% (w / w), about 7% (w / w), about 8% (w / w), about 9% (w / w), about 10% (w / w), about 11% (w / w), about 12% (w / w), about 13% (w / w), about 14% (w / w), about 15% (w / w), about 16% (w / w), about 17% (w / w), about 18% (w / w), about 19% (w / w), about 20% (w / w), about 21% (w / w), about 22% (w / w), about 23% (w / w), about 24% (w / w), or about 25% (w / w). In some embodiments, the silk fibroin fragments comprise one or more of the molecular weight, polydispersity, and / or modifications, substitutions, and / or substitutions at specific amino acid positions as defined in any one of the above embodiments. In some embodiments, the solution is stabilized as defined in any one of the above embodiments.

[0013] In any of the above embodiments, the particles have a reconstitution yield of greater than 90%.In any of the above embodiments, the particles have a reconstitution yield in DI water of greater than 90%.

[0014] In some embodiments, at least a 90% reconstitution rate is maintained after a stability test comprising simulated aging of a plurality of substantially solid silk fibroin particles of any one of claims 1-92, wherein the aging comprises storage at a temperature of about 40° C. to about 60° C. for a period ranging from about 400 days to about 650 days. In some embodiments, the simulated aging is in the range of about 4 years to about 15 years. [Brief explanation of the drawings]

[0015] The presently disclosed embodiments will be further described with reference to the accompanying drawings, in which the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0016] [Figure 1] Ion exchange fractionation scheme for isolation of populations constituting low- and medium-skid silk / modified polypeptide compositions. Low- and medium-skid silk / modified polypeptide compositions contain silk / modified polypeptides that are negatively or positively charged, or neutral. Q anion exchange chromatography (A) was used to isolate these populations. [Figure 2] Chromatogram of low-skid silk / modified polypeptide composition loaded onto a Q-Sepharose HP column (Cytiva). The flow-through contains silk / modified polypeptides that were not trapped within the column and lack negatively charged amino acids. After loading the column with low-skid or medium-skid silk / modified polypeptide composition and collecting the flow-through, the column is washed until the UV-280 absorbance is below 200 AU. The trapped negatively charged silk / modified polypeptides are eluted with a high salt concentration (1 M NaCl) and constitute AS11 and AS22. Chromatography is performed in a Tris-containing buffer, but the flow-through and Q eluate are finally dialyzed into water. [Figure 3] Analytical size exclusion chromatography of low-skid silk, medium-skid silk / modified silk compositions and their constituent AS compositions. Average molecular weight and polydispersity measurements in kDa are shown. [Figure 4A-4B]Analytical size exclusion chromatography of low-skid and medium-skid silk / modified peptide compositions and their constituents (see Table 1 for details). Figure 4A: Molecular weights of various activated silk novel compositions described in this study. Figure 4B: Polydispersity (PDI) of various activated silk novel compositions described in this study. AS24 reconstitutes the average molecular weight and polydispersity of low-skid silk / modified peptide compositions and is composed of 50% AS12 and 50% AS22 (see Table 1 for details). AS6 reconstitutes the average molecular weight and polydispersity of medium-skid silk / modified peptide compositions and is composed of 50% AS1 and 50% AS11 (see Table 1 for details). [Figure 5] Isoelectric focusing of low-skid silk / modified polypeptide compositions. Lanes 2 and 7: Low-skid silk loaded in different amounts. Lanes 3, 5, 8, and 10: AS12 silk loaded in different amounts in different preparations. Lanes 4, 6, 9, and 11: AS22 silk loaded in different amounts in different preparations. [Figures 6A-6B] Self-assembly reactions of low-skid and medium-skid silk / modified peptide compositions and their constituents (see Table 1 for details). Both graphs illustrate the kinetic parameters of gel formation during silk self-assembly. Graph A shows the calculation of three self-assembly kinetic parameters: t, A, and SARF. See text for details. [Figure 7A-7C] Self-assembly kinetics of low-skid and medium-skid silk / modified peptide compositions and their constituents (see Table 1 for details). Figure 7A: "Self-assembly rate coefficient" indicates how fast the self-assembly reaction proceeds once the self-assembly reaction is initiated and the self-assembly nuclei are assembled. Figure 7B: "Maximum gel yield" indicates the density of the silk gel after self-assembly is complete. Figure 7C: "Time" is the time required for the self-assembly reaction to produce half of the maximum gel volume. [Figure 8]Low-skid and medium-skid silk / modified peptide compositions and their components (see Table 1 for details). The "self-assembly coefficient" reflects the average tendency of silk to self-assemble to form a gel. The above-identified figures illustrate embodiments disclosed herein; however, other embodiments are contemplated, as noted in the discussion. This disclosure presents exemplary embodiments that are representative and not limiting. Numerous other modifications and embodiments can be derived by those skilled in the art that fall within the scope and spirit of the principles of the presently disclosed embodiments. [Figure 9] FIG. 1 is a graph of weight average molecular weight (i.e., average molecular weight average or average MW) using size exclusion chromatography with refractive index detector (SEC-RI) plotted as a function of time for solubilized fibroin in 9.3 M LiBr at 100° C. to 103° C. (i.e., a temperature gradient of 3 degrees Celsius between 100° C. and 103° C.). [Figure 10] FIG. 1 is a graph of weight average molecular weight (i.e., average molecular weight average or average MW) using size exclusion chromatography with refractive index detector (SEC-RI) plotted as a function of time for solubilized fibroin in 9.3 M LiBr at 122°C to 125°C (i.e., a temperature gradient of 3 degrees Celsius between 122°C and 125°C). [Figure 11] 1 is a graph illustrating the percentage of amino acid modifications in silk. [Figures 12A-12C] 12A and 12B are graphs illustrating the proportion of amino acid modifications in low- and medium-skid silks. Figure 12A illustrates heavy chain modifications, Figure 12B illustrates light chain modifications, and Figure 12C illustrates fibrohexamerin modifications. N is asparagine, which becomes aspartic acid, and Q is glutamine, which is deamidated. M corresponds to methionine, which is oxidized. The number after each amino acid indicates its position along the amino acid chain from the corresponding protein. [Figures 13A-13B]13A and 13B are graphs illustrating the percentage of amino acid modifications produced and lyophilized in low- and medium-skid silk. Figure 13A illustrates heavy chain modifications, and Figure 13B illustrates light chain modifications. N is asparagine, which becomes aspartic acid, and Q is glutamine, which is deamidated. M corresponds to methionine, which is oxidized. The number after each amino acid indicates its position along the amino acid chain from the corresponding protein. [Figures 14A-14B] 14A and 14B are graphs illustrating the percentage of amino acid modifications in low-skid silk produced at Walpole and Medford using the skid process with different process parameters and varying levels. Figure 14A illustrates heavy chain modifications, and Figure 14B illustrates light chain modifications. N is asparagine, which becomes aspartic acid, and Q is glutamine, which is deamidated. M corresponds to methionine, which is oxidized. The number after each amino acid indicates its position along the amino acid chain from the corresponding protein. [Figures 15A-15D] 1 is a graph illustrating the percentage of amino acid modifications produced in low and medium silk by the skid and benchtop processes. N is asparagine, which becomes aspartic acid, and Q is glutamine, which is deamidated. M corresponds to methionine, which is oxidized. The number after each amino acid indicates its position along the amino acid chain from the corresponding protein. [Figure 16] 1 is a description of the method used to calculate the percentage ratio of modified amino acids at specific locations along the sequence of each peptide. [Figure 17]Figure 1 illustrates anion exchange and size exclusion chromatography schemes for the isolation of low-skid silk / modified peptide compositions. Low-skid silk / modified polypeptide compositions are composed of a diverse population of peptides with a wide range of sizes and charges. Using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step, distinct populations of low-skid silk / modified polypeptide compositions were separated. The Q-Sepharose eluate was loaded onto HiLoad Superdex 200 size exclusion chromatography, which resulted in size-fractionated negatively charged silk compositions / modified peptides. [Figures 18A-18B] Chromatograms of anion exchange chromatography of a low-skid silk / modified polypeptide composition and subsequent size-exclusion chromatography of the eluate (Q eluate). Figure 18A: Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). The low-skid silk / modified peptide composition was separated by anion exchange chromatography into an uncharged peptide population (flow-through—light blue background) and the eluted negatively charged silk composition (eluate—light pink background). The light yellow background represents a column wash with 50 mM Tris pH=8.0 prior to elution of the charged peptide population. Figure 18B: The negatively charged eluate was loaded onto a Superdex 200 column and run through the column using 50 mM Tris, 200 mM CaCl2, pH=8.0. When UV-280 absorbance began to increase for the fractions, the low-skid silk / modified peptide composition was collected and separated by size. The relative elution volumes of silk compositions AS77 and AS81 are depicted on the chromatogram. [Figures 19A-19B] Analytical size exclusion chromatography of low-skid silk / modified silk compositions and their constituent AS compositions is illustrated. Figure 19A: Average molecular weights in kDa of low-skid silk (LS) and AS77-AS81. Figure 19B: Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 7. [Figure 20]SDS polyacrylamide gel electrophoresis of low-skid silk / modified polypeptide compositions. Lanes are represented by fraction number in order of elution from a Superdex 200 column and their respective silk compositions: fraction 6 is AS77, fraction 7 is AS78, fraction 8 is AS79, fraction 9 is AS80, and fraction 10 is AS81. [Figures 21A-21B] Figure 21A is a graph illustrating the self-assembly reaction of a low-skid silk / modified peptide composition. A medium-skid silk reaction was used as a positive control. Figure 21A illustrates the kinetic parameters of gel formation during silk self-assembly. Self-assembly parameters for medium-skid silk: Amax is 0.6780 (Abs), SARF is 8.676, T0.5 is 3.668 hours, and FSAF is 3.08 (Abs / min). Figure 21B is a snapshot of the same self-assembly assay at a later time point, 12 days after the assay was set up. None of the fractions tested self-assembled over time. [Figures 22A-22B] Figure 22 illustrates the characterization of low-skid silk compositions by dynamic light scattering. Low-skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 22A illustrates the intensity diameter particle size distribution measured for silk compositions AS77, AS78, AS79, AS80, and AS81. Figure 22B illustrates the correlogram functions of silk compositions AS77, AS78, AS79, AS80, and AS81. [Figure 23] 1 illustrates a size-exclusion chromatography scheme for the isolation of a low-skid silk / modified peptide composition. The low-skid silk / modified polypeptide composition is composed of various peptide populations across a wide range of sizes. Using HiLoad Superdex 200 size-exclusion chromatography, distinct populations of the low-skid silk / modified polypeptide composition were separated. [Figure 24]This is a chromatogram of a low-skid silk / modified polypeptide composition loaded onto a Superdex 200 gel filtration column. The low-skid silk / modified peptide composition was loaded onto a Superdex 200 column and run through the column using 50 mM Tris, 200 mM CaCl2, pH = 8.0. The low-skid silk / modified peptide composition was collected and separated by size when the UV-280 absorbance began to increase for the fractions. The relative elution volumes of silk compositions AS82, AS86, and AS87 are shown on the chromatogram. [Figures 25A-25B] Analytical size exclusion chromatography of low-skid silk / modified silk compositions and their constituent AS compositions is illustrated. Figure 25A: Average molecular weights in kDa of low-skid silk (LS) and AS82-AS89 are illustrated. Figure 25B: Polydispersity index (PDI) measurements are shown. Numerical data are presented in Table 9. [Figure 26] SDS polyacrylamide gel electrophoresis of low-skid silk / modified polypeptide compositions. Lanes are represented by fraction number in order of elution from a Superdex 200 column and their respective silk compositions: fraction 6 is AS82, fraction 7 is AS83, fraction 8 is AS84, fraction 9 is AS85, and fraction 10 is AS86. [Figures 27A-27B] Figure 27A illustrates the self-assembly reaction of a low-skid silk / modified peptide composition. A medium-skid silk reaction was used as a positive control. Figure 27A illustrates the kinetic parameters of gel formation during silk self-assembly. Self-assembly parameters for medium-skid silk: Amax 0.6978 (Abs), SARF 8.591, T0.5 3.361 hours, FSAF 3.46 (Abs / min). Figure 27B illustrates a snapshot of the same self-assembly assay at a later time point, 18 days after the assay was set up. AS87, AS88, and AS89 demonstrate gel formation, which was already observed after 5 days of assay (LS, low-skid silk; MS, medium-skid silk). [Figures 28A-28C]Figures 28A and 28B show graphs characterizing low-skid silk compositions by dynamic light scattering. Low-skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed using a Zetasizer Pro to estimate the particle size of each silk composition. Figure 28A shows the intensity particle size distribution measured for silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89. Figure 28B shows the intensity particle size distribution measured for silk composition AS82, the low-skid silk / modified peptide composition (LS), and the medium-skid silk / modified peptide composition (MS). Figure 28C shows the correlogram functions for silk compositions AS82, AS83, AS84, AS85, AS86, AS87, AS88, AS89, the low-skid silk / modified peptide composition (LS), and the medium-skid silk / modified peptide composition (MS). [Figure 29] Figure 1 shows anion exchange chromatography (Q), hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC) schemes for the isolation of low-skid silk / modified peptide compositions. Low-skid silk / modified polypeptide compositions consist of a diverse population of peptides with a wide range of sizes and charges. Separate populations of low-skid silk / modified polypeptide compositions were isolated using Q-Sepharose anion exchange chromatography as the first step, Butyl ImpRes hydrophobic interaction resin as the second step, and HiLoad Superdex 200 size exclusion chromatography as the third purification step. The Q-Sepharose eluate was loaded onto a Butyl ImpRes (HIC) column, and the HIC eluate was loaded onto HiLoad Superdex 200 size exclusion chromatography, which resulted in the fractionation of negatively charged silk compositions / modified peptides, sized according to their hydrophobicity. The Q-Sepharose eluate contained negatively charged peptides of all sizes. These peptides were resolved on a Butyl ImpRes column, resulting in the elution of high molecular weight, negatively charged, slightly hydrophobic silk components / modified peptides. Smaller, negatively charged peptides washed through and did not bind to the Butyl ImpRes column. The Q-HIC (elution) was loaded onto a Superdex 200 and separated by size. [Figures 30A-30E]Figure 30A shows chromatograms of anion exchange chromatography, hydrophobic interaction chromatography, and size exclusion chromatography of a low-skid silk / modified polypeptide composition. Figure 30A illustrates that anion exchange chromatography was performed using a Q-Sepharose column. The low-skid silk / modified peptide composition was separated by anion exchange chromatography into an uncharged peptide population (flow-through—light blue background) and the eluted negatively charged silk composition (eluate—light pink background). The light yellow background represents a column wash with 50 mM Tris pH=8.0 prior to elution of the charged peptide population. Figure 30B illustrates that the negatively charged eluate (Q eluate) was loaded onto a Butyl ImpRes column in the presence of 300 mM ammonium sulfate [(NH4)2SO4], exposing the hydrophobic domains of the silk peptides, which allow binding to the column. The highly charged peptide population did not bind to the column (flow-through) and is highlighted in light blue. The column was washed until the OD280 decreased to approximately 100 units (light yellow). The bound silk peptides (Q-HIC (elution)) were then eluted using 50 mM Tris, pH 8.0, without ammonium sulfate (light pink). Figure 30C illustrates that Q-HIC (elution) was further fractionated by size exclusion chromatography (SEC) using a Superdex 200 gel filtration column. The Q-HIC (elution) fraction was run through the column using 50 mM Tris, 200 mM CaCl2, pH 8.0. When the UV-280 absorbance of the fractions began to increase, the low-skid silk / modified peptide compositions were collected and separated by size. The relative elution volumes of silk compositions AS90 and AS94 are shown on the chromatogram. Figure 30D illustrates that the Q-HIC (flow-through) fraction was further fractionated by SEC using a Superdex 200 column, following the same procedure as (VC). The relative elution volumes of silk compositions AS95 and AS100 are depicted on the chromatogram. Figure 30E illustrates the overlay of chromatograms (VC) and (VD). The Q-HIC (elution) fraction has a higher molecular weight range and a lower molecular weight range compared to the Q-HIC (flow-through) fraction, which elutes later in the SEC. [Figure 31A-31B] 31A-31B are graphs showing analytical size exclusion chromatography of low-skid silk / modified silk compositions and their constituent AS compositions. Figure 31A: Average molecular weights in kDa of low-skid silk (LS) and AS90-AS100. Figure 31B: Polydispersity (PDI) measurements. Numerical data are presented in Table 11. [Figure 32A-32B] SDS polyacrylamide gel electrophoresis of low-skid silk / modified polypeptide compositions. Figure 32A: Q-HIC (elution) SEC fractions. Figure 32B: Q-HIC (flow-through) SEC fractions. Lanes are represented by fraction number in order of elution from the Superdex 200 column and their respective silk compositions: in Figure 32A, fraction 6 is AS90, fraction 7 is AS91, fraction 8 is AS92, fraction 9 is AS93, and fraction 10 is AS94. In Figure 32B, fraction 8 is AS95, fraction 9 is AS96, fraction 10 is AS97, fraction 11 is AS98, fraction 12 is AS99, and fraction 13 is AS100. [Figure 33] The self-assembly reaction of a low-skid silk / modified peptide composition is illustrated. A medium-skid silk reaction was used as a positive control. Kinetic parameters of gel formation during silk self-assembly. Q-HIC (elution) is the elution fraction eluted from the Butyl ImpRes column before SEC purification. LS, low-skid silk; MS, medium-skid silk. Self-assembly parameters for medium-skid silk: Amax is 0.6974 (Abs), SARF is 8.661, T0.5 is 3.834 h, and FSAF is 3.03 (Abs / min). [Figures 34A-34F]Figure 34 illustrates the characterization of low-skid silk compositions by dynamic light scattering. Low-skid and medium-skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed using a Zetasizer Pro to estimate the diameter particle size of each silk composition. Figure 34A: Intensity diameter particle size distributions measured for silk composition AS90, the Q-HIC (elution) fraction (before fractionation by SEC), low-skid silk (LS), and medium-skid silk (MS). Figure 34B: Correlogram functions of the silk compositions presented in (34A). Figure 34C: Intensity diameter particle size distributions measured for silk compositions AS90-AS94 derived from the Q-HIC (elution)-SEC fractionation process. Figure 34D: Correlogram functions of the silk compositions presented in (34C). Figure 34E: Intensity diameter particle size distributions measured for silk compositions AS95-AS100 derived from the Q-HIC (flow-through)-SEC fractionation process. Figure 34F: Correlogram function of the silk composition presented in (34E). [Figure 35] 1 illustrates a size-exclusion chromatography scheme for the isolation of a medium-skid silk / modified peptide composition. The medium-skid silk / modified polypeptide composition is composed of various peptide populations across a wide range of sizes. Using HiLoad Superdex 200 size-exclusion chromatography, distinct populations of the medium-skid silk / modified polypeptide composition could be separated. [Figure 36] This is a chromatogram of a medium-skid silk / modified polypeptide composition loaded onto a Superdex 200 gel filtration column. The medium-skid silk / modified peptide composition was loaded onto a Superdex 200 column and run through the column using 50 mM Tris, 200 mM CaCl2, pH = 8.0. The medium-skid silk / modified peptide composition was collected and separated by size when the UV-280 absorbance began to increase for the fractions. The relative elution volumes of silk compositions AS107 and AS111 are shown on the chromatogram. [Figure 37A-37B]Analytical size exclusion chromatography of medium-skid silk, medium-skid silk / modified silk compositions, and their constituent AS compositions are illustrated in Figure 37A. Average molecular weights in kDa of medium-skid silk (MS) and AS106-AS111 are shown. Figure 37B. Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 14. [Figure 38] SDS polyacrylamide gel electrophoresis of medium-skid silk / modified polypeptide compositions. Lanes are represented by fraction number in order of elution from a Superdex 200 column and their respective silk compositions: fraction 6 is AS107, fraction 7 is AS108, fraction 8 is AS109, fraction 9 is AS110, and fraction 10 is AS111. [Figure 39] Figure 1 illustrates the self-assembly reaction of medium-skid silk / modified peptide compositions. Kinetic parameters of gel formation during silk self-assembly. The red dashed line shows how the self-assembly parameters Amax, SARF, and T0.5 were calculated for unfractionated medium-skid silk (MS). These numerically calculated parameters for silk compositions AS106-AS111 can be found in Table 16. Low-skid silk (LS) was used as a negative control. LS, low-skid silk; MS, medium-skid silk. [Figure 40A-40B] Figure 40 illustrates the characterization of medium-skid silk compositions by dynamic light scattering. The medium-skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by Zetasizer Pro to estimate the particle size of each silk composition. Figure 40A: Intensity particle size distribution measured for silk compositions AS106, AS107, AS108, AS109, AS110, AS111, and medium-skid (MS). Figure 50B: Correlation functions for the silk compositions presented in (40A). [Figure 41]

[0039] Figure 1 illustrates anion exchange and size exclusion chromatography schemes for the isolation of medium-skid silk / modified peptide compositions. The medium-skid silk / modified polypeptide composition is composed of a diverse population of peptides with a wide range of sizes and charges. Using Q-Sepharose anion exchange chromatography as the first step and HiLoad Superdex 200 size exclusion chromatography as the second purification step, distinct populations of the medium-skid silk / modified polypeptide composition were separated. The Q-Sepharose eluate was loaded onto HiLoad Superdex 200 size exclusion chromatography, which resulted in size-fractionated negatively charged silk compositions / modified peptides. [Figure 42A-42B] Chromatograms of anion exchange chromatography of the medium-skid silk / modified polypeptide composition and subsequent size-exclusion chromatography of the eluate (Q eluate). Figure 42A: Anion exchange chromatography was performed using a Q-Sepharose column (Cytiva). The medium-skid silk / modified peptide composition was separated by anion exchange chromatography into an uncharged peptide population (flow-through—light blue background) and the eluted negatively charged silk composition (eluate—light pink background). The light yellow background represents a column wash with 50 mM Tris pH=8.0 prior to elution of the charged peptide population. Figure 42B: The negatively charged eluate (Q eluate) was loaded onto a Superdex 200 column and run through the column using 50 mM Tris, 200 mM CaCl2, pH=8.0. When UV-280 absorbance began to increase for the fractions, the medium-skid silk / modified peptide composition was collected and separated by size. The relative elution volumes of silk compositions AS101 and AS105 are depicted on the chromatogram. [Figure 43A-43B] Analytical size exclusion chromatography of medium-skid silk, medium-skid silk / modified silk compositions, and their constituent AS compositions are illustrated in Figure 43A. Average molecular weights in kDa of medium-skid silk (MS) and AS101-AS105 are shown. Figure 43B. Polydispersity (PDI) measurements are shown. Numerical data are presented in Table 16. [Figure 44A]SDS polyacrylamide gel electrophoresis of medium-skid silk / modified polypeptide compositions. Lanes are represented by fraction number in order of elution from a Superdex 200 column and their respective silk compositions: fraction 6 is AS101, fraction 7 is AS102, fraction 8 is AS103, fraction 9 is AS104, and fraction 10 is AS105. [Figure 44B] Figure 1 illustrates the self-assembly reaction of a medium-skid silk / modified peptide composition. A low-skid silk reaction was used as a negative control. Kinetic parameters for gel formation during silk self-assembly are shown. The red dotted lines are shown for clarity in the calculation of the Amax, SARF (self-assembly rate factor), and T0.5 parameters in Table 17. [Figures 45A-45C] 45A-45C are graphs illustrating the characterization of medium-skid silk compositions by dynamic light scattering. Medium-skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by a Zetasizer Pro (Malvern) to estimate the diameter particle size of each silk composition. Figure 45A: Intensity diameter particle size distribution by intensity measured for silk compositions AS101, AS102, AS103, AS104, and AS105. Figure 45B: Intensity diameter particle size distribution by intensity measured for silk compositions AS101, AS105, and medium-skid silk (MS) to highlight the size difference between AS101 and AS105. Figure 45C: Correlogram functions for silk compositions AS101, AS102, AS103, AS104, AS105, and medium-skid silk (MS). [Figure 46] Illustrative values ​​for the three molar mass moments (Mn, Mw, and Mz), which relate to the molar mass and the number of molecules at each molar mass. This example is applicable to polydisperse samples; for monodisperse samples, Mn = Mw = Mz. [Figure 47A-47B] Analytical SEC-MALS of low, medium, and high molecular weight silks. Figure 47A: Weight average molecular weight in kDa for low, medium, and high molecular weight silks. Figure 47B: Polydispersity index (PDI) measurements for low, medium, and high molecular weight silks. [Figure 48A-48B]Analytical SEC-MALS of low, medium, and high molecular weight silks comprised of silk types produced by different process parameters and variable levels. Individual data points are shown, and the mean is represented by the height of the box. Bars encompass one standard deviation. Figure 48A: Weight average molecular weight ranges for low, medium, and high molecular weight silks. Figure 48B: PDI ranges for low, medium, and high molecular weight silks. [Figure 49A-49B] Analytical SEC-MALS of low-skid silk / modified silk compositions and constituent AS compositions separated by Q-SEC (Q eluent). Figure 49A: Average molecular weights in kDa of low-skid silk (LS) and AS77-AS81. Figure 49B: Polydispersity (PDI) measurements. Numerical data are presented in Table 24. [Figure 50A-50B] Analytical SEC-MALS of low-skid silk / modified silk compositions and constituent AS compositions separated by SEC. Figure 50A: Average molecular weights in kDa of low-skid silk (LS) and AS82-AS89. Figure 50B: Polydispersity (PDI) measurements. Numerical data are presented in Table 25. [Figure 51A-51B] Analytical SEC-MALS of low-skid silk / modified silk compositions and constituent AS compositions separated by Q-HIC-SEC (Q-HIC-eluent). Figure 51A: Average molecular weights in kDa of low-skid silk (LS) and AS90-AS94. Figure 51B: Polydispersity (PDI) measurements. Numerical data are presented in Table 26. [Figure 52A-52B] Analytical SEC-MALS of low-skid silk / modified silk compositions and constituent AS compositions separated by Q-HIC-SEC (Q-HIC-flow-through). Figure 52A: Average molecular weights in kDa of low-skid silk (LS) and AS95-AS100. Figure 52B: Polydispersity (PDI) measurements. Numerical data are presented in Table 26. [Figure 53A-53B]Analytical SEC-MALS of medium-skid silk / modified silk compositions and constituent AS compositions separated by Q-SEC (Q-flow-through). Figure 53A: Average molecular weights in kDa of medium-skid silk (MS) and AS101-AS105. Figure 53B: Polydispersity (PDI) measurements. Numerical data are presented in Table 27. [Figure 54A-54B] Analytical SEC-MALS of medium-skid silk / modified silk compositions and constituent AS compositions separated by SEC. Figure 54A: Average molecular weights in kDa of medium-skid silk (MS) and AS106-AS111. Figure 54B: Polydispersity (PDI) measurements. Numerical data are presented in Table 28. [Figures 55A-55C] The sequence listing for the fibroin heavy chain is shown. [Figure 56] The sequence listing for the fibroin light chain is shown. [Figure 57] The sequence listing for fibrohexamerin is shown. [Figure 58] Three chromatographic principles of silk fractionalization are illustrated. [Figure 59] 1 illustrates anion exchange chromatography followed by size exclusion chromatography of silk fractionation. [Figure 60] Anion exchange chromatography followed by hydrophobic interaction chromatography and size exclusion chromatography is exemplified. [Figure 61] 1 is a chart containing assays for characterizing silk fractions. [Figure 62] Figure 4 shows a graph presenting the data from Tables 44 and 45. Data is shown with standard deviation. "Nanoclay" refers to Elementis Bentone Hydroclay. [Figure 63] FIG. 1 is a diagram of a typical bentonite clay structure. [Figure 64] SEM image of the cross section of a film cast from Elementis Bentone Hydroclay 2001 shows highly ordered stacking of the clay layers. [Figure 65]1 is an SEM image of the cross section of a film cast with pure RSF. [Figure 66] SEM image of the cross section of a 1:1 RSF / 2001 film cast under neutral (pH 7.0) conditions. The layered structure of the clay is preserved. [Figure 67] FIG. 10 is an SEM image of the cross section of a 1:1 RSF / 2001 film cast under acidic (pH 3.5) conditions. Note the ribbon-like structure. [Figure 68] Illustrates the increased diffusion paths created by the RSF / nanoclay composite. [Figure 69] FTIR scan of the amide I region of RSF / 2001 film cast under neutral conditions. Nanoclay concentration is varied from 0% (red) to 70% (yellow). As the nanoclay content increases, the amide I peak shifts to the left, away from the beta-sheet region. [Figure 70] 1 is a flow chart illustrating various embodiments for producing pure silk fibroin protein fragments (SPF) of the present disclosure. [Figure 71] 1 is a flow chart illustrating various parameters that can be modified during the process of producing the SPF of the present disclosure during the extraction and lysis steps. [Figure 72-73] 1 is a graph depicting the effect of extraction volume on % mass loss. [Figure 74] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under the conditions of 100° C. extraction temperature, 100° C. LiBr, and 100° C. oven dissolution (varying oven / dissolution times). [Figure 75] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, boiling LiBr, and 60° C. oven dissolution (with oven / dissolution times varied). [Figure 76] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, 60° C. LiBr, and 60° C. oven dissolution (varying oven / dissolution times). [Figure 77] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, 80° C. LiBr, and 80° C. oven dissolution (varying oven / dissolution times). [Figure 78] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, 80° C. LiBr, and 60° C. oven dissolution (with oven / dissolution times varied). [Figure 79] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, 100° C. LiBr, and 60° C. oven dissolution (with oven / dissolution times varied). [Figure 80] 1 is a graph summarizing the effect of extraction time on the molecular weight of silk processed under conditions of 100° C. extraction temperature, 140° C. LiBr, and 140° C. oven dissolution (varying oven / dissolution times). [Figure 81] 1 is a graph summarizing the effect of extraction temperature on the molecular weight of silk processed under conditions of 60 minute extraction time, 100° C. LiBr, and 100° C. oven dissolution (with oven / dissolution times varied). [Figure 82] 1 is a graph summarizing the effect of LiBr temperature on the molecular weight of silk processed under conditions of 60 minutes extraction time, 100° C. extraction temperature, and 60° C. oven dissolution (varying oven / dissolution times). [Figure 83] 1 is a graph summarizing the effect of LiBr temperature on the molecular weight of silk processed under conditions of 30 minutes extraction time, 100° C. extraction temperature, and 60° C. oven dissolution (varied oven / dissolution times). [Figure 84] 1 is a graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk treated under conditions of 100°C extraction temperature, 30 minutes extraction time, and 100°C lithium bromide (varying oven / dissolution times). [Figure 85]1 is a graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk treated under conditions of 100°C extraction temperature, 60 minutes extraction time, and 100°C lithium bromide (varying oven / dissolution times). [Figure 86] 1 is a graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk treated under conditions of an extraction temperature of 100°C, an extraction time of 60 minutes, and lithium bromide at 140°C (with varying oven / dissolution times). [Figure 87] 1 is a graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk treated under conditions of an extraction temperature of 100°C, an extraction time of 30 minutes, and lithium bromide at 140°C (with varying oven / dissolution times). [Figure 88] 1 is a graph summarizing the effect of oven / dissolution temperature on the molecular weight of silk treated under conditions of 100°C extraction temperature, 60 minutes extraction time, and 80°C lithium bromide (varying oven / dissolution times). [Figure 89] 1 is a graph summarizing the molecular weight of silk processed under varying conditions, including extraction time, extraction temperature, lithium bromide (LiBr) temperature, oven temperature for dissolution, and oven time for dissolution. [Figure 90] 1 is a graph summarizing the molecular weight of silk processed under conditions where the oven / melt temperature is equal to the LiBr temperature. [Figures 91A-91C] Figures 91A and 91B illustrate low MW silk solids resulting from the freeze-drying process described herein at different stages of grinding. Figure 91A illustrates the coarse particles of low MW silk solids immediately after removal from the freeze-drying bottle. Figure 91B illustrates particles of reduced size midway through grinding. Figure 91C illustrates fine particles with a uniform size distribution upon completion of grinding. [Figure 92] Illustrates solid particles of medium MW silk solids. [Figure 93] 1 illustrates examples of solid silk particles of two different particle sizes formed during thin film evaporation as described herein. [Figure 94A-94B] 1 illustrates examples of microparticles prepared by the solution precipitation process described herein. [Figure 95] 1 illustrates a milled silk powder for use as described herein. [Figure 96A-96B] 96A and 96B illustrate pouches containing solid formulations comprising silk fibroin fragments as described herein. Figure 96A illustrates a pouch described herein containing loose silk fibroin fragments. Figure 96B illustrates a pouch described herein containing a disk of cryo-pelletized silk fibroin fragments. [Figure 97A] Illustrated is a freeze-dried silk pellet. [Figure 97B] 1 is a graph showing activated silk reconstitution yield. [Figure 98A] 1 shows freeze-dried silk. [Figure 98B] 1 is a graph showing freeze-drying temperatures. [Figure 99A] 1 shows cryogenically pelleted silk. [Figure 99B] 1 is a graph showing freeze-drying temperatures. [Figure 100] Lyophilized beads of various concentrations are illustrated. [Figures 101A-101G] Figure 101A shows SEM images of freeze-dried silk under different processing conditions. Figure 101A shows freeze-dried silk at 6% without annealing. Figure 101B shows freeze-dried silk at 6% with annealing. Figure 101C shows freeze-dried silk at 6% without annealing. Figure 101D shows freeze-dried silk at 6% with annealing. Figure 101E shows 16% silk without annealing. Figure 101F shows 16% silk annealed for 4 hours. Figure 101G shows 16% silk annealed for 20 hours. [Figure 102A-102B] 102A and 102B show images of powdered lyophilized silk from densified lyophilized pellets. Figure 102A shows 10% medium Mw Activated Silk™ ground from densified pellets. Figure 102B shows 16% low Mw Activated Silk™ ground from densified pellets. [Figure 103]1 is an image showing how particle size and reconstitution method affect reconstitution yield: More finely milled silk powders remained above the liquid level and were less wetted when reconstituted using static methods compared to more coarsely milled ones. [Figures 104A-104C] 104A and 104B are images showing the ability to spray Activated Silk™. Fig. 104A is a close-up of the aerosol nozzle during spraying. Fig. 104B illustrates the spray mist stream. Fig. 104C illustrates the spray pattern using an aerosol can 6 inches above a surface. [Figure 105A] 10 is an image showing the bulk "floating" off the tray during freeze drying. [Figure 105B] 1 is an image showing meltback. [Figure 106] 1 is a graph showing the profile of an example bulk freeze-drying. [Figure 107] 1 is an image of the product from bulk freeze-freeze drying. [Figure 108] This is an image of the product meltback. [Figure 109] 10 is an image showing the discoloration of the final product. [Figure 110] 10 is a graph showing the results of Profile 5. [Figures 111A-111C] 11A and 11B are images of cryo-lyophilized pellets. FIG. 111A is a 6% cryo-lyophilized pellet. FIG. 111B is a 10% cryo-lyophilized pellet. FIG. 111C is a 17% cryo-lyophilized pellet. [Figure 112] 1 is a graph showing a BET isotherm plot. [Figure 113] 1 is an SEM image of cryogenically pelleted lyophilized Activated Silk™. [Figure 114] 1 is a graph showing "z-average" particle size. [Figure 115] 1 is an image showing a sample of silk solution. [Figure 116]Graphs showing sample output for both intensity and Z-average particle size. Over time, the particle size distribution shifts towards the higher size regime, increasing the intensity peak in that region (left). This is summarized by an increase in Z-average (right). The Python program allows for quick comparison of data across time points and samples. [Figure 117] 10 is an image showing the dynamic light scattering workflow of data processing in Python and JMP. [Figure 118] Figure 118 is a graph illustrating the silk-only control, where 27P is, as expected, an order of magnitude more stable than 33B across a range of temperatures and concentrations (bottom left, top right). However, the faster aggregation of 33B makes it a useful model system for studying the effect of excipients on aggregation rate (bottom right). (n=3 replicates unless otherwise noted). Figure 119 is a graph showing baseline system aggregation for both 27P and 33B. The graph shows that 27P is an order of magnitude more stable than 33B across a range of temperatures and concentrations. [Figure 119A] 1 is a graph showing baseline aggregation of 27P. [Figure 119B] 1 is a graph showing the main effect on aggregation rate of a baseline system. [Figure 120] 1 is a graph showing the z-average of various silk solutions. [Figure 121] 1 is a graph showing the z-average of various silk solutions. [Figure 122] 1 is a graph showing the z-average of various silk solutions. [Figure 123] 1 is an image showing soluble silk, gelled silk, and particles in solution. [Figure 124] 1 is a graph showing the z-average of various silk solutions. [Figure 125] 1 is a graph showing the z-average of various silk solutions. [Figure 126] 1 is a graph showing the z-average of various silk solutions. [Figures 127A-127C]Figures 127A and 127B are graphs showing the stability of concentrated 33B solutions. Figure 127A shows the results of DLS testing of 33B samples. Figure 127B shows the z-average of various silk solutions at 4°C. Figure 127C shows the incubation curves at 70°C. These graphs highlight the significant differences in aggregation profiles with temperature, further demonstrating the superior performance of PBS buffer in slowing down aggregation. [Figure 128A-128B] Excipient results from the 40C test are shown. Figure 128A: Arginine HCl, MgCl2 > NaCl, KCl > PBS > CaCl2. Figure 128B: The two best performers (ArgHCl, MgCl2) showed no change in pH or flocculation after 1085 hours (45 days). The equivalent 33B / DI water gelled completely after 10 days at this temperature. [Figure 129A-129B] Scans taken from solutions stored at 40° C. for approximately 45 days (FIG. 129A) show a similar aggregation profile to solutions scanned continuously at 70° C. for 1 hour (FIG. 129B). [Figures 130A-130C] 1 is a graph showing how normalized relative aggregation rates correlate across temperatures. [Figure 131A-131B] 1 is a graph showing the best performing excipients at 40C, showing a significant improvement at 70C compared to DI water. [Figure 132] The equation used to model 70C aggregation in prism is shown below. [Figures 133A-133C] Shown are 70C curves analyzed via the One-Phase Association model in Prism software to extract better quantitative data from the samples. [Figure 134] 1 is a graph showing the results of a preservative screening, specifically, consecutive z-average measurements at 70° C. [Figure 135] 1 is a bar graph showing the effect of various preservatives on aggravation. [Figure 136] 1 is a graph showing that salt has the potential to stabilize solutions that would otherwise be prone to flocculation. [Figure 137]1 is a bar graph showing that salt has the potential to stabilize solutions that would otherwise be prone to flocculation. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present disclosure provides lyophilized or nebulizable peptide or protein fragments comprising a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W, wherein at least one of the amino acids is modified, substituted, or substituted as disclosed herein.

[0018] The present disclosure provides a pouch with a plurality of holes, the pouch enclosing a substantially solid formulation comprising silk fibroin fragments. The present disclosure further provides a method for reconstituting the substantially solid formulation comprising silk fibroin fragments in a solvent. The present disclosure also provides a method for making a pouch enclosing a substantially solid formulation of silk fibroin fragments.

[0019] Silk is a natural polymer produced by various insects and spiders. Silk produced by Bombyx mori (the silkworm) contains a filament core protein, silk fibroin, and a colloidal coating consisting of the nonfilamentous protein sericin. Silk fibroin is an FDA-approved, edible, nontoxic, and relatively inexpensive protein derived from silkworm cocoons. The structure and content of amino acids in silk fibroin protein are very similar to those in human body tissues.

[0020] Methods for producing silk fibroin or silk fibroin-based protein fragments are known and are described, for example, in U.S. Pat. Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177, all of which are incorporated herein in their entireties.

[0021] definition Unless otherwise defined, as used in the above section, and throughout the remainder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents and publications mentioned herein are incorporated by reference in their entirety.

[0022] Unless otherwise specified, all percentages, parts, and ratios are based on the total weight of the eye care compositions of the present disclosure. All such weights relating to listed ingredients are based on the active level and therefore do not include solvents or by-products that may be included in commercially available materials, unless otherwise specified. The term "weight percent" may be referred to herein as "wt %" or % w / w.

[0023] As used herein, the terms "a," "an," or "the" are generally interpreted to encompass both the singular and the plural.

[0024] As used herein, the term "about" generally refers to a particular numerical value within a range of variation and acceptable error as determined by one of ordinary skill in the art, which will depend in part on how the numerical value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean zero variation and a range of ±20%, ±10%, or ±5% of the given numerical value.

[0025] As used herein, the term "dermatologically acceptable carrier" means a carrier that is suitable for use in contact with mammalian keratinous tissue without causing any adverse effects, such as, for example, undue toxicity, incompatibility, instability, allergic response, etc. Dermatologically acceptable carriers may include, without limitation, water, liquid or solid emollients, humectants, solvents, and the like.

[0026] As used herein, the term "hydrophilic-lipophilic balance" (HLB) of a surfactant is a measure of the degree to which it is hydrophilic or hydrophobic, determined by calculating the values ​​of different regions of the molecule, according to Griffin's method: HLB=20*M h / M(where M h HLB values ​​are described by the HLB (where M is the molecular mass of the hydrophilic portion of the surfactant and M is the molecular mass of the entire surfactant molecule) and give results on a scale of 0 to 20. An HLB value of 0 corresponds to a completely lipophilic molecule, and a value of 20 corresponds to a completely hydrophilic molecule. HLB values ​​can be used to predict a molecule's surfactant properties: HLB < 10: lipid soluble (water insoluble), HLB > 10: water soluble (lipid insoluble), HLB = 1-3: antifoaming agent, 3-6: W / O (water-in-oil) emulsifier, 7-9: wetting agent and spreading agent, 8-16: O / W (oil-in-water) emulsifier, 13-16: surfactant, 16-18: solubilizer or hydrotrope.

[0027] As used herein, "average weight average molecular weight" refers to the average of two or more values ​​of weight average molecular weight of silk fibroin or fragments thereof of the same composition, the two or more values ​​being determined by two or more separate experimental readings.

[0028] As used herein, the term "polydispersity (PD)" of a polymer is generally used as a measure of the broadness of the molecular weight distribution of a polymer, and polydispersity

number

[0029] As used herein, the term "substantially homogeneous" can refer to silk fibroin-based protein fragments that are distributed in a normal distribution around an identified molecular weight. As used herein, the term "substantially homogeneous" can refer to, for example, an even distribution of components or additives, such as silk fibroin fragments, dermatologically acceptable carriers, etc., throughout the compositions of the present disclosure.

[0030] As used herein, the terms "silk fibroin peptide," "silk fibroin protein fragment," and "silk fibroin fragment" are used interchangeably. Molecular weight or number of amino acid units is defined when molecular size is the critical parameter.

[0031] As used herein, the term "fast dissolving solid form" refers to fast dissolving solid forms including lyophilized forms (cakes, wafers, films) and compressed tablets.

[0032] As used herein, the term "peptide" or "protein" refers to a chain of amino acids held together by peptide bonds (also called amide bonds). The fundamental distinguishing factors between proteins and peptides are size and structure. Peptides are smaller than proteins. Conventionally, peptides are defined as molecules consisting of 2 to 50 amino acids, while proteins are composed of 50 or more amino acids. In addition, peptides tend to be less well-defined in structure than proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures.

[0033] As used herein, the term "fibroin" or "silk protein" refers to a type of structural protein produced by certain silk-producing spider and insect species (see definitions provided in the WIPO Pearl-WIPO's Multilingual Terminology Portal database, https: / / wipopearl.wipo.int / en / linguistic). Fibroin may include silkworm fibroin, insect or spider silk proteins (e.g., spidroins), recombinant spider proteins, silk proteins present in other spider silk types, such as tubular gland silk proteins (TuSPs), flagellate gland silk proteins, ampullate gland silk proteins, grape-like gland silk proteins, piriform gland silk proteins, agglutinating gland silk glue, silkworm fibroin produced by transgenic silkworms, or recombinant silkworm fibroin.

[0034] As used herein, the term "silk fibroin" refers to silkworm fibroin, silk fibroin produced by genetically modified silkworms, or recombinant silkworm fibroin (see (1) Narayan Ed., Encyclopedia of Biomedical Engineering, Vol. 2, Elsevier, 2019; (2) Kobayashi et al. Eds., Encyclopedia of Polymeric Nanomaterials, Springer, 2014, https: / / link.springer.com / referenceworkentry / 10.1007%2F978-3-642-36199-9_323-1). In one embodiment, the silk fibroin is obtained from Bombyx mori.

[0035] As used herein, the term "solid solution" refers to an active agent molecularly dissolved in a solid excipient matrix, such as a hydrophobic polymer, wherein the active agent is miscible with the polymer matrix excipient.

[0036] As used herein, the term "solid dispersion" refers to an active agent dispersed as crystalline or amorphous particles, wherein the active agent is dispersed in an amorphous polymer and randomly distributed among the polymer matrix excipients.

[0037] As used herein, the term "substantially homogeneous" can refer to silk fibroin-based protein fragments that are distributed in a normal distribution around a specified molecular weight. As used herein, the term "substantially homogeneous" can also refer to, for example, an even distribution of components or additives, such as silk fibroin-based protein fragments, dermatologically acceptable carriers, etc., throughout a silk composition or formulation.

[0038] As used herein, the term "surface tension" refers to the tendency of a fluid surface to contract to the smallest possible surface area. At a liquid-air interface, surface tension results from the greater attraction of liquid molecules to each other than to molecules in the air (due to adhesive forces). The net effect is an inward force at the liquid's surface that causes it to behave as if it were covered by a stretched elastic film. Because of the relatively high attraction of water molecules to each other through a network of hydrogen bonds, water has a higher surface tension (72.8 mN / m at 20°C) than most other liquids.

[0039] SPF definition and characteristics As used herein, "silk protein fragments" (SPFs) include one or more of the following, but are not limited to: "silk fibroin fragments" as defined herein, "recombinant silk fragments" as defined herein, "spider silk fragments" as defined herein, "silk fibroin-like protein fragments" as defined herein, "chemically modified silk fragments" as defined herein, and / or "sericin or sericin fragments" as defined herein. SPFs can have any molecular weight value or range described herein and any polydispersity value or range described herein. As used herein, in some embodiments, the term "silk protein fragment" also refers to a silk protein comprising or consisting of at least two identical repeating units, each independently selected from a natural silk polypeptide or a variation thereof, the amino acid sequence of a natural silk polypeptide, or a combination of both.

[0040] SPF molecular weight and polydispersity In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 to about 70 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 70 to about 75 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 75 to about 80 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 80 to about 85 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 85 to about 90 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 90 to about 95 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 95 to about 100 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 to about 170 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 170 to about 175 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 175 to about 180 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 180 to about 185 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 185 to about 190 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 190 to about 195 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 195 to about 200 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 200 to about 205 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 205 to about 210 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 210 to about 215 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 215 to about 220 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 220 to about 225 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 225 to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 to about 255 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 to about 260 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 to about 270 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 270 to about 275 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 275 to about 280 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 280 to about 285 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 285 to about 290 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 to about 300 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 to about 310 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 to about 330 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 330 to about 335 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 335 to about 340 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 340 to about 345 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 345 to about 350 kDa.

[0041] In some embodiments, the compositions of the present disclosure include SPF compositions selected from compositions #1001-#3500, having a weight average molecular weight selected from about 1 kDa to about 250 kDa, and having a polydispersity selected from 1 to about 5 (including, but not limited to, a polydispersity of 1), 1 to about 1.5 (including, but not limited to, a polydispersity of 1), about 1.5 to about 2, about 1.5 to about 3, about 2 to about 2.5, about 2.5 to about 3, about 3 to about 3.5, about 3.5 to about 4, about 4 to about 4.5, and about 4.5 to about 5: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0042] As used herein, "low molecular weight," "low MW," or "low-MW" SPFs can include SPFs having a weight average molecular weight or average weight average molecular weight selected from about 5 kDa to about 38 kDa, about 14 kDa to about 30 kDa, or about 6 kDa to about 17 kDa. In some embodiments, the target low molecular weight of a particular SPF is about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, or about 22 kDa. The weight average molecular weight of the polymer may be about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, or about 38 kDa.

[0043] As used herein, "medium molecular weight," "medium MW," or "mid-MW" SPF can include SPFs having a weight average molecular weight or average weight average molecular weight selected from between about 31 kDa and about 55 kDa, or between about 39 kDa and about 54 kDa. In some embodiments, the target mid-molecular weight for a particular SPF can be a weight average molecular weight of about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, or about 55 kDa.

[0044] As used herein, "high molecular weight," "high MW," or "high-MW" SPF can include SPFs having a weight average molecular weight, or an average weight average molecular weight, selected from between about 55 kDa and about 150 kDa. In some embodiments, the target high molecular weight for a particular SPF can be about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, about 75 kDa, about 76 kDa, about 77 kDa, about 78 kDa, about 79 kDa, or about 80 kDa.

[0045] In some embodiments, the molecular weights described herein (e.g., low molecular weight silk, medium molecular weight silk, high molecular weight silk) can be converted to the approximate number of amino acids contained within each SPF, as would be understood by one skilled in the art. For example, the average weight of an amino acid may be about 110 Daltons (i.e., 110 g / mol). Thus, in some embodiments, dividing the molecular weight of a linear protein by 110 Daltons can be used to approximate the number of amino acid residues contained therein.

[0046] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between 1 and about 5.0, including, but not limited to, a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 1.5 and about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between 1 and about 1.5, including, but not limited to, a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 1.5 and about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 2.0 and about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 2.5 and about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 3.0 and about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 3.5 and about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

[0047] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.6.In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 5.0.

[0048] In some embodiments, in compositions described herein having a combination of low, medium, and / or high molecular weight SPFs, such low, medium, and / or high molecular weight SPFs may have the same or different polydispersities.

[0049] Silk fibroin fragments Methods for producing silk fibroin or silk fibroin protein fragments and their uses in various fields are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177, 10,287,728, and 10,301,768, all of which are incorporated herein in their entireties. Raw silk from the silkworm Bombyx mori is composed of two major proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin is a fibrous protein with a semi-crystalline structure that provides rigidity and strength. As used herein, the term "silk fibroin" refers to the fiber of Bombyx mori cocoons, which has a weight-average molecular weight of approximately 370,000 Da. Silkworm crude fiber consists of double strands of fibroin. The adhesive substance that holds these double strands together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with large hydrophobic domains that account for the majority of the polymer's high molecular weight. The hydrophobic regions are interrupted by small hydrophilic spacers, and the N- and C-termini of the chains are also highly hydrophilic. The hydrophobic domains of the H chains contain a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and a repeating Gly-Ala / Ser / Tyr dipeptide, which can form stable antiparallel sheet crystallites. The amino acid sequence of the L chain is non-repetitive, making it more hydrophilic and relatively elastic. The hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) chain segments in the silk fibroin molecule are alternately arranged to allow the silk fibroin molecule to self-assemble.

[0050] Provided herein are methods for producing pure and highly scalable silk fibroin-protein fragment mixture solutions that may be used across multiple industries for a variety of applications. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to the fragmentation of any SPF described herein, including, but not limited to, recombinant silk proteins and silk-like or fibroin-like proteins.

[0051] As used herein, the term "fibroin" includes silkworm fibroin and insect or spider silk proteins. In one embodiment, the fibroin is obtained from Bombyx mori. Raw silk from Bombyx mori is composed of two major proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin is a fibrous protein with a semi-crystalline structure that provides rigidity and strength. As used herein, the term "silk fibroin" refers to Bombyx mori cocoon fibers having a weight-average molecular weight of approximately 370,000 Da. Conversion of these insoluble silk fibroin fibrils into water-soluble silk fibroin protein fragments requires the addition of concentrated neutral salt (e.g., 8-10 M lithium bromide), which interferes with the intermolecular and intramolecular ionic and hydrogen bonds that would otherwise render the fibroin protein insoluble in water. Methods for making silk fibroin protein fragments and / or compositions thereof are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0052] Raw silk cocoons from the silkworm Bombyx mori were cut into small pieces. The small pieces were treated in an aqueous solution of Na2CO3 at approximately 100°C for approximately 60 minutes to remove sericin (degumming). The volume of water used was approximately 0.4 times the raw silk weight, and the amount of Na2CO3 was approximately 0.848 times the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The volume of rinse water for each cycle was 0.2 L x the weight of the raw silk cocoon pieces. Excess water was removed from the degummed silk cocoon pieces. After the DI water washing step, the wet, degummed silk cocoon pieces were dried at room temperature. Degummed silk cocoon pieces were mixed with a LiBr solution, and the mixture was heated to approximately 100°C. The warmed mixture was placed in a drying oven and heated at approximately 100°C for approximately 60 minutes to achieve complete dissolution of the native silk protein. The resulting silk fibroin solution was filtered and dialyzed for 72 hours using tangential flow filtration (TFF) and a 10 kDa membrane against deionized water. The resulting aqueous silk fibroin solution had a concentration of approximately 8.5% by weight. The 8.5% silk solution was then diluted with water to yield a 1.0% w / v silk solution. TFF can then be used to further concentrate the pure silk solution to a concentration of 20.0% w / w silk in water.

[0053] Dialyzing silk through a series of water changes is a manual, time-intensive process that can be accelerated by varying certain parameters, e.g., diluting the silk solution before dialysis. The dialysis process can be scaled up for production by using semi-automated equipment, e.g., tangential flow filtration systems.

[0054] In some embodiments, silk solutions are prepared under various preparation condition parameters, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3 M LiBr was prepared and left at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in an oven at 60°C. Samples from each set were removed at 4, 6, 8, 12, 24, 168, and 192 hours.

[0055] In some embodiments, silk solutions are prepared under various preparation condition parameters, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3 M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of the hot LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples from each set were removed at 1, 4, and 6 hours.

[0056] In some embodiments, silk solutions are prepared under various preparation condition parameters. For example, four different silk extraction combinations were used: 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5mL of the hot LiBr solution was added to 1.25g of silk and placed in an oven at the same temperature of LiBr. Samples from each set were removed at 1, 4, and 6 hours. 1mL of each sample was added to 7.5mL of 9.3M LiBr and refrigerated for viscosity testing.

[0057] In some embodiments, SPF is achieved by dissolving raw undegummed, partially degummed, or degummed silkworm fiber with a neutral lithium bromide salt. The raw silkworm silk is treated to remove any sericin and to obtain a desired weight average molecular weight (M WThe silk fibroin protein fragments are processed under selected temperatures and other conditions to achieve a desired size and polydispersity (PD). The selection of process parameters can be varied to achieve different final silk protein fragment characteristics, depending on the intended use. The resulting final fragment solution is pure silk fibroin protein fragments and water with undetectable levels of process contaminants, parts per million (ppm), levels acceptable in the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further varied depending on the desired use and performance requirements.

[0058] Methods for producing silk protein fragments used in the compositions of the present disclosure are described in U.S. Patent Application Publication Nos. 2015 / 00933340, 2015 / 0094269, 2016 / 0193130, 2016 / 0022560, 2016 / 0022561, 2016 / 0022562, 2016 / 0022563, and 2016 / 0022564. and US Patent Nos. 2016 / 0222579, 2016 / 0281294, and U.S. Patent Nos. 9,187,538, 9,522,107, 9,517,191, 9,522,108, 9,511,012, and 9,545,369, the entire contents of which are incorporated herein by reference. However, an exemplary method is demonstrated in Figure 70, which is a flow chart illustrating various embodiments for producing pure silk fibroin-based protein fragments (SPF) of the present disclosure. It should be understood that not all of the illustrated steps are necessarily required to produce all of the silk solutions of the present disclosure. As illustrated in Figure 70, step A, silk cocoons (heat-treated or non-heat-treated), silk fibers, silk powder, or spider silk can be used as the silk source. Starting with raw silk cocoons from Bombyx mori, the cocoons can be cut into small pieces, e.g., pieces of approximately equal size (step B1). The raw silk is then extracted and rinsed to remove sericin (step C1a). This results in raw silk that is substantially free of sericin. In one embodiment, water is heated to a temperature between 84°C and 100°C (ideally boiling), and then Na2CO3 (sodium carbonate) is added to the boiling water until the Na2CO3 is completely dissolved. The raw silk is added to the boiling water / Na2CO3 (100°C) and allowed to soak for approximately 15 to 90 minutes, with longer boiling times resulting in smaller silk protein fragments. In one embodiment, the volume of water is equal to about 0.4 x the weight of the raw silk, and the volume of Na2CO3 is equal to about 0.848 x the weight of the raw silk. In one embodiment, the volume of water is equal to 0.1 x the weight of raw silk and the volume of Na2CO3 is maintained at 2.12 g / L.This is demonstrated in Figures 72 and 73: silk mass (x-axis) was varied in the same volume of extraction solution (i.e., same volume of water and concentration of Na2CO3), and sericin removal (substantially free of sericin) was achieved, as demonstrated by an overall silk mass loss (y-axis) of 26 to 31 percent.

[0059] The aqueous NaCO solution is then drained, and excess water / NaCO is removed from the silk fibroin fibers (e.g., by ringing out the fibroin extract using manual or mechanical dehydration cycles). The resulting silk fibroin extract is rinsed with warm to hot water, typically at a temperature ranging from about 40°C to about 80°C, with at least one volume change of water (repeated as many times as necessary) to remove any remaining adsorbed sericin or contaminants. The resulting silk fibroin extract is silk fibroin substantially depleted of sericin. In one embodiment, the resulting silk fibroin extract is rinsed with water at a temperature of about 60°C. In one embodiment, the amount of rinse water for each cycle is equal to 0.1 L to 0.2 L times the weight of the raw silk. To maximize the rinsing effect, it may be advantageous to agitate, rotate, or circulate the rinse water. After rinsing, excess water is removed from the extracted silk fibroin fibers (e.g., manually or mechanically ringing out the fibroin extract). Alternatively, methods known to those skilled in the art, such as pressure, temperature, or other reagents, or a combination thereof, can be used for the purpose of sericin extraction. Alternatively, silk glands (100% sericin-free silk protein) can be directly removed from the worm. This can result in sericin-free liquid silk protein without any change in protein structure.

[0060] The extracted fibroin fibers are then completely dried. Once dry, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at ambient to boiling temperatures (step C1b). In one embodiment, the solvent is a solution of lithium bromide (LiBr) (the boiling point for LiBr is 140°C). Alternatively, the extracted fibroin fibers are not dried, but are wet and placed in a solvent; the solvent concentration can then be varied to achieve a concentration similar to that achieved when dry silk is added to the solvent. The final LiBr solvent concentration can range from 0.1 M to 9.3 M. Table D summarizes the molecular weight of silk dissolved from different concentrations of lithium bromide (LiBr) and from different extraction and dissolution sizes. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature along with the concentration of the dissolution solvent. Other solvents can be used, including, but not limited to, phosphate phosphoric acid, calcium nitrate, calcium chloride solution, or concentrated aqueous solutions of other inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed in the already heated solvent solution and then maintained at a temperature ranging from about 60° C. to about 140° C. for 1 to 168 hours. In one embodiment, the silk fibers should be fully immersed in the solvent solution and then placed in a drying oven at a temperature of about 100° C. for about 1 hour. [Table 2]

[0061] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an effect on the time required to completely dissolve the fibroin, as well as on the molecular weight and polydispersity of the resulting final SPF mixed solution. In one embodiment, the silk solvent solution concentration is less than or equal to 20% w / v. In addition, stirring during addition or dissolution may be used to promote dissolution at varying temperatures and concentrations. The temperature of the LiBr solution provides control over the molecular weight and polydispersity of the resulting silk protein fragment mixture. In one embodiment, higher temperatures dissolve the silk more quickly, resulting in enhanced process scalability and mass production of the silk solution. In one embodiment, using a LiBr solution heated to a temperature between 80°C and 140°C reduces the time required in the oven to achieve complete dissolution. Varying the time and temperature of the dissolving solvent at 60°C or above changes and controls the MW and polydispersity of the SPF mixed solution formed from native silk fibroin protein of the original molecular weight.

[0062] Alternatively, whole cocoons can be placed directly into a solvent, such as LiBr, bypassing the extraction (step B2). This requires subsequent filtration of the silkworm particles from the silk and solvent solution and removal of sericin (step C2) using methods known in the art to separate hydrophobic and hydrophilic proteins, such as column separation and / or chromatography, ion exchange, chemical precipitation with salt and / or pH, and / or enzymatic digestion and filtration or extraction (all methods are general examples of standard protein separation methods, but are not limited to these). Non-heat-treated cocoons from which the silkworms have been removed can instead be placed directly into a solvent, such as LiBr, bypassing the extraction. The methods described above can be used for sericin isolation, with the advantage that non-heat-treated cocoons contain significantly less silkworm debris.

[0063] Dialysis can be used to remove the dissolving solvent from the resulting dissolved fibroin protein fragment solution by dialyzing the solution against a volume of water (step E1). Pre-filtration before dialysis is useful to remove any debris (i.e., silkworm remnants) from the silk and LiBr solution (step D). In one example, a 0.1% to 1.0% silk-LiBr solution is filtered using a 3 μm or 5 μm filter at a flow rate of 200 to 300 mL / min, if desired, prior to dialysis and potential concentration. A method disclosed herein, as described above, is to use time and / or temperature to reduce the concentration from 9.3 M LiBr to a range of 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering creating a scalable process method. Alternatively, the 9.3 M LiBr-silk protein fragment solution may be diluted with water to facilitate debris filtration and dialysis without the use of additional time or temperature. The result of dissolution, upon filtration for the desired time and temperature, is a translucent, particle-free, room-temperature storage-stable LiBr solution of silk protein fragments of known MW and polydispersity. It is advantageous to periodically change the dialysis water (e.g., change the water after 1 hour, 4 hours, then every 12 hours for a total of six changes) until the solvent is removed. The total number of water volume changes can be varied based on the resulting concentration of the solvent used for dissolving and fragmenting the silk protein. After dialysis, the final silk solution can be further filtered to remove any remaining debris (i.e., silkworm remnants).

[0064] Alternatively, tangential flow filtration (TFF), a rapid and efficient method for the separation and purification of biomolecules, can be used to remove solvent from the resulting dissolved fibroin solution (step E2). TFF provides highly pure aqueous silk protein fragment solutions and allows for process scalability to produce large volumes of solution in a controlled and repeatable manner. The silk and LiBr solution can be diluted (down from 20% silk to 0.1% silk in either water or LiBr) prior to TFF. Prefiltration, as described above, prior to TFF processing can maintain filter efficiency and potentially avoid the formation of a silk gel boundary layer on the surface of the filter as a result of the presence of debris particles. Prefiltration prior to TFF also helps remove any remaining debris (i.e., silkworm remnants) from the silk and LiBr solution, which could cause spontaneous or long-term gelation of the resulting water-only solution (step D). TFF can be used, with recirculation or single pass, to produce water-silk protein fragment solutions ranging from 0.1% silk to 30.0% silk (more preferably 0.1%-6.0% silk). Different cutoff size TFF membranes may be required based on the desired concentration, molecular weight, and polydispersity of the silk protein fragment mixture in solution. Membranes ranging from 1 to 100 kDa may be required to vary the molecular weight silk solutions produced, for example, by varying the length of extraction boiling time or the time and temperature in the dissolving solvent (e.g., LiBr). In one embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution to achieve the final desired silk-to-water ratio. Additionally, TFF single-pass, TFF, and other methods known in the art, such as falling film evaporators, can be used to concentrate the solution (resulting in a desired concentration ranging from 0.1% to 30% silk) following removal of the dissolving solvent (e.g., LiBr). This can be used as an alternative to the standard HFIP concentration method known in the art to create water-based solutions. Larger pore membranes can also be utilized to filter out small silk protein fragments and create higher molecular weight silk solutions with and / or without tighter polydispersity values.

[0065] Table C summarizes the molecular weights of several embodiments of silk protein solutions of the present disclosure. The processing conditions for the silk protein solutions were as follows: 100°C extraction for 20 minutes, room temperature rinse, LiBr for 4-6 hours in a 60°C oven. The TFF processing conditions for the water-soluble films were as follows: 100°C extraction for 60 minutes, 60°C rinse, 100°C LiBr for 60 minutes in a 100°C oven. Figures 93-104 further demonstrate the extraction times, LiBr dissolution conditions, and TFF processing operations, as well as the resulting exemplary molecular weights and polydispersities. These examples are not intended to be limiting, but rather to demonstrate the feasibility of identifying parameters for silk fragment solutions of specific molecular weights. [Table 3]

[0066] Assays for LiBr and Na2CO3 detection were performed using an HPLC system equipped with an evaporative light scattering detector (ELSD). Calculations were performed by linear regression of the resulting peak areas for the analytes plotted against concentration. Two or more samples of various formulations of the present disclosure were used for sample preparation and analysis. Typically, four samples of different formulations were weighed directly into a 10 mL volumetric flask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and maintained at 2-8 °C with occasional shaking for 2 hours to extract the analytes from the film. After 2 hours, the solution was diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask was transferred into an HPLC vial and injected into the HPLC-ELSD system for estimation of sodium carbonate and lithium bromide.

[0067] The analytical method developed for the quantification of Na2CO3 and LiBr in silk protein preparations was found to be linear within the range of 10-165 μg / mL, with RSDs of 2% and 1% for area and 0.38% and 0.19% for retention time for sodium carbonate and lithium bromide, respectively. This analytical method can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein preparations.

[0068] The final silk protein fragment solution is pure silk protein fragments and water with PPM to non-detectable levels of particulate debris and / or process contaminants, including LiBr and Na2CO3. Tables A and B summarize the LiBr and Na2CO3 concentrations in the solutions of the present disclosure. In Table A, the processing conditions included 100°C extraction for 60 minutes, a 60°C rinse, and 100°C LiBr for 60 minutes in a 100°C oven. TFF conditions, including pressure differential and number of diafiltration volumes, were varied. In Table B, the processing conditions included 100°C boiling for 60 minutes, a 60°C rinse, and LiBr for 4-6 hours in a 60°C oven. [Table 4] [Table 5]

[0069] The aqueous silk fragment solution, the lyophilized silk protein fragment mixture, or any other composition containing SPF can be sterilized according to standard methods in the art, including but not limited to filtration, heating, radiation, or electron beam. Due to its shorter protein polymer length, the silk protein fragment mixture is expected to withstand sterilization better than the intact silk protein solution described in the art. Furthermore, silk articles made from the SPF mixture described herein can be sterilized to suit their intended use.

[0070] 71 is a flow chart illustrating various parameters that can be modified during the extraction and dissolution steps of the process to produce a silk protein fragment solution of the present disclosure. Selected method parameters can be altered to achieve specific final solution characteristics, such as molecular weight and polydispersity, depending on the intended use. It should be understood that not all of the illustrated steps are necessarily required to produce all silk solutions of the present disclosure.

[0071] In one embodiment, the process for producing a silk protein fragment solution of the present disclosure includes forming silk cocoon pieces from Bombyx mori silkworms; extracting the pieces in a solution of water and Na2CO3 at about 100°C for about 60 minutes, where the volume of water is equal to about 0.4 x the raw silk weight and the amount of Na2CO3 is about 0.848 x the weight of the pieces to form a silk fibroin extract; rinsing the silk fibroin extract three times in a volume of rinse water at about 60°C for about 20 minutes per rinse, where the rinse water for each cycle is equal to about 0.2 L x the weight of the pieces; The process includes removing excess water; drying the silk fibroin extract; dissolving the dried silk fibroin extract in a LiBr solution (where the LiBr solution is first heated to about 100°C to create and maintain a silk and LiBr solution); placing the silk and LiBr solution in a drying oven at about 100°C for about 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into a mixture with the desired molecular weight and polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to produce a 1% silk solution; and removing the solvent from the solution using tangential flow filtration (TFF). In one embodiment, a 10 kDa membrane is used to purify the silk solution to produce the final desired silk-to-water ratio. TFF can then be used to further concentrate the pure silk solution to a concentration of 2% silk in water.

[0072] Each process step, from raw cocoons to dialysis, can be scaled up to increase production efficiency. Whole cocoons are currently purchased as raw materials, but pre-cleaned or non-heat-treated cocoons are also used (silkworm removal leaves traces of debris). Cutting and cleaning the cocoons is a manual process, but for scalability, the process could be made less labor-intensive by, for example, using an automated machine in combination with compressed air to remove worms and any particulates, or by using a cutting mill to cut the cocoons into smaller pieces. The extraction step is currently performed in small batches and can be completed in larger vessels, such as industrial washing machines, that can maintain temperatures between 60°C and 100°C or thereabouts. The rinsing step can also be completed in industrial washing machines, eliminating the manual rinse cycle. Dissolution of silk in LiBr solution can occur in vessels other than convection ovens, such as stirred-tank reactors. Dialyzing silk through a series of water changes is a manual, time-intensive process that can be accelerated by varying certain parameters, e.g., diluting the silk solution before dialysis. The dialysis process can be scaled for manufacturing by using semi-automated equipment, e.g., tangential flow filtration systems.

[0073] While not wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent and silk solutions with different viscosities, uniformities, and colors. Also, while not wishing to be bound by any particular theory, increasing the temperature for extraction, extending the extraction time, using a higher temperature LiBr solution when dissolving the silk (e.g., in an oven as shown herein or an alternative heat source), both initially and over a longer period of time, and increasing the time at temperature all resulted in lower viscosity, more uniform solvent and silk solutions. While nearly all parameters resulted in viable silk solutions, methods that achieve complete dissolution in less than 4-6 hours are preferred for process scalability.

[0074] In one embodiment, a solution of silk fibroin protein fragments having a weight average selected from between about 6 kDa and about 17 kDa is prepared according to the following steps: degumming the silk source by adding the silk source to an aqueous solution of boiling (100°C) sodium carbonate for a treatment time of about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract containing non-detectable levels of sericin; draining the solution from the silk fibroin extract; and heating the silk fibroin extract at about 60°C to about 140°C. The method includes dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of 140°C, maintaining the silk fibroin-lithium bromide solution in an oven having a temperature of about 140°C for up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk protein fragments, the aqueous solution comprising fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm of sodium carbonate residue as measured using a high-performance liquid chromatography sodium carbonate assay. The aqueous solution of silk fibroin protein fragments may be freeze-dried. In some embodiments, the silk fibroin protein fragment solution may be further processed into various forms, including gels, powders, and nanofibers.

[0075] In one embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from between about 17 kDa and about 39 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes to effect degumming; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C upon placing the silk fibroin extract in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in a drying oven having a temperature in the range of about 60°C to about 100°C for a period of up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk fibroin protein fragments, the aqueous solution of silk fibroin protein fragments comprising about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments comprising about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of silk fibroin protein fragments comprising fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay.

[0076] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight average molecular weight selected from between about 6 kDa and about 17 kDa comprises the following steps: degumming a silk source by adding the silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes and about 60 minutes; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; and removing the silk fibroin extract from the solution containing undetectable levels of sericin. The method includes dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature ranging from 0°C to about 140°C, maintaining the silk fibroin-lithium bromide solution in an oven having a temperature of about 140°C for a period of at least 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk protein fragments, the aqueous solution comprising fragments having an average weight-average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin protein fragments may contain less than 100 ppm of sodium carbonate residue as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin protein fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin protein fragments.The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding at least one of zinc oxide or titanium dioxide. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The gel may contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

[0077] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight average molecular weight selected from between about 17 kDa and about 39 kDa comprises the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes to effect degumming, removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin, draining the solution from the silk fibroin extract, dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C, and then dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 60°C to about 80°C. maintaining the silk fibroin-lithium bromide solution in an oven having a temperature ranging from about 10 ppm to about 100°C for a period of at least 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of pure silk fibroin protein fragments (the aqueous solution of pure silk fibroin protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments comprises about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of pure silk fibroin protein fragments comprises fragments having an average weight-average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0). The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin protein fragments may contain less than 100 ppm of residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof.The aqueous solution of pure silk fibroin protein fragments may be freeze-dried. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding at least one of zinc oxide or titanium dioxide. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel can be made from an aqueous solution of the pure silk fibroin protein fragments produced by the present method. The gel can contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel can have a silk content of at least 2% and a vitamin content of at least 20%.

[0078] In one embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from between about 39 kDa and about 80 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to effect degumming; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; and placing the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C. The method includes the steps of dissolving the silk fibroin extract and maintaining the silk fibroin-lithium bromide solution in a drying oven having a temperature in the range of about 60°C to about 100°C for a period of up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk fibroin protein fragments, the aqueous solution of silk fibroin protein fragments comprising about 10 ppm to about 300 ppm of lithium bromide residue, about 10 ppm to about 100 ppm of sodium carbonate residue, fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa, and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm sodium carbonate residual as measured using a high performance liquid chromatography sodium carbonate assay. In some embodiments, the method may further comprise adding an active agent (e.g., a therapeutic agent) to the aqueous solution of pure silk fibroin protein fragments. The method may further comprise adding an active agent selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin protein fragments may be lyophilized.The method may further include adding an alpha-hydroxy acid to the aqueous solution of pure silk fibroin protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The gel may contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% by weight and a vitamin content of at least 20% by weight.

[0079] The molecular weight of the silk protein fragments can be controlled based on specific parameters utilized during the extraction step, including extraction time and temperature; the dissolution step, including the LiBr temperature during the silk immersion in lithium bromide and the time the solution is maintained at a specific temperature; and the filtration step. By controlling process parameters using the disclosed method, it is possible to create SPF mixture solutions with polydispersities equal to or less than 2.5 at a variety of different molecular weights, ranging from 1 kDa to 250 kDa, 5 kDa to 200 kDa, 5 kDa to 150 kDa, 10 kDa to 150 kDa, or 10 kDa to 80 kDa. By varying process parameters to achieve silk solutions with different molecular weights, it is possible to target a range of final fragment mixture products with desirable polydispersities equal to or less than 2.5 based on desired performance requirements. For example, lower molecular weight silk films containing drugs may have faster release rates compared to higher molecular weight SPF preparations. Additionally, SPF mixed solutions with polydispersities greater than 2.5 can be achieved. Furthermore, two solutions with different average molecular weights and polydispersities can be mixed to create a combined solution. Alternatively, liquid silk glands (100% sericin-free silk protein) extracted directly from silkworms can be used in combination with any of the SPF mixed solutions disclosed herein. The molecular weights of pure silk fibroin-based protein fragment compositions were determined using high-pressure liquid chromatography (HPLC) with a refractive index detector (RID). Polydispersities were calculated using Cirrus GPC Online GPC / SEC software version 3.3 (Agilent).

[0080] Differences in processing parameters can result in regenerated silk fibroin that varies in molecular weight and peptide chain size distribution (polydispersity, PD), which in turn affects the performance of the regenerated silk fibroin, including mechanical strength, water solubility, etc.

[0081] Parameters were varied during the processing of raw silk cocoons into silk solution. Varying these parameters affected the MW of the resulting silk solution. The manipulated parameters included (i) extraction time and temperature, (ii) LiBr temperature, (iii) dissolution oven temperature, and (iv) dissolution time. The molecular weight was determined by mass spectrometry, as shown in Figures 74-90.

[0082] Experiments were conducted to determine the effect of varying extraction time. Figures 74-90 are graphs showing these results, and Tables A-G summarize the results. The following is a summary: A sericin extraction time of 30 minutes resulted in a higher molecular weight than a sericin extraction time of 60 minutes. -The molecular weight decreases over time in the oven. -140°C LiBr and oven resulted in a lower confidence interval limit below a molecular weight of 9500 Da. - The 30 minute extractions at 1 hour and 4 hours have undigested silk. A 30 minute extraction at -1 hour resulted in a significantly higher molecular weight with the lower limit of the confidence interval being 35,000 Da. The molecular weight range within which the upper limit of the confidence interval was reached was 18,000 to 216,000 Da (which is important for providing a solution with a specific upper limit). [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12]

[0083] Experiments were conducted to determine the effect of varying the extraction temperature. Figure 74 is a graph showing these results, and Table H summarizes the results. The following is a summary: Sericin extraction at -90°C resulted in a higher MW than sericin extracted at 100°C. Both -90°C and 100°C show a decrease in MW over time in the oven. [Table 13]

[0084] Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) when added to silk. Figures 82-83 are graphs showing these results, and Tables I-J summarize the results. The following is a summary: - No effect on molecular weight or confidence intervals (all CI approximately 10500-6500 Da) Studies have shown that when LiBr is added and begins to dissolve, the temperature of the LiBr-silk melt drops rapidly below the original LiBr temperature due to the majority of its mass being silk at room temperature. [Table 14] [Table 15]

[0085] Experiments were conducted to determine the effect of oven / melt temperature. Figures 84-88 are graphs showing these results, and Tables K-O summarize the results. The following is a summary: - Oven temperature has less effect on silk extracted for 60 minutes than on silk extracted for 30 minutes. Without wishing to be bound by theory, it is believed that the 30 minute silk is less degraded during extraction and therefore oven temperature has more effect on the larger MW and less degraded portion of the silk. For the -60°C vs. 140°C oven, silk extracted for 30 minutes showed a very significant effect of the lower MW at the higher oven temperature, while silk extracted for 60 minutes had an effect, but it was much less. The -140°C oven yielded a lower limit in the confidence interval at approximately 6000 Da. [Table 16] [Table 17] [Table 18] [Table 19] [Table 20]

[0086] In one embodiment, the methods disclosed herein result in solutions with characteristics that can be controlled during production, including, but not limited to, MW, which can be varied by varying extraction and / or dissolution time, as well as temperature (e.g., LiBr temperature), pressure, and filtration (e.g., size exclusion chromatography). Structure—removal or cleavage of heavy or light chains of fibroin protein polymers; purity—hot water rinse temperature for improved sericin removal or filterability for improved particle removal that adversely affects the storage stability of the silk fragment protein mixture solution; color—solution color can be controlled, along with, for example, LiBr temperature and time; viscosity; clarity; and solution stability. The pH of the resulting solution is typically about 7, but can be modified using acid or base to suit storage requirements.

[0087] Raw silk cocoons from the silkworm Bombyx mori were cut into small pieces. The raw silk cocoon pieces were boiled in an aqueous solution of Na2CO3 (approximately 100°C) for approximately 30 to 60 minutes to remove sericin (degumming). The volume of water used was approximately 0.4 times the weight of the raw silk, and the amount of Na2CO3 was approximately 0.848 times the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The volume of rinse water for each cycle was 0.2 L times the weight of the raw silk cocoon pieces. Excess water was removed from the degummed silk cocoon pieces. After the DI water washing step, the wet, degummed silk cocoon pieces were allowed to dry at room temperature. Degummed silk cocoon pieces were mixed with a LiBr solution, and the mixture was heated to approximately 100°C. The warmed mixture was placed in a dry oven and heated at a temperature ranging from approximately 60°C to approximately 140°C for approximately 60 minutes to achieve complete dissolution of the native silk proteins. The resulting solution was cooled to room temperature and then dialyzed using a 3,500 Da MWCO membrane to remove the LiBr salt. Multiple exchanges were performed using octane bromide (Br - ) Br as determined in a hydrolyzed fibroin solution lead on a dual-junction ion-selective electrode. - It was carried out in Di water until the ions were below 1 ppm.

[0088] The resulting silk fibroin aqueous solution has a concentration of approximately 8.0% w / v containing pure silk fibroin protein fragments with an average weight-average molecular weight selected from between about 6 kDa to about 16 kDa, about 17 kDa to about 39 kDa, and about 39 kDa to about 80 kDa and a polydispersity of about 1.5 to about 3.0. The 8.0% w / v was diluted with DI water to provide 1.0% w / v, 2.0% w / v, 3.0% w / v, 4.0% w / v, and 5.0% w / v coating solutions.

[0089] Various % silk concentrations have been produced through the use of tangential flow filtration (TFF). In all cases, a 1% silk solution was used as the input feed. A range of 750-18,000 mL of 1% silk solution was used as the starting volume. The solution is diafiltered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, the solution undergoes ultrafiltration to increase concentration through water removal. See example below. Six silk solutions were utilized in a standard silk construction with the following results: Solution #1 has a silk concentration of 5.9 wt %, an average MW of 19.8 kDa, and a PDI of 2.2 (made by 60 min boiling extraction, LiBr dissolution at 100° C. for 1 hour). Solution #2 is 6.4 wt% silk concentration (made by 30 min boiling extraction, LiBr dissolution at 60° C. for 4 h). Solution #3 is 6.17 wt% silk concentration (made by 30 min boiling extraction, LiBr dissolution at 100° C. for 1 hour). Solution #4 has a silk concentration of 7.30% by weight: The 7.30% silk solution was produced from a 30-minute extraction batch of 100 g of silk cocoons per batch. The extracted silk fibers were then dissolved in 9.3 M LiBr at 100°C for 1 hour in a 100°C oven. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 15,500 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of approximately 1300 mL. 1262 mL of 7.30% silk was then recovered. Water was added to the feed solution to help remove the remaining solution, and 547 mL of 3.91% silk was then recovered. Solution #5 has a silk concentration of 6.44 wt%: The 6.44 wt% silk solution was produced by starting with 60-minute extraction batches of a mixture of 25, 33, 50, 75, and 100 g of silk cocoons per batch. The extracted silk fibers were then dissolved in 9.3 M LiBr at 100°C for 1 hour in an oven. 35, 42, 50, and 71 g of silk fibers per batch were dissolved and combined to create 20% silk in LiBr. The dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 17,000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of approximately 3000 mL. 1490 mL of 6.44% silk was then recovered. Water was added to the feed solution to help remove any remaining solution, and 1454 mL of 4.88% silk was then recovered. Solution #6 has a silk concentration of 2.70% by weight: A 2.70% silk solution was produced starting with a 60-minute extraction batch of 25 g of silk cocoons per batch. The extracted silk fibers were then dissolved in a 100°C oven for 1 hour using 9.3 M LiBr at 100°C. 35.48 g of silk fibers were dissolved per batch to produce 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 1000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of around 300 mL. 312 mL of 2.7% silk was then recovered.

[0090] The preparation of higher molecular weight silk fibroin solutions is shown in Table O. [Table 21]

[0091] Silk aqueous coating compositions for application to textiles are given in Tables P and Q below. [Table 22] [Table 23]

[0092] Three silk solutions were utilized in film preparation with the following results: Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and 2.2 PD (made by boiling extraction for 60 minutes, LiBr dissolution at 100° C. for 1 hour). Solution #2 is a 6.4% silk concentration (made by boiling extraction for 30 minutes, LiBr dissolution at 60°C for 4 hours). Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes, 100° C. LiBr dissolution for 1 hour).

[0093] Films were prepared according to Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published online September 22, 2011; doi:10.1038 / nprot.2011.379). Four milliliters of 1% or 2% (weight / volume) aqueous silk solution was added to a 100 mm Petri dish (the volume of silk can vary for thicker or thinner films and is not critical) and allowed to dry uncovered overnight. The bottom of a vacuum desiccator was filled with water. The dried film was placed in the desiccator, and a vacuum was applied to allow the film to water-anneal for 4 hours before removal from the dish. Films cast from Solution #1 did not yield structurally continuous films. The film cracked into several small pieces. These small pieces dissolved in water despite the water-annealing treatment.

[0094] Silk solutions of various molecular weights and / or combinations of molecular weights can be optimized for gel applications. The following provides an example of this process, but it is not intended to be limiting in application or formulation. Three silk solutions were utilized in gel creation with the following results: Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and 2.2 PD (made by boiling extraction for 60 minutes, LiBr dissolution at 100° C. for 1 hour). Solution #2 is a 6.4% silk concentration (made by boiling extraction for 30 minutes, LiBr dissolution at 60°C for 4 hours). Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes, 100° C. LiBr dissolution for 1 hour).

[0095] "Egel" is an electrogelation process as described in Rockwood et al. Briefly, 10 ml of aqueous silk solution was added to a 50 ml conical tube, and a pair of platinum wire electrodes was immersed in the silk solution. A potential of 20 volts was applied to the platinum electrodes for 5 minutes, and the power supply was turned off to collect the gel. Solution #1 did not form EGEL over the 5 minutes of applied current.

[0096] Solutions #2 and #3 were gelled according to published horseradish peroxidase (HRP) protocols, and behavior appeared to be typical of published solutions.

[0097] Materials and Methods: The following equipment and materials were used in the determination of silk molecular weight: Agilent 1100 with chemstation software version 10.01, refractive index detector (RID), analytical balance, volumetric flasks (1000 mL, 10 mL, and 5 mL), HPLC-grade water, ACS-grade sodium chloride, ACS-grade sodium phosphate dibasic heptahydrate, phosphoric acid, dextran MW standards—nominal molecular weights of 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa, 50 mL PET or polypropylene disposable centrifuge tubes, graduated pipettes, amber glass HPLC vials with Teflon caps; Phenomenex PolySep GFC P-4000 column (size: 7.8 mm x 300 mm).

[0098] Procedure steps: A) Preparation of 1 L mobile phase (0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer) Take a clean, dry 250 mL beaker, place it on a balance, and tare it. Add approximately 3.3509 g of sodium phosphate dibasic heptahydrate to the beaker. Note the exact weight of sodium phosphate dibasic. Dissolve the weighed sodium phosphate by adding 100 mL of HPLC water to the beaker. Be careful not to spill the contents of the beaker. Carefully transfer the solution into a clean, dry 1000 mL volumetric flask. Rinse the beaker and transfer the rinse into the volumetric flask. Repeat the rinse 4-5 times. Accurately weigh approximately 5.8440 g of sodium chloride into a separate, clean, dry 250 mL beaker. Dissolve the weighed sodium chloride in 50 mL of water and transfer the solution to the sodium phosphate solution in the volumetric flask. Rinse the beaker and transfer the rinse into the volumetric flask. Adjust the pH of the solution to 7.0 ± 0.2 using phosphoric acid. Bring the volume in the volumetric flask to 1000 mL with HPLC water and stir vigorously to mix the solution uniformly. Filter the solution through a 0.45 μm polyamide membrane filter. Transfer the solution to a clean, dry solvent bottle and label the bottle. The volume of this solution can be varied as needed by correspondingly varying the amounts of sodium phosphate dibasic heptahydrate and sodium chloride.

[0099] B) Preparation of dextran molecular weight standard solution At least five different molecular weight standards are used for each batch of samples run so that the expected values ​​of the samples being tested are bracketed by the values ​​of the standards used. Six 20 mL scintillation glass vials are labeled with each molecular weight standard. Approximately 5 mg of each dextran molecular weight standard is accurately weighed and the weight is recorded. The dextran molecular weight standards are dissolved in 5 mL of mobile phase to make a 1 mg / mL standard solution.

[0100] C) Preparation of sample solution When preparing the sample solution, if the amount of sample available is limited, the preparation may be adjusted as long as the ratio is maintained. Depending on the sample type and silk protein content in the sample, weigh enough sample into a 50 mL disposable centrifuge tube on an analytical balance to create a 1 mg / mL sample solution for analysis. Dissolve the sample in an equal volume of mobile phase to create a 1 mg / mL solution. Cap the tube tightly and mix the sample (in solution). Allow the sample solution to stand at room temperature for 30 minutes. Mix the sample solution gently again for 1 minute and centrifuge at 4000 RPM for 10 minutes.

[0101] D) HPLC analysis of the samples Transfer 1.0 mL of all standard and sample solutions into individual HPLC vials. Inject molecular weight standards (single injection each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions: [Table 24]

[0102] Data Analysis and Calculations - Calculation of Average Molecular Weight Using Cirrus Software Chromatographic data files of standards and analytical samples are uploaded into the Cirrus SEC data collection and molecular weight analysis software. For each injection of sample, the weight average molecular weight (M w ), number average molecular weight (M n ), peak average molecular weight (M p ), and polydispersity are calculated.

[0103] Spider silk fragments Spider silk is a natural polymer composed of three domains: a repetitive middle core domain that occupies the majority of the protein chain, and nonrepetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX (SEQ ID NO: 6)) polypeptides, alternate. Dragline silk is a protein complex composed of major ampullate gland dragline silk protein 1 (MaSp1) and major ampullate gland dragline silk protein 2 (MaSp2). Both silks are approximately 3500 amino acids long. MaSp1 can be found in the fiber core and periphery, while MaSp2 clusters in a specific core region. The large central domains of MaSp1 and MaSp2 are organized in a block copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX (SEQ ID NO: 6)) polypeptides, alternate in the core domain. Specific secondary structures have been assigned to the poly(A) / (GA), GGX, and GPGXX (SEQ ID NO: 6) motifs, containing β-sheets, α-helices, and β-spirals, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain are responsible for the mechanical properties of spider silk, whereas the non-repetitive N- and C-terminal domains are essential for the storage of the liquid silk dope in the lumen and for fiber formation in the spinning ducts.

[0104] The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which account for 15% of the total amino acid content in MaSp2, whereas MaSp1 lacks them. By calculating the number of proline residues in N. clavipes dragline silk, it is possible to estimate the presence of the two proteins in the fiber: 81% MaSp1 and 19% MaSp2. Different spiders have different ratios of MaSp1 and MaSp2. For example, dragline silk fibers from the orb weaver Argiope aurantia contain 41% MaSp1 and 59% MaSp2. Such changes in the ratio of major ampullate gland silk can affect the performance of the silk fiber.

[0105] At least seven different types of silk proteins are known for spiders of the Araneidae family. Silks differ in primary sequence, physical properties, and function. For example, dragline silk, used to construct frames, radii, and lifelines, is known for its remarkable mechanical properties, including strength, toughness, and elasticity. On an equal weight basis, spider silk has higher toughness than steel and Kevlar. Flagellate silk, found in spiral weft threads, has an extensibility of up to 500%. Minor ampullate silk, found in the auxiliary spirals of spherical spider webs and wrapped around prey, has high toughness and strength similar to major ampullate silk, but does not hypercontract in water.

[0106] Spider silk is known for its high tensile strength and toughness. Recombinant silk proteins also confer advantageous properties to cosmetic or dermatological compositions, such as improved moisturizing or softening properties, good film-forming properties, and low surface density, among others. The diverse and unique biomechanical properties, along with biocompatibility and a slow degradation rate, make spider silk an excellent candidate for tissue engineering, guided tissue regeneration, and drug delivery biomaterials, cosmetic products (e.g., nail and hair strengtheners, skin care products), and industrial materials (e.g., nanowires, nanofibers, surface coatings).

[0107] In one embodiment, the silk protein may comprise a polypeptide derived from a natural spider silk protein. The polypeptide is not particularly limited as long as it is derived from a natural spider silk protein. Examples of the polypeptide include natural spider silk proteins and recombinant spider silk proteins, such as mutants, analogs, derivatives, or the like of natural spider silk proteins. For superior tenacity, the polypeptide may be derived from a major dragline silk protein produced in the major ampullate gland of spiders. Examples of major ampullate gland spidroins MaSp1 and MaSp2 from Nephila clavipes and ADF3 and ADF4 from Araneus diadematus. Examples of polypeptides derived from major dragline silk proteins include mutants, analogs, derivatives, or the like of major dragline silk proteins. Furthermore, the polypeptide may be derived from a flagellate gland silk protein produced in the flagellate gland of spiders. Examples of flagellate gland silk proteins include flagellate gland silk proteins derived from Nephila clavipes.

[0108] Examples of polypeptides derived from major dragline silk proteins include polypeptides containing two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, polypeptides derived from major dragline silk proteins may be polypeptides containing units of the amino acid sequence represented by Formula 1:REP1-REP2(1), which have at their C-terminus an amino acid sequence represented by any of SEQ ID NOS:52-54 in U.S. Patent No. 9,051,453, the entirety of which is incorporated by reference, or an amino acid sequence having 90% or greater identity to any of SEQ ID NOS:52-54 set forth in U.S. Patent No. 9,051,453, the entirety of which is also incorporated by reference. In polypeptides derived from major dragline silk proteins, the units of the amino acid sequence represented by Formula 1:REP1-REP2(1) may be identical or different from each other. When recombinant proteins are produced using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from the major drugline silk protein is, from the viewpoint of productivity, 500 kDa or less, or 300 kDa or less, or 200 kDa or less.

[0109] In formula (1), REP1 represents polyalanine. In REP1, the number of consecutive alanine residues is preferably 2 or more, more preferably 3 or more, even more preferably 4 or more, and particularly preferably 5 or more. Furthermore, in REP1, the number of consecutive alanine residues is preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and particularly preferably 10 or less. In formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine, and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more of the total number of amino acid residues contained therein.

[0110] In the primary dragline silk, REP1 corresponds to the crystalline region in the fiber where crystalline β-sheets are formed, while REP2 corresponds to the amorphous region in the fiber where the majority of the components lack regular organization and have more flexibility. Furthermore, [REP1-REP2] corresponds to the repeat region (repeat sequence) composed of crystalline and amorphous regions, which is a characteristic sequence of dragline silk proteins.

[0111] Recombinant silk fragments In some embodiments, recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide, or a recombinant mussel silk polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a recombinant spider silk polypeptide of the Araneidae or Araneoids, or a recombinant insect silk polypeptide of Bombyx mori. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a recombinant spider silk polypeptide of the Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a block copolymer having repeating units derived from a naturally occurring spider silk polypeptide of the Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a block copolymer having synthetic repeating units derived from a spider silk polypeptide of the Araneidae or Araneoids and non-repeating units derived from naturally occurring repeating units of a spider silk polypeptide of the Araneidae or Araneoids.

[0112] Recent advances in genetic engineering have provided a route to producing various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein that is heterologously produced in a prokaryotic or eukaryotic expression system using genetic engineering methods.

[0113] Various methods for synthesizing recombinant silk peptides are known and are described by Ausubel et al., Current Protocols in Molecular Biology § 8 (John Wiley & Sons 1987, (1990)), which is incorporated herein by reference. Escherichia coli, a gram-negative rod-shaped bacterium, is a well-established host for industrial-scale production of proteins. Therefore, the majority of recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively low cost, and can be scaled up to produce larger amounts of protein.

[0114] Recombinant silk proteins can be produced by transformed prokaryotic or eukaryotic systems containing cDNAs encoding silk proteins, fragments of these proteins, or analogs of such proteins. The recombinant DNA approach allows for the production of recombinant silks with programmed sequences, secondary structures, architectures, and precise molecular weights. There are four major steps in this process: (i) the design and assembly of synthetic silk-like genes into genetic "cassettes," (ii) the insertion of these segments into DNA recombinant vectors, (iii) the transformation of these recombinant DNA molecules into host cells, and (iv) the expression and purification of selected clones.

[0115] The term "recombinant vector," as used herein, includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, or plants) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify a specific desired DNA fragment and may lack functional sequences necessary for expression of the desired DNA fragment.

[0116] Prokaryotic systems include gram-negative or gram-positive bacteria. Prokaryotic expression vectors may contain an origin of replication recognizable by the host organism, a homologous or heterologous promoter that is functional in the host, and a DNA sequence encoding a spider silk protein, a fragment of this protein, or a similar protein. Non-limiting examples of prokaryotic expression organisms are cells of Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus (e.g., Bacillus subtilis), Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Propionibacterium, Staphylococcus, or Streptomyces.

[0117] Eukaryotic systems include yeast and insect, mammalian, or plant cells, in which case the expression vector may comprise a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding the spider silk protein, a fragment, or a similar protein, a polyadenylation sequence, a transcription termination site, and finally, a selection gene. Non-limiting examples of eukaryotic expression organisms include yeast (such as Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous, ascosporogenous), filamentous fungi (such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum, Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia (e.g., Pichia pastoris), or Yarrowia cells), mammalian cells (such as HeLa cells, COS cells, CHO cells), insect cells (such as Sf9 cells, MEL cells), and "insect host cells" (such as Spodoptera frugiperda or Trichoplusia ni cells). SF9 cells, SF-21 cells, or High-Five cells (SF-9 and SF-21 are ovary cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia ni) are "plant host cells," such as tobacco cells, potato cells, or pea cells.

[0118] Various heterologous host systems have been explored to produce different types of recombinant silk. Recombinant partial spidroins as well as engineered silks have been cloned and expressed in bacteria (Escherichia coli), yeast (Pichia pastoris), insects (Bombyx mori larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT / hamster), and transgenic animals (mouse, goat). The majority of silk proteins are produced with N- or C-terminal His tags to simplify purification and produce sufficient amounts of protein.

[0119] In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems may include transgenic animals and plants. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include E. coli. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include transgenic B. mori silkworms generated using genome editing techniques (e.g., CRISPR). Recombinant silk proteins in the present disclosure include synthetic proteins based on repeating units of natural silk proteins. In addition to synthetic repetitive silk protein sequences, they can additionally contain one or more naturally occurring non-repetitive silk protein sequences.

[0120] In some embodiments, "recombinant silk protein" refers to recombinant silkworm silk protein or a fragment thereof. Recombinant production of silk fibroin and silk sericin has been reported. A variety of hosts are used for production, including Escherichia coli, Sacchromyces cerevisiae, Pseudomonas sp., Rhodopseudomonas sp., Bacillus sp., and Strepomyces. See EP0230702, incorporated herein by reference in its entirety.

[0121] Also provided herein is the design and biological synthesis of silk fibroin protein-like multiblock polymers containing the GAGAGX (SEQ ID NO: 1) hexapeptide (X is A, Y, V, or S) derived from the repeating domain of the B. mori silk heavy chain (H chain).

[0122] In some embodiments, the present disclosure provides silk protein-like multiblock polymers derived from the repeat domain of B. mori silk heavy chain (H chain) containing GAGAGS (SEQ ID NO: 2) hexapeptide repeat units. GAGAGS (SEQ ID NO: 2) hexapeptide repeat units are the core units of the H chain and play an important role in the formation of crystalline domains. Silk protein-like multiblock polymers containing GAGAGS (SEQ ID NO: 2) hexapeptide repeat units spontaneously aggregate into a β-sheet structure, similar to natural silk fibroin protein, and the silk protein-like multiblock polymers have any weight-average molecular weight described herein.

[0123] In some embodiments, the present disclosure provides a silk peptide-like multiblock copolymer composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat fragment derived from the heavy chain of a B. mori silk chain and a mammalian elastin VPGVG (SEQ ID NO: 3) motif produced by E. coli. In some embodiments, the present disclosure provides a fusion silk fibroin protein composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat fragment derived from the heavy chain of a B. mori silk chain and a GVGVP (SEQ ID NO: 4) motif produced by E. coli, wherein the silk protein-like multiblock polymer has any weight-average molecular weight described herein.

[0124] In some embodiments, the present disclosure provides (GAGAGS) 16 (SEQ ID NO: 55) In some embodiments, the present disclosure provides a B. mori silkworm recombinant protein composed of a repeat fragment (GAGAGS) produced by E. coli. 16 (SEQ ID NO: 55) Repetitive and non-repetitive (GAGAGS) fragments 16-F-COOH (SEQ ID NO: 56), (GAGAGS) 16 -FF-COOH (SEQ ID NO: 57), (GAGAGS) 16 -FFF-COOH (SEQ ID NO: 58), (GAGAGS) 16 -FFFF-COOH (SEQ ID NO: 59), (GAGAGS) 16 -FFFFFFFF-COOH (SEQ ID NO: 60), (GAGAGS) 16 -FFFFFFFFFFFF-COOH (SEQ ID NO: 61), wherein F has the following amino acid sequence: SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG (SEQ ID NO: 5), and the silk protein-like multiblock polymer has any weight average molecular weight described herein.

[0125] In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or fragment thereof. The production of recombinant spider silk proteins based on partial cDNA clones has been reported. Recombinant spider silk proteins produced as such contain portions of repeat sequences derived from the drug-line spider silk protein spidroin 1 from the spider Nephila clavipes. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87:7120-7124 (1990). A cDNA clone encoding a portion of the repeat sequence of spidroin 2, a second fibroin protein from Nephila clavipes dragline silk, and its recombinant synthesis are described in J. Biol. Chem., 1992, volume 267, pp. 19320-19324. Recombinant synthesis of spider silk proteins, including Nephila clavipes protein fragments and variants, from transformed E. coli is described in U.S. Patents 5,728,810 and 5,989,894. A cDNA clone encoding a small ampullate gland spider silk protein and its expression are described in U.S. Patents 5,733,771 and 5,756,677. A cDNA clone encoding a flagellate gland silk protein from a spider that spins a spherical spider web is described in U.S. Patents 5,733,771 and 5,756,677. No. 6,268,169 describes the recombinant synthesis of spider silk-like proteins derived from repetitive peptide sequences found in the natural spider drug line of Nephila clavipes using E. coli, Bacillus subtilis, and Pichia pastoris recombinant expression systems.WO 03 / 020916 describes cDNA clones encoding spider silk proteins with repetitive sequences derived from the major ampullate glands of Nephila madagascariensis, Nephila senegalensis, Tetragnatha kauaiensis, Tetragnatha versicolor, Argiope aurantia, Argiope trifasciata, Gasteracantha mammosa, and Latrodectus geometricus, the flagellate glands of Argiope trifasciata, the ampullate glands of Dolomedes tenebrosus, two pairs of silk glands from Plectreurys tristis, and the silk glands of the mygalomorph Euagrus chisoseus, and their recombinant production. Each of the above references is incorporated herein by reference in its entirety.

[0126] In some embodiments, the recombinant spider silk protein is a hybrid protein of spider silk protein and insect silk protein, spider silk protein and collagen, spider silk protein and resilin, or spider silk protein and keratin. The spider silk repeating unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that occurs repeatedly within naturally occurring major ampullate gland polypeptides (such as dragline spider silk polypeptides), minor ampullate gland polypeptides, flagellate gland polypeptides, condensed gland spider silk polypeptides, botryoid gland spider silk polypeptides, or piriform gland spider silk polypeptides.

[0127] In some embodiments, the recombinant spider silk proteins of the present disclosure comprise repeating units of natural spider silk proteins, consensus sequences, and optionally synthetic spider silk proteins derived from one or more natural non-repetitive spider silk protein sequences. The repeating units of natural spider silk polypeptides may comprise dragline spider silk polypeptides or flagellate gland spider silk polypeptides of the Araneidae or Araneoids.

[0128] As used herein, a spider silk "repeating unit" comprises or consists of at least one peptide motif that occurs repeatedly in a naturally occurring major ampullate gland polypeptide (such as a dragline spider silk polypeptide), minor ampullate gland polypeptide, flagellate gland polypeptide, agglutinate gland spider silk polypeptide, botryoid gland spider silk polypeptide, or piriform gland spider silk polypeptide. A "repeating unit" refers to a region in amino acid sequence that corresponds to (i.e., an identical amino acid sequence) or to (i.e., a variant amino acid sequence) a region that comprises or consists of (i.e., an identical amino acid sequence) at least one peptide motif (e.g., AAAAAA (SEQ ID NO: 20)) or GPGQQ (SEQ ID NO: 15)) that occurs repeatedly in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag). A "repeat unit" having an amino acid sequence that is "substantially similar" to a corresponding amino acid sequence in a naturally occurring silk polypeptide (i.e., a wild-type repeat unit) is also similar in terms of its properties; for example, a silk protein comprising a "substantially similar repeat unit" will remain insoluble and retain its insolubility. For example, a "repeat unit" having an amino acid sequence that is "identical" to that of a naturally occurring silk polypeptide can be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50), and / or ADF-4 (SEQ ID NO: 51). For example, a "repeat unit" having an amino acid sequence that is "substantially similar" to that of a naturally occurring silk polypeptide can be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50), and / or ADF-4 (SEQ ID NO: 51), but with one or more amino acid substitutions at specific amino acid positions.

[0129] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing a frequently occurring amino acid at a certain position (e.g., G), with other amino acids not yet determined being replaced by the placeholder "X." In some embodiments, the consensus sequence is selected from the group consisting of: (i) GPGXX (SEQ ID NO: 6), where X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, where X is an amino acid selected from Y, P, R, S, A, T, N, and Q, preferably Y, P, and Q; (iii) A x , (wherein x is an integer of 5 to 10).

[0130] The consensus peptide sequences GPGXX (SEQ ID NO: 6) and GGX, i.e., glycine-rich motifs, provide flexibility to silk polypeptides and, thus, to sutures formed from silk proteins containing these motifs. Specifically, the repeated GPGXX (SEQ ID NO: 6) motif forms a turn helix structure, imparting elasticity to silk polypeptides. Both major ampullate and flagellate gland silks contain the GPGXX (SEQ ID NO: 6) motif. The repeated GGX motif is associated with a helical structure with three amino acids per turn and is found in most spider silks. The GGX motif may provide additional elastic properties to silk. The repeated polyalanine Ax (peptide) motif forms a crystalline β-sheet structure that provides strength to silk polypeptides, as described, for example, in WO 03 / 057727.

[0131] In some embodiments, the recombinant spider silk protein of the present disclosure comprises two identical repeating units, each comprising at least one, and preferably one, amino acid sequence selected from the group consisting of GGRPSDTYG (SEQ ID NO: 7) and GGRPSSSYG (SEQ ID NO: 8), which are derived from resilin, an elastomeric protein found in most arthropods, providing low stiffness and high strength.

[0132] As used herein, a "non-repetitive unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repetitive (carboxy-terminal) amino acid sequence (i.e., the wild-type non-repetitive (carboxy-terminal) unit) in a naturally occurring dragline polypeptide, preferably ADF-3 (SEQ ID NO: 50), ADF-4 (SEQ ID NO: 51), NR3 (SEQ ID NO: 62), or NR4 (SEQ ID NO: 63) of the spider Araneus diadematus, which is also a C16 peptide (spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk) containing 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), an amino acid sequence adapted from the native sequence of ADF4 from A. diadematus, as described in U.S. Patent No. 9,217,017, which is incorporated herein by reference in its entirety. Non-repetitive ADF-4 and its variants exhibit efficient assembly behavior.

[0133] Among the synthetic spider silk proteins, the recombinant silk proteins in the present disclosure include, in some embodiments, the C16 protein having the polypeptide sequence SEQ ID NO: 64, which is also described in U.S. Patent No. 8,288,512, which is incorporated herein by reference in its entirety. In addition to the polypeptide sequence set forth in SEQ ID NO: 64, functional equivalents, functional derivatives, and salts of this sequence, among others, are also included.

[0134] As used herein, "functional equivalents" refers to variants of the above amino acid sequences which, at at least one sequence position, have an amino acid other than the amino acid specifically mentioned.

[0135] In some embodiments, the recombinant spider silk proteins of the present disclosure are spidroin major 1 as described by Xu et al., PNAS, USA, 87, 7120, (1990), spidroin major 2 as described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), U.S. Patent Application No. 2016 / 0222174, and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,803 ... The composition of the present invention comprises an effective amount of at least one natural or recombinant silk protein, including the recombinant spider silk proteins described in US Pat. Nos. 5,097,583, 8,030,024, 7,754,851, 7,148,039, and 7,060,260, or alternatively, spider silk proteins corresponding to the minor spidroins described in Patent Application WO 95 / 25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spider silk proteins suitable for the recombinant RSPF of the present disclosure include ADF3 and ADF4 from the "major ampullate" gland of Araneus diadematus.

[0136] Recombinant silk is also described in other patents and patent applications which are incorporated herein by reference: US2004 / 590196, US7,754,851, US2007 / 654470, US7,951,908, US2010 / 785960, US8,034,897, US2009 / 0263430, US2008 / 226854, US2009 / 0123967, US2005 / 712095, US2007 / 991037, US2009 / 0162896, US2008 / 85266, US8,372,436, US2007 / 989907, US2009 / 267596, US2010 / 319542, US2009 / 265344, US2012 / 684607, US2004 / 583227, US8,030,024, US2006 / 643569, US7,868,146, US2007 / 991916, US8,097,583, US2006 / 643200, US8,729,238, US8,877,903, US2019 / 0062557, US2016 / 0280960, US2011 / 0201783, US2008 / 991916, US2011 / 986662, US2012 / 697729, US2015 / 0328363, US9,034,816, US2013 / 0172478, US9,217,017, US2017 / 0202995, US8,721,991, US2008 / 227498, US9,233,067, U S8,288,512, US2008 / 161364, US7,148,039, US1999 / 247806, US2001 / 861597, US2004 / 887100, US9,481,719, US8,765,688, US2008 / 80705, US2010 / 809102, US8,367,803, US2010 / 664902, US7,569,660, US1999 / 138833, US2000 / 591632, US2012 / 0065126, US2010 / 0278882, US2008 / 161352, US2010 / 0015070, US2009 / 513709, US2009 / 0194317, US2004 / 559286, US2005 / 89551, US2008 / 187824, US2005 / 0266242, US2005 / 0227322, and US20044418.

[0137] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US2019 / 0062557, US2015 / 0284565, US2013 / 0225476, US2013 / 0172478, US2013 / 0136779, US2013 / 0109762, US2012 / 0252294, US2011 / 0230911, US2011 / 0201783, US2010 / 029 8877, US10,478,520, US10,253,213, US10,072,152, US9,233,067, US9,217,017, US9,034,816, US8,877,903, US8,729,238, US8,721,991, US8,097,583, US8,034,897, US8,030,024, US7,951,908, US7,868,146, and US7,754,851.

[0138] In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of 2 to 80 repeating units, each of which is selected from the group consisting of GPGXX (SEQ ID NO: 6), GGX, and A, as defined herein. x are independently selected from

[0139] In some embodiments, the recombinant spider silk protein of the present disclosure is selected from the group consisting of GPGAS (SEQ ID NO: 10), GPGSG (SEQ ID NO: 11), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13), GPGGA (SEQ ID NO: 14), GPGQQ (SEQ ID NO: 15), GPGGG (SEQ ID NO: 16), GPGQG (SEQ ID NO: 17), GPGGS (SEQ ID NO: 18), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 19), AAAAAA (SEQ ID NO: 20), AAAAAAA (SEQ ID NO: 21), ), AAAAAAAA (SEQ ID NO: 22), AAAAAAAAA (SEQ ID NO: 23), AAAAAAAAAA (SEQ ID NO: 24), GGRPSDTYG (SEQ ID NO: 7), and GGRPSSSYG (SEQ ID NO: 8), (i) GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 25), (ii) GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), (iii) GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 26), (iv) GPGGAGGPYGPGGAGGPYGP GGAGGPY (SEQ ID NO: 27), (v) GGTTIIEDLDITIDGADGPITISEELTI (SEQ ID NO: 28), (vi) PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 29), (vii) SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), (viii) GGAGGAGGAGGSGGAGGS (SEQ ID NO: 31), (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 32), ( (x) GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 33), (xi) GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 34), (xii) GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 35), (xiii) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 36), (xiv) GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO: 37), or U.S. Patent No. 8,877,903, and variants thereof, such as synthetic spider peptides having the sequence GPGAS (SEQ ID NO: 10), GGY, GPGSG (SEQ ID NO: 11) in the peptide chain, or the sequence AAAAAAAA (SEQ ID NO: 22), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13) in the peptide chain, or the sequence AAAAAAAA (SEQ ID NO: 22), GPGQG (SEQ ID NO: 17), GGR in the peptide chain.

[0140] In some embodiments, the present disclosure provides silk protein-like multi-block peptides that mimic amino acid repeat units derived from natural spider silk proteins, such as spidroin major 1 domain, spidroin major 2 domain, or spidroin minor 1 domain, and profiles of variations between the repeat units without modifications of their three-dimensional conformation, wherein these silk protein-like multi-block peptides comprise amino acid repeat units corresponding to one of the following sequences (I), (II), (III), and / or (IV): [(XGG) w (XGA)(GXG) x (AGA) y (G) z A.G.] p (SEQ ID NO: 38) formula (I), wherein X corresponds to tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer between 1 and 3, y is an integer between 5 and 7, z is an integer equal to 1 or 2, and p is an integer, and has any weight average molecular weight described herein; and / or [(GPG2YGPGQ2) a (X')2S(A) b ] p (SEQ ID NO: 39) formula (II), wherein X' corresponds to the amino acid sequence GPS or GPG, a is equal to 2 or 3, b is an integer between 7 and 10, and p is an integer, and has any weight average molecular weight described herein; and / or [(GR)(GA) l (A) m(GGX) n (GA) l (A) m ] p (SEQ ID NO: 40) Formula (III) and / or [(GGX") n (GA) m (A) l ] p (SEQ ID NO: 41) Formula (IV), wherein X" corresponds to tyrosine, glutamine, or alanine, 1 is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer.

[0141] In some embodiments, the recombinant spider silk protein or analogue of the spider silk protein comprises an amino acid repeat unit of the following sequence (V): [(Xaa Gly Gly) w (Xaa Gly Ala)(Gly Xaa Gly) x (Ala Gly Ala) y (Gly) z Ala Gly] p It includes formula (V) (wherein Xaa is tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer).

[0142] In some embodiments, the recombinant spider silk protein of the present disclosure is selected from the group consisting of ADF-3 or a mutant thereof, ADF-4 or a mutant thereof, MaSpI or a mutant thereof, and MaSpII or a mutant thereof, as described in U.S. Pat. No. 9,217,017.

[0143] In some embodiments, the present disclosure provides water-soluble recombinant spider silk proteins produced in mammalian cells. The solubility of spider silk proteins produced in mammalian cells is due to the presence of COOH-terminal amino acids in these proteins, which make them more hydrophilic. These COOH-terminal amino acids are not present in spider silk proteins expressed in microbial hosts.

[0144] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a water-soluble recombinant spider silk protein C16 modified at the amino or carboxyl terminus selected from the amino acid sequences consisting of GCGGGGGG (SEQ ID NO: 42), GKGGGGGG (SEQ ID NO: 43), GCGGSGGGGSGGGG (SEQ ID NO: 44), GKGGGGGGSGGGG (SEQ ID NO: 45), and GCGGGGGSGGGG (SEQ ID NO: 46). ... 16 NR4, C 32 NR4, C16, C32, NR4C 16 NR4, NR4C 32 NR4, NR3C 16 NR3 or NR3C 32 NR3, which brings the molecular weight of the protein into the ranges described herein. In some embodiments, the recombinant spider silk protein of the present disclosure comprises a recombinant spider silk protein having a synthetic repeat peptide segment and amino acid sequence adapted from the native sequence of ADF4 from A. diadematus, as described in U.S. Patent No. 8,877,903. In some embodiments, the RSPF of the present disclosure comprises a recombinant spider silk protein having a repeat peptide unit derived from a native spider silk protein, such as a spidroin major 1 domain, a spidroin major 2 domain, or a spidroin minor 1 domain, wherein the repeat peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 47) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), as described in U.S. Patent No. 8,367,803, which is incorporated herein by reference in its entirety.

[0145] In some embodiments, the disclosure provides a recombinant spider protein composed of a GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 32) repeat fragment and having a molecular weight as described herein.

[0146] As used herein, the term "recombinant silk" refers to recombinant spider and / or silkworm silk proteins or fragments thereof. In one embodiment, the spider silk protein is selected from the group consisting of entrapment silk (bacilliform gland silk), egg capsule silk (cylindrical gland silk), egg sheath silk (canalicular silk), non-sticky dragline silk (ampullate gland silk), attachment silk (pyriform gland silk), adhesive silk core fiber (flap-like gland silk), and adhesive outer silk fiber (aggregate gland silk). For example, recombinant spider silk proteins include those described in U.S. Patent Application No. 2016 / 0222174 and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734, as described herein.

[0147] Some organisms produce multiple silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb-weaving spiders have six unique types of glands that produce different silk polypeptide sequences that are polymerized into fibers tailored to their environment or life cycle niche. Fibers are named after the gland from which they originate, and polypeptides are labeled with the gland abbreviation (e.g., "Ma") and "Sp" for spidroin (short for spider fibroin). In orb-weaving spiders, these types include the major ampullate gland (MaSp, also called dragline), minor ampullate gland (MiSp), flagellate gland (Flag), botryoid gland (AcSp), small tubular gland (TuSp), and piriform gland (PySp). This combination of polypeptide sequences across fiber types, domains, and variations among organisms of different genera and species leads to a vast array of potential properties that can be exploited through the commercial production of recombinant fibers. To date, the majority of research using recombinant silk has focused on major ampullate spidroins (MaSp).

[0148] Acroplasty-like gland (AcSp) silks tend to have high toughness, resulting from moderately high strength combined with moderately high extensibility. AcSp silks are characterized by large block ("ensemble repeat") sizes, often incorporating polyserine and GPX motifs. Tubulous gland (TuSp or cylindrical) silks tend to have large diameters and have moderate strength and high extensibility. TuSp silks are characterized by their polyserine and polythreonine content and short tubules of polyalanine. Major ampullate gland (MaSp) silks tend to have high strength and moderate extensibility. MaSp silks can be one of two subtypes: MaSp1 and MaSp2. MaSp1 silks are generally less extensible than MaSp2 silks and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silks are characterized by polyalanine, GGX, and GPX motifs. MiSp silks tend to have moderate strength and moderate extensibility. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagellate silks tend to have very high extensibility and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0149] Silk polypeptides are characteristically composed of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repeat domains exhibit a hierarchical structure. The repeat domains contain a series of blocks (also called repeat units). The blocks are repeated sometimes perfectly and sometimes imperfectly (forming quasi-repetitive domains) throughout the silk repeat domain. The length and composition of the blocks vary among different silk types and across different species. Table 1 of U.S. Published Application No. 2016 / 0222174 (incorporated herein in its entirety) lists examples of block sequences from selected species and silk types, while further examples are provided in Rising, A. et al., "Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications," Cell Mol. Life Sci., 68:2, pp. 169-184 (2011), and Gatesy, J. et al., "Extreme diversity, conservation, and convergence of spider silk fibroin sequences," Science, 291:5513, pp. 2603-2605 (2001). In some cases, blocks may be arranged in a regular pattern to form larger macrorepeats that appear multiple times (usually 2-8 times) within the repeat domain of the silk sequence. Repeated blocks within a repeat domain or macrorepeat, and repeated macrorepeats within a repeat domain, may be separated by spacing elements.

[0150] The structure of certain spider silk block copolymer polypeptides from block and / or macrorepeat domains according to certain embodiments of the present disclosure is exemplified in U.S. Patent Application Publication No. 2016 / 0222174.

[0151] Recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art. In a preferred embodiment, commercially available expression / secretion systems can be used, whereby the recombinant polypeptide is expressed and then secreted from the host cell and easily purified from the surrounding medium. If an expression / secretion vector is not used, an alternative approach involves purifying the recombinant block copolymer polypeptide from cell lysates (cell remains after disruption of cellular integrity) derived from prokaryotic or eukaryotic cells in which the polypeptide was expressed. Methods for producing such cell lysates are known to those skilled in the art. In some embodiments, the recombinant block copolymer polypeptide is isolated from the cell culture supernatant.

[0152] Recombinant block copolymer polypeptides may be purified by affinity separation, such as immunological interaction with an antibody that specifically binds to the recombinant polypeptide or a nickel column for isolation of recombinant polypeptides tagged with 6 to 8 histidine residues at their N- or C-termini; alternative tags may comprise the FLAG epitope or hemagglutinin epitope. Such methods are commonly used by those skilled in the art.

[0153] Solutions of such polypeptides (ie, recombinant silk proteins) may then be prepared and used as described herein.

[0154] In another embodiment, recombinant silk proteins may be prepared according to the methods described in U.S. Pat. No. 8,642,734, which is incorporated herein in its entirety, and used as described herein.

[0155] In one embodiment, a recombinant spider silk protein is provided. Spider silk proteins typically consist of 170 to 760 amino acid residues, such as 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, such as 300 to 400 amino acid residues, and more preferably 340 to 380 amino acid residues. A small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require the use of harsh solvents for solubilization and polymerization. The recombinant spider silk protein may contain more than 760 residues, particularly when the spider silk protein contains three or more fragments derived from the N-terminal portion of the spider silk protein, including an N-terminal fragment (NT) derived from the corresponding portion of the spider silk protein and a repeat fragment (REP) derived from the corresponding internal fragment of the spider silk protein. Optionally, the spider silk protein includes a C-terminal fragment (CT) derived from the corresponding fragment of the spider silk protein. Spider silk proteins typically comprise a single fragment (NT) derived from the N-terminal portion of the spider silk protein, however, in a preferred embodiment, the N-terminal fragment comprises at least two, such as two fragments (NT) derived from the N-terminal portion of the spider silk protein. Spidroins are therefore of the formula NT m -REP, and alternatively NT m Spidroins can be represented schematically by the formula NT2-REP or NT-REP, and alternatively NT2-REP-CT or NT-REP-CT, where m is an integer of 1 or more, such as 2 or more, preferably in the range of 1-2, 1-4, 1-6, 2-4, or 2-6. Preferred spidroins can be represented schematically by the formula NT2-REP or NT-REP, and alternatively NT2-REP-CT or NT-REP-CT. The protein fragments are typically covalently linked via peptide bonds. In one embodiment, the spider silk protein consists of an NT fragment linked to a REP fragment, which is optionally linked to a CT fragment.

[0156] In one embodiment, the first step of the method for producing isolated spider silk protein polymers involves the expression of a polynucleic acid molecule encoding the spider silk protein in a suitable host, such as E. coli. The resulting protein is then isolated using standard procedures. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage.

[0157] In the second step of the method for producing isolated spider silk protein polymers, a solution of spider silk protein in a liquid medium is provided. The terms "soluble" and "in solution" mean that the protein does not visibly aggregate or precipitate from the solvent at 60,000 x g. The liquid medium may be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as 10-50 mM Tris-HCl buffer or phosphate buffer. The liquid medium has a pH of 6.4 or greater and / or an ionic composition that prevents polymerization of the spider silk protein. That is, the liquid medium has either a pH of 6.4 or greater, an ionic composition that prevents polymerization of the spider silk protein, or both.

[0158] Ion compositions that prevent spider silk protein polymerization can be easily prepared by those skilled in the art using the methods disclosed herein.Preferred ion compositions that prevent spider silk protein polymerization have an ionic strength of more than 300 mM.Specific examples of ion compositions that prevent spider silk protein polymerization include more than 300 mM NaCl, 100 mM phosphate, and combinations of these ions that have the desired preventive effect on spider silk protein polymerization, such as a combination of 10 mM phosphate and 300 mM NaCl.

[0159] The presence of NT fragments improves the stability of the solution and prevents polymer formation under these conditions. This can be advantageous when immediate polymerization is undesirable, for example, during protein purification, large batch preparation, or when other conditions need to be optimized. The pH of the liquid medium is preferably adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, for example, up to 10.5, to achieve high solubility of the spider silk protein. It can also be advantageous to adjust the pH of the liquid medium to a range of 6.4 to 6.8, which provides sufficient solubility of the spider silk protein, but facilitates subsequent pH adjustment to 6.3 or lower.

[0160] In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or less and an ionic composition that permits polymerization. That is, if the liquid medium in which the spider silk protein is dissolved has a pH of 6.4 or more, the pH is reduced to 6.3 or less. Those skilled in the art are well aware of various methods for achieving this, typically involving the addition of a strong or weak acid. If the liquid medium in which the spider silk protein is dissolved has an ionic composition that prevents polymerization, the ionic composition is changed to permit polymerization. Those skilled in the art are well aware of various methods for achieving this, such as dilution, dialysis, or gel filtration. If necessary, this step involves both reducing the pH of the liquid medium to 6.3 or less and changing the ionic composition to permit polymerization. Preferably, the pH of the liquid medium is adjusted to 6.2 or less, such as 6.0 or less. Specifically, it may be advantageous from a practical standpoint to limit the pH drop from 6.4 or 6.4-6.8 in the previous step to 6.3 or 6.0-6.3, e.g., 6.2, in this step. In a preferred embodiment, the pH of the liquid medium in this step is greater than or equal to 3, such as greater than or equal to 4.2. The resulting pH range, for example, 4.2 to 6.3, promotes rapid polymerization.

[0161] In the fourth step, spider silk proteins are polymerized in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk proteins. The presence of the NT fragments improves the solubility of the spider silk proteins at pHs above 6.4 and / or accelerates polymer formation at pHs below 6.3, where the ionic composition allows polymerization of the spider silk proteins. The resulting polymers are preferably solid and macroscopic, and they are formed in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk proteins. In a preferred embodiment, the pH of the liquid medium in this step is 3 or greater, such as 4.2 or greater. The resulting pH range, for example, 4.2 to 6.3, promotes rapid polymerization. The resulting polymers may be provided in the molecular weights described herein or prepared in solution form, which may be used as needed for article coating.

[0162] Ionic compositions that allow the polymerization of spider silk proteins can be easily prepared by those skilled in the art using the methods disclosed herein. Preferred ionic compositions that allow the polymerization of spider silk proteins have an ionic strength of less than 300 mM. Specific examples of ionic compositions that allow the polymerization of spider silk proteins include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack a preventive effect on the polymerization of spider silk proteins, such as a combination of 10 mM phosphate or 20 mM phosphate and 150 mM NaCl. The ionic strength of the liquid medium is preferably adjusted to a range of 1 to 250 mM.

[0163] Without wishing to be limited to any particular theory, it is postulated that NT fragments have oppositely charged poles and that environmental changes in pH affect the charge balance on the surface of the protein, leading to polymerization, while salt inhibits the same event.

[0164] At neutral pH, one might expect that the energetic cost of compensating for the excess negative charge at the acidic pole would prevent polymerization. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces eventually dominate, explaining the observed salt- and pH-dependent polymerization behavior of NTs and NT-containing minispidroins. In some embodiments, it is proposed that the pH-induced NT polymerization and increased efficiency of NT-minispidroin fiber assembly are due to changes in the surface electrostatic potential, and that clustering of acidic residues at one pole of NTs shifts their charge balance, causing the polymerization transition to occur at pH values ​​below 6.3.

[0165] In a fifth step, the resulting, preferably solid, spider silk protein polymer is isolated from the liquid medium, optionally involving the active removal of lipopolysaccharides and other pyrogens from the spidroin polymer.

[0166] Without wishing to be limited to any particular theory, it has been observed that the formation of spidroin polymers proceeds via the formation of water-soluble spidroin dimers. Therefore, the present disclosure also provides a method for producing isolated spider silk protein dimers, wherein the first two method steps are as described above. The spider silk protein exists as a dimer in a liquid medium with a pH of 6.4 or higher and / or an ionic composition that prevents polymerization of the spider silk protein. The third step involves isolating the dimer obtained in the second step and, optionally, removing lipopolysaccharides and other pyrogens. In a preferred embodiment, the spider silk protein polymer of the present disclosure consists of polymerized protein dimers. The present disclosure therefore provides a novel use of spider silk proteins, preferably those disclosed herein, for producing spider silk protein dimers.

[0167] According to another aspect, the present disclosure provides a polymer of a spider silk protein as disclosed herein. In one embodiment, the protein polymer is obtainable by any one of the methods according to the present disclosure. Thus, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing polymers of spider silk proteins as recombinant silk-based coatings. According to one embodiment, the present disclosure provides a novel use of a spider silk protein dimer, preferably those disclosed herein, for producing polymers of isolated spider silk proteins as recombinant silk-based coatings. For these uses, the polymer is preferably produced in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk protein. In one embodiment, the pH of the liquid medium is 3 or greater, such as 4.2 or greater. The resulting pH range, e.g., 4.2 to 6.3, promotes rapid polymerization.

[0168] Using the methods of the present disclosure, it is possible to control the polymerization process, which allows for the optimization of parameters to obtain silk polymers with desired properties and shapes.

[0169] In one embodiment, the recombinant silk proteins described herein include those described in US Pat. No. 8,642,734, which is incorporated by reference in its entirety.

[0170] In another embodiment, the recombinant silk proteins described herein may be prepared according to the methods described in U.S. Patent No. 9,051,453, the entirety of which is incorporated herein by reference.

[0171] The amino acid sequence represented by SEQ ID NO:52, also described in U.S. Patent No. 9,051,453, is identical to the amino acid sequence consisting of 50 amino acid residues of the amino acid sequence of ADF3 at the C-terminus (NCBI accession number: AAC47010, GI:1263287). The amino acid sequence represented by SEQ ID NO:53, also described in U.S. Patent No. 9,051,453, is identical to the amino acid sequence represented by SEQ ID NO:52, also described in U.S. Patent No. 9,051,453, with 20 residues removed from the C-terminus. The amino acid sequence represented by SEQ ID NO:54, also described in U.S. Patent No. 9,051,453, is identical to the amino acid sequence represented by SEQ ID NO:52, with 29 residues removed from the C-terminus.

[0172] An example of a polypeptide comprising a unit of the amino acid sequence represented by Formula 1: REP1-REP2(1) and having at its C-terminus an amino acid sequence represented by any one of SEQ ID NOs: 52 to 54 or an amino acid sequence having 90% or more homology to any one of SEQ ID NOs: 52 to 54 also described in U.S. Pat. No. 9,051,453 is a polypeptide having the amino acid sequence represented by SEQ ID NO: 65 also described in U.S. Pat. No. 9,051,453, the entirety of which is incorporated herein by reference. A polypeptide having an amino acid sequence represented by SEQ ID NO: 65, also described in U.S. Patent No. 9,051,453, can be obtained by the following mutations: an amino acid sequence (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) consisting of an initiation codon, a His10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site is added to the N-terminus of the amino acid sequence of ADF3 (NCBI Accession No. AAC47010, GI: 1263287), the repeat region from residues 1 to 13 is approximately doubled, and translation terminates at amino acid residue 1154. In the polypeptide having an amino acid sequence represented by SEQ ID NO: 65, also described in U.S. Patent No. 9,051,453, the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 54.

[0173] Furthermore, a polypeptide comprising a unit of the amino acid sequence represented by Formula 1: REP1-REP2(1) and having at its C-terminus an amino acid sequence represented by any one of SEQ ID NOS: 52 to 54 also described in U.S. Pat. No. 9,051,453, or an amino acid sequence having 90% or more homology to any one of SEQ ID NOS: 52 to 54 also described in U.S. Pat. No. 9,051,453, may be a protein having an amino acid sequence represented by SEQ ID NOS: 65 also described in U.S. Pat. No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and having a repeat region composed of crystalline and amorphous regions.

[0174] Furthermore, an example of a polypeptide containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2(1) is a recombinant protein derived from ADF4 having the amino acid sequence represented by SEQ ID NO: 67, which is also described in U.S. Patent No. 9,051,453, the entirety of which is incorporated herein by reference. The amino acid sequence represented by SEQ ID NO: 67, also described in U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon, a His10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of a partial amino acid sequence of ADF4 obtained from the NCBI database (NCBI accession number: AAC47011, GI: 1263289). Furthermore, a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1) may be a polypeptide having an amino acid sequence represented by SEQ ID NO:67, also described in U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and having a repeat region composed of crystalline and amorphous regions. Furthermore, an example of a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1) is a recombinant protein derived from MaSp2 having an amino acid sequence represented by SEQ ID NO:68, also described in U.S. Patent No. 9,051,453, the entire contents of which are incorporated herein by reference. The amino acid sequence represented by SEQ ID NO: 68, which is also described in U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon, a His10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of the partial sequence of MaSp2 obtained from the NCBI web database (NCBI accession number: AAT75313, GI: 50363147).Furthermore, a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1: REP1-REP2(1) may be a polypeptide having an amino acid sequence represented by SEQ ID NO: 68, also described in U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and having a repeat region composed of crystalline and amorphous regions.

[0175] Examples of polypeptides derived from flagellate gland silk proteins include polypeptides containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2), preferably polypeptides containing 20 or more units thereof, and more preferably polypeptides containing 30 or more units thereof. In cases where recombinant proteins are produced using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from flagellate gland silk protein is preferably 500 kDa or less, more preferably 300 kDa or less, and even more preferably 200 kDa or less, from the viewpoint of productivity.

[0176] In formula (2), REP3 represents an amino acid sequence consisting of Gly-Pro-Gly-Gly-X (SEQ ID NO: 69), where X represents an amino acid selected from the group consisting of Ala, Ser, Tyr, and Val.

[0177] A key feature of spider silk is that it does not have crystalline regions, but rather has repeating regions composed of amorphous regions. Because primary dragline silks and their analogs have repeating regions composed of crystalline and amorphous regions, they are expected to have both high stress and stretchability. On the other hand, the stress of the primary dragline silk is inferior to that of the primary dragline silk, but the stretchability is high. The reason for this is thought to be that the majority of the primary dragline silk is composed of amorphous regions.

[0178] An example of a polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2:REP3(2) is a recombinant protein derived from flagellate gland silk protein having the amino acid sequence represented by SEQ ID NO:70, which is also described in U.S. Patent No. 9,051,453, the entire contents of which are incorporated herein by reference. The amino acid sequence represented by SEQ ID NO: 70, which is also described in U.S. Patent No. 9,051,453, was obtained by combining a partial sequence of the flagellate gland silk protein of Nephila clavipes obtained from the NCBI database (NCBI accession number: AAF36090, GI: 7106224), specifically the amino acid sequence from residues 1220 to 1659 from the N-terminus corresponding to the repeat portion and motif (referred to as the PR1 sequence), with a partial sequence of the flagellate gland silk protein of Nephila clavipes obtained from the NCBI database (NCBI accession number: AAC38847, GI: 2833649), specifically the C-terminal amino acid sequence from residues 816 to 907 from the C-terminus, and then adding an amino acid sequence consisting of a start codon, a His10 tag, and an HRV3C protease recognition site (SEQ ID NO: 66, also described in U.S. Patent No. 9,051,453) to the N-terminus of the combined sequence. Furthermore, a polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2:REP3(2) may be a polypeptide having an amino acid sequence represented by SEQ ID NO:70, also described in U.S. Pat. No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and having a repeat region made up of amorphous regions.

[0179] Polypeptides can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, but it can be produced by amplifying a gene encoding a natural spider silk protein from cells derived from a spider by cloning it using polymerase chain reaction (PCR), or it can be chemically synthesized. The method for chemically synthesizing the gene is also not particularly limited, but it can be synthesized as follows: based on the amino acid sequence of a natural spider silk protein obtained from the NCBI web database, oligonucleotides automatically synthesized using AKTA OligoPilot Plus 10 / 100 (GE Healthcare Japan Corporation) are ligated by PCR. At this point, to facilitate protein purification and observation, a gene encoding a protein having the above amino acid sequence can be synthesized with an amino acid sequence consisting of an initiation codon and a His 10 tag added to the N-terminus.

[0180] Examples of expression vectors include plasmids, phages, viruses, and the like, which are capable of expressing proteins based on DNA sequences. Plasmid-type expression vectors are not particularly limited, as long as they can express target genes in host cells and amplify themselves. For example, when Escherichia coli Rosetta (DE3) is used as a host, pET22b(+) plasmid vectors, pCold plasmid vectors, and the like can be used. Among these, the pET22b(+) plasmid vector is preferred from the viewpoint of protein productivity. Examples of hosts include animal cells, plant cells, and microorganisms.

[0181] The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, which is one of the two major dragline silk proteins of Araneus diadematus. This polypeptide has the advantages of essentially high strength, elongation, and toughness, and is easily synthesized.

[0182] Accordingly, the recombinant silk proteins (e.g., recombinant spider silk-based proteins) used in accordance with the embodiments, articles, and / or methods described herein may be any of the silk proteins described above or in any of the U.S. Patent Nos. 8,173,772, 8,278,416, 8,618,255, 8,642,734, 8,691,581, 8,729,235, 9,115, 9,278,416, 9,618,255, 9,642,734, 9,691,581, 9,278,416 ... ,204, 9,157,070, 9,309,299, 9,644,012, 9,708,376, 9,051,453, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332;and U.S. Patent Publication Nos. 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066, 2014 / 0079674, 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 0141618, 2015 / 0291673, 2015 / 0291674, and 2015 / 023958 No. 7, No. 2015 / 0344542, No. 2015 / 0361144, No. 2015 / 0374833, No. 2015 / 037 No. 6247, No. 2016 / 0024464, No. 2017 / 0066804, No. 2017 / 0066805, No. 2015 / No. 0293076, No. 2016 / 0222174, No. 2017 / 0283474, No. 2017 / 0088675, No. 20 No. 19 / 0135880, No. 2015 / 0329587, No. 2019 / 0040109, No. 2019 / 0135881, No. 2019 / 0177363, 2019 / 0225646, 2019 / 0233481, 2019 / 0031842 , same No. 2018 / 0355120, same No. 2019 / 0186050, same No. 2019 / 0002644, same No. 2020 / 00318 No. 87, No. 2018 / 0273590, No. 20191 / 094403, No. 2019 / 0031843, No. 2018 / 02 No. 51501, No. 2017 / 0066805, No. 2018 / 0127553, No. 2019 / 0329526, No. 2020 / 0031886, 2018 / 0080147, 2019 / 0352349, 2020 / 0043085, 2019 / 0144819, 2019 / 0228449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191;

[0183] Silk fibroin-like protein fragments The recombinant silk proteins of the present disclosure include synthetic proteins based on the repeating units of natural silk proteins. In addition to synthetic repeating silk protein sequences, they may additionally contain one or more natural non-repeating silk protein sequences. As used herein, "silk fibroin-like protein fragments" refer to protein fragments having a molecular weight and polydispersity as defined herein and a degree of homology to a protein selected from natural silk proteins, fibroin heavy chains, fibroin light chains, or any protein containing one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeating units. In some embodiments, the degree of homology is selected from about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, or less than 75%.

[0184] As described herein, proteins, such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contain between about 9% and about 45% glycine, or about 9% glycine, or about 10% glycine, about 43% glycine, about 44% glycine, about 45% glycine, or about 46% glycine. As described herein, proteins, such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contain between about 13% and about 30% alanine, or about 13% alanine, or about 28% alanine, or about 29% alanine, or about 30% alanine, or about 31% alanine. As described herein, a protein, such as a natural silk protein, a fibroin heavy chain, a fibroin light chain, or any protein containing one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contains between 9% and about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0185] In some embodiments, the silk fibroin-like proteins described herein are about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about Contains 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% glycine. In some embodiments, the silk fibroin-like proteins described herein contain about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, or about 39% alanine. In some embodiments, the silk fibroin-like proteins described herein contain about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, or about 22% serine. In some embodiments, the silk fibroin-like proteins described herein may independently include any amino acid known to be contained in native fibroin. In some embodiments, the silk fibroin-like proteins described herein may independently exclude any amino acid known to be contained in native fibroin. In some embodiments, an average of 2 out of 6 amino acids, 3 out of 6 amino acids, or 4 out of 6 amino acids in the silk fibroin-like proteins described herein are glycines. In some embodiments, an average of 1 in 6 amino acids, 2 in 6 amino acids, or 3 in 6 amino acids in the silk fibroin-like proteins described herein is alanine.In some embodiments, on average, 0 out of 6 amino acids, 1 out of 6 amino acids, or 2 out of 6 amino acids in the silk fibroin-like proteins described herein are serine.

[0186] Sericin or sericin fragment The main body of raw silk is silk fibroin fiber, which is coated with an adhesive substance called silk sericin. Sericin is a colloidal silk protein that covers the surface of silk threads and is composed of chemically reactive bulky amino acids such as serine, threonine, and aspartic acid in addition to glycine and alanine. In various processes for producing silk from raw silk, sericin is important for controlling the solubility of silk and producing high-quality silk. Furthermore, sericin plays a very important role as an adhesive functional protein. When silk fibers are used as clothing materials, most of the silk sericin covering the silk threads is removed and discarded, making sericin a valuable unused resource.

[0187] In some embodiments, the silk protein fragments described herein comprise sericin or sericin fragments. Methods for preparing sericin or sericin fragments and their uses in various fields are known and described herein, and also in, for example, U.S. Patent Nos. 7,115,388, 7,157,273, and 9,187,538, all of which are incorporated herein by reference in their entireties.

[0188] In some embodiments, sericin removed from raw silk cocoons, such as during a degumming process, can be collected and used in the methods described herein. Sericin can also be reconstituted from a powder and used in the compositions and methods of the present disclosure.

[0189] Other properties of SPF The compositions of the present disclosure are "biocompatible," meaning that they are compatible with living tissues or systems by being non-toxic, non-intoxicating, or physiologically non-reactive, and by not eliciting immunological rejection or an inflammatory response. Such biocompatibility can be demonstrated by participants applying the compositions of the present disclosure topically to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. For example, in some embodiments, the coatings described herein are biocompatible coatings.

[0190] In some embodiments, the compositions described herein, which may be biocompatible compositions (e.g., biocompatible coatings including silk), may be evaluated using and may comply with the international standard ISO 10993-1, entitled "Biological evaluation of medical devices—Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein, which may be biocompatible compositions, may be evaluated under ISO 10993-1 for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantability, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation.

[0191] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0192] In one embodiment, the composition of the present disclosure has a stability of about 1 day. In one embodiment, the composition of the present disclosure has a stability of about 2 days. In one embodiment, the composition of the present disclosure has a stability of about 3 days. In one embodiment, the composition of the present disclosure has a stability of about 4 days. In one embodiment, the composition of the present disclosure has a stability of about 5 days. In one embodiment, the composition of the present disclosure has a stability of about 6 days. In one embodiment, the composition of the present disclosure has a stability of about 7 days. In one embodiment, the composition of the present disclosure has a stability of about 8 days. In one embodiment, the composition of the present disclosure has a stability of about 9 days. In one embodiment, the composition of the present disclosure has a stability of about 10 days.

[0193] In one embodiment, the stability of the compositions of the present disclosure is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days.

[0194] In one embodiment, the composition of the present disclosure has a stability of 10 days to 6 months. In one embodiment, the composition of the present disclosure has a stability of 6 months to 12 months. In one embodiment, the composition of the present disclosure has a stability of 12 months to 18 months. In one embodiment, the composition of the present disclosure has a stability of 18 months to 24 months. In one embodiment, the composition of the present disclosure has a stability of 24 months to 30 months. In one embodiment, the composition of the present disclosure has a stability of 30 months to 36 months. In one embodiment, the composition of the present disclosure has a stability of 36 months to 48 months. In one embodiment, the composition of the present disclosure has a stability of 48 months to 60 months.

[0195] In one embodiment, the SPF composition of the present disclosure is not soluble in aqueous solution due to the crystallinity of the protein. In one embodiment, the SPF composition of the present disclosure is soluble in aqueous solution. In one embodiment, the SPF of the composition of the present disclosure comprises about two-thirds crystalline fraction and about one-third amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises about half crystalline fraction and about half amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 99% crystalline fraction and 1% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 95% crystalline fraction and 5% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 90% crystalline fraction and 10% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 85% crystalline fraction and 15% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 80% crystalline fraction and 20% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 75% crystalline fraction and 25% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 70% crystalline fraction and 30% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 65% crystalline fraction and 35% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 60% crystalline fraction and 40% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 50% crystalline fraction and 50% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 40% crystalline fraction and 60% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 35% crystalline fraction and 65% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 30% crystalline fraction and 70% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 25% crystalline fraction and 75% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 20% crystalline fraction and 80% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 15% crystalline fraction and 85% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 10% crystalline fraction and 90% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 5% crystalline fraction and 90% amorphous region.In one embodiment, the SPF of the composition of the present disclosure comprises 1% crystalline fraction and 99% amorphous region.

[0196] As used herein, the term "substantially free of inorganic residue" means that a composition exhibits 0.1% (w / w) or less residue. In one embodiment, "substantially free of inorganic residue" refers to a composition exhibiting 0.05% (w / w) or less residue. In one embodiment, "substantially free of inorganic residue" refers to a composition exhibiting 0.01% (w / w) or less residue. In one embodiment, the amount of inorganic residue is from 0 ppm ("non-detectable" or "ND") to 1000 ppm. In one embodiment, the amount of inorganic residue is from ND to about 500 ppm. In one embodiment, the amount of inorganic residue is from ND to about 400 ppm. In one embodiment, the amount of inorganic residue is from ND to about 300 ppm. In one embodiment, the amount of inorganic residue is from ND to about 200 ppm. In one embodiment, the amount of inorganic residue is from ND to about 100 ppm. In one embodiment, the amount of inorganic residue is from 10 ppm to 1000 ppm.

[0197] As used herein, the term "substantially free of organic residue" means that a composition exhibits 0.1% (w / w) or less of residue; in one embodiment, "substantially free of organic residue" refers to a composition exhibiting 0.05% (w / w) or less of residue. In one embodiment, "substantially free of organic residue" refers to a composition exhibiting 0.01% (w / w) or less of residue. In one embodiment, the amount of organic residue is from 0 ppm ("non-detectable" or "ND") to 1000 ppm. In one embodiment, the amount of organic residue is from ND to about 500 ppm. In one embodiment, the amount of organic residue is from ND to about 400 ppm. In one embodiment, the amount of organic residue is from ND to about 300 ppm. In one embodiment, the amount of organic residue is from ND to about 200 ppm. In one embodiment, the amount of organic residue is from ND to about 100 ppm. In one embodiment, the amount of organic residue is from 10 ppm to 1000 ppm.

[0198] The compositions of the present disclosure are "biocompatible," meaning that the compositions are compatible with living tissues or systems by being non-toxic, non-intoxicating, or physiologically reactive, and by not causing immunological rejection. Such biocompatibility can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days, in one embodiment, the extended period is about 14 days, and in one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0199] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0200] The following are non-limiting examples of suitable ranges for various parameters in and for preparing silk solutions of the present disclosure. Silk solutions of the present disclosure may include one or more (but not necessarily all) of these parameters and may be prepared using various combinations of such parameter ranges.

[0201] In one embodiment, the percent SPF in the solution is less than 30.0% by weight. In one embodiment, the percent SPF in the solution is less than 25.0% by weight. In one embodiment, the percent SPF in the solution is less than 20.0% by weight. In one embodiment, the percent SPF in the solution is less than 19.0% by weight. In one embodiment, the percent SPF in the solution is less than 18.0% by weight. In one embodiment, the percent SPF in the solution is less than 17.0% by weight. In one embodiment, the percent SPF in the solution is less than 16.0% by weight. In one embodiment, the percent SPF in the solution is less than 15.0% by weight. In one embodiment, the percent SPF in the solution is less than 14.0% by weight. In one embodiment, the percent SPF in the solution is less than 13.0% by weight. In one embodiment, the percent SPF in the solution is less than 12.0% by weight. In one embodiment, the percent SPF in the solution is less than 11.0% by weight. In one embodiment, the percent SPF in the solution is less than 10.0% by weight. In one embodiment, the percent SPF in the solution is less than 9.0% by weight. In one embodiment, the percent SPF in the solution is less than 8.0% by weight. In one embodiment, the percent SPF in the solution is less than 7.0% by weight. In one embodiment, the percent SPF in the solution is less than 6.0% by weight. In one embodiment, the percent SPF in the solution is less than 5.0% by weight. In one embodiment, the percent SPF in the solution is less than 4.0% by weight. In one embodiment, the percent SPF in the solution is less than 3.0% by weight. In one embodiment, the percent SPF in the solution is less than 2.0% by weight. In one embodiment, the percent SPF in the solution is less than 1.0% by weight. In one embodiment, the percent SPF in the solution is less than 0.9% by weight. In one embodiment, the percent SPF in the solution is less than 0.8% by weight. In one embodiment, the percent of SPF in the solution is less than 0.7% by weight. In one embodiment, the percent of SPF in the solution is less than 0.6% by weight. In one embodiment, the percent of SPF in the solution is less than 0.5% by weight. In one embodiment, the percent of SPF in the solution is less than 0.4% by weight.In one embodiment, the percent of SPF in the solution is less than 0.3% by weight. In one embodiment, the percent of SPF in the solution is less than 0.2% by weight. In one embodiment, the percent of SPF in the solution is less than 0.1% by weight.

[0202] In one embodiment, the percent of SPF in the solution is greater than 0.1% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.2% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.3% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.4% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.5% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.6% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.7% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.8% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.9% by weight. In one embodiment, the percent of SPF in the solution is greater than 1.0% by weight. In one embodiment, the percent of SPF in the solution is greater than 2.0% by weight. In one embodiment, the percent of SPF in the solution is greater than 3.0% by weight. In one embodiment, the percent SPF in the solution is greater than 4.0% by weight. In one embodiment, the percent SPF in the solution is greater than 5.0% by weight. In one embodiment, the percent SPF in the solution is greater than 6.0% by weight. In one embodiment, the percent SPF in the solution is greater than 7.0% by weight. In one embodiment, the percent SPF in the solution is greater than 8.0% by weight. In one embodiment, the percent SPF in the solution is greater than 9.0% by weight. In one embodiment, the percent SPF in the solution is greater than 10.0% by weight. In one embodiment, the percent SPF in the solution is greater than 11.0% by weight. In one embodiment, the percent SPF in the solution is greater than 12.0% by weight. In one embodiment, the percent SPF in the solution is greater than 13.0% by weight. In one embodiment, the percent SPF in the solution is greater than 14.0% by weight. In one embodiment, the percent SPF in the solution is greater than 15.0% by weight. In one embodiment, the percent SPF in the solution is greater than 16.0% by weight. In one embodiment, the percent SPF in the solution is greater than 17.0% by weight. In one embodiment, the percent SPF in the solution is greater than 18.0% by weight. In one embodiment, the percent SPF in the solution is greater than 19.0% by weight. In one embodiment, the percent SPF in the solution is greater than 20.0% by weight.In one embodiment, the percent SPF in the solution is greater than 25.0% by weight.

[0203] In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 30.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 25.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 20.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 15.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 9.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 8.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 7.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 5.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 5.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 4.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 4.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.4% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 5.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 4.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 4.0% by weight.In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 4.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.4% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.0% by weight.

[0204] In one embodiment, the percentage of SPF in the solution ranges from about 20.0% to about 30.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 2% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 8.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 9.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 10.0% to about 20.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 11.0% to about 19.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 12.0% to about 18.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 13.0% to about 17.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 14.0% to about 16.0% by weight. In one embodiment, the percentage of SPF in the solution is about 1.0% by weight. In one embodiment, the percentage of SPF in the solution is about 0.5% by weight. In one embodiment, the percentage of SPF in the solution is about 1.5% by weight. In one embodiment, the percentage of SPF in the solution is about 2.0% by weight. In one embodiment, the percentage of SPF in the solution is about 2.4% by weight. In one embodiment, the percent of SPF in the solution is about 3.0% by weight. In one embodiment, the percent of SPF in the solution is about 3.5% by weight. In one embodiment, the percent of SPF in the solution is about 4.0% by weight. In one embodiment, the percent of SPF in the solution is about 4.5% by weight. In one embodiment, the percent of SPF in the solution is about 5.0% by weight. In one embodiment, the percent of SPF in the solution is about 5.5% by weight.In one embodiment, the percent of SPF in the solution is about 6.0% by weight. In one embodiment, the percent of SPF in the solution is about 6.5% by weight. In one embodiment, the percent of SPF in the solution is about 7.0% by weight. In one embodiment, the percent of SPF in the solution is about 7.5% by weight. In one embodiment, the percent of SPF in the solution is about 8.0% by weight. In one embodiment, the percent of SPF in the solution is about 8.5% by weight. In one embodiment, the percent of SPF in the solution is about 9.0% by weight. In one embodiment, the percent of SPF in the solution is about 9.5% by weight. In one embodiment, the percent of SPF in the solution is about 10.0% by weight.

[0205] In one embodiment, the percentage of sericin in the solution is undetectable to 25.0% by weight. In one embodiment, the percentage of sericin in the solution is undetectable to 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 1.0% by weight. In one embodiment, the percentage of sericin in the solution is 2.0% by weight. In one embodiment, the percentage of sericin in the solution is 3.0% by weight. In one embodiment, the percentage of sericin in the solution is 4.0% by weight. In one embodiment, the percentage of sericin in the solution is 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 10.0% by weight. In one embodiment, the percentage of sericin in the solution is 25.0% by weight.

[0206] In some embodiments, the silk fibroin protein fragments of the present disclosure have a storage stability of 10 days to 3 years (when stored in aqueous solution, they do not slowly or spontaneously gel, and the fragments do not aggregate, thus resulting in no increase in molecular weight over time), depending on storage conditions, percent SPF, and number of shipments and shipping conditions. Additionally, the pH may be altered to extend shelf life and / or support shipping conditions by preventing premature folding and aggregation of the silk. In one embodiment, the stability of the LiBr-silk fragments solution is 0-1 year. In one embodiment, the stability of the LiBr-silk fragments solution is 0-2 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-3 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-4 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-5 years. In one embodiment, the stability of the LiBr-silk fragments solution is 1-2 years. In one embodiment, the stability of the LiBr-silk fragments solution is 1-3 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 3 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 4 to 5 years.

[0207] In one embodiment, the composition of the present disclosure has a stability of 10 days to 6 months. In one embodiment, the composition of the present disclosure has a stability of 6 months to 12 months. In one embodiment, the composition of the present disclosure has a stability of 12 months to 18 months. In one embodiment, the composition of the present disclosure has a stability of 18 months to 24 months. In one embodiment, the composition of the present disclosure has a stability of 24 months to 30 months. In one embodiment, the composition of the present disclosure has a stability of 30 months to 36 months. In one embodiment, the composition of the present disclosure has a stability of 36 months to 48 months. In one embodiment, the composition of the present disclosure has a stability of 48 months to 60 months.

[0208] In one embodiment, compositions of the present disclosure having an SPF have undetectable levels of LiBr residual. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is from 10 ppm to 1000 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is from 10 ppm to 300 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 25 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 50 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 75 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 100 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 200 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 300 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 500 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 600 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 700 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 800 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 300 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 250 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 200 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 150 ppm.In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from undetectable to 100 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 100 ppm to 200 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 200 ppm to 300 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 300 ppm to 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is from 400 ppm to 500 ppm.

[0209] In one embodiment, the disclosed compositions having SPF have undetectable levels of Na2CO3 residue. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 100 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 200 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 300 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 400 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 500 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 600 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 700 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 800 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 300 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 250 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 200 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 150 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 100 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is 100 ppm to 200 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is 200 ppm to 300 ppm.In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is 300 ppm to 400 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is 400 ppm to 500 ppm.

[0210] A unique feature of the SPF compositions of the present disclosure is their storage stability (when stored in aqueous solution, they do not slowly or spontaneously gel, and the fragments do not aggregate, thus resulting in no increase in molecular weight over time), ranging from 10 days to 3 years, depending on storage conditions, percent silk, and number of shipments and shipping conditions. Additionally, the pH may be altered to extend shelf life by preventing premature folding and aggregation of the silk and / or to support shipping conditions. In one embodiment, the SPF solution composition of the present disclosure has storage stability at room temperature (RT) for up to 2 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 4 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 6 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 8 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 10 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 12 weeks. In one embodiment, the SPF solution composition of the present disclosure has a storage stability at RT ranging from about 4 weeks to about 52 weeks. Table R below shows storage stability test results for embodiments of the SPF compositions of the present disclosure. [Table 25]

[0211] In some embodiments, the water solubility of silk films derived from silk fibroin protein fragments as described herein can be modified by solvent annealing (water annealing or methanol annealing), chemical cross-linking, enzymatic cross-linking, and heat treatment.

[0212] In some embodiments, the annealing process may involve inducing beta-sheet formation in a silk fibroin protein fragment solution used as a coating material. Techniques have been described to promote annealing (e.g., increasing crystallinity) or otherwise "molecular packing" of silk fibroin protein-based fragments. In some embodiments, amorphous silk films are annealed to introduce beta-sheets in the presence of a solvent selected from the group consisting of water or organic solvents. In some embodiments, amorphous silk films are annealed to introduce beta-sheets in the presence of water (aqueous annealing process). In some embodiments, amorphous silk fibroin protein fragment films are annealed to introduce beta-sheets in the presence of methanol. In some embodiments, annealing (e.g., beta-sheet formation) is induced by the addition of an organic solvent. Suitable organic solvents include, but are not limited to, methanol, ethanol, acetone, isopropanol, or combinations thereof.

[0213] In some embodiments, annealing is carried out by so-called "water annealing" or "water vapor annealing," in which water vapor is used as an intermediate plasticizer or catalyst to promote beta-sheet packing. In some embodiments, the water annealing process may be carried out under vacuum. Suitable such methods are described in Jin HJ et al. (2005), Water-stable Silk Films with Reduced Beta-Sheet Content, Advanced Functional Materials, 15:1241-1247, and Xiao H. et al. (2011), Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing, Biomacromolecules, 12(5):1686-1696.

[0214] A key feature of the water annealing process is that it drives the formation of crystalline beta sheets in the silk fibroin protein fragment peptide chains, allowing the silk fibroin to self-assemble into a continuous film. In some embodiments, the crystallinity of the silk fibroin protein fragment film is controlled by controlling the temperature of the water vapor and the duration of the annealing. In some embodiments, the annealing is carried out at a temperature ranging from about 65°C to about 110°C. In some embodiments, the temperature of the water is maintained at about 80°C. In some embodiments, the annealing is carried out at a temperature selected from the group consisting of about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, about 100°C, about 105°C, and about 110°C.

[0215] In some embodiments, the annealing process may be performed for about 1 minute to about 40 minutes, about 1 minute to about 50 minutes, about 1 minute to about 60 minutes, about 1 minute to about 70 minutes, about 1 minute to about 80 minutes, about 1 minute to about 90 minutes, about 1 minute to about 100 minutes, about 1 minute to about 110 minutes, about 1 minute to about 120 minutes, about 1 minute to about 130 minutes, about 5 minutes to about 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 150 minutes, about 15 minutes to about 200 minutes, about 15 minutes to about 250 minutes, about 15 minutes to about 260 minutes, about 15 minutes to about 270 minutes, about 15 minutes to about 280 minutes, about 15 minutes to about 290 minutes, about 15 minutes to about 300 minutes, about 15 minutes to about 310 minutes, about 15 minutes to about 320 minutes, about 15 minutes to about 330 minutes, about 15 minutes to about 340 minutes, about 15 minutes to about 350 minutes, about 15 minutes to about 360 minutes, about 15 minutes to about 370 minutes, about 15 minutes to about 380 minutes, about 15 minutes to about 390 minutes, about 15 minutes to about 400 minutes Approximately 70 minutes, approximately 10 minutes to approximately 80 minutes, approximately 10 minutes to approximately 90 minutes, approximately 10 minutes to approximately 100 minutes, approximately 10 minutes to approximately 110 minutes, approximately 10 minutes to approximately 120 minutes, approximately 10 minutes to approximately 130 minutes, approximately 15 minutes to approximately 40 minutes, approximately 15 minutes to approximately 50 minutes, approximately 15 minutes to approximately 60 minutes, approximately 15 minutes to approximately 70 minutes, approximately 15 minutes to approximately 80 minutes, approximately 15 minutes to Approximately 90 minutes, approximately 15 minutes to approximately 100 minutes, approximately 15 minutes to approximately 110 minutes, approximately 15 minutes to approximately 120 minutes, approximately 15 minutes to approximately 130 minutes, approximately 20 minutes to approximately 40 minutes, approximately 20 minutes to approximately 50 minutes, approximately 20 minutes to approximately 60 minutes, approximately 20 minutes to approximately 70 minutes, approximately 20 minutes to approximately 80 minutes, approximately 20 minutes to approximately 90 minutes, approximately 20 minutes to approximately 100 minutes, approximately 20 minutes ~approx. 110 minutes, ~approx. 20 minutes ~approx. 120 minutes, ~approx. 20 minutes ~approx. 130 minutes, ~approx. 25 minutes ~approx. 40 minutes, ~approx. 25 minutes ~approx. 50 minutes, ~approx. 25 minutes ~approx. 60 minutes, ~approx. 25 minutes ~approx. 70 minutes, ~approx. 25 minutes ~approx. 80 minutes, ~approx. 25 minutes ~approx. 90 minutes, ~approx. 25 minutes ~approx. 100 minutes, ~approx. 25 minutes ~approx. 110 minutes, ~approx. 25 minutes ~approx. 120 minutes, ~approx. 5 minutes to approximately 130 minutes, approximately 30 minutes to approximately 40 minutes, approximately 30 minutes to approximately 50 minutes, approximately 30 minutes to approximately 60 minutes, approximately 30 minutes to approximately 70 minutes, approximately 30 minutes to approximately 80 minutes, approximately 30 minutes to approximately 90 minutes, approximately 30 minutes to approximately 100 minutes, approximately 30 minutes to approximately 110 minutes, approximately 30 minutes to approximately 120 minutes, approximately 30 minutes to approximately 130 minutes, approximately 35 minutes to approximately 40 minutes, approximately 35 minutes to approximately 50 minutes, approximately 35 minutes to approximately 60 minutes, approximately 35 minutes to approximately 70 minutes, approximately 35 minutes to approximately 80 minutes, approximately 35 minutes to approximately 90 minutes, approximately 35 minutes to approximately 100 minutes, approximately 35 minutes to approximately 110 minutes, approximately 35 minutes to approximately 120 minutes, approximately 35 minutes to approximately 130 minutes, approximately 40 minutes to approximately 50 minutes, approximately 40 minutes to approximately 60 minutes, approximately 40 minutes to approximately 70 minutes, approximately 40 minutes to approximately 80 minutes, approximately 40 minutes to approximately 90 minutes, approximately 40 minutes to approximately 100 minutes, approximately 40 minutes to approximately 110 minutes, approximately 40 minutes to approximately 120 minutes, approximately 40 minutes to approximately 130 minutes, approximately 45 minutes to approximately 50 minutes, approximately 45 minutes to approximately 60 minutes, approximately 45 minutes to approximately 70 minutes, approximately 45 minutes to approximately 80 minutes, approximately 45 minutes to approximately 90 minutes, approximately 45 minutes to approximately 100 minutes,The annealing process lasts for a period selected from the group consisting of about 45 minutes to about 110 minutes, about 45 minutes to about 120 minutes, and about 45 minutes to about 130 minutes. In some embodiments, the annealing process lasts for a period ranging from about 1 minute to about 60 minutes. In some embodiments, the annealing process lasts for a period ranging from about 45 minutes to about 60 minutes. Longer water annealing after processing has been associated with increased crystallinity of the silk fibroin protein fragments.

[0216] In some embodiments, the annealed silk fibroin protein fragment film is prepared by immersing the wet silk fibroin protein fragment film in 100% methanol at room temperature for 60 minutes. Methanol annealing changes the composition of the silk fibroin protein fragment film from a predominantly amorphous random coil to a crystalline antiparallel beta sheet structure.

[0217] In some embodiments, SPF microparticles can be prepared by precipitation with methanol using SPF as described herein. Alternatively, flash drying, fluidized bed drying, spray drying, or vacuum drying can be applied to remove water from the silk solution. The SPF powder can then be stored and handled without refrigeration or other special handling procedures. In some embodiments, the SPF powder comprises low molecular weight silk fibroin protein fragments. In some embodiments, the SPF powder comprises medium molecular weight silk fibroin protein fragments. In some embodiments, the SPF powder comprises a mixture of low molecular weight and medium molecular weight silk fibroin protein fragments.

[0218] As used herein, the terms "substantially free of sericin" or "substantially devoid of sericin" refer to silk fibers from which the majority of the sericin protein has been removed. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 10.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 9.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 8.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 7.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 6.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 5.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.05% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.1% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.5% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 1.0% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 1.5% to about 4.0% by weight of sericin, and in one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 2.0% to about 4.0% by weight of sericin.In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 2.5% to about 4.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content of about 0.01% to about 0.1% by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.1% by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.05% by weight. In one embodiment, when a silk source is added to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes, a degumming loss of about 26.0% to about 31.0% by weight is obtained.

[0219] The following are non-limiting examples of suitable ranges for various parameters in and for preparing silk solutions of the present disclosure. Silk solutions of the present disclosure may include one or more (but not necessarily all) of these parameters and may be prepared using various combinations of such parameter ranges.

[0220] In one embodiment, the percent SPF in the solution is less than 30.0% by weight. In one embodiment, the percent SPF in the solution is less than 25.0% by weight. In one embodiment, the percent SPF in the solution is less than 20.0% by weight. In one embodiment, the percent SPF in the solution is less than 19.0% by weight. In one embodiment, the percent SPF in the solution is less than 18.0% by weight. In one embodiment, the percent SPF in the solution is less than 17.0% by weight. In one embodiment, the percent SPF in the solution is less than 16.0% by weight. In one embodiment, the percent SPF in the solution is less than 15.0% by weight. In one embodiment, the percent SPF in the solution is less than 14.0% by weight. In one embodiment, the percent SPF in the solution is less than 13.0% by weight. In one embodiment, the percent SPF in the solution is less than 12.0% by weight. In one embodiment, the percent SPF in the solution is less than 11.0% by weight. In one embodiment, the percent SPF in the solution is less than 10.0% by weight. In one embodiment, the percent SPF in the solution is less than 9.0% by weight. In one embodiment, the percent SPF in the solution is less than 8.0% by weight. In one embodiment, the percent SPF in the solution is less than 7.0% by weight. In one embodiment, the percent SPF in the solution is less than 6.0% by weight. In one embodiment, the percent SPF in the solution is less than 5.0% by weight. In one embodiment, the percent SPF in the solution is less than 4.0% by weight. In one embodiment, the percent SPF in the solution is less than 3.0% by weight. In one embodiment, the percent SPF in the solution is less than 2.0% by weight. In one embodiment, the percent SPF in the solution is less than 1.0% by weight. In one embodiment, the percent SPF in the solution is less than 0.9% by weight. In one embodiment, the percent SPF in the solution is less than 0.8% by weight. In one embodiment, the percent of SPF in the solution is less than 0.7% by weight. In one embodiment, the percent of SPF in the solution is less than 0.6% by weight. In one embodiment, the percent of SPF in the solution is less than 0.5% by weight. In one embodiment, the percent of SPF in the solution is less than 0.4% by weight.In one embodiment, the percent of SPF in the solution is less than 0.3% by weight. In one embodiment, the percent of SPF in the solution is less than 0.2% by weight. In one embodiment, the percent of SPF in the solution is less than 0.1% by weight.

[0221] In one embodiment, the percent of SPF in the solution is greater than 0.1% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.2% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.3% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.4% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.5% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.6% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.7% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.8% by weight. In one embodiment, the percent of SPF in the solution is greater than 0.9% by weight. In one embodiment, the percent of SPF in the solution is greater than 1.0% by weight. In one embodiment, the percent of SPF in the solution is greater than 2.0% by weight. In one embodiment, the percent of SPF in the solution is greater than 3.0% by weight. In one embodiment, the percent SPF in the solution is greater than 4.0% by weight. In one embodiment, the percent SPF in the solution is greater than 5.0% by weight. In one embodiment, the percent SPF in the solution is greater than 6.0% by weight. In one embodiment, the percent SPF in the solution is greater than 7.0% by weight. In one embodiment, the percent SPF in the solution is greater than 8.0% by weight. In one embodiment, the percent SPF in the solution is greater than 9.0% by weight. In one embodiment, the percent SPF in the solution is greater than 10.0% by weight. In one embodiment, the percent SPF in the solution is greater than 11.0% by weight. In one embodiment, the percent SPF in the solution is greater than 12.0% by weight. In one embodiment, the percent SPF in the solution is greater than 13.0% by weight. In one embodiment, the percent SPF in the solution is greater than 14.0% by weight. In one embodiment, the percent SPF in the solution is greater than 15.0% by weight. In one embodiment, the percent SPF in the solution is greater than 16.0% by weight. In one embodiment, the percent SPF in the solution is greater than 17.0% by weight. In one embodiment, the percent SPF in the solution is greater than 18.0% by weight. In one embodiment, the percent SPF in the solution is greater than 19.0% by weight. In one embodiment, the percent SPF in the solution is greater than 20.0% by weight.In one embodiment, the percent SPF in the solution is greater than 25.0% by weight.

[0222] In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 30.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 25.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 20.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 15.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 9.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 8.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 7.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 5.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 5.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 4.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 4.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 2.4% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 5.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 4.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 4.0% by weight.In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.5% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 4.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 3.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 3.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.5% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.4% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 2.0% by weight.

[0223] In one embodiment, the percentage of SPF in the solution ranges from about 20.0% to about 30.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 1.0% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 2% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 0.1% to about 6.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 10.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 8.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 6.0% to about 9.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 10.0% to about 20.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 11.0% to about 19.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 12.0% to about 18.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 13.0% to about 17.0% by weight. In one embodiment, the percentage of SPF in the solution ranges from about 14.0% to about 16.0% by weight. In one embodiment, the percentage of SPF in the solution is about 1.0% by weight. In one embodiment, the percentage of SPF in the solution is about 1.5% by weight. In one embodiment, the percentage of SPF in the solution is about 2.0% by weight. In one embodiment, the percentage of SPF in the solution is about 2.4% by weight. In one embodiment, the percentage of SPF in the solution is about 3.0% by weight. In one embodiment, the percent of SPF in the solution is about 3.5% by weight. In one embodiment, the percent of SPF in the solution is about 4.0% by weight. In one embodiment, the percent of SPF in the solution is about 4.5% by weight. In one embodiment, the percent of SPF in the solution is about 5.0% by weight. In one embodiment, the percent of SPF in the solution is about 5.5% by weight. In one embodiment, the percent of SPF in the solution is about 6.0% by weight.In one embodiment, the percent of SPF in the solution is about 6.5% by weight. In one embodiment, the percent of SPF in the solution is about 7.0% by weight. In one embodiment, the percent of SPF in the solution is about 7.5% by weight. In one embodiment, the percent of SPF in the solution is about 8.0% by weight. In one embodiment, the percent of SPF in the solution is about 8.5% by weight. In one embodiment, the percent of SPF in the solution is about 9.0% by weight. In one embodiment, the percent of SPF in the solution is about 9.5% by weight. In one embodiment, the percent of SPF in the solution is about 10.0% by weight.

[0224] In one embodiment, the percentage of sericin in the solution is undetectable to 25.0% by weight. In one embodiment, the percentage of sericin in the solution is undetectable to 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 1.0% by weight. In one embodiment, the percentage of sericin in the solution is 2.0% by weight. In one embodiment, the percentage of sericin in the solution is 3.0% by weight. In one embodiment, the percentage of sericin in the solution is 4.0% by weight. In one embodiment, the percentage of sericin in the solution is 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 10.0% by weight. In one embodiment, the percentage of sericin in the solution is 25.0% by weight.

[0225] In some embodiments, the silk fibroin-based protein fragments of the present disclosure have a storage stability (when stored in aqueous solution, they do not slowly or spontaneously gel, and the fragments do not aggregate, thus resulting in no increase in molecular weight over time) of 10 days to 3 years, depending on storage conditions, percent SPF, and number of shipments and shipping conditions. Additionally, the pH may be altered to extend shelf life by preventing premature folding and aggregation of the silk and / or to support shipping conditions. In one embodiment, the stability of the LiBr-silk fragments solution is 0-1 year. In one embodiment, the stability of the LiBr-silk fragments solution is 0-2 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-3 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-4 years. In one embodiment, the stability of the LiBr-silk fragments solution is 0-5 years. In one embodiment, the stability of the LiBr-silk fragments solution is 1-2 years. In one embodiment, the stability of the LiBr-silk fragments solution is 1-3 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 3 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 4 to 5 years.

[0226] In one embodiment, the composition of the present disclosure has a stability of 10 days to 6 months. In one embodiment, the composition of the present disclosure has a stability of 6 months to 12 months. In one embodiment, the composition of the present disclosure has a stability of 12 months to 18 months. In one embodiment, the composition of the present disclosure has a stability of 18 months to 24 months. In one embodiment, the composition of the present disclosure has a stability of 24 months to 30 months. In one embodiment, the composition of the present disclosure has a stability of 30 months to 36 months. In one embodiment, the composition of the present disclosure has a stability of 36 months to 48 months. In one embodiment, the composition of the present disclosure has a stability of 48 months to 60 months.

[0227] In one embodiment, selected properties of an SPF coated article that may be enhanced compared to an uncoated article include dimensional stability to laundering, dimensional stability to dry cleaning, appearance after laundering, appearance after dry cleaning, colorfastness to laundering, colorfastness to dry cleaning, colorfastness to non-chlorine bleach, seam torque / spirality (on knitted fabrics), colorfastness to crocking, colorfastness to abrasion, colorfastness to water, colorfastness to light, colorfastness to sweat, colorfastness to chlorinated pool water, colorfastness to saltwater, tensile strength, seam slippage, tear strength, seam break strength, abrasion resistance, pilling resistance, elongation These may include one or more of recovery, burst strength, colorfastness to dye transfer during storage (label), colorfastness to ozone, pile retention, flex and skew, colorfastness to saliva, snagging resistance, wrinkle resistance (e.g., garment appearance, fabr...

Claims

1. A plurality of substantially solid silk fibroin particles comprising silk fibroin fragments, the particles being characterized by at least one of bulk density, surface area, pore size, pore volume, aspect ratio, and / or Hausner ratio.

2. 10. The plurality of substantially solid silk fibroin particles of claim 1, wherein the substantially solid silk fibroin particles are annealed.

3. 3. The plurality of substantially solid silk fibroin particles of claim 1 or 2, wherein the substantially solid silk fibroin particles are comminuted.

4. the substantially solid silk fibroin particles have a viscosity of less than 0.03 g / ml, less than 0.04 g / ml, less than 0.05 g / ml, less than 0.06 g / ml, less than 0.07 g / ml, less than 0.08 g / ml, less than 0.09 g / ml, less than 0.10 g / ml, less than 0.11 g / ml, less than 0.12 g / ml, less than 0.13 g / ml, less than 0.14 g / ml, less than 0.15 g / ml, less than 0.16 g / ml, less than 0.17 g / ml, less than 0.18 g / ml, less than 0.19 g / ml, less than 0.20 g / ml 4. The plurality of substantially solid silk fibroin particles of claim 1, having a bulk density of less than 0.21 g / ml, less than 0.22 g / ml, less than 0.23 g / ml, less than 0.24 g / ml, or less than 0.25 g / ml, less than 0.26 g / ml, less than 0.27 g / ml, less than 0.28 g / ml, less than 0.29 g / ml, less than 0.30 g / ml, less than 0.31 g / ml, less than 0.32 g / ml, less than 0.33 g / ml, less than 0.34 g / ml, or less than 0.35 g / ml.

5. The substantially solid silk fibroin particles have a viscosity of about 0.03 g / ml, about 0.04 g / ml, or about 0.05 g / ml, 4. The plurality of substantially solid silk fibroin particles of claim 1, having an average bulk density of about 9 g / ml, about 0.20 g / ml, about 0.21 g / ml, about 0.22 g / ml, about 0.23 g / ml, about 0.24 g / ml, about 0.25 g / ml, about 0.26 g / ml, about 0.27 g / ml, about 0.28 g / ml, about 0.29 g / ml, about 0.30 g / ml, about 0.31 g / ml, about 0.32 g / ml, about 0.33 g / ml, about 0.34 g / ml, or about 0.35 g / ml.

6. 6. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 5, wherein the substantially solid silk fibroin particles have a Hausner ratio of 1.00 to 1.11, 1.12 to 1.18, 1.19 to 1.25, 1.26 to 1.34, or 1.35 to 1.

45.

7. 7. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 6, wherein the substantially solid silk fibroin particles have an average diameter of about 3 mm to about 10 mm.

8. 7. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 6, wherein the substantially solid silk fibroin particles have an average diameter of about 3 mm, about 4 mm, about 5 mm, about 6 mm, about 7 mm, about 8 mm, about 9 mm, or about 10 mm.

9. 9. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 8, wherein the substantially solid silk fibroin particles have an aspect ratio of 1 to about 1.

45.

10. 9. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 8, wherein the substantially solid silk fibroin particles have an aspect ratio of 1, about 1.10, about 1.15, about 1.20, about 1.25, about 1.30, about 1.35, about 1.40, or about 1.

45.

11. 9. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 8, wherein the substantially solid silk fibroin particles are substantially spherical.

12. 12. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 11, wherein the substantially solid silk fibroin particles are mesoporous.

13. The substantially solid silk fibroin particles are about 2.50 m ２ / g ~ approx. 6.50m ２ 12. The plurality of substantially solid silk fibroin particles according to any one of claims 1 to 11, having a BET (Brunauer-Emmett-Teller) surface area of ​​1 / g.

14. The substantially solid silk fibroin particles are about 2.50 m ２ / g ~ approx. 3.00m ２ / g, approx. 3.00m ２ / g ~ approx. 3.50m ２ / g, approx. 3.50m ２ / g ~ approx. 4.00m ２ / g, approx. 4.00m ２ / g ~ approx. 4.50m ２ / g, approx. 4.50m ２ / g ~ approx. 5.00m ２ / g, approx. 5.00m ２ / g ~ approx. 5.50m ２ / g, approx. 5.50m ２ / g ~ approx. 6.00m ２ / g, or about 6.00 m ２ / g ~ approx. 6.50m ２ 12. The plurality of substantially solid silk fibroin particles according to any one of claims 1 to 11, having a BET (Brunauer-Emmett-Teller) surface area of ​​1 / g.

15. 15. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 14, wherein the substantially solid silk fibroin particles have an average pore size of about 25 Å to about 500 Å.

16. The substantially solid silk fibroin particles have a thickness of about 25 Å to about 30 Å, about 30 Å to about 35 Å, about 35 Å to about 40 Å, about 40 Å to about 45 Å, about 45 Å to about 50 Å, about 50 Å to about 55 Å, about 55 Å to about 60 Å, about 60 Å to about 65 Å, about 65 Å to about 70 Å, about 70 Å to about 75 Å, about 75 Å to about 80 Å, about 80 Å to about 85 Å, about 85 Å to about 90 Å, about 90 Å to about 95 Å, about 95 Å to about 100 Å 15. The plurality of substantially solid silk fibroin particles according to any one of claims 1 to 14, having an average pore diameter of about 100 Å to about 105 Å, about 105 Å to about 110 Å, about 110 Å to about 115 Å, about 115 Å to about 120 Å, about 120 Å to about 125 Å, about 125 Å to about 130 Å, about 130 Å to about 135 Å, about 135 Å to about 140 Å, about 140 Å to about 145 Å, or about 145 Å to about 150 Å.

17. 17. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 16, wherein the substantially solid silk fibroin particles comprise a plurality of radially oriented microchannels.

18. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise an emulsifier, a surfactant, a buffer, an amino acid, or a sugar.

19. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a polysaccharide, polysorbate, glycoside, PBS, arginine, trehalose, glucose, or sucrose.

20. 18. The plurality of substantially solid silk fibroin particles according to any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a surfactant selected from sucrose esters, cetearyl glucoside, caprylyl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof.

21. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise an additional protein or peptide, a C12 to C24 fatty alcohol, a glycolipid, or a lipid.

22. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination.

23. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a sugar fatty acid ester, a sugar fatty acid monoester, a sugar fatty diester, a sugar fatty triester, or a sugar fatty and polyester.

24. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise a sucrose fatty acid ester, a sorbitan or sorbitol fatty acid ester, an alkyl glucoside, an alkyl polyglucoside, or a combination thereof.

25. 18. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 17, wherein the substantially solid silk fibroin particles further comprise KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, capryl glucoside, sucrose, histidine, glycine, or arginine.

26. A silk fibroin nanoclay composite or film comprising silk fibroin fragments and clay, wherein the % (w / w) of clay in the composite is from about 1% to about 99%.

27. 27. The nanoclay composite or film of claim 26, wherein the clay is a bentonite clay.

28. 28. The nanoclay composite or film of claim 26 or 27, wherein the concentration of clay in the composite or film is from about 20% (w / w) to about 33% (w / w), from about 33% (w / w) to about 50% (w / w), or from about 50% (w / w) to about 67% (w / w).

29. 29. The nanoclay composite or film of any one of claims 26 to 28, wherein the water vapor transmission rate (WVP) of the composite is inversely proportional to the concentration of clay in the composite.

30. The water vapor transmission rate (WVP, g / m ２ 29. The nanoclay composite or film of any one of claims 26 to 28, wherein the modulus of elasticity (MPa x Pa x 24 hr) is from about 0.20 to about 0.30, from about 0.30 to about 0.35, from about 0.35 to about 0.40, from about 0.40 to about 0.45, from about 0.45 to about 0.50, from about 0.50 to about 0.55, from about 0.55 to about 0.60, from about 0.60 to about 0.65, from about 0.65 to about 0.70, from about 0.70 to about 0.75, from about 0.75 to about 0.80, or from about 0.80 to about 0.

85.

31. A stabilized silk fibroin solution comprising silk fibroin fragments and a stabilizer, i) the solution has a lower z-average value than a substantially similar silk fibroin solution containing silk fibroin fragments but excluding the stabilizer; and / or ii) A stabilized silk fibroin solution, wherein said solution has a lower z-average plateau value than a substantially similar silk fibroin solution comprising silk fibroin fragments but excluding said stabilizer.

32. 32. The stabilized silk fibroin solution of claim 31, wherein the z-average is measured after a period of time after co-combining the silk fibroin fragments with the stabilizer, the period of time being in the range of 1 hour to 250 hours, 1 hour to 350 hours, 1 hour to 450 hours, 1 hour to 550 hours, 1 hour to 650 hours, or 1 hour to 1000 hours.

33. 32. The stabilized silk fibroin solution of claim 31 , wherein the z-average is measured after a period of time after co-combining the silk fibroin fragments with the stabilizer, the period of time being in the range of 1 minute to 10 minutes, 1 minute to 20 minutes, 1 minute to 30 minutes, 1 minute to 40 minutes, 1 minute to 50 minutes, 1 minute to 60 minutes, 1 minute to 70 minutes, or 1 minute to 80 minutes.

34. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is an emulsifier, a surfactant, a buffer, an amino acid, or a sugar.

35. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is a polysaccharide, polysorbate, glycoside, PBS, arginine, trehalose, glucose, or sucrose.

36. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is a surfactant selected from sucrose esters, cetearyl glucoside, caprylyl / capryl glucoside, sucrose laurate, sucrose palmitate, sucrose stearate, sucrose cocoate, sorbitan monostearate, and combinations thereof.

37. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is an additional protein or peptide, a C12 to C24 fatty alcohol, a glycolipid, or a lipid.

38. The stabilized silk fibroin solution of any one of claims 31 to 33, wherein the stabilizer is a protein, a peptide, a sugar surfactant, a biosurfactant, a lipid, or a combination.

39. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is a sugar fatty acid ester, a sugar fatty acid monoester, a sugar fatty diester, a sugar fatty triester, or a sugar fatty and polyester.

40. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is a sucrose fatty acid ester, a sorbitan or sorbitol fatty acid ester, an alkyl glucoside, an alkyl polyglucoside, or a combination thereof.

41. The stabilized silk fibroin solution according to any one of claims 31 to 33, wherein the stabilizer is KCl, NaCl, MgCl2, CaCl2, PBS, Tris, polysorbate 20, polysorbate 80, capryl glucoside, sucrose, histidine, glycine, or arginine.

42. The stabilized silk fibroin solution of any one of claims 31 to 41, wherein the solution is sprayable.

43. 43. A liquid-in-air suspension comprising a plurality of droplets comprising the stabilized silk fibroin solution of claim 42, wherein the liquid-in-air suspension is sufficiently stable for the period of time required for the droplets to reach a surface after being sprayed.

44. 42. A plurality of droplets or small droplets comprising the stabilized silk fibroin solution of any one of claims 31 to 41, wherein the droplets or small droplets are sufficiently stable after formation for a period of time required to reach a surface.

45. the silk fibroin fragments are selected from the group consisting of about 1 kDa to about 5 kDa, about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 10 kDa to about 15 kDa, about 14 kDa to about 30 kDa, about 15 kDa to about 20 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, and about 55 kDa to about 60 kDa; 46. ​​A method for manufacturing a silk fibroin nanoclay composite or film according to claim 45, a method for manufacturing a silk fibroin nanoclay composite or film according to claim 45, a method for manufacturing a silk fibroin nanoclay composite or film according to claim 46 ...

46. 46. ​​The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 45, wherein the polydispersity is from 1 to about 1.5, from about 1.5 to about 2.0, from about 2.0 to about 2.5, from about 2.5 to about 3.0, from about 3.0 to about 3.5, from about 3.5 to about 4.0, from about 4.0 to about 4.5, or from about 4.5 to about 5.

0.

47. 46. ​​The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 45, further comprising about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin fragments.

48. 48. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets according to any one of claims 45 to 47, wherein the silk fibroin fragments do not spontaneously or gradually gel or undergo a visual change in color or turbidity when in aqueous solution for at least 10 days before being incorporated into the substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, or stabilized silk fibroin solution.

49. 45. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 25, the silk fibroin nanoclay composite or film of any one of claims 26 to 30, the stabilized silk fibroin solution of any one of claims 31 to 42, the liquid-in-air suspension of claim 43, or the plurality of droplets or droplets of claim 44, wherein the silk fibroin fragments comprise a plurality of amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W, and at least one of the amino acids is modified, substituted, or substituted.

50. 50. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 49, wherein the fibroin is a fibroin heavy chain, a fibroin light chain, or fibrohexamerin.

51. 51. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 49 or 50, wherein the silk fibroin fragments comprise from about 2 to about 100 amino acids.

52. 52. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claims 49-51, wherein the silk fibroin fragments comprise 1 to 5 modifications, substitutions, and / or substitutions.

53. 53. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets according to any one of claims 49 to 52, wherein the modification, substitution, and / or substitution is selected from modification, substitution, and / or substitution of asparagine with aspartic acid, modification, substitution, and / or substitution of glutamine with glutamic acid, and modification, substitution, and / or substitution of methionine with methionine oxide.

54. 54. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 49 to 53, wherein the fibroin is a fibroin heavy chain and the modification, substitution, and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain.

55. 55. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 54, wherein the modification, substitution, and / or substitution is in Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, and / or N5262.

56. 54. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 49 to 53, wherein the fibroin is a fibroin light chain and the modification, substitution, and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain.

57. 57. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 56, wherein the modification, substitution, and / or substitution is in N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, and / or Q255.

58. 54. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 49 to 53, wherein the fibroin is fibrohexamerin (p25) and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the fibrohexamerin (p25).

59. 59. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 58, wherein the modification, substitution, and / or substitution is in Q62, N93, M120, N149, N172, N174, and / or N202.

60. 60. A plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets according to any one of claims 49 to 59, wherein each modification, substitution, and / or substitution independently ranges from about 1% to about 99% of the silk fibroin fragment portion of the substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, or stabilized silk fibroin solution composition.

61. 62. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 61 , wherein the % modification, substitution, and / or substitution is defined as (the number of peptides or protein fragments containing a modification, substitution, and / or substitution at a particular position divided by the total number of peptides or protein fragments containing said particular position, regardless of whether they contain a modification, substitution, and / or substitution) x 100.

62. 45. A plurality of substantially solid silk fibroin particles according to any one of claims 1 to 25, a silk fibroin nanoclay composite or film according to any one of claims 26 to 30, a stabilized silk fibroin solution according to any one of claims 31 to 42, a liquid-in-air suspension according to claim 43, or a plurality of droplets or droplets according to claim 44, wherein the silk fibroin fragments are contained in one or more fractions, each fraction independently comprising a plurality of fibroin heavy chain fragments, a plurality of fibroin light chain fragments, and / or a plurality of fibrohexamerin (p25) fragments.

63. The silk fibroin fragments have a weight average molecular weight (M) selected from about 1 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, or about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 250 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.7, and a polydispersity of 1 to about 1.

7.

64. The silk fibroin fragments have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, or about 160 kDa to about 180 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, or 1 to about 1.

2.

65. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, or 1 to about 1.

2.

66. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 120 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, and

67. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 10 kDa to about 20 kDa, about 20 kDa to about 40 kDa, about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, or 1 to about 1.

2.

68. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, and

69. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 20 kDa to about 40 kDa, or about 40 kDa to about 60 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, or 1 to about 1.

2.

70. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS77, AS78, AS79, AS80, and AS81.

71. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS82, AS83, AS84, AS85, AS86, AS87, AS88, and AS89.

72. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS90, AS91, AS92, AS93, and AS94.

73. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS95, AS96, AS97, AS98, AS99, and AS100.

74. The silk fibroin fragments have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 220 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.7, and a polydispersity of 1 to about 1.

7.

75. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, or about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.2, or 1 to about 1.

3.

76. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 40 kDa to about 60 kDa, about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, or about 100 kDa to about 110 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.1, or 1 to about 1.

2.

77. The silk fibroin fragments in the fraction have a weight average molecular weight (M) selected from about 60 kDa to about 80 kDa, about 80 kDa to about 100 kDa, about 100 kDa to about 120 kDa, about 120 kDa to about 140 kDa, about 140 kDa to about 160 kDa, about 160 kDa to about 180 kDa, about 180 kDa to about 200 kDa, or about 200 kDa to about 210 kDa. ｗ 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, having a polydispersity of 1 to about 1.2, or 1 to about 1.

3.

78. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS101, AS102, AS103, AS104, and AS105.

79. 63. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 62, wherein the one or more fractions are selected from AS106, AS107, AS108, AS109, AS110, and AS111.

80. 80. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 62 to 79, wherein the silk fibroin fragments comprise a modification, substitution, or replacement of one or more amino acids selected from M, R, V, K, T, F, I, L, C, A, Q, Y, N, D, E, G, S, H, P, and W.

81. 81. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 62 to 80, wherein the silk fibroin fragments comprise from about 2 to about 100 amino acids.

82. 82. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 80 or 81, wherein the silk fibroin fragments comprise 1 to 5 modifications, substitutions, and / or alternatives.

83. 83. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80 to 82, wherein the fibroin is a fibroin heavy chain and the modification, substitution, and / or replacement is at a position corresponding to any one of positions 1 to 5263 of the fibroin heavy chain.

84. 83. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80 to 82, wherein the fibroin is a fibroin light chain and the modification, substitution, and / or replacement is at a position corresponding to any one of positions 1 to 262 of the fibroin light chain.

85. 83. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80 to 82, wherein the fibroin is a fibrohexamerin (p25) chain and the modification, substitution and / or replacement is at a position corresponding to any one of positions 1 to 220 of the fibrohexamerin (p25) chain.

86. 86. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets according to any one of claims 80 to 85, wherein the modification, substitution, and / or substitution is selected from modification, substitution, and / or substitution of asparagine with aspartic acid, modification, substitution, and / or substitution of glutamine with glutamic acid, and modification, substitution, and / or substitution of methionine with methionine oxide.

87. 87. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80-86, wherein the modification, substitution, and / or substitution is at a fibroin heavy chain position selected from Q58, M64, N68, N70, N77, M80, N93, M103, Q125, N132, Q139, Q275, N4191, Q5216, and / or N5262.

88. 87. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80-86, wherein the modification, substitution, and / or substitution is at a fibroin light chain position selected from N23, Q24, N28, M69, N105, N108, N118, N136, N138, Q149, N186, N200, Q202, N204, N240, N248, and / or Q255.

89. 87. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80-86, wherein the modification, substitution, and / or substitution is at a fibrohexamerin (p25) position selected from Q62, N93, M120, N149, N172, N174, and / or N202.

90. 90. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 80-89, wherein each modification, substitution, and / or substitution independently ranges from about 1% to about 99% in the composition.

91. 91. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of claim 90, wherein the % modification, substitution, and / or substitution is defined as (the number of peptides or protein fragments that contain a modification, substitution, and / or substitution at a particular position divided by the total number of peptides or protein fragments that contain said particular position, regardless of whether they contain a modification, substitution, and / or substitution) x 100.

92. 92. The plurality of substantially solid silk fibroin particles, silk fibroin nanoclay composite or film, stabilized silk fibroin solution, liquid-in-air suspension, or plurality of droplets or droplets of any one of claims 45 to 91, wherein the molecular weight is determined by MALS.

93. A pouch or laundry pod comprising a plurality of substantially solid silk fibroin particles according to any one of claims 1 to 92.

94. 94. The laundry pod of claim 93, wherein the plurality of substantially solid silk fibroin particles are compressed into a multi-particle pack.

95. 95. The laundry pod of claim 93 or 94, further comprising a dissolvable enclosure comprising polyvinyl alcohol (PVA) or a derivative of PVA.

96. 94. The pouch of claim 93, further comprising an enclosure comprising one or more of nylon, polyglycolide (PGA), polylactic acid (PLA), poly(lactide-co-glycolide) (PLGA), polycaprolactone (PCL), poly(butylene succinate) (PBS), polybutylene succinate adipate, poly(p-dioxanone) (PPDO), poly(butylene adipate-co-terephthalate) (PBAT), copolyesters of terephthalic acid and lactic acid, copolyesters of terephthalic acid and glycolic acid, copolyesters of terephthalic acid and succinic acid, poly(hydroxybutyrate), poly(hydroxyvalerate), polyhydroxyhexanoate, poly(hydroxyalkanoates) (PHA), polymethylene adipate / terephthalate.

97. 93. A method for making a plurality of substantially solid silk fibroin particles according to any one of claims 1 to 92, the method comprising dropping a solution comprising a plurality of the silk fibroin fragments into liquid nitrogen.

98. 98. The method of claim 97, further comprising a freeze-drying step.

99. 99. The method of claim 97 or 98, wherein the concentration of silk fibroin fragments in the solution is from about 3% (w / w) to about 50% (w / w).

100. 100. The method of any one of claims 97 to 99, wherein the silk fibroin fragment comprises one or more of the molecular weight, polydispersity, and / or modifications, substitutions, and / or substitutions at specific amino acid positions defined in any one of claims 45 to 91.

101. 101. The method of any one of claims 97 to 100, wherein the solution is stabilized as defined in any one of claims 31 to 41.

102. 93. A method for reconstituting a silk fibroin fragment solution, comprising dissolving a plurality of substantially solid silk fibroin particles according to any one of claims 1 to 92 in a solvent, wherein the particles have a reconstitution yield of greater than 90%.

103. 93. The plurality of substantially solid silk fibroin particles of any one of claims 1 to 92, wherein the particles have a reconstitution yield in DI water of greater than 90%.

104. 104. The method of claim 102; or the plurality of substantially solid silk fibroin particles of claim 103, wherein at least 90% reconstitution rate is maintained after a stability test comprising simulated aging of the plurality of substantially solid silk fibroin particles of any one of claims 1 to 92, wherein the aging comprises storage at a temperature of about 40°C to about 60°C for a period ranging from about 400 days to about 650 days.

105. 105. The method of claim 104, wherein the simulated aging ranges from about 4 years to about 15 years.

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