Composite polymeric materials, and articles and methods of preparation thereof

Abstract

The present disclosure provides a leather article and a method for making a leather article, comprising a substrate and a coating comprising a cellulose derivative having improved color fastness to rubbing. The cellulose derivative can be methyl cellulose, ethyl cellulose, ethyl methyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxyethyl cellulose, cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate, microcrystalline cellulose, cellulose nitrate, or cellulose sulfate.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 315,945, filed March 2, 2022, U.S. Provisional Application No. 63 / 355,412, filed June 24, 2022, U.S. Provisional Application No. 63 / 376,219, filed September 19, 2022, U.S. Provisional Application No. 63 / 376,224, filed September 19, 2022, U.S. Provisional Application No. 63 / 376,229, filed September 19, 2022, U.S. Provisional Application No. 63 / 383,196, filed November 10, 2022, and U.S. Provisional Application No. 63 / 479,947, filed January 13, 2023. The contents of each of these applications are incorporated herein by reference in their entirety.

[0002] The present disclosure relates to composite polymeric materials comprising, in part, cellulosic coating compositions, optionally including silk fibroin protein or fragments thereof and various additional agents, for coating various substrates. [Background technology]

[0003] Silk is a natural polymer produced by various insects and spiders, and is composed of a filament core protein, silk fibroin, and a colloidal coating made of the non-fibrous protein, sericin. Silk fibers are lightweight, breathable, and hypoallergenic. Summary of the Invention

[0004] The present disclosure provides a method for producing a steroid hormone receptor agonist (SHR) having a steroid activity 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, Provided is an article comprising one or more surfaces coated with a silk fibroin protein or fragment thereof having an average weight-average molecular weight selected from 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, or about 80 kDa to about 144 kDa, and having a polydispersity ranging from 1 to about 5, a cellulose derivative, a plasticizer, and a crosslinker. In some embodiments, the cellulose derivative is selected from methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. In some embodiments, the plasticizer is selected from triethyl citrate, dibutyl sebacate, triacetin, glycerol, 1,3-propanediol, propylene glycol, pentylene glycol, epoxidized vegetable oils, isosorbide esters, succinic acid derivatives, and acetate esters of monoglycerides. In some embodiments, the crosslinker is selected from polyisocyanates, polycarbodiimides, polyaziridines, polyureas, glutaraldehyde, and starch dialdehyde. In some embodiments, the silk fibroin protein or fragment thereof has a polydispersity of 1 to about 1.5, about 1.5 to about 2, 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, or about 4.5 to about 5. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin protein or fragment thereof.In some embodiments, the silk fibroin protein or fragment thereof does not spontaneously or gradually gel and shows no visible change in color or turbidity when present in aqueous solution for at least 10 days prior to addition to the article. In some embodiments, a portion of the silk fibroin protein or fragment thereof is coated on the surface of a leather substrate. In some embodiments, a portion of the silk fibroin protein or fragment thereof is impregnated into a layer of the leather substrate. In some embodiments, a portion of the silk fibroin protein or fragment thereof is present in a recess in the leather substrate. In some embodiments, the article further comprises one or more polysaccharides selected from starch, cellulose, gum arabic, guar gum, xanthan gum, alginate, pectin, chitin, chitosan, carrageenan, inulin, and gellan gum. In some embodiments, the gellan gum comprises a low acyl content gellan gum.In some embodiments, the w / w ratio between the silk fibroin protein or fragment thereof and the polysaccharide is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, about 76:24, about 75:25, about 74:26, about 73:27, Approximately 72:28, approximately 71:29, approximately 70:30, approximately 69:31, approximately 68:32, approximately 67:33, approximately 66:34, approximately 65:35, approximately 64:36, approximately 63:37, approximately 62:38, approximately 61:39, approximately 60:40, approximately 59:41, approximately 58:42, approximately 57:43, approximately 56:44, approximately 45:55, approximately 54:46, approximately 53:47, approximately 52:48, approximately 51:49, approximately 40:50, approximately 49:51, approximately 48:52, approximately 47:53, approximately 46:54, approximately 45:55, approximately 44:56, approximately 43:57, approximately 42:58, approximately 41:59, approximately 40 :60, approximately 39:61, approximately 38:62, approximately 37:63, approximately 36:64, approximately 35:75, approximately 34:66, approximately 33:67, approximately 32:68, approximately 31:69, approximately 30:70, approximately 29:71, approximately 28:72, approximately 27:73, approximately 26:74, approximately 25:85, approximately 24:76, approximately 23:77, approximately 22:78, approximately 21:79, approximately 20:80, approximately 19:81, approximately 18:82, approximately 17:83, approximately 16:84, approximately 15:95, approximately 14:86, approximately 13:87, approximately 12:88, approximately 11:89, approximately 10:90, approximately 9:91, approximately 8:92, approximately 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99, about 100:1, about 50:1, about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, and about 1:5.In some embodiments, the w / w ratio between the silk fibroin protein or fragment thereof and the polysaccharide is about 12:1, about 11.9:1, about 11.8:1, about 11.7:1, about 11.6:1, about 11.5:1, about 11.4:1, about 11.3:1, about 11.2:1, about 11.1:1, about 11:1, about 10.9:1, about 10.8:1, about 10.7:1, about 10.6:1, about 10.5:1, about 10.4:1, about 10.3:1, about 10.2:1, about 10.1:1, about 10:1, about 9.9:1, about 9.8:1, about 9.7:1, about 9.6:1, about 9.5:1, about 9.4:1, about 9.3:1, about 9.2:1, about 9.1:1, about 9:1, about 8.9:1, about 8.8:1, about 8.7:1, about 8.6:1, about 8.5:1, about 8.4:1, about 8.3:1, about 8.2:1, about 8.1:1, about 8:1, about 7.9:1, about 7.8:1, about 7.7:1, about 7.6:1, about 7.5:1, about 7.4:1, about 7.3:1, about 7.2:1, about 7.1:1, about 7:1, about 6.9:1, about 6.8:1, about 6.7:1, about 6.6:1, about 6.5:1 , about 6.4:1, about 6.3:1, about 6.2:1, about 6.1:1, about 6:1, about 5.9:1, about 5.8:1, about 5.7:1, about 5.6:1, about 5.5:1, about 5.4:1, about 5.3:1, about 5.2:1, about 5.1:1, about 5:1, about 4.9:1, about 4.8:1, about 4.7:1, about 4.6:1, about 4.5:1, about 4.4:1, about 4.3:1, about 4.2:1, about 4.1:1, about 4:1, about 3.9:1, about 3.8:1, about 3.7:1, about 3.6:1, about 3.5:1, about 3.4:1, about 3.3:1, about 3.2 1, about 3.1:1, about 3:1, about 2.9:1, about 2.8:1, about 2.7:1, about 2.6:1, about 2.5:1, about 2.4:1, about 2.3:1, about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 0.9:1, about 0.8:1, about 0.7:1, about 0.6:1, about 0.5:1, about 0.4:1, about 0.3:1, about 0.2:1, and about 0.1:1. In some embodiments, the article further comprises one or more polyols and / or one or more polyethers.In some embodiments, the polyol comprises one or more of glycol, glycerol, sorbitol, D-sorbitol, glucose, sucrose, mannitol, D-mannitol, and dextrose. In some embodiments, the polyether comprises one or more polyethylene glycols (PEGs). In some embodiments, the w / w ratio between the silk fibroin protein or fragment thereof and the one or more polyols and / or one or more polyethers is about 5:1, about 4.9:1, about 4.8:1, about 4.7:1, about 4.6:1, about 4.5:1, about 4.4:1, about 4.3:1, about 4.2:1, about 4.1:1, about 4:1, about 3.9:1, about 3.8:1, about 3.7:1, about 3.6:1, about 3.5:1, about 3.4:1, about 3. 3:1, approximately 3.2:1, approximately 3.1:1, approximately 3:1, approximately 2.9:1, approximately 2.8:1, approximately 2.7:1, approximately 2.6:1, approximately 2.5:1, approximately 2.4:1, approximately 2.3:1, approximately 2.2:1, approximately 2.1:1, approximately 2:1, approximately 1.9:1, approximately 1.8:1, approximately 1.7:1, approximately 1.6:1, approximately 1.5:1, approximately 1.4:1, approximately 1.3:1, approximately 1.2:1, approximately 1.1:1, approximately 1:1, approximately 0.9:1, approximately 0.8:1, approximately 0.7:1, approximately 0.6:1, approximately 0 0.5:1, approx. 0.4:1, approx. 0.3:1, approx. 0.2:1, approx. 0.1:1, approx. 1:0.1, approx. 1:0.2, approx. 1:0.3, approx. 1:0.4, approx. 1:0.5, approx. 1:0.6, approx. 1:0.7, approx. 1:0.8, approx. 1:0.9, approx. 1:1.1, approx. 1:1.2, approx. 1:1.3, approx. 1:1.4, approx. 1:1.5, approx. 1:1.6, approx. 1:1.7, approx. 1:1.8, approx. 1:1.9, approx. 1:2, approx. 1:2.1, approx. 1:2.2, approx. 1:2.3, approx. 1: The ratio is selected from about 1:2.4, about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4, about 1:3.5, about 1:3.6, about 1:3.7, about 1:3.8, about 1:3.9, about 1:4, about 1:4.1, about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about 1:4.7, about 1:4.8, about 1:4.9, and about 1:5. In some embodiments, the article further comprises one or more of a silicone, a dye, a pigment, and a polyurethane. In some embodiments, the article further comprises one or more of a crosslinker, a crosslinker adduct, or a crosslinker reactive derivative.In some embodiments, the article is made of an isocyanate, an isocyanate adduct, and / or an isocyanate-reactive derivative; a polydiisocyanate, a polydiisocyanate adduct, and / or a polydiisocyanate-reactive derivative; an aziridine, an aziridine adduct, and / or an aziridine-reactive derivative; a carbodiimide, a carbodiimide adduct, and / or a carbodiimide-reactive derivative; an aldehyde, an aldehyde adduct, and / or an aldehyde-reactive derivative; a polyisocyanate, a polyisocyanate adduct, and / or a polyisocyanate-reactive derivative; a polyaziridine, a polyaziridine adduct, and / or a polyaziridine-reactive derivative; a polycarbodiimide polyaldehydes, polyaldehyde adducts and / or polyaldehyde reaction derivatives; polyurethanes, polyurethane adducts and / or polyurethane reaction derivatives; polyacrylates, polyacrylate adducts and / or polyacrylate reaction derivatives; polyesters, polyester adducts and / or polyester reaction derivatives; waxes, wax adducts and / or wax reaction derivatives; proteins, protein adducts and / or protein reaction derivatives; or alcohols, alcohol adducts and / or alcohol reaction derivatives.

[0005] In some embodiments, one or more surfaces of the article have greater colorfastness to rubbing than one or more surfaces of a similar article that is similarly uncoated. In some embodiments, the article comprises leather.

[0006] The present disclosure also provides a method of coating one or more surfaces of an article, the method comprising: coating one or more surfaces of an article with a densitometer 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 54 kDa, about 40 kDa to about 54 kDa, about 54 ... The method includes applying a first composition comprising a silk fibroin protein or a fragment thereof having an average weight-average molecular weight selected from 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, or about 80 kDa to about 144 kDa, and having a polydispersity ranging from 1 to about 5; and applying a second composition comprising one or more components selected from a cellulose derivative and a plasticizer. In some embodiments, the first composition further comprises a crosslinker. In some embodiments, the crosslinker is selected from a polyisocyanate, a polycarbodiimide, a polyaziridine, a polyurea, a glutaraldehyde, and a starch dialdehyde. In some embodiments, the cellulose derivative is selected from methyl cellulose, ethyl cellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose. In some embodiments, the plasticizer is selected from triethyl citrate, dibutyl sebacate, triacetin, glycerol, 1,3-propanediol, propylene glycol, pentylene glycol, epoxidized vegetable oils, isosorbide esters, succinic acid derivatives, and acetate esters of monoglycerides. In some embodiments, the silk fibroin protein or fragment thereof has a polydispersity of 1 to about 1.5, about 1.5 to about 2, 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, or about 4.5 to about 5.In some embodiments, the first component further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin protein or fragment thereof. In some embodiments, the silk fibroin protein or fragment thereof does not spontaneously or gradually gel and shows no visible change in color or turbidity when present in aqueous solution for at least 10 days prior to incorporation into the composition and application to one or more surfaces of the article. In some embodiments, the article comprises leather. In some embodiments, a portion of the silk formulation is coated on the surface of the leather substrate, and / or a portion of the silk formulation penetrates into a layer of the leather substrate, and / or a portion of the silk formulation penetrates into recesses in the leather substrate. In some embodiments, the silk formulation further comprises a rheology modifier. In some embodiments, the rheology modifier comprises one or more polysaccharides selected from starch, cellulose, gum arabic, guar gum, xanthan gum, alginate, pectin, chitin, chitosan, carrageenan gum, inulin, and gellan gum. In some embodiments, the gellan gum comprises low acyl content gellan gum. In some embodiments, the w / w ratio between the silk fibroin protein or fragment thereof and the rheology modifying agent in the silk formulation is selected from about 25:1, about 24:1, about 23:1, about 22:1, about 21:1, about 20:1, about 19:1, about 18:1, about 17:1, about 16:1, about 15:1, about 14:1, about 13:1, about 12:1, about 11:1, about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, and about 1:5.In some embodiments, the w / w ratio between the silk fibroin protein or fragment thereof and the rheology modifying agent in the silk formulation is about 12:1, about 11.9:1, about 11.8:1, about 11.7:1, about 11.6:1, about 11.5:1, about 11.4:1, about 11.3:1, about 11.2:1, about 11.1:1, about 11:1, about 10.9:1, about 10.8:1, about 10.7:1, about 10.6:1, about 10.5:1, about 10.4:1, about 10.3:1, about 10.2:1, about 10.1:1, about 10:1, about 9.9:1, about 10 ... :1, about 9.8:1, about 9.7:1, about 9.6:1, about 9.5:1, about 9.4:1, about 9.3:1, about 9.2:1, about 9.1:1, about 9:1, about 8.9:1, about 8.8:1, about 8.7:1, about 8.6:1, about 8.5:1, about 8.4:1, about 8.3:1, about 8.2:1, about 8.1:1, about 8:1, about 7.9:1, about 7.8:1, about 7.7:1, about 7.6:1, about 7.5:1, about 7.4:1, about 7.3:1, about 7.2:1, about 7.1:1, about 7:1, about 6.9:1, about 6.8:1, about 6.7:1, about 6.6:1 , about 6.5:1, about 6.4:1, about 6.3:1, about 6.2:1, about 6.1:1, about 6:1, about 5.9:1, about 5.8:1, about 5.7:1, about 5.6:1, about 5.5:1, about 5.4:1, about 5.3:1, about 5.2:1, about 5.1:1, about 5:1, about 4.9:1, about 4.8:1, about 4.7:1, about 4.6:1, about 4.5:1, about 4.4:1, about 4.3:1, about 4.2:1, about 4.1:1, about 4:1, about 3.9:1, about 3.8:1, about 3.7:1, about 3.6:1, about 3.5:1, about 3.4:1, about 3.3:1, The ratio is selected from about 3.2:1, about 3.1:1, about 3:1, about 2.9:1, about 2.8:1, about 2.7:1, about 2.6:1, about 2.5:1, about 2.4:1, about 2.3:1, about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 0.9:1, about 0.8:1, about 0.7:1, about 0.6:1, about 0.5:1, about 0.4:1, about 0.3:1, about 0.2:1, and about 0.1:1. In some embodiments, the w / v concentration of the rheology modifier in the silk formulation is from about 0.01% to about 5%, or from about 0.1% to about 1%. In some embodiments, the silk formulation further comprises a plasticizer.In some embodiments, the plasticizer comprises one or more polyols and / or one or more polyethers. In some embodiments, the polyol is selected from one or more of glycol, glycerol, sorbitol, D-sorbitol, glucose, sucrose, mannitol, D-mannitol, and dextrose. In some embodiments, the polyether is one or more polyethylene glycols (PEGs). In some embodiments, the w / w ratio between silk fibroin protein or fragments thereof and the plasticizer in the silk formulation is about 5:1, about 4.9:1, about 4.8:1, about 4.7:1, about 4.6:1, about 4.5:1, about 4.4:1, about 4.3:1, about 4.2:1, about 4.1:1, about 4:1, about 3.9:1, about 3.8:1, about 3.7:1, about 3.6:1, about 3.5:1, about 3.4:1, about 3.3:1, about 3.2:1, About 3.1:1, about 3:1, about 2.9:1, about 2.8:1, about 2.7:1, about 2.6:1, about 2.5:1, about 2.4:1, about 2.3:1, about 2.2:1, about 2.1:1, about 2:1, about 1.9:1, about 1.8:1, about 1.7:1, about 1.6:1, about 1.5:1, about 1.4:1, about 1.3:1, about 1.2:1, about 1.1:1, about 1:1, about 0.9:1, about 0.8:1, about 0.7:1, about 0.6:1, about 0.5:1, about 0 0.4:1, approximately 0.3:1, approximately 0.2:1, approximately 0.1:1, approximately 1:0.1, approximately 1:0.2, approximately 1:0.3, approximately 1:0.4, approximately 1:0.5, approximately 1:0.6, approximately 1:0.7, approximately 1:0.8, approximately 1:0.9, approximately 1:1.1, approximately 1:1.2, approximately 1:1.3, approximately 1:1.4, approximately 1:1.5, approximately 1:1.6, approximately 1:1.7, approximately 1:1.8, approximately 1:1.9, approximately 1:2, approximately 1:2.1, approximately 1:2.2, approximately 1:2.3, approximately 1:2.4, In some embodiments, the w / v concentration of the plasticizer in the silk formulation is selected from about 1:2.5, about 1:2.6, about 1:2.7, about 1:2.8, about 1:2.9, about 1:3, about 1:3.1, about 1:3.2, about 1:3.3, about 1:3.4, about 1:3.5, about 1:3.6, about 1:3.7, about 1:3.8, about 1:3.9, about 1:4, about 1:4.1, about 1:4.2, about 1:4.3, about 1:4.4, about 1:4.5, about 1:4.6, about 1:4.7, about 1:4.8, about 1:4.9, and about 1:5. In some embodiments, the w / v concentration of the plasticizer in the silk formulation is from about 0.01% to about 10%.In some embodiments, the silk formulation further comprises an anti-foaming agent at a concentration of about 0.001% to about 1%. In some embodiments, the anti-foaming agent comprises a silicone. In some embodiments, the silk formulation further comprises one or more of an isocyanate, a polydiisocyanate, an aziridine, a carbodiimide, an aldehyde, a polyisocyanate, a polyaziridine, a polycarbodiimide, a polyaldehyde, a polyurethane, a polyacrylate, a polyester, a wax, a protein, and / or an alcohol. In some embodiments, the silk formulation is a liquid, a gel, a paste, a wax, or a cream. In some embodiments, the silk formulation comprises one or more sub-formulations to be applied simultaneously or at different times. In some embodiments, the concentration of silk fibroin protein or fragments thereof in the silk formulation is about 0.1% w / v to about 15% w / v. In some embodiments, the concentration of silk fibroin protein or fragments thereof in the silk formulation is about 0.5% w / v to about 12% w / v. In some embodiments, the concentration of silk fibroin protein or fragment thereof in the silk formulation is about 1% w / v, about 1.5% w / v, about 2% w / v, about 2.5% w / v, about 3% w / v, about 3.5% w / v, about 4% w / v, about 4.5% w / v, about 5% w / v, about 5.5% w / v, about 6% w / v, about 6.5% w / v, about 7% w / v, about 7.5% w / v, about 8% w / v, about 8.5% w / v, about 9% w / v, about 9.5% w / v, or about 10% w / v. In some embodiments, the concentration of silk fibroin protein or fragment thereof in the silk formulation is about 3% w / v, about 3.25% w / v, about 3.5% w / v, about 3.75% w / v, about 4% w / v, about 4.25% w / v, about 4.5% w / v, about 4.75% w / v, about 5% w / v, about 5.25% w / v, about 5.5% w / v, about 5.75% w / v, about 6% w / v, about 6.5 ... % w / v, about 6.25% w / v, about 6.5% w / v, about 6.75% w / v, about 7% w / v, about 7.25% w / v, about 7.5% w / v, about 7.75% w / v, about 8% w / v, about 8.25% w / v, about 8.5% w / v, about 8.75% w / v, about 9% w / v, about 9.25% w / v, about 9.5% w / v, about 9.75% w / v, or about 10% w / v.In some embodiments, the concentration of silk fibroin protein or fragment thereof in the silk formulation is about 5 mg / mL to about 125 mg / mL. In some embodiments, the concentration of silk fibroin protein or fragment thereof in the silk formulation is about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL 9mg / mL, approximately 50mg / mL, approximately 51mg / mL, approximately 52mg / mL, approximately 53mg / mL, approximately 54mg / mL, approximately 55mg / mL, approximately 56mg / mL, approximately 57mg / mL, approximately 58mg / mL, approximately 59mg / mL, approximately 60mg / mL, approximately 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 m. g / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, or about 90 mg / mL. In some embodiments, the method further comprises one or more additional steps selected from dyeing, drying, water annealing, mechanical stretching, trimming, polishing, applying a pigment, applying a colorant, applying an acrylic formulation, applying a urethane formulation, chemical fixing, stamping, applying a silicone finish, applying a Uniflex treatment, and / or applying a Finiflex treatment, wherein the step of applying the silk formulation to the surface of the leather occurs before, during, or after the one or more additional steps. In some embodiments, treating a leather substrate with a silk blend results in one or more of increased gloss, enhanced saturation, color enhancement, improved color fixation, reduced dye usage, and / or improved colorfastness. In some embodiments, the improvement is relative to a leather substrate that has not been similarly treated with a silk blend. The composition is incorporated into a composition and applied to one or more surfaces of an article. In some embodiments, the article comprises leather.

[0007] Disclosed herein are silk-coated leather products and methods for their preparation. The silk and silk protein fragments, and silk and silk protein fragment (SPF) compositions described herein can be used as a surface treatment to fix color, in place of or in addition to any chemicals used during any chemical treatment process, to alter the appearance, feel, texture, and / or quality of leather.

[0008] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to finish leather, for example, to alter the sheen or luster of leather and / or to achieve a matte, glossy, mirror-like, embossed, etc. finish.

[0009] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to repair, mask, or conceal defects in leather or hide, such as hair follicle defects or other mechanical defects, whether on the surface or within the leather or hide.

[0010] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to alter and / or improve the appearance of leather, hides, and / or leather products, or to change the grade of leather or hide, thereby expanding the range of applicable market areas for a given leather type.

[0011] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to improve the handle of leather, for example, its feel or softness.

[0012] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used as a pigment delivery system to fix color, adjust final coloration, or alter pigment chemistry or improve colorant delivery in a finishing stage or other suitable process step.

[0013] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used before or after any mechanical processing step typical of leather processing, such as, but not limited to, Uniflex processing, Finiflex processing, heat stamping, sanding, skin trimming, or before or after drying. In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used before any mechanical process described herein. In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used in a finishing or dyeing process. In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used before any pressing process described herein.

[0014] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be applied by spraying onto leather.

[0015] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be applied to leather by stamping.

[0016] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be incorporated into and onto leather.

[0017] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions described herein can be used before, during, or after the leather processing step (e.g., finishing process) in place of any chemistry used to stabilize and modify gloss, luster, color, shade, tone, finish, hand, weight, etc.

[0018] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions described herein can be used before, during, or after the leather processing step (e.g., finishing process), in addition to any chemicals used to stabilize and modify gloss, luster, color, shade, tone, finish, hand, etc.

[0019] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to perform one or more chemical functions during the tanning and dyeing stages of leather processing.

[0020] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to perform one or more mechanical functions during the tanning and dyeing stages of leather processing.

[0021] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to perform one or more functions during the tanning and dyeing stages of leather processing.

[0022] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used to alter the contact angle of solvents applied to semi-finished or finished leather before, during, or after a leather processing step (e.g., a finishing process).

[0023] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions described herein can be used as defect fillers in skins before, during, or after leather processing steps (e.g., finishing processes), either before or after dyeing. In some embodiments, such uses include combinations with pigments, dyes, admixtures, softeners, rheology modifiers, and the like.

[0024] In some embodiments, the silk and silk protein fragments, and silk and silk protein fragment compositions described herein can be used for any of the purposes described herein before, during, or after any of the processes described herein, and such uses are enhanced by the additional use of one or more physicochemical processing treatments, such as, but not limited to, the use of O2 plasma, crosslinking agents, photocrosslinking agents, or ultraviolet light treatment.

[0025] In some embodiments, the silk and silk protein fragments and / or silk and silk protein fragment compositions described herein can be mixed with or substituted for classes of materials such as, but not limited to, water-based lacquers, waxes, oils, proteins or other binders, fillers, feel modifiers, leveling agents, solvent lacquers, water-based lacquers, penetrating agents, acrylic resins, butadiene resins, compact resins, hybrid resins, impregnating resins, rheology modifiers, solvent dullers, solvent urethanes, water-based dullers, water-based topcoats, chrome, dye dispersants, acid dyes, basic dyes, chrome-based or other dyes, and / or colorants.

[0026] In some embodiments, the leather preparation process can include treating leather with a silk and / or SPF composition described herein. In some embodiments, the silk and / or SPF composition can include one or more chemical agents (e.g., silicone, polyurethane, etc.) described below.

[0027] In embodiments, the present disclosure provides methods of treating leather with the silk and / or SPF compositions described herein, which may include dyeing the leather, mechanically stretching the leather, trimming the leather, sanding the leather, applying a pigment and / or acrylic coating to the leather (optionally by spray application), chemically fixing the leather, stamping the leather, applying a silicone or other finish to the leather, applying a Uniflex treatment to the leather, and / or filling defects on the surface or within the leather with the silk or SPF composition, wherein one or more of the foregoing steps include applying the silk and / or SPF composition to the leather before, during, or after the described steps.

[0028] In embodiments, the present disclosure provides methods of treating leather with the silk and / or SPF compositions described herein, which may include dyeing the leather, drying the leather, mechanically stretching the leather, trimming the leather, first sanding the leather, applying a colorant and / or acrylic to the leather (optionally by spray application), second sanding the leather, treating the leather with a Finiflex treatment, and / or filling defects on the surface or within the leather with the silk or SPF composition, wherein one or more of the foregoing steps include applying a silk composition to the leather before, during, or after the described steps.

[0029] In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles by any of the methods described herein, but also by hand spraying, spraying using a mechanical spray setup, applying with a brush, bath coating, rubbing, wet mixing, washing, drumming, dipping, extruding, pouring, plastering, roller coating, and / or filling.

[0030] In some embodiments, the silk and / or SPF compositions described herein, alone or mixed with one or more chemicals (e.g., chemical agents), can be applied multiple times, in one coat or multiple coats, using a variety of application methods, to leather that has or has not been dyed, chrome-treated, sprayed with pigments, acrylics, fixatives, finishes, and / or colorants. In some embodiments, the silk and / or SPF compositions described herein can be applied to finished leather or leather articles, mechanically treated leather or leather articles, or drummed leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied to defects in finished leather or leather articles, mechanically treated leather or leather articles, or drummed leather or leather articles.

[0031] In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers before dyeing and finishing. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers after dyeing and before finishing. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers after dyeing and finishing.

[0032] In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied by hand. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied by finger. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied using a brush-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied using a marker-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied using a pen-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied using a pipette-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers and are applied using a syringe-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers, and application is performed using an eyeliner brush-type applicator and any brush or brush-like applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers, and application is performed using a heated stamp device applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers, and application is performed using a sponge applicator.In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers, and application is performed using a roller coater. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather articles as defect fillers, and application is performed with an applicator such as a "glue gun."

[0033] In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to bovine skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to sheep skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to lamb skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to horse skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to crocodile skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to alligator skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to avian skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to animal skin leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied to split leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to suede leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to wet blue leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to altered leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to aniline leather or leather articles as defect fillers.In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to bonded leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to brushed leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to buffed leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to bycast leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to chamois leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to plongé leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to chrome-tanned leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to mixed-tanned leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied to cordovan leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to collected grain leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to crackproof leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to drummed leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to embossed leather or leather articles as defect fillers.In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to enhanced grain leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to grain leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to metallized leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to naked leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to natural grain leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to nubuck leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to patent leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied as defect fillers to pearlized leather or leather articles. In some embodiments, the silk and / or SPF compositions described herein can be applied to plated leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to printed leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to protected leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to pure aniline leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to tanned / retanned leather or leather articles as defect fillers.In some embodiments, the silk and / or SPF compositions described herein can be applied to round hand leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to saddle leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to semi-aniline leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to shrunken grain leather or leather articles as defect fillers. In some embodiments, the silk and / or SPF compositions described herein can be applied to side leather or leather articles as defect fillers.

[0034] In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the liming process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the deliming and / or baching process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the pickling process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the tanning process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the neutralization, dyeing, and / or emulsion fatliquoring process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after the drying process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather before or after a finishing process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used during or as part of a finishing process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used in a separate silk and / or SPF treatment step.

[0035] In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the liming process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the deliming and / or baching process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the pickling process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the tanning process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the neutralization, dyeing, and / or emulsion fatliquoring processes. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the drying process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during the finishing process. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used during or as part of a finishing process.

[0036] In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during a process that includes one or more steps, such as one or more dyeing steps. In some embodiments, the silk and / or SPF compositions can be used before, during, or after a dyeing step. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during a process that includes one or more steps, such as one or more mechanical processing steps. In some embodiments, the silk and / or SPF compositions can be used before, during, or after a mechanical processing step. Mechanical processing steps include, but are not limited to, drying, sanding, stamping, Uniflex and / or Finiflex, drawing, and / or trimming. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during a process that includes one or more steps, such as one or more polishing steps. In some embodiments, the silk and / or SPF compositions can be used before, during, or after a polishing step. In some embodiments, the silk and / or SPF compositions described herein (with or without one or more chemical agents) can be used to treat leather during one or more steps, for example, a process that includes one or more chemical treatment steps. In some embodiments, the silk and / or SPF compositions can be used before, during, or after a chemical treatment step. Chemical treatment steps include, but are not limited to, one or more pigment treatment steps, one or more acrylic, silicone, and / or polyurethane treatment steps, and / or one or more chemical fixation treatment steps.

[0037] In embodiments, methods are provided for treating leather with silk fibroin and / or SPF, which may include silk-based proteins or fragments thereof, to provide silk fibroin-treated leather. In some embodiments, the methods may include preparing a silk fibroin solution or other composition that may include one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin at a concentration of less than about 1% by weight (w / w), or less than about 0.1% by weight (w / w), or less than about 0.01% by weight (w / w), or less than about 0.001% by weight (w / w).In some embodiments, the method comprises dissolving one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin in an amount of less than about 1% by weight (w / w), or less than about 2% by weight (w / w), or less than about 3% by weight (w / w), or less than about 4% by weight (w / w), or less than about 5% by weight (w / w), or less than about 6% by weight (w / w), or less than about 7% by weight (w / w), or less than about 8% by weight (w / w), or less than about 9% by weight (w / w), or less than about 10% by weight (w / w), or less than about 11% by weight (w / w). / w), or less than about 12% by weight (w / w), or less than about 13% by weight (w / w), or less than about 14% by weight (w / w), or less than about 15% by weight (w / w), or less than about 16% by weight (w / w), or less than about 17% by weight (w / w), or less than about 18% by weight (w / w), or less than about 19% by weight (w / w), or less than about 20% by weight (w / w), or less than about 21% by weight (w / w), or less than about 22% by weight (w / w), or less than about 23% by weight (w / w), or less than about 24% by weight (w / w), or less than about 25% by weight ( % (w / w), or less than about 26% (w / w), or less than about 27% (w / w), or less than about 28% (w / w), or less than about 29% (w / w), or less than about 30% (w / w), or less than about 31% (w / w), or less than about 32% (w / w), or less than about 33% (w / w), or less than about 34% (w / w), or less than about 35% (w / w), or less than about 36% (w / w), or less than about 37% (w / w), or less than about 38% (w / w), or less than about 39% The method may include preparing a silk fibroin solution or other composition that may contain silk fibroin at a concentration of less than about 40% by weight (w / w), or less than about 41% by weight (w / w), or less than about 42% by weight (w / w), or less than about 43% by weight (w / w), or less than about 44% by weight (w / w), or less than about 45% by weight (w / w), or less than about 46% by weight (w / w), or less than about 47% by weight (w / w), or less than about 48% by weight (w / w), or less than about 49% by weight (w / w), or less than about 50% by weight (w / w).In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after any processing step. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after pigment delivery. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after color fixation. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after final color adjustment. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after pigment chemical modification. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after colorant delivery improvement. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after Uniflex treatment. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after the Finiflex process. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after the heat stamping process. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after the sanding process. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after skin trimming. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after a finishing process. In some embodiments, the method can include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after tanning.In some embodiments, the method may comprise treating the surface of the leather material with a silk fibroin solution or composition before, during, or after dyeing. In some embodiments, the method may comprise treating the surface of the leather material with a silk fibroin solution or composition before, during, or after stretching. In some embodiments, the method may comprise treating the surface of the leather material with a silk fibroin solution or composition before, during, or after drying. In some embodiments, the method may comprise treating the surface of the leather material with a silk fibroin solution or composition before, during, or after trimming. In some embodiments, the method may comprise treating the surface of the leather material with a silk fibroin solution or composition before, during, or after sanding.

[0038] In embodiments, methods are provided for coating leather with silk fibroin and / or SPF, which may include silk-based proteins or fragments thereof, to provide silk fibroin-coated leather. In some embodiments, the methods may include preparing a silk fibroin solution or other composition that may include one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin at a concentration of less than about 1% by weight (w / w), or less than about 0.1% by weight (w / w), or less than about 0.01% by weight (w / w), or less than about 0.001% by weight (w / w).In some embodiments, the method comprises dissolving one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin in an amount of less than about 1% by weight (w / w), or less than about 2% by weight (w / w), or less than about 3% by weight (w / w), or less than about 4% by weight (w / w), or less than about 5% by weight (w / w), or less than about 6% by weight (w / w), or less than about 7% by weight (w / w), or less than about 8% by weight (w / w), or less than about 9% by weight (w / w), or less than about 10% by weight (w / w), or less than about 11% by weight (w / w). / w), or less than about 12% by weight (w / w), or less than about 13% by weight (w / w), or less than about 14% by weight (w / w), or less than about 15% by weight (w / w), or less than about 16% by weight (w / w), or less than about 17% by weight (w / w), or less than about 18% by weight (w / w), or less than about 19% by weight (w / w), or less than about 20% by weight (w / w), or less than about 21% by weight (w / w), or less than about 22% by weight (w / w), or less than about 23% by weight (w / w), or less than about 24% by weight (w / w), or less than about 25% by weight ( % (w / w), or less than about 26% (w / w), or less than about 27% (w / w), or less than about 28% (w / w), or less than about 29% (w / w), or less than about 30% (w / w), or less than about 31% (w / w), or less than about 32% (w / w), or less than about 33% (w / w), or less than about 34% (w / w), or less than about 35% (w / w), or less than about 36% (w / w), or less than about 37% (w / w), or less than about 38% (w / w), or less than about 39% The method may include preparing a silk fibroin solution or other composition that may contain silk fibroin at a concentration of less than about 40% by weight (w / w), or less than about 41% by weight (w / w), or less than about 42% by weight (w / w), or less than about 43% by weight (w / w), or less than about 44% by weight (w / w), or less than about 45% by weight (w / w), or less than about 46% by weight (w / w), or less than about 47% by weight (w / w), or less than about 48% by weight (w / w), or less than about 49% by weight (w / w), or less than about 50% by weight (w / w).In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after any processing step. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after pigment delivery. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after color fixation. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after final color adjustment. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after pigment chemical alteration. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after colorant delivery. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after Uniflex treatment. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after the Finiflex process. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after the heat stamping process. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after the sanding process. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after the skin trimming process. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after the finishing process.In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after tanning. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after dyeing. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after stretching. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after drying. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after trimming. In some embodiments, the method can include coating the surface of the leather material with a silk fibroin solution or composition before, during, or after burnishing.

[0039] In some embodiments, the method can include filling and / or repairing defects in the surface of the leather material with a silk fibroin composition, such as a silk fibroin glue, paste, gel, wax, putty, etc. In embodiments, methods are provided for repairing leather with silk fibroin and / or SPF, which can include silk-based proteins or fragments thereof, to provide a silk fibroin-repaired leather. In some embodiments, the method can include preparing a silk fibroin solution or other composition that can include one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin, at a concentration of less than about 1% by weight (w / w), or less than about 0.1% by weight (w / w), or less than about 0.01% by weight (w / w), or less than about 0.001% by weight (w / w).In some embodiments, the method comprises dissolving one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin in an amount of less than about 1% by weight (w / w), or less than about 2% by weight (w / w), or less than about 3% by weight (w / w), or less than about 4% by weight (w / w), or less than about 5% by weight (w / w), or less than about 6% by weight (w / w), or less than about 7% by weight (w / w), or less than about 8% by weight (w / w), or less than about 9% by weight (w / w), or less than about 10% by weight (w / w), or less than about 11% by weight (w / w). / w), or less than about 12% by weight (w / w), or less than about 13% by weight (w / w), or less than about 14% by weight (w / w), or less than about 15% by weight (w / w), or less than about 16% by weight (w / w), or less than about 17% by weight (w / w), or less than about 18% by weight (w / w), or less than about 19% by weight (w / w), or less than about 20% by weight (w / w), or less than about 21% by weight (w / w), or less than about 22% by weight (w / w), or less than about 23% by weight (w / w), or less than about 24% by weight (w / w), or less than about 25% by weight ( % (w / w), or less than about 26% (w / w), or less than about 27% (w / w), or less than about 28% (w / w), or less than about 29% (w / w), or less than about 30% (w / w), or less than about 31% (w / w), or less than about 32% (w / w), or less than about 33% (w / w), or less than about 34% (w / w), or less than about 35% (w / w), or less than about 36% (w / w), or less than about 37% (w / w), or less than about 38% (w / w), or less than about 39% The method may include preparing a silk fibroin solution or other composition that may contain silk fibroin at a concentration of less than about 40% by weight (w / w), or less than about 41% by weight (w / w), or less than about 42% by weight (w / w), or less than about 43% by weight (w / w), or less than about 44% by weight (w / w), or less than about 45% by weight (w / w), or less than about 46% by weight (w / w), or less than about 47% by weight (w / w), or less than about 48% by weight (w / w), or less than about 49% by weight (w / w), or less than about 50% by weight (w / w).In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after any processing step. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after pigment delivery. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after color fixation. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after final color adjustment. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after pigment chemical alteration. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after improved colorant delivery. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after Uniflex treatment. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the Finiflex treatment. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the heat stamping treatment. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the sanding treatment. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the skin trimming treatment.In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the finishing process. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after tanning. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after dyeing. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after stretching. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after drying. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after trimming. In some embodiments, the method can include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after sanding.

[0040] In embodiments, methods are provided for coating leather with silk fibroin and / or SPF, which may include silk-based proteins or fragments thereof, to provide silk fibroin-coated leather, wherein the silk fibroin coated on the silk fibroin-coated leather may be heat-resistant to a selected temperature. In some embodiments, the methods may include preparing a silk fibroin solution or other composition that may include one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin at a concentration of less than about 1% by weight (w / w), or less than about 0.1% by weight (w / w), or less than about 0.01% by weight (w / w), or less than about 0.001% by weight (w / w).In some embodiments, the method comprises dissolving one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin in an amount of less than about 1% by weight (w / w), or less than about 2% by weight (w / w), or less than about 3% by weight (w / w), or less than about 4% by weight (w / w), or less than about 5% by weight (w / w), or less than about 6% by weight (w / w), or less than about 7% by weight (w / w), or less than about 8% by weight (w / w), or less than about 9% by weight (w / w), or less than about 10% by weight (w / w), or less than about 11% by weight (w / w). / w), or less than about 12% by weight (w / w), or less than about 13% by weight (w / w), or less than about 14% by weight (w / w), or less than about 15% by weight (w / w), or less than about 16% by weight (w / w), or less than about 17% by weight (w / w), or less than about 18% by weight (w / w), or less than about 19% by weight (w / w), or less than about 20% by weight (w / w), or less than about 21% by weight (w / w), or less than about 22% by weight (w / w), or less than about 23% by weight (w / w), or less than about 24% by weight (w / w), or less than about 25% by weight ( % (w / w), or less than about 26% (w / w), or less than about 27% (w / w), or less than about 28% (w / w), or less than about 29% (w / w), or less than about 30% (w / w), or less than about 31% (w / w), or less than about 32% (w / w), or less than about 33% (w / w), or less than about 34% (w / w), or less than about 35% (w / w), or less than about 36% (w / w), or less than about 37% (w / w), or less than about 38% (w / w), or less than about 39% The method may include preparing a silk fibroin solution or other composition that may contain silk fibroin at a concentration of less than about 40% by weight (w / w), or less than about 41% by weight (w / w), or less than about 42% by weight (w / w), or less than about 43% by weight (w / w), or less than about 44% by weight (w / w), or less than about 45% by weight (w / w), or less than about 46% by weight (w / w), or less than about 47% by weight (w / w), or less than about 48% by weight (w / w), or less than about 49% by weight (w / w), or less than about 50% by weight (w / w).In some embodiments, the method can include coating a surface of a leather material with a silk fibroin solution. In some embodiments, the method can include drying the surface of the leather material coated with the silk fibroin solution or composition to provide a silk fibroin-coated leather material, where drying the surface of the leather material includes heating the surface of the material without substantially reducing silk fibroin coating performance. In some embodiments, the method can include filling defects in the surface of the leather material with a silk fibroin composition, such as a silk fibroin glue, paste, gel, wax, putty, etc.

[0041] In embodiments, the silk fibroin processed leather material of the present disclosure can be processed with one or more of low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk to provide a coated leather material with increased hydrophobicity or hydrophilicity. In embodiments, the silk fibroin coated leather material of the present disclosure can be coated with one or more of low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk to provide a coated leather material with increased hydrophobicity or hydrophilicity. In embodiments, the silk fibroin repaired leather material of the present disclosure has one or more defects repaired, masked, or concealed with one or more of low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk to provide a leather material with improved properties, such as an improved quality grade.

[0042] In embodiments, the silk fibroin processed leather material of the present disclosure can be processed with a composition comprising low molecular weight silk and medium molecular weight silk. In embodiments, the silk fibroin coated leather material of the present disclosure can be coated with a composition comprising low molecular weight silk and medium molecular weight silk. In embodiments, the silk fibroin defect repair leather material of the present disclosure can be repaired with a composition comprising low molecular weight silk and medium molecular weight silk. In some embodiments, the w / w ratio between the low molecular weight silk and the medium molecular weight silk is about 99:1 to about 1:99, about 95:5 to about 5:95, about 90:10 to about 10:90, about 75:25 to about 25:75, about 65:35 to about 35:65, or about 55:45 to about 45:55. In some embodiments, the w / w ratio between the low molecular weight silk and the medium molecular weight silk is about 99:1 to about 55:45, about 95:5 to about 45:55, about 90:10 to about 35:65, about 75:25 to about 15:85, about 65:35 to about 10:90, or about 55:45 to about 1:99.In embodiments, the w / w ratio between the low molecular weight silk and the medium molecular weight silk is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, approximately 76:24, approximately 75:25, approximately 74:26, approximately 73:27, approximately 72:28, approximately 71:29, approximately 70:30, approximately 69:31, approximately 68:32, approximately 67:33, approximately 66:34, approximately 65:35, approximately 64:36, approximately 63:37, approximately 62:38, approximately 61:39, approximately 60:40, approximately 59:41, approximately 58:42, approximately 57:43, approximately 56:44, approximately 55:45, approximately 54:46, approximately 53:47, approximately 52: 48, approximately 51:49, approximately 50:50, approximately 49:51, approximately 48:52, approximately 47:53, approximately 46:54, approximately 45:55, approximately 44:56, approximately 43:57, approximately 42:58, approximately 41:59, approximately 40:60, approximately 39:61, approximately 38:62, approximately 37:63, approximately 36:64, approximately 35:75, approximately 34:66, approximately 33:67, approximately 32:68, approximately 31:69, approximately 30:70, approximately 29:71, approximately 28:72, approximately 27:73, about 26:74, about 25:85, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:95, about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99.

[0043] In embodiments, the silk fibroin processed leather material of the present disclosure can be processed with a composition comprising low molecular weight silk and high molecular weight silk. In embodiments, the silk fibroin coated leather material of the present disclosure can be coated with a composition comprising low molecular weight silk and high molecular weight silk. In embodiments, the silk fibroin defect repair leather material of the present disclosure can be repaired with a composition comprising low molecular weight silk and high molecular weight silk. In some embodiments, the w / w ratio between the low molecular weight silk and the high molecular weight silk is about 99:1 to about 1:99, about 95:5 to about 5:95, about 90:10 to about 10:90, about 75:25 to about 25:75, about 65:35 to about 35:65, or about 55:45 to about 45:55. In some embodiments, the w / w ratio between the low molecular weight silk and the high molecular weight silk is about 99:1 to about 55:45, about 95:5 to about 45:55, about 90:10 to about 35:65, about 75:25 to about 15:85, about 65:35 to about 10:90, or about 55:45 to about 1:99.In embodiments, the w / w ratio between the low molecular weight silk and the high molecular weight silk is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, approximately 76:24, approximately 75:25, approximately 74:26, approximately 73:27, approximately 72:28, approximately 71:29, approximately 70:30, approximately 69:31, approximately 68:32, approximately 67:33, approximately 66:34, approximately 65:35, approximately 64:36, approximately 63:37, approximately 62:38, approximately 61:39, approximately 60:40, approximately 59:41, approximately 58:42, approximately 57:43, approximately 56:44, approximately 45:55, approximately 54:46, approximately 53:47, approximately 52: 48, approximately 51:49, approximately 50:50, approximately 49:51, approximately 48:52, approximately 47:53, approximately 46:54, approximately 45:55, approximately 44:56, approximately 43:57, approximately 42:58, approximately 41:59, approximately 40:60, approximately 39:61, approximately 38:62, approximately 37:63, approximately 36:64, approximately 35:75, approximately 34:66, approximately 33:67, approximately 32:68, approximately 31:69, approximately 30:70, approximately 29:71, approximately 28:72, approximately 27:73, about 26:74, about 25:85, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:95, about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99.

[0044] In embodiments, the silk fibroin processed leather material of the present disclosure can be processed with a composition comprising medium molecular weight silk and high molecular weight silk. In embodiments, the silk fibroin coated leather material of the present disclosure can be coated with a composition comprising medium molecular weight silk and high molecular weight silk. In embodiments, the silk fibroin defect repair leather material of the present disclosure can be repaired with a composition comprising medium molecular weight silk and high molecular weight silk. In some embodiments, the w / w ratio between the medium molecular weight silk and the high molecular weight silk is about 99:1 to about 1:99, about 95:5 to about 5:95, about 90:10 to about 10:90, about 75:25 to about 25:75, about 65:35 to about 35:65, or about 55:45 to about 45:55. In some embodiments, the w / w ratio between the medium molecular weight silk and the high molecular weight silk is about 99:1 to about 55:45, about 95:5 to about 45:55, about 90:10 to about 35:65, about 75:25 to about 15:85, about 65:35 to about 10:90, or about 55:45 to about 1:99.In embodiments, the w / w ratio between medium molecular weight silk and high molecular weight silk is about 99:1, about 98:2, about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20, about 79:21, about 78:22, about 77:23, approximately 76:24, approximately 75:25, approximately 74:26, approximately 73:27, approximately 72:28, approximately 71:29, approximately 70:30, approximately 69:31, approximately 68:32, approximately 67:33, approximately 66:34, approximately 65:35, approximately 64:36, approximately 63:37, approximately 62:38, approximately 61:39, approximately 60:40, approximately 59:41, approximately 58:42, approximately 57:43, approximately 56:44, approximately 45:55, approximately 54:46, approximately 53:47, approximately 52: 48, approximately 51:49, approximately 50:50, approximately 49:51, approximately 48:52, approximately 47:53, approximately 46:54, approximately 45:55, approximately 44:56, approximately 43:57, approximately 42:58, approximately 41:59, approximately 40:60, approximately 39:61, approximately 38:62, approximately 37:63, approximately 36:64, approximately 35:75, approximately 34:66, approximately 33:67, approximately 32:68, approximately 31:69, approximately 30:70, approximately 29:71, approximately 28:72, approximately 27:73, about 26:74, about 25:85, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:95, about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about 2:98, or about 1:99.

[0045] In embodiments, the silk fibroin processed leather material of the present disclosure can be processed with a composition comprising low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk. In embodiments, the silk fibroin coated leather material of the present disclosure can be coated with a composition comprising low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk. In embodiments, the silk fibroin defect repair leather material of the present disclosure can be repaired with a composition comprising low-molecular-weight silk, medium-molecular-weight silk, and high-molecular-weight silk. In embodiments, the w / w ratio of low molecular weight silk, medium molecular weight silk, and high molecular weight silk is about 1:1:8, 1:2:7, 1:3:6, 1:4:5, 1:5:4, 1:6:3, 1:7:2, 1:8:1, 2:1:7, 2:2:6, 2:3:5, 2:4:4, 2:5:3, 2:6:2, 2:7:1, 3:1:6, 3:2:5, 3:3:4, 3:4:3, 3:5:2, 3:6:1, 4:1:5, 4:2:4, 4:3:3, 4:4:2, 4:5:1, 5:1:4, 5:2:3, 5:3:2, 5:4:1, 6:1:3, 6:2:2, 6:3:1, 7:1:2, 7:2:1, or 8:1:1.

[0046] In embodiments, the present disclosure provides a silk and / or SPF treated leather article, wherein the treatment comprises a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a silk and / or SPF coated leather article, wherein the coating comprises a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a silk and / or SPF defect repair leather article, wherein the defect filling comprises a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0047] In embodiments, the present disclosure provides a silk and / or SPF treated leather article, wherein the treatment comprises a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a silk and / or SPF coated leather article, wherein the coating comprises a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a silk and / or SPF defect repair leather article, wherein the defect filling comprises a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa.

[0048] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having an average number of amino acid residues of about 1-400, 1-300, 1-200, 1-100, 1-50, 5-25, or 10-20. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average number of amino acid residues of about 1-400, 1-300, 1-200, 1-100, 1-50, 5-25, or 10-20. In embodiments, the present disclosure provides a leather article comprising one or more leather defect fillers, wherein the composition comprises a silk-based protein or fragment thereof having an average number of amino acid residues of about 1-400, 1-300, 1-200, 1-100, 1-50, 5-25, or 10-20.

[0049] In embodiments, the present disclosure provides a leather article treated with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a leather defect filler composition, the composition comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0050] In embodiments, the present disclosure provides a leather article treated with a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition, the composition comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa.

[0051] In embodiments, the present disclosure provides a leather article processed with a silk protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) of sericin. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) of sericin. In embodiments, the present disclosure provides a leather article comprising a leather defect filler composition comprising a silk-based protein or fragment thereof having a weight average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) sericin.

[0052] In embodiments, the present disclosure provides a leather article processed with a silk protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) of sericin. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) of sericin. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a silk fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) of sericin.

[0053] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof. In embodiments, the present disclosure provides a leather article comprising a leather defect filler composition comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof.

[0054] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof.

[0055] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and a combination thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and a combination thereof. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and a combination thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and a combination thereof. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof, and the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and combinations thereof.

[0056] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and a combination thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and a combination thereof. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and a combination thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and a combination thereof. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and combinations thereof, and the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and combinations thereof.

[0057] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of a natural silk-based protein or fragment thereof, a recombinant silk-based protein or fragment thereof, and a combination thereof; the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof selected from the group consisting of a spider silk-based protein or fragment thereof, a silkworm silk-based protein or fragment thereof, and a combination thereof; the natural silk-based protein or fragment is a silkworm silk-based protein or fragment; and the silkworm silk-based protein or fragment is a Bombyx mori silk-based protein or fragment thereof. In embodiments, the present disclosure provides a leather article having a coating comprising a silk base protein or fragment thereof having a weight average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk base protein or fragment thereof is selected from the group consisting of a natural silk base protein or fragment thereof, a recombinant silk base protein or fragment thereof, and a combination thereof; the silk base protein or fragment thereof is a natural silk base protein or fragment thereof selected from the group consisting of a spider silk base protein or fragment thereof, a silkworm silk base protein or fragment thereof, and a combination thereof; the natural silk base protein or fragment is a silkworm silk base protein or fragment; and the silkworm silk base protein or fragment is a Bombyx mori silk base protein or fragment.In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk base protein or fragment thereof having a weight average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk base protein or fragment thereof is selected from the group consisting of a natural silk base protein or fragment thereof, a recombinant silk base protein or fragment thereof, and a combination thereof; the silk base protein or fragment thereof is a natural silk base protein or fragment thereof selected from the group consisting of a spider silk base protein or fragment thereof, a silkworm silk base protein or fragment thereof, and a combination thereof; the natural silk base protein or fragment is a silkworm silk base protein or fragment; and the silkworm silk base protein or fragment is a Bombyx mori silk base protein or fragment.

[0058] In embodiments, the present disclosure provides a leather article processed with a silk base protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa, wherein the silk base protein or fragment thereof is selected from the group consisting of a natural silk base protein or fragment thereof, a recombinant silk base protein or fragment thereof, and a combination thereof; the silk base protein or fragment thereof is a natural silk base protein or fragment thereof selected from the group consisting of a spider silk base protein or fragment thereof, a silkworm silk base protein or fragment thereof, and a combination thereof; the natural silk base protein or fragment is a silkworm silk base protein or fragment; and the silkworm silk base protein or fragment is a Bombyx mori silk base protein or fragment. In embodiments, the present disclosure provides a leather article having a coating comprising a silk base protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa, wherein the silk base protein or fragment thereof is selected from the group consisting of a natural silk base protein or fragment thereof, a recombinant silk base protein or fragment thereof, and combinations thereof; the silk base protein or fragment thereof is a natural silk base protein or fragment thereof selected from the group consisting of a spider silk base protein or fragment thereof, a silkworm silk base protein or fragment thereof, and combinations thereof; the natural silk base protein or fragment is a silkworm silk base protein or fragment; and the silkworm silk base protein or fragment is a Bombyx mori silk base protein or fragment.In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition comprising a silk base protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa, wherein the silk base protein or fragment thereof is selected from the group consisting of a natural silk base protein or fragment thereof, a recombinant silk base protein or fragment thereof, and combinations thereof; the silk base protein or fragment thereof is a natural silk base protein or fragment thereof selected from the group consisting of a spider silk base protein or fragment thereof, a silkworm silk base protein or fragment thereof, and combinations thereof; the natural silk base protein or fragment is a silkworm silk base protein or fragment; and the silkworm silk base protein or fragment is a Bombyx mori silk base protein or fragment.

[0059] In embodiments, the present disclosure provides a leather article treated with a composition comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a defect-filling composition comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0060] In embodiments, the present disclosure provides a leather article treated with a composition comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a defect-filling composition comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0061] In embodiments, the present disclosure provides a leather article treated with a composition comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a defect-filling composition comprising a silk-based protein or fragment thereof and a polymer and / or copolymer, wherein the silk-based protein or fragment thereof has a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0062] In embodiments, the present disclosure provides a leather article treated with a composition comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a defect-filling composition comprising a silk-based protein or fragment thereof and a pigment and / or colorant, wherein the silk-based protein or fragment thereof has an average weight-average molecular weight of about 5 kDa to about 144 kDa.

[0063] In embodiments, the present disclosure provides a leather article processed with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or protein fragment thereof has an average weight-average molecular weight range selected from the group consisting of about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof has a polydispersity of about 1.5 to about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel and does not show a visible change in color or turbidity when present in solution for at least 10 days prior to processing into a leather article. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or protein fragment thereof has an average weight-average molecular weight range selected from the group consisting of about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof has a polydispersity of about 1.5 to about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel and does not show a visible change in color or turbidity when present in solution for at least 10 days prior to coating the leather article.In embodiments, the present disclosure provides a leather article comprising a leather defect filler composition comprising a silk-based protein or fragment thereof having a weight average molecular weight range of about 5 kDa to about 144 kDa, wherein the silk-based protein or protein fragment thereof has an average weight average molecular weight range selected from the group consisting of about 5 kDa to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof has a polydispersity of about 1.5 to about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel and does not show a visible change in color or turbidity when present in solution for at least 10 days prior to repairing the leather article.

[0064] In embodiments, the present disclosure provides a leather article treated with a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a leather defect filler composition, the composition comprising a silk-based protein or fragment thereof having a weight-average molecular weight range of about 5 kDa to about 144 kDa.

[0065] In embodiments, the present disclosure provides a leather article treated with a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article having a coating comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa. In embodiments, the present disclosure provides a leather article comprising a leather defect filling composition, the composition comprising a silk-based protein or fragment thereof having an average weight-average molecular weight of about 5 kDa to about 144 kDa.

[0066] The presently disclosed embodiments will be further described with reference to the accompanying drawings, in which the drawings are not necessarily to scale, emphasis generally being placed upon illustrating the principles of the presently disclosed embodiments. [Brief explanation of the drawings]

[0067] [Figure 1] 1 is a flow chart illustrating various embodiments for producing pure silk fibroin-based protein fragments (SPF) of the present disclosure. [Figure 2] 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 3] Illustrates common processes used in leather processing. [Figure 4] Photographs of felt pads (and associated leather samples) after 600 consecutive cycles of Wet Veslic Rubbing, comparing leather samples treated with a silk fibroin fragment composition (bottom sample - entry B2) to leather samples treated with polyurethane (top two samples). Note the damage to the polyurethane sample after 600 cycles and the loss of dye from the leather to the felt. [Figure 5] Photographs of felt pads after 10 cycles of Wet Veslic Rubbing for leather samples treated with entries A1, A2, B1, and B2 (Table 1). [Figure 6] This photograph shows a water droplet placed on a sample treated with either a silk fibroin fragment or a cross-linked polyurethane coating system after wet vein rubbing. In the case of the silk fibroin fragment (entry B2), the sample was subjected to 600 cycles of rubbing, while the polyurethane sample only withstood 10 cycles. The photograph was taken 5 minutes after the water droplet was placed. Note the penetration of water into the leather matrix when using a commercially available reference material system designed as a topcoat. [Figure 7A]8A is a graphical analysis illustrating the results of Water Vapor Transmission Test #1 for coated leather (8A) and uncoated leather (8B). [Figure 7B] 8A is a graphical analysis illustrating the results of Water Vapor Transmission Test #1 for coated leather (8A) and uncoated leather (8B). [Figure 8A] 9A is a graphical analysis illustrating the results of Water Vapor Transmission Test #2 for coated leather (9A) and uncoated leather (9B). [Figure 8B] 9A is a graphical analysis illustrating the results of Water Vapor Transmission Test #2 for coated leather (9A) and uncoated leather (9B). [Figure 9A] 1 is a graphical analysis illustrating the results of Water Vapor Transmission Test #3 for coated leather (10A) and uncoated leather (10B). [Figure 9B] 1 is a graphical analysis illustrating the results of Water Vapor Transmission Test #3 for coated leather (10A) and uncoated leather (10B). [Figure 10] A to B are photographs of plain uncoated leather. [Figure 11A] Figure 1 shows an FTIR analysis of plain uncoated leather. [Figure 11B] Figure 1 shows an FTIR analysis of plain uncoated leather. [Figure 12] AB are photographs of leather treated with an adhesive coating of the coating system disclosed herein. [Figure 13A] 1 shows an FTIR analysis of leather treated with an adhesive coating of the coating system disclosed herein. [Figure 13B] 1 shows an FTIR analysis of leather treated with an adhesive coating of the coating system disclosed herein. [Figure 14A]1 is a photograph of treated leather finished with a topcoat of the coating system disclosed herein. [Figure 14B] 1 is a photograph of treated leather finished with a topcoat of the coating system disclosed herein. [Figure 14C] 1 shows an FTIR analysis of treated leather finished with a topcoat of the coating system disclosed herein. [Figure 14D] 1 shows an FTIR analysis of treated leather finished with a topcoat of the coating system disclosed herein. [Figure 15A] 1 is an IR spectrum of a leather sample treated with a coating system disclosed herein by an LN-MCT detector. [Figure 15B] 1 shows macroscopic ATR imaging of a leather sample treated with an adhesive basecoat of a coating system disclosed herein. [Figure 15C] 1 shows macroscopic ATR imaging of a leather sample treated with a topcoat of a coating system disclosed herein. [Figure 16A] 1 is a photograph illustrating the results of stain release tests using various sources of soiling on leather treated with the coating system disclosed herein. [Figure 16B] 1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with a coating system disclosed herein. Water. [Figure 16C] 1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with a coating system disclosed herein. Mustard. [Figure 16D] 1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with a coating system disclosed herein: Corn oil. [Figure 16E]1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with the coating system disclosed herein: Wine. [Figure 16F] 1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with the coating system disclosed herein: Ketchup. [Figure 16G] Photographs illustrating the results of stain release tests using various stain sources on leather treated with the coating system disclosed herein. French dressing. [Figure 16H] 1 is a photograph illustrating the results of stain release tests using various stain sources on leather treated with a coating system disclosed herein: Coffee. [Figure 17] Photographs of leather samples treated with the coating system disclosed herein used in commercial trials are shown in FIGS. [Figure 18A] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18B] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18C] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18D] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18E] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18F] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18G] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18H] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 18I] 18A and 18B are photographs of felt pads (and associated leather samples treated with the coating system disclosed herein) after 600 consecutive cycles of Wet Veslic Rubbing (Note: FIG. 18H only underwent 360 cycles). [Figure 19A] 1 is a photograph illustrating the results of a Bally Flex Test performed on various leather samples treated with the coating system disclosed herein. [Figure 19B] 1 is a photograph illustrating the results of a Bally Flex Test performed on various leather samples treated with the coating system disclosed herein. [Figure 19C] 1 is a photograph illustrating the results of a Bally Flex Test performed on various leather samples treated with the coating system disclosed herein. [Figure 19D]1 is a photograph illustrating the results of a Bally Flex Test performed on various leather samples treated with the coating system disclosed herein. [Figure 20A] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20B] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20C] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20D] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20E] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20F] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20G] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20H] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 20I] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with an adhesive coating system. [Figure 21] 1 is a photograph illustrating the difference between leather samples treated with the adhesive coating system disclosed herein before and after milling. [Figure 22A]1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22B] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22C] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22D] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22E] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22F] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22G] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22H] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 22I] 1 is a photograph illustrating the results of a sticky tape test performed on various leather samples treated with the adhesive coating system disclosed herein. [Figure 23] 1 is a photograph illustrating the difference between leather samples treated with the adhesive coating system disclosed herein before and after milling. [Figure 24]AB are photographs illustrating the difference in adhesive tape tests performed on leather samples treated with the adhesive coating system disclosed herein before and after milling. [Figure 25] 1A-C are microscopic cross-sectional images of leather surfaces treated with the coating system disclosed herein. [Figure 26] 1A-C are microscopic top view images of leather surfaces treated with the coating system disclosed herein. [Figure 27] 1A-C are images under a digital microscope showing a wet blue leather strip treated with a coating system disclosed herein: A: side view, B: top grain view, C: fresh view. [Figure 28] 1A-C are images under a digital microscope showing a piece of paper treated with the coating system disclosed herein: A: top view, B: side view, C: back view. [Figure 29] 1A-C are images under a digital microscope showing a piece of fabric treated with a coating system disclosed herein: A: top view, B: side view, C: back view. [Figure 30] 1A-C are images under a digital microscope showing a piece of fabric with blue tape treated with a coating system disclosed herein: A: top view, B: side view, C: back view. [Figure 31] Photographs of the tensile testing process of AS-104 + 2% glycerol + 50 mM magnesium sulfate film are shown. [Figure 32] 1 shows a proposed formulation scheme incorporating AS-104, 2% glycerol, and various concentrations of salt. [Figure 33A] The elongation at break for AS-104, 2% glycerol, and guanidinium hydrochloride (5, 10, 25, and 50 mM) is shown. [Figure 33B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and guanidinium hydrochloride (5, 10, 25, and 50 mM). [Figure 34A]The elongation at break for AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM) is shown. [Figure 34B] The ultimate tensile strength of AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM) is shown. [Figure 35A] The elongation at break for AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM) is shown. [Figure 35B] The ultimate tensile strength of AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM) is shown. [Figure 36A] 1 shows the elongation at break for AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM). [Figure 36B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM). [Figure 37A] 1 shows the elongation at break for AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM). [Figure 37B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM). [Figure 38A] The elongation at break for AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM) is shown. [Figure 38B] The ultimate tensile strength of AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM) is shown. [Figure 39A] The elongation at break for AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM) is shown. [Figure 39B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM). [Figure 40A] The elongation at break for AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM) is shown. [Figure 40B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM). [Figure 41A] 1 shows the elongation at break for AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM). [Figure 41B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM). [Figure 42A] 1 shows the elongation at break for AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM). [Figure 42B] 1 shows the ultimate tensile strength of AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM). [Figure 43] All data regarding elongation at break is compiled. [Figure 44] Compile all data on ultimate tensile strength. [Figure 45] Figure 1 shows Veslic wet and dry test results for Bodin basic black leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10mM CaCl2, 17% AS-104-5% Melio-9S11-50mM MgSO4, and 17% AS-104-5% Melio-9S11-25mM L-arginine hydrochloride. [Figure 46] 1 shows Veslic wet and dry test results for Bodin brown leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. [Figure 47]Figure 1 shows the Veslic scores for Bodin Basic Black leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. [Figure 48] Figure 1 shows the Veslic scores of Bodin brown leather coated with 17% AS-104-5% Melio-9S11, 17% AS-104-5% Melio-9S11-10 mM CaCl2, 17% AS-104-5% Melio-9S11-50 mM MgSO4, and 17% AS-104-5% Melio-9S11-25 mM L-arginine hydrochloride. [Figure 49A] Topographic traces of a GG-silk coated leather sample are shown before and after coating. A point-filled coating of silk + 0.5 wt% GG is shown before (Figure 23A) and after (Figure 23B). Traces were captured using a Taylor Hobson CCI HD optical profilometer. [Figure 49B] Topographic traces of a GG-silk coated leather sample are shown before and after coating. A point-filled coating of silk + 0.5 wt% GG is shown before (Figure 23A) and after (Figure 23B). Traces were captured using a Taylor Hobson CCI HD optical profilometer.

[0068] While the above-identified drawings illustrate embodiments of the presently disclosed invention, other embodiments are contemplated, as noted in the discussion. The present disclosure presents exemplary embodiments that are representative and not limiting. Numerous other variations and embodiments can be devised by those skilled in the art that fall within the scope and spirit of the principles of the presently disclosed embodiments. DETAILED DESCRIPTION OF THE INVENTION

[0069] In some embodiments, the present disclosure provides a composition comprising a coating comprising two components. In some embodiments, the second component is impregnated onto the first component. In some embodiments, the second component undergoes a phase change (e.g., without limitation, Tg, polymerization, etc.). The first coating described herein can include, without limitation, a polymer or any protein disclosed herein, such as biodegradable polyurethane, silk protein, collagen, casein, elastin, etc. The second coating described herein can include, without limitation, a cellulose derivative disclosed herein. The order of the first and second coatings should not be limited, as any coating disclosed herein can be replaced with any other coating disclosed herein. While ethylcellulose is typically brittle and can crack, in some embodiments, the present disclosure provides a flexible ethylcellulose coating. The present disclosure provides, without limitation, coating any surface, for example, leather, fabric, wood, protective coatings for food (fruits, vegetables, etc.). In some embodiments, the coatings disclosed herein are made of two or more films (possibly starting with one film made of two polymers) with a monolayer distribution for coating onto a substrate. As disclosed herein, the composites and / or coatings disclosed herein can be based, without limitation, on molecular entanglement where the EC does not involve a crosslinker. In some embodiments, all layers are fixed together through molecular interactions. In some embodiments, all molecular interactions are cured, set, or polymerized. In some embodiments, the molecular interactions of the two layers result in a film that hardens and the molecules form a larger polymeric structure. In some embodiments, the outer layer described herein comprises 1% to 100% EC on the surface.In some embodiments, the first layer (in application to the surface to be coated) involves molecular entanglement, such as between the first and second layers to be adhered; the first layer can adhere to heterogeneous surfaces; the first layer is thermoplastic, self-assembles, and soluble in the solvent used for the second layer; the first layer polymerizes through cross-linking and self-assembly. In some embodiments, the first layer is soluble and can be hardened. In some embodiments, polymers or proteins, such as, without limitation, silk proteins, play a role in the first layer. In some embodiments, the second layer (deposited on top of the first and outer layers) is made of ethyl cellulose (EC) or biomaterials or polymers in a molecular dispersion; in some embodiments, this layer in a solvent contains about 1-5 grams / L of EC by volume. In some embodiments, this layer can deliver dyes, silk, or other molecules to modify optical, tactile, and mechanical properties. In some embodiments, the EC is a protective barrier that can enhance the performance and properties of the first layer. In some embodiments, the EC is mechanically resilient and enhances water resistance. In some embodiments, the EC can adhere to a dynamic first layer substrate. In some embodiments, the EC can adhere to an uneven first layer surface. In some embodiments, the majority of the EC faces outward toward the external environment / forces. In some embodiments, proteins or polymers, such as, without limitation, silk, play a role in the second layer.

[0070] Silk-coated leather articles and methods for their manufacture are described in WO2020 / 018821 and WO2021 / 146654, each of which is incorporated herein by reference in its entirety.

[0071] Leather is a material produced by subjecting skins removed from animals to a series of physical, mechanical, and chemical processes, followed by tanning. Leather materials consist of woven collagen fiber bundles and traces of elastic and reticular fibers, of which collagen accounts for 95-98 percent. In the natural woven structure of collagen fibers in natural leather, thick fiber bundles sometimes split into several thinner fiber bundles, and the resulting thinner fiber bundles may incorporate other fiber bundles to form larger fiber bundles.

[0072] Leather in its natural state is a nonwoven material in which fiber fibrils grow together. The silk fibroin protein and collagen fibers in leather are natural proteins consisting of 22 proteinogenic amino acids. Silk protein has a high affinity for leather fibers (collagen fibers) (e.g., physical entanglement through the formation of hydrogen bonds between silk protein fragments and leather fibers) due to the presence of hydrophilic amino acid residues in silk fibroin protein, such as -OH groups from serine, guanidine groups from arginine, free amine groups from lysine, and -COOH groups from aspartic acid and glutamic acid.

[0073] In some embodiments, the silk fibroin-based protein fragments and solutions described herein find use as color performance enhancers for leather or leather articles. In some embodiments, the present disclosure provides silk-treated leather or leather articles that exhibit good dyeability, excellent colorfastness, and improved saturation.

[0074] Treatment of leather and leather articles with silk fibroin-based protein fragments and solutions improves the quality and aesthetic properties of natural leather using non-toxic, sustainable, natural silk-based compositions. The silk treatment process disclosed herein advances leather products without disrupting the leather tanning and crafting process, while respecting its traditions and techniques.

[0075] SPF definition and characteristics As used herein, "silk protein fragments" (SPFs) include, without limitation, one or more of "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 naturally occurring silk polypeptide or variant thereof, the amino acid sequence of a naturally occurring silk polypeptide, or a combination of both.

[0076] SPF molecular weight and polydispersity In an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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 an 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.

[0077] In some embodiments, the compositions of the present disclosure comprise an SPF composition selected from compositions #1001 to #2450, which have a weight average molecular weight selected from about 1 kDa to about 145 kDa and 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]

[0078] As used herein, "low molecular weight," "low MW," or "low-MW" SPF 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 for 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, or about 25 kDa. The weight average molecular weight can be about 21 kDa, 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.

[0079] 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 about 31 kDa to about 55 kDa or about 39 kDa to 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.

[0080] As used herein, "high molecular weight," "high MW," or "high-MW" SPF may include an SPF having a weight average molecular weight, or average weight average molecular weight, selected from about 55 kDa to 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.

[0081] 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 into the approximate number of amino acids contained in each SPF, as understood by those skilled in the art. For example, the average weight of an amino acid can be about 110 Daltons (i.e., 110 g / mol). Therefore, in some embodiments, the molecular weight of a linear protein can be divided by 110 Daltons to roughly estimate the number of amino acid residues contained therein.

[0082] In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 1 to about 5.0 (including but not limited to, a polydispersity of 1). In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 1.5 to about 3.0. In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 1 to about 1.5 (including but not limited to, a polydispersity of 1). In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 1.5 to about 2.0. In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 2.0 to about 2.5. In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 2.5 to about 3.0. In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 3.0 to about 3.5. In embodiments, the SPF in the composition of the present disclosure has a polydispersity selected from 3.5 to about 4.0. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

[0083] In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of 1. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.1. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.2. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.3. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.4. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.5. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.6. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.7. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.8. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 1.9. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.0. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.1. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.2. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.3. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.4. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.5. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.6. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.7. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.8. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 2.9. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.0. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.1. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.2. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.3. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.4. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.5. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.6. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.7.In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.8. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 3.9. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.0. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.1. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.2. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.3. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.4. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.5. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.6. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.7. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.8. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 4.9. In embodiments, the SPF in the compositions of the present disclosure has a polydispersity of about 5.0.

[0084] 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 can have the same or different polydispersities.

[0085] 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. Pat. 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 by reference 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 semicrystalline 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. Natural silkworm fiber is composed of two strands of fibroin. The adhesive substance that holds these two 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 a high molecular weight, in which large hydrophobic domains dominate the polymer. 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 repeating hexapeptide sequences of Gly-Ala-Gly-Ala-Gly-Ser and repeating Gly-Ala / Ser / Tyr dipeptides, which can form stable antiparallel sheet crystallites. Because the amino acid sequence of the L chain is non-repetitive, the L chain is more hydrophilic and relatively elastic. The hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) chain segments in silk fibroin molecules are arranged alternately, allowing the silk fibroin molecules to self-assemble.

[0086] Provided herein are methods for producing pure, highly scalable silk fibroin-protein fragment mixture solutions that can 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.

[0087] As used herein, the term "fibroin" includes silkworm fibroin and insect or spider silk proteins. In embodiments, 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 the fibers of Bombyx mori cocoons, which have a weight-average molecular weight of approximately 370,000 Da. Conversion of these insoluble silk fibroin fibrils to water-soluble silk fibroin protein fragments requires the addition of concentrated neutral salt (e.g., 8-10 M lithium bromide), which disrupts the inter- and intramolecular ionic and hydrogen bonds that 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. Pat. Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0088] Raw silk cocoons from silkworms (Bombyx mori) were shredded into small pieces. These silk cocoon fragments were treated in an aqueous Na2CO3 solution at approximately 100°C for approximately 60 minutes to remove sericin (degumming). The amount 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 fragments. The resulting degummed silk cocoon fragments were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The amount of rinsing water for each cycle was 0.2 L times the weight of the raw silk cocoon fragments. Excess water was removed from the degummed raw silk cocoon fragments. After the DI water washing step, the wet-degummed silk cocoon fragments were dried at room temperature. The degummed silk cocoon fragments 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 against deionized water for 72 hours using tangential flow filtration (TFF) and a 10 kDa membrane. 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 obtain a 1.0% w / v silk solution. The pure silk solution can then be further concentrated to a concentration of 20.0% w / w silk in water using TFF.

[0089] Dialysis of silk through a series of water exchanges is a manual, time-intensive process, but can be accelerated by modifying certain parameters, e.g., by 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.

[0090] In some embodiments, silk solutions were 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 allowed to stand at room temperature for at least 30 minutes. 5 mL of the 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.

[0091] In some embodiments, silk solutions were 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.

[0092] In some embodiments, silk solutions were prepared under various preparation parameters, as follows: 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, 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 an oven at the same temperature as the LiBr. Samples from each set were removed at 1, 4, and 6 hours. 1 mL of each sample was added to 7.5 mL of 9.3 M LiBr and refrigerated for viscosity testing.

[0093] In some embodiments, SPF is obtained by dissolving raw, unrefined, partially refined, or refined silkworm fiber in a neutral lithium bromide salt. The sericin is removed, and the desired weight average molecular weight (M W Raw silkworm silk is processed under selected temperatures and other conditions to achieve a desired size and polydispersity (PD). The selection of process parameters can be modified to achieve distinct final silk protein fragment properties depending on the intended use. The resulting final fragment solution is silk fibroin protein fragments and water with parts per million (ppm) to non-detectable levels of process contaminants, which are acceptable for the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further modified depending on the desired application and performance requirements.

[0094] FIG. 1 is a flowchart illustrating various embodiments for producing pure silk fibroin 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 step A of FIG. 1, cocoons (heat-treated or non-heat-treated), silk fibers, silk powder, spider silk, or recombinant spider silk can be used as silk sources. When starting with raw silk cocoons from Bombyx mori, the cocoons can be shredded into small pieces, e.g., pieces of approximately equal size (step B1). Next, in step C1a, the raw silk is extracted and rinsed to remove sericin. This results in substantially sericin-free raw silk. In an embodiment, water is heated to a temperature of 84°C to 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 soaked for approximately 15 to 90 minutes. In an embodiment, the amount of water is equal to about 0.4 x raw silk weight, and the amount of Na2CO3 is equal to about 0.848 x raw silk weight. In an embodiment, the amount of water is equal to 0.1 x raw silk weight, and the amount of Na2CO3 is maintained at 2.12 g / L.

[0095] The aqueous NaCO solution is then drained, and excess water / NaCO is removed from the silk fibroin fibers (e.g., by manually or mechanically rinsing the fibroin extract). The resulting silk fibroin extract is rinsed with warm to hot water, typically at temperatures ranging from about 40°C to about 80°C, with at least one change in the amount of water (and multiple times as necessary) to remove any remaining adsorbed sericin or contaminants. The resulting silk fibroin extract is silk fibroin from which sericin has been substantially removed. In embodiments, the resulting silk fibroin extract is rinsed with water at a temperature of about 60°C. In embodiments, 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 effectiveness of the rinsing, 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., by manually or mechanically rinsing the fibroin extract). Alternatively, methods known to those skilled in the art (e.g., 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 extracted from silkworms. As a result, liquid sericin-free silk protein can be obtained without changing the protein structure.

[0096] The extracted fibroin fibers are then completely dried. Once dried, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at a temperature between ambient temperature and its boiling point (step C1b). In an embodiment, the solvent is a solution of lithium bromide (LiBr) (the boiling point of LiBr is 140°C). Alternatively, the extracted fibroin fibers are placed in the solvent in a wet state without being dried. 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 concentration can range from 0.1 M to 9.3 M. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature, along with the concentration of the dissolving solvent. Other solvents can be used, including, but not limited to, phosphoric acid phosphate, calcium nitrate, calcium chloride solution, or other concentrated aqueous solutions of inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed in the preheated solvent solution and then maintained at a temperature ranging from about 60°C to about 140°C for 1 to 168 hours. In an embodiment, the silk fibers should be completely immersed in the solvent solution and then placed in a drying oven at a temperature of about 100° C. for about 1 hour.

[0097] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) affects the time required to completely dissolve the fibroin and the resulting molecular weight and polydispersity of the final SPF mixture solution. In embodiments, the concentration of the silk solvent solution is 20% w / v or less. In addition, stirring during addition or dissolution can be used to facilitate dissolution at various 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 embodiments, higher temperatures dissolve the silk more quickly, improving the process scalability and mass production of the silk solution. In embodiments, 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 dissolution solvent above 60°C can change and control the MW and polydispersity of the SPF mixture solution formed from the original molecular weight of the natural silk fibroin protein.

[0098] Alternatively, whole cocoons can be placed directly into a solvent such as LiBr, thereby avoiding 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 for separating hydrophobic and hydrophilic proteins, such as column separation and / or chromatography, ion exchange, chemical precipitation using salt and / or pH, and / or enzymatic digestion and filtration or extraction, all of which are general examples and are not limited to standard protein separation methods. Alternatively, unheat-treated cocoons from which the silkworm has been removed can be placed into a solvent such as LiBr, thereby avoiding extraction. This method can be used for sericin isolation and has the advantage that unheat-treated cocoons contain significantly less silkworm debris.

[0099] Dialysis can be used to remove the dissolving solvent from the resulting dissolved fibroin protein fragment solution by dialyzing it against a volume of water (step E1). Pre-filtration before dialysis is useful to remove all debris (i.e., silkworm remnants) from the silk and LiBr solution (step D). In one example, a 3 μm or 5 μm filter is used at a flow rate of 200-300 mL / min to filter the 0.1%-1.0% silk-LiBr solution prior to dialysis and potential concentration, if desired. As mentioned above, the method disclosed herein utilizes time and / or temperature to reduce the concentration from 9.3 M LiBr to a range of 0.1 M-9.3 M to facilitate filtration and downstream dialysis, particularly when considering the creation of a scalable process. Alternatively, the 9.3 M LiBr-silk protein fragment solution may be diluted with water without additional time or temperature to facilitate filtration and dialysis of the debris. Dissolution and thermal filtration for the desired time results in a translucent, particle-free, room temperature shelf-stable silk protein fragment-LiBr solution of known MW and polydispersity. It is advantageous to periodically change the dialysis water until the solvent is removed (e.g., change the water after 1 hour, 4 hours, and then every 12 hours for a total of six water changes). The total number of water changes can be varied based on the resulting concentration of the solvent used to dissolve and fragment the silk protein. After dialysis, the final silk solution can be further filtered to remove any remaining debris (i.e., silkworm remnants).

[0100] Alternatively, tangential flow filtration (TFF), a rapid and efficient method for biomolecule separation and purification, can be used to remove the solvent from the resulting dissolved fibroin solution (step E2). TFF provides highly pure silk protein fragment aqueous solutions and allows for process scalability for producing large quantities of the solution in a controlled and reproducible manner. The silk and LiBr solution can be diluted (from 20% silk to 0.1% silk in either water or LiBr) before TFF. Prefiltration as described above before TFF can maintain filter efficiency and potentially avoid the formation of a silk gel boundary layer on the filter surface as a result of the presence of debris particles. Prefiltration before TFF is also useful to 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 (circulating or single-pass) can be used to produce water-silk protein fragment solutions with concentrations of 0.1% to 30.0% silk (more preferably 0.1% to 6.0% silk). Depending on the desired concentration, molecular weight, and polydispersity of the silk protein fragment mixture in solution, different cutoff sizes of TFF membranes may be required. Membranes ranging from 1 to 100 kDa may be necessary to vary the molecular weight silk solution, for example, by varying the length of extraction boiling time or the time and temperature in the dissolving solvent (e.g., LiBr). In embodiments, TFF 5 or 10 kDa membranes are used to purify the silk protein fragment mixture solution to create the final desired silk-to-water ratio. Similarly, other methods known in the art, such as TFF single-pass, TFF, and falling film evaporators, can be used to concentrate the solution after removal of the dissolving solvent (e.g., LiBr) (to obtain desired concentrations ranging from 0.1% to 30% silk). This can be used as an alternative to the standard HFIP concentration method known in the art for creating water-based solutions. Larger pore membranes can also be utilized to filter out smaller silk protein fragments and create higher molecular weight silk solutions with or without tighter polydispersity values.

[0101] Assays for LiBr and Na2CO3 detection can be 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 the concentrations. Two or more samples of several formulations of this disclosure were used for sample preparation and analysis. Generally, four samples of different formulations were weighed directly into 10 mL volumetric flasks. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and maintained at 2-8 °C for 2 hours with occasional shaking 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 to an HPLC vial and injected into the HPLC-ELSD system for estimation of sodium carbonate and lithium bromide.

[0102] The analytical method developed for the quantification of Na2CO3 and LiBr in silk protein formulations was found to be linear in the range of 10-165 μg / mL, with RSDs for precision of injection 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 to the quantification of sodium carbonate and lithium bromide in silk protein formulations.

[0103] 2 is a flow chart illustrating various parameters that can be varied during the extraction and dissolution steps of the process for producing a silk protein fragment solution of the present disclosure. Selected process parameters can be varied to achieve specific final solution properties, 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 of the silk solutions of the present disclosure.

[0104] In embodiments, a silk protein fragment solution useful for a wide variety of applications is prepared according to the following steps: forming silk cocoon pieces from Bombyx mori silkworms; extracting the pieces in an aqueous Na2CO3 solution at about 100°C for about 60 minutes to form a silk fibroin extract (wherein the amount 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); rinsing the silk fibroin extract three times in an amount of rinse water at about 60°C for about 20 minutes per rinse (wherein the rinse water for each cycle is equal to 0.2 L x the weight of the pieces); removing excess water from the silk fibroin extract; drying the silk fibroin extract; Dissolving the dried silk fibroin extract in a LiBr solution (wherein the LiBr solution is first heated to and maintained at approximately 100°C to create the silk and LiBr solution); placing the silk and LiBr solution in a drying oven at approximately 100°C for approximately 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into a mixture of the desired molecular weight and polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to obtain a 1.0 wt% silk solution; and removing the solvent from the solution using tangential flow filtration (TFF). In an embodiment, the silk solution is purified using a 10 kDa membrane to create the final desired silk-to-water ratio. TFF can then be used to further concentrate the silk solution to a concentration of 2.0 wt% silk in water.

[0105] While not wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature at which the LiBr solution is added to the silk fibroin extract (or vice versa)), and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions of different viscosity, homogeneity, and color. Similarly, while not wishing to be bound by any particular theory, increasing the temperature for extraction, lengthening the extraction time, using a hotter LiBr solution during immersion and over time in dissolving the silk, and increasing the time at temperature (e.g., in an oven or alternative heat source as shown herein) all result in solvent and silk solutions that are less viscous and more homogeneous.

[0106] The extraction process can be completed in larger vessels, such as industrial washing machines capable of maintaining temperatures between 60°C and 100°C or thereabouts. The rinsing process can also be completed in industrial washing machines, eliminating manual rinsing cycles. Dissolution of silk in LiBr solution can be performed in vessels other than convection ovens, such as stirred tank reactors. Dialysis of silk through a series of water exchanges is a manual, time-intensive process, but can be accelerated by modifying certain parameters, such as diluting the silk solution before dialysis. The dialysis process can be scaled to accommodate manufacturing needs by using semi-automated equipment, such as tangential flow filtration systems.

[0107] Varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature at which the LiBr solution is added to the silk fibroin extract (or vice versa)), and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, homogeneities, and colors. Increasing the temperature for extraction, lengthening the extraction time, using a hotter LiBr solution during immersion and over time to dissolve the silk, and increasing the time at temperature (e.g., in an oven or alternative heat source as demonstrated herein) all result in solvent and silk solutions with lower viscosities and more homogeneous properties. While nearly all parameters resulted in viable silk solutions, for process scalability, methods that allow complete dissolution to be achieved in less than 4-6 hours are preferred.

[0108] In an embodiment, a solution of silk fibroin protein fragments having a weight average selected from about 6 kDa to about 17 kDa is prepared according to the following steps: degumming the silk source by adding the silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to 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; dissolving the silk fibroin extract in a lithium bromide solution, the starting temperature of which ranges from about 60°C to about 140°C when the silk fibroin extract is placed in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in an oven at a temperature of about 140°C for up to 1 hour; removing the lithium bromide from the silk fibroin extract; 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 can further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments can 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 can 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 can be freeze-dried. In some embodiments, the silk fibroin protein fragment solution can be further processed into various forms, including gels, powders, and nanofibers.

[0109] In an embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa is prepared by the following steps: adding the silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to about 60 minutes to degumm the silk source; 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 when the silk fibroin extract is placed in the lithium bromide solution; and heating the solution to about 60°C to about 140°C. maintaining the silk fibroin-lithium bromide solution in a drying oven at a temperature in the range of 100°C for up to 1 hour; removing lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk fibroin protein fragments, wherein the aqueous solution of silk fibroin protein fragments contains about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments contains about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of silk fibroin protein fragments contains 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 can 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 residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay.

[0110] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight-average molecular weight selected from about 6 kDa to about 17 kDa includes the following steps: degumming the silk source by adding the silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to 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; dissolving the silk fibroin extract in a lithium bromide solution, the starting temperature of which when the silk fibroin extract is placed in the lithium bromide solution being in the range of about 60°C to about 140°C; maintaining the silk fibroin-lithium bromide solution in an oven at a temperature of about 140°C for at least 1 hour; removing the lithium bromide from the silk fibroin extract; 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 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 prepared from the aqueous solution of pure silk fibroin protein fragments produced by this 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 prepared from the aqueous solution of pure silk fibroin protein fragments produced by this 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%.

[0111] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa includes the following steps: degumming the silk source by adding it to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to 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; 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 when the silk fibroin extract is placed in the lithium bromide solution; and heating the solution to about 60°C to about 100°C. maintaining the silk fibroin-lithium bromide solution in a drying oven at a temperature in the range of 0°C for at least 1 hour; removing lithium bromide from the silk fibroin extract; and producing an aqueous solution of pure silk fibroin protein fragments, wherein the aqueous solution of pure silk fibroin protein fragments contains about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments contains about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of pure silk fibroin protein fragments includes 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 can 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 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 this 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 this 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%.

[0112] In an embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa is prepared by the following steps: adding a silk source to a boiling (100°C) aqueous sodium carbonate solution for a treatment time of about 30 minutes to degumm it; 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, the starting temperature of which is in the range of about 80°C to about 140°C. maintaining the silk fibroin-lithium bromide solution in a drying oven at a temperature ranging from about 60°C to about 100°C for up to 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk fibroin protein fragments, wherein the aqueous solution of silk fibroin protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residue, about 10 ppm to about 100 ppm of sodium carbonate residue, and 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 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 of residual sodium carbonate as measured using a high-performance liquid chromatography sodium carbonate assay. In some embodiments, the method may further include adding an active agent (e.g., a therapeutic agent) to the aqueous solution of pure silk fibroin protein fragments. The method may further include 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 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 can be freeze-dried. The method can further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin protein fragments. The alpha hydroxy acid can be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method can 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 can be made from the aqueous solution of pure silk fibroin protein fragments produced by this method. The film can contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film can have a water content ranging from about 2.0% to about 20.0% by weight. The film can contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel can be made from the aqueous solution of pure silk fibroin protein fragments produced by this 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% by weight and a vitamin content of at least 20% by weight.

[0113] The molecular weight of the silk protein fragments can be controlled based on specific parameters utilized during the extraction process, including extraction time and temperature; specific parameters utilized during the dissolution process, including the LiBr temperature during immersion of the silk in lithium bromide and the time the solution is maintained at a specific temperature; and specific parameters utilized during the filtration process. By controlling the process parameters using the disclosed method, it is possible to create silk fibroin protein fragment solutions with a polydispersity of 2.5 or less at a wide variety of molecular weights selected from 5 kDa to 200 kDa or 10 kDa to 80 kDa. By varying the process parameters to achieve silk solutions with different molecular weights, a range of final fragment mixture products with a desired polydispersity of 2.5 or less can be targeted based on desired performance requirements. For example, a film of higher molecular weight silk containing an ophthalmic drug can have a controlled, sustained release rate compared to a lower molecular weight film, making it ideal as a delivery vehicle in eye care products. In addition, silk fibroin protein fragment solutions with a polydispersity 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 silk fibroin protein fragment solutions disclosed herein. The molecular weight of the pure silk fibroin protein fragment compositions was determined using high-pressure liquid chromatography (HPLC) equipped with a refractive index detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC / SEC Software Version 3.3 (Agilent).

[0114] Different processing parameters result in regenerated silk fibroin with different molecular weights and peptide chain size distributions (polydispersity, PD), which in turn affect the performance of the regenerated silk fibroin, including mechanical strength, water solubility, etc.

[0115] 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 parameters manipulated included (i) extraction time and temperature, (ii) LiBr temperature, (iii) melting oven temperature, and (iv) melting time. Experiments were conducted to determine the effect of varying the extraction time. Tables A-G summarize the results. The following is a summary: The molecular weight of sericin extracted for -30 minutes was larger than that of sericin extracted for 60 minutes. -Molecular weight decreases with time in the oven. For LiBr and oven at -140°C, the lower end of the confidence interval was below 9500 Da molecular weight. - In the 30 minute extraction at 1 hour and 4 hour time points, there is undigested silk. The 30-minute extraction at -1 hour time point resulted in a significantly higher molecular weight with a lower confidence interval of 35,000 Da. The range of molecular weights that reached the upper limit of the confidence interval was 18,000 to 216,000 Da (important for providing solutions with a specified upper limit). [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0116] Experiments were conducted to determine the effect of varying the extraction temperature. Table G summarizes the results. The summary is as follows: The sericin extracted at -90°C had a higher MW than the sericin extracted at 100°C. Both -90°C and 100°C show a decreasing MW with time in the oven. [Table 8]

[0117] Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) when added to silk. Tables H-I summarize the results. The following is a summary: - No effect on molecular weight or confidence intervals (all CIs approximately 10500-6500 Da). - Studies have shown that the temperature of the LiBr-silk solution drops rapidly below the original LiBr temperature once LiBr is added and begins to dissolve, as most of the mass is room temperature silk. [Table 9] [Table 10]

[0118] Experiments were conducted to determine the effect of oven / melt temperature. Tables J-N summarize the results. The following is a summary: - Oven temperature has less effect on 60 minute extracted silk than 30 minute extracted silk. Without wishing to be bound by theory, it is believed that the 30 minute silk decomposes less during extraction and therefore oven temperature has more effect on the larger MW, less decomposed silk fractions. When comparing the -60°C oven with the 140°C oven, the 30 min extracted silk showed a highly significant effect of being less MW at the higher oven temperature, while the 60 min extracted silk had an effect, but it was much less. The -140°C oven resulted in a lower confidence interval of approximately 6000 Da. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0119] Raw silk cocoons from silkworms (Bombyx mori) were shredded into small pieces. The raw silk cocoon fragments were boiled in an aqueous Na2CO3 solution (approximately 100°C) for approximately 30 to 60 minutes to remove sericin (degumming). The amount 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 fragments. The resulting degummed silk cocoon fragments were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The amount of rinsing water for each cycle was 0.2 L times the weight of the raw silk cocoon fragments. Excess water was removed from the degummed raw silk cocoon fragments. After the DI water washing step, the wet-degummed silk cocoon fragments 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 temperatures 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. Oakton Bromide (Br - Br in hydrolyzed fibroin solutions read with a dual-junction ion-selective electrode - Multiple exchanges with Di water were performed until the ions were below 1 ppm.

[0120] 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 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 solution was diluted with DI water to provide coating solutions of 1.0% w / v, 2.0% w / v, 3.0% w / v, 4.0% w / v, and 5.0% w / v.

[0121] Tangential flow filtration (TFF) was used to produce various % silk concentrations. In each case, a 1% silk solution was used as the input feed. Starting volumes ranging from 750 to 18,000 mL of 1% silk solution were used. The solution was diafiltered in the TFF to remove lithium bromide. Once the residual LiBr was below a specified level, the solution underwent ultrafiltration to increase the concentration by removing water. See the examples below.

[0122] The six silk solutions were applied to standard silk constructs 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, 1 hr LiBr dissolution at 100° C.).

[0123] Solution #2 has a silk concentration of 6.4 wt% (made by boiling extraction for 30 min and dissolving in LiBr at 60°C for 4 h).

[0124] Solution #3 has a silk concentration of 6.17 wt% (made by boiling extraction for 30 min and dissolving in LiBr at 100°C for 1 h).

[0125] Solution #4 has a silk concentration of 7.30% by weight. Starting with a 30-minute extraction batch of 100 g of silk cocoons per batch, a 7.30% silk solution was produced. The extracted silk fibers were then dissolved in a 100°C oven for 1 hour using 9.3 M LiBr at 100°C. 100 g of silk fibers per batch were dissolved to create a 20% silk solution in LiBr. The LiBr-dissolved silk 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. After removing the LiBr, the 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 to aid in the removal of the remaining solution, and 547 mL of 3.91% silk was then recovered.

[0126] Solution #5 has a silk concentration of 6.44 wt%. Starting with 60-minute extraction batches of a mixture of 25, 33, 50, 75, and 100 g of silk cocoons per batch, a 6.44 wt% silk solution was produced. The extracted silk fibers were then dissolved in a 100°C oven for 1 hour using 9.3 M LiBr at 100°C. 35, 42, 50, and 71 g of silk fibers per batch were dissolved to create a 20% silk solution in LiBr and combined. The LiBr-dissolved silk 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. After removing the LiBr, the 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 to aid in removing residual solution, and then 1454 mL of 4.88% silk was recovered.

[0127] Solution #6 has a silk concentration of 2.70% by weight. Starting with a 60-minute extraction batch of 25 g of silk cocoons per batch, a 2.70% silk solution was produced. 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 a 20% silk solution in LiBr. The LiBr-dissolved silk 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. After removing the LiBr, the solution was ultrafiltered to a volume of approximately 300 mL. 312 mL of 2.7% silk was then recovered.

[0128] The preparation of silk fibroin solutions with higher molecular weights is shown in Table O. [Table 16]

[0129] Silk aqueous coating compositions for application to fabric are shown in Tables P and Q below. [Table 17] [Table 18]

[0130] Three silk solutions were used to prepare films with the following results:

[0131] Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and a PD of 2.2 (made by 60 min boiling extraction, 1 hr LiBr dissolution at 100° C.).

[0132] Solution #2 is a 6.4% silk concentration (made by 30 min boiling extraction, 4 h LiBr dissolution at 60°C).

[0133] Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes and dissolving in LiBr at 100°C for 1 hour).

[0134] 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% (wt / vol) aqueous silk solution was added to a 100 mm Petri dish (the volume of silk may be varied to accommodate thicker or thinner films, but this is not critical) and allowed to dry overnight, uncovered. The bottom of a vacuum desiccator was filled with water. The dried film was placed in the desiccator, vacuum applied, and the film was allowed to water-anneal for 4 hours before being removed from the dish. The film cast from Solution #1 did not form a structurally continuous film; the film broke into several pieces. These pieces dissolved in water despite the water-annealing treatment.

[0135] Silk solutions of various molecular weights and / or molecular weight combinations can be optimized for gel applications. The following provides an example of this process, but is not intended to limit the application or formulation. Three silk solutions were utilized in gel creation, with the following results:

[0136] Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and a PD of 2.2 (made by 60 min boiling extraction, 1 hr LiBr dissolution at 100° C.).

[0137] Solution #2 is a 6.4% silk concentration (made by 30 min boiling extraction, 4 h LiBr dissolution at 60°C).

[0138] Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes and dissolving in LiBr at 100°C for 1 hour).

[0139] "Egel" is an electrogelation process described by Rockwood et al. [1]. 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, the power was turned off, and the gel was collected. Solution #1 did not form an edge over the 5-minute period.

[0140] Solutions #2 and #3 were gelled according to published horseradish peroxidase (HRP) protocols, and behavior appeared typical of published solutions.

[0141] Materials and Methods: The following equipment and materials were used in determining 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; and a Phenomenex PolySep GFC P-4000 column (size: 7.8 mm x 300 mm).

[0142] Procedure steps: A) Preparation of 1 L of 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 the balance, and tare the weight. Add approximately 3.3509 g of sodium phosphate dibasic heptahydrate to the beaker. Note the exact weight of the sodium phosphate dibasic weighed out. Add 100 mL of HPLC water to the beaker to dissolve the weighed out sodium phosphate. Be careful not to spill the contents of the beaker. Carefully transfer the solution to a clean, dry 1000 mL volumetric flask. Rinse the beaker and transfer the rinse to the volumetric flask. Repeat the rinsing process 4 to 5 times. Accurately weigh out approximately 5.8440 g of sodium chloride into another clean, dry 250 mL beaker. Dissolve the weighed out 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 to the volumetric flask. Adjust the pH of the solution to 7.0 ± 0.2 with phosphoric acid. Bring the volume in the volumetric flask to 1000 mL with HPLC water and shake 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 the solution can be varied as needed by changing the amounts of sodium phosphate dibasic heptahydrate and sodium chloride accordingly.

[0143] B) Preparation of dextran molecular weight standard solutions. Use at least five different molecular weight standards 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. Label six 20 mL scintillation glass vials each for a molecular weight standard. Accurately weigh approximately 5 mg of each dextran molecular weight standard and record the weight. Dissolve the dextran molecular weight standards in 5 mL of mobile phase to make a 1 mg / mL standard solution.

[0144] C) Preparation of sample solution When preparing the sample solution, if the amount of sample available is limited, the preparation volume can be scaled 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. Tightly cap the centrifuge tube and mix the sample (in solution). Allow the sample solution to stand at room temperature for 30 minutes. Gently mix the sample solution again for 1 minute and centrifuge at 4000 RPM for 10 minutes.

[0145] D) HPLC analysis of the samples Transfer 1.0 mL of all standard and sample solutions to individual HPLC vials. Inject molecular weight standards (one each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions: [Table 19]

[0146] E) Data Analysis and Calculations—Calculation of Average Molecular Weight Using Cirrus Software Chromatographic data files of standards and analytical samples are uploaded to 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.

[0147] spider silk fragments Spider silk is a natural polymer composed of three domains: a repetitive central core domain that dominates the protein chain, a nonrepetitive N-terminal domain, and a nonrepetitive C-terminal domain. The large core domain is organized in a block copolymer-like arrangement, with two basic sequences, crystalline [poly(A) or poly(GA)] and poorly crystalline (GGX or GPGXX) polypeptides, alternating. Dragline silk is a protein complex composed of major ampullate dragline silk protein 1 (MaSp1) and major ampullate dragline silk protein 2 (MaSp2). Both silks are approximately 3500 amino acids long. MaSp1 is 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, with two basic sequences, crystalline [poly(A) or poly(GA)] and poorly crystalline (GGX or GPGXX) polypeptides, alternating in the core domain. The poly(A) / (GA), GGX, and GPGXX motifs have been assigned specific secondary structures, including β-sheet, α-helix, and β-spiral, 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 liquid silk dope in the lumen and fiber formation in the spinning duct.

[0148] The main difference between MaSp1 and MaSp2 is that MaSp1 is proline-free, whereas MaSp2 contains proline (P) residues, accounting for 15% of the total amino acid content. By calculating the number of proline residues in the dragline silk of the American orb weaver (N. clavipes), it is possible to estimate the presence of two proteins in the fiber: 81% MaSp1 and 19% MaSp2. The ratio of MaSp1 to MaSp2 varies among spiders. For example, dragline silk fibers from the orb weaver (Argiope aurantia) contain 41% MaSp1 and 59% MaSp2. Such variations in the ratio of ampullate silk affect the performance of the silk fiber.

[0149] At least seven different types of silk proteins are known for one orb-weaver spider species. Silks differ in primary sequence, physical properties, and function. For example, dragline silk, used to construct frames, radii, and lifelines, is known for its exceptional mechanical properties, including strength, toughness, and elasticity. Weight for weight, spider silk has higher toughness than steel and Kevlar®. Flagellate silk, found in capture spirals, has an extensibility of up to 500%. Minor ampullate silk, found in the minor spirals and prey wrappings of orb-webs, has high toughness and strength similar to major ampullate silk, but does not hypercontract in water.

[0150] Spider silk is known for its high tensile strength and toughness. Recombinant silk proteins can also impart advantageous properties to cosmetic or dermatological compositions, particularly improved hydration or softening, good film-forming properties, and low surface density. Spider silk's diverse and unique biomechanical properties, along with its biocompatibility and slow degradation rate, make it an excellent candidate for use as a biomaterial for tissue engineering, guided tissue repair, and drug delivery, in cosmetics (e.g., nail and hair strengtheners, skin care products), and industrial materials (e.g., nanowires, nanofibers, surface coatings).

[0151] In embodiments, the silk protein may include 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, for example, natural spider silk proteins and recombinant spider silk proteins, such as mutants, analogs, and derivatives of natural spider silk proteins. From the viewpoint of superior tenacity, the polypeptide may be derived from a major dragline silk protein produced in the major ampullate gland of spiders. Examples of major dragline silk proteins include the major ampullate spidroins MaSp1 and MaSp2 from Nephila clavipes and ADF3 and ADF4 from Araneus diadematus. Polypeptides derived from major dragline silk proteins include mutants, analogs, and derivatives of the major dragline silk protein. Furthermore, the polypeptide may be derived from a flagellate silk protein produced in the flagellate gland of spiders. Examples of flagellate silk proteins include the flagellate silk protein from Nephila clavipes.

[0152] 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 five or more units, and more preferably ten or more units. Alternatively, polypeptides derived from major dragline silk proteins may be polypeptides containing the amino acid sequence represented by Formula 1:REP1-REP2(1) and, at the C-terminus, a unit of an amino acid sequence represented by any of SEQ ID NOS: 1-3 in U.S. Pat. No. 9,051,453, or an amino acid sequence having 90% or greater identity to any of SEQ ID NOS: 1-3 in U.S. Pat. No. 9,051,453. In polypeptides derived from major dragline silk proteins, the units of the amino acid sequence represented by Formula 1:REP1-REP2(1) may be the same 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 dragline silk protein is 500 kDa or less, 300 kDa or less, or 200 kDa or less from the viewpoint of productivity.

[0153] 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.

[0154] 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 that, for the most part, lacks regular organization and is more flexible. 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.

[0155] 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 recombinant Araneidae or Araneoids spider silk polypeptides, or recombinant Bombyx mori insect silk polypeptides. In some embodiments, the recombinant silk protein fragments disclosed herein comprise recombinant Araneidae or Araneoids spider silk polypeptides. In some embodiments, the recombinant silk protein fragments disclosed herein comprise block copolymers having repeating units derived from natural Araneidae or Araneoids spider silk polypeptides. In some embodiments, the recombinant silk protein fragments disclosed herein comprise block copolymers having synthetic repeating units derived from Araneidae or Araneoids spider silk polypeptides and non-repeating units derived from natural repeating units of Araneidae or Araneoids spider silk polypeptides.

[0156] Recent advances in genetic engineering have provided a route to produce 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 the synthetic protein that is heterologously produced in prokaryotic or eukaryotic expression systems using genetic engineering methods.

[0157] 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. The gram-negative bacillus Escherichia coli (E. coli) is an established host for industrial-scale production of proteins. Therefore, the majority of recombinant silk is 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.

[0158] Recombinant silk proteins can be produced by transformed prokaryotic or eukaryotic systems containing cDNA encoding silk proteins, fragments of these proteins, or analogs of such proteins. The recombinant DNA approach allows for the production of recombinant silk with programmed sequence, secondary structure, architecture, and precise molecular weight. The process involves four major steps: (i) designing a synthetic silk-like gene and constructing a gene "cassette," (ii) inserting this segment into a DNA recombinant vector, (iii) transforming this recombinant DNA molecule into a host cell, and (iv) expressing and purifying selected clones.

[0159] As used herein, the term "recombinant vector" 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, including plasmid vectors and viral vectors, generally contain a desired coding sequence and appropriate DNA sequences required for expression of the 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 specific, desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0160] Prokaryotic systems include gram-negative or gram-positive bacteria. Prokaryotic expression vectors can include a replication origin that can be recognized 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 include 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 cells.

[0161] Eukaryotic systems include yeast and insect, mammalian, or plant cells, in which case the expression vector can include a yeast plasmid replication origin or autonomously replicating sequence, a promoter, a DNA sequence encoding the spider silk protein, fragment, or similar protein, a polyadenylation sequence, a transcription termination site, and finally a selection gene. Non-limiting examples of eukaryotic expression organisms include yeast (e.g., Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous, ascosporogenous), filamentous fungi (e.g., 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, etc.), mammalian cells (e.g., HeLa cells, COS cells, CHO cells, etc.), insect cells (e.g., Sf9 cells, MEL cells, etc.), "insect host cells" (e.g., Spodoptera frugiperda or Trichoplusia ni cells). SF9 cells, SF-21 cells or High-Five cells (wherein SF-9 and SF-21 are ovary cells of Spodoptera frugiperda and High-Five cells are egg cells of Trichoplusia ni) are "plant host cells", such as, for example, tobacco, potato or pea cells.

[0162] Various heterologous host systems have been explored to produce different types of recombinant silk. Recombinant partial spidroins and 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). Most silk proteins are produced with N- or C-terminal His tags to simplify purification and produce sufficient protein quantities.

[0163] In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems 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 Escherichia coli (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).

[0164] Recombinant silk proteins in the present disclosure include synthetic proteins based on repeating units of natural silk proteins. In addition to the synthetic repeating silk protein sequences, they can further include one or more natural non-repeating silk protein sequences.

[0165] 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 E. coli, Sacchromyces cerevisiae, Pseudomonas sp., Rhodopseudomonas sp., Bacillus sp., and Strepomyces. See EP 0230702, incorporated herein by reference in its entirety.

[0166] Also provided herein is the design and biological synthesis of silk fibroin protein-like multiblock polymers containing 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).

[0167] In some embodiments, the present disclosure provides silk protein-like multiblock polymers derived from the repeat domain of B. mori silk heavy chains (H chains) containing GAGAGS (SEQ ID NO: 2) hexapeptide repeat units. GAGAGS (SEQ ID NO: 2) hexapeptide repeat units are the core units of the H chains 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 beta-sheet structure similar to that of natural silk fibroin protein, where the silk protein-like multiblock polymers have any weight-average molecular weight described herein.

[0168] In some embodiments, the present disclosure provides a silk-peptide-like multiblock copolymer composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat fragment from the heavy chain of B. mori silk 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 from the heavy chain of B. mori silk 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.

[0169] In some embodiments, the present disclosure provides (GAGAGS) 16 In some embodiments, the present disclosure provides a B. mori silkworm recombinant protein composed of a repeat fragment (GAGAGS). 16 Repetitive and non-repetitive (GAGAGS) fragments produced by E. coli 16 -F-COOH, (GAGAGS) 16 -FF-COOH, (GAGAGS) 16 -FFF-COOH, (GAGAGS) 16 -FFFF-COOH, (GAGAGS) 16 -FFFFFFFF-COOH, (GAGAGS) 16 -FFFFFFFFFFFF-COOH, 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.

[0170] In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or a fragment thereof. The production of recombinant spider silk proteins based on partial cDNA clones has been reported. The recombinant spider silk protein thus produced contains a portion of the repeat sequence from the dragline spider silk protein spidroin 1 of 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 the second fibroin protein spidroin 2 of the dragline silk of Nephila clavipes and its recombinant synthesis are described in J. Biol. Chem., 1992, vol. 267, pp. 19320-19324. The production of recombinant spider silk proteins from transformed Escherichia coli (E. coli) containing the spider silk protein spidroin 1 has been reported. Recombinant synthesis of spider silk proteins, including Nephila clavipes protein fragments and variants, is described in U.S. Patent Nos. 5,728,810 and 5,989,894. cDNA clones encoding ampullate spider silk proteins and their expression are described in U.S. Patent Nos. 5,733,771 and 5,756,677. cDNA clones encoding flagellate silk proteins from orb-web spinning spiders are described in U.S. Patent No. 5,994,099. U.S. Patent No. 6,268,169 describes the recombinant synthesis of spider silk-like proteins derived from repetitive peptide sequences found in the native spider dragline of Nephila clavipes using E. coli, Bacillus subtilis, and Pichia pastoris recombinant expression systems.WO 03 / 020916 describes cDNA clones and recombinant production encoding spider silk proteins with repetitive sequences from the large 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. Each of the above references is incorporated herein by reference in its entirety.

[0171] 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 repeat unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that is repeatedly present in naturally occurring major ampullate gland polypeptides, such as Dragulain spider silk polypeptides, minor ampullate gland polypeptides, flagellate polypeptides, aggregated spider silk polypeptides, acinar spider silk polypeptides, or pear-shaped spider silk polypeptides.

[0172] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a synthetic spider silk protein derived from a repeating unit of a natural spider silk protein, a consensus sequence, and optionally one or more natural non-repetitive spider silk protein sequences. The repeating unit of a natural spider silk polypeptide can comprise a dragline spider silk polypeptide or a flagellate spider silk polypeptide of the Araneidae or Araneoids.

[0173] As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that is repeatedly present in a naturally occurring major ampullate gland polypeptide, such as a Dragulina spider silk polypeptide, a minor ampullate gland polypeptide, a flagellate polypeptide, an aggregated spider silk polypeptide, an acinar spider silk polypeptide, or a pear-shaped spider silk polypeptide. A "repeat unit" refers to a region in amino acid sequence that comprises or consists of at least one peptide motif (e.g., AAAAAA or GPGQQ) that is repeatedly present in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., an identical amino acid sequence), or a region that corresponds to a substantially similar amino acid sequence (i.e., a variant amino acid sequence). A "repeat unit" having an amino acid sequence that is "substantially similar" to the 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" is still insoluble and retains its insolubility. A "repeating unit" having an amino acid sequence that is "identical" to the amino acid sequence of a naturally occurring silk polypeptide can be, for example, a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3, and / or ADF-4. A "repeating unit" having an amino acid sequence that is "substantially similar" to the amino acid sequence of a naturally occurring silk polypeptide can be, for example, a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3, and / or ADF-4, but with one or more amino acid substitutions at specific amino acid positions.

[0174] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence that includes a frequently occurring amino acid (e.g., "G") at a particular position, where other amino acids not yet determined are substituted with a placeholder "X." In some embodiments, the consensus sequence is: (i) GPGXX (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 (where x is an integer from 5 to 10).

[0175] The consensus peptide sequences GPGXX and GGX, i.e., glycine-rich motifs, confer flexibility to silk polypeptides and, therefore, to threads formed from silk proteins containing these motifs. Specifically, the repeated GPGXX motif forms a turn-helix structure that confers elasticity to silk polypeptides. Both ampullate and flagellate silks contain the GPGXX 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 can confer additional elastic properties to silk. The repeated polyalanine Ax (peptide) motif forms a crystalline β-sheet structure that confers strength to silk polypeptides, as described, for example, in WO 03 / 057727.

[0176] 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) from resilin, an elastic protein found in most arthropods that provides low stiffness and high strength.

[0177] 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 in a naturally occurring dragline polypeptide (i.e., a wild-type non-repetitive (carboxy-terminal) unit), preferably ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2), NR3 (SEQ ID NO: 41), NR4 (SEQ ID NO: 42), ADF-4 from the spider Araneus diadematus, described in U.S. Patent No. 8,367,803, and a C16 peptide (spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk) consisting of 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), an amino acid sequence adapted from the naturally occurring sequence of ADF4 from A. diadematus. Non-repetitive ADF-4 and its variants exhibit efficient assembly behavior.

[0178] Among the synthetic spider silk proteins, the recombinant silk proteins of the present disclosure, in some embodiments, include the C16 protein having the polypeptide sequence (SEQ ID NO: 1) set forth in U.S. Patent No. 8,288,512. In addition to the polypeptide sequence set forth in SEQ ID NO: 1, functional equivalents, functional derivatives, and salts of this sequence, among others, are also included.

[0179] 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.

[0180] In some embodiments, the recombinant spider silk protein of the present disclosure is Spidroin major 1 as described in Xu et al., PNAS, USA, 87, 7120, (1990), Spidroin major 2 as described in Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), 2. The composition contains an effective amount of at least one natural or recombinant silk protein, including the recombinant spider silk proteins 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, 8,642,734, 8,367,803, 8,097,583, 8,030,024, 7,754,851, 7,148,039, and 7,060,260, or the spider silk proteins corresponding to the small spidroins described in Patent Application No. WO95 / 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.

[0181] Recombinant silk is also described in other patents and patent applications which are incorporated herein by reference: US2004590196, US7,754,851, US2007654470, US7,951,908, US2010785960, US8,034,897, US20090263430, US2008226854, US20090123967, US2005712095, US2007991037, US20090162896, US200885266, US8,372,436, US200798 9907, US2009267596, US2010319542, US2009265344, US2012684607, US2004583227, US8,030,024, US2006643569, US7,868,146, US20079919 16, US8,097,583, US2006643200, US8,729,238, US8,877,903, US20190062557, US20160280960, US20110201783, US2008991916, US20119866 62, US2012697729, US20150328363, US9,034,816, US20130172478, US9,217,017, US20170202995, US8,721,991, US2008227498, US9,233,06 7, US8,288,512, US2008161364, US7,148,039, US1999247806, US2001861597, US2004887100, US9,481,719, US8,765,688, US200880705, US2 010809102, US8,367,803, US2010664902, US7,569,660, US1999138833, US2000591632, US20120065126, US20100278882, US2008161352, US20100015070, US2009513709, US20090194317, US2004559286, US200589551, US2008187824, US20050266242, US20050227322, and US20044418.

[0182] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US20190062557, US20150284565, US20130225476, US20130172478, US20130136779, US20130109762, US20120252294, US20110230911, US20110201783, US20100298877 , 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.

[0183] In some embodiments, the recombinant spider silk proteins of the present disclosure are GPGXX, GGX, and A as defined herein. x The repeating unit may comprise or consist of 2 to 80 repeating units each independently selected from:

[0184] In some embodiments, a recombinant spider silk protein of the disclosure comprises or consists of repeat units, each independently 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), AAAAA A (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, and GGRPSSSYG, (i) GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 25), (ii) GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), (iii) GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 26): (iv) GPGGAGGPYGPGGA GGPYGPGGAGGPY (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) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 36), or U.S. Patent No. 8,877,903, for example, 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.

[0185] In some embodiments, the present disclosure provides silk protein-like multi-block peptides that mimic the amino acid repeat units and inter-repeat unit variation profiles from natural spider silk proteins, such as spidroin major 1 domain, spidroin major 2 domain, or spidroin minor 1 domain, without altering their three-dimensional conformation, and 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):

[0186] [(XGG) w (XGA)(GXG) x (AGA) y (G) z A.G.] p 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

[0187] [(GPG2YGPGQ2) a (X')2S(A) b ] p 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

[0188] [(GR)(GA) l (A) m (GGX) n (GA) l (A) m ] p Formula (III) and / or [(GGX) n (GA) m (A) l ] p Formula (IV): In the formula, X'' corresponds to tyrosine, glutamine or alanine, l is an integer of 1 to 6, m is an integer of 0 to 4, n is an integer of 1 to 4, and p is an integer.

[0189] In some embodiments, the recombinant spider silk protein or analog of the spider silk protein comprises an amino acid repeat unit of the following sequence (V):

[0190] [(Xaa Gly Gly) w (Xaa Gly Ala)(Gly Xaa Gly) x (Ala Gly Ala) y (Gly) z Ala Gly] p 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.

[0191] In some embodiments, the recombinant spider silk protein of the present disclosure is selected from the group consisting of ADF-3 or a variant thereof, ADF-4 or a variant thereof, MaSpI (SEQ ID NO: 43) or a variant thereof, MaSpII (SEQ ID NO: 44) or a variant thereof, as described in U.S. Patent No. 8,367,803.

[0192] In some embodiments, the present disclosure provides water-soluble recombinant spider silk protein produced in mammalian cells.The solubility of spider silk protein produced in mammalian cells is due to the presence of COOH-terminal amino acids in these proteins, which make these proteins more hydrophilic.These COOH-terminal amino acids are not present in spider silk protein expressed in microbial hosts.

[0193] In some embodiments, a 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: 37), GKGGGGGG (SEQ ID NO: 38), GCGGSGGGGSGGGG (SEQ ID NO: 39), GKGGGGGGSGGGG (SEQ ID NO: 40), and GCGGGGGSGGGG (SEQ ID NO: 41). In some embodiments, the recombinant spider silk protein of the present disclosure is C16 modified at the amino or carboxyl terminus such that the molecular weight of the protein is in the range described herein. 16 NR4, C 32 NR4, C16, C32, NR4C 16 NR4, NR4C 32 NR4, NR3C 16 NR3 or NR3C 32 Includes NR3.

[0194] 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 an 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 the spidroin major 1 domain, the spidroin major 2 domain, or the spidroin minor 1 domain, wherein the repeat peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 42) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), as described in U.S. Patent No. 8,367,803.

[0195] 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.

[0196] As used herein, the term "recombinant silk" refers to recombinant spider and / or silkworm silk proteins or fragments thereof. In embodiments, the spider silk protein is selected from the group consisting of entrapment silk (acniform gland silk), egg capsule silk (cylindrical gland silk), egg sheath silk (tubular silk), non-adhesive dragline silk (ampullate gland silk), attachment silk (pear-shaped gland silk), adhesive silk core fiber (flagellate gland silk), and adhesive silk outer fiber (aggregate gland silk). For example, recombinant spider silk proteins described herein 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.

[0197] 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 environmental or life cycle niche. Fibers are named after the glands from which they originate, and polypeptides are labeled with an abbreviation for the gland (e.g., "Ma") and "Sp" for spidroin (an abbreviation for spider fibroin). In orb-weaving spiders, these types include large ampullate (MaSp, also known as dragline), small ampullate (MiSp), flagellate (Flag), acinar (AcSp), tubular (TuSp), and pear-shaped (PySp). This combination of polypeptide sequences across fiber types, domains, and differences between different genera and species of organisms results in a vast number of potential properties that can be exploited for commercial production of recombinant fibers. To date, the majority of research with recombinant silk has focused on major ampullate spidroins (MaSps).

[0198] Acinar (AcSp) silks tend to have high toughness, resulting from a combination of moderately high strength and moderately high extensibility. AcSp silks are characterized by large block ("ensemble repeat") sizes that often incorporate polyserine and GPX motifs. Tubular (TuSp or cylindrical) silks tend to have large diameters, along with moderate strength and high extensibility. TuSp silks are characterized by their polyserine and polythreonine content and short tracts of polyalanine. Major ampullate (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 extensibility. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high extensibility and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0199] Silk polypeptides are characteristically composed of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In embodiments, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repeat domains exhibit a hierarchical structure. They contain a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (forming quasi-repeat domains), throughout the silk repeat domain. The length and composition of the blocks vary between different silk types and across different species. Table 1 of U.S. Published Application No. 2016 / 0222174 (incorporated herein by reference in its entirety) lists examples of block sequences from selected species and silk types, and 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 can be arranged in regular patterns to form larger macrorepeats that occur multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Repeated blocks within a repeat domain or macrorepeat, and repeated macrorepeats within a repeat domain, can be separated by spacing elements.

[0200] The construction of certain spider silk block copolymer polypeptides from block and / or macrorepeat domains according to certain embodiments of the present disclosure is shown in U.S. Published Patent Application No. 2016 / 0222174.

[0201] 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, a commercially available expression / secretion system can be used, whereby the recombinant polypeptide is expressed and then secreted from the host cell, allowing it to be 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 (the remains of cells after the integrity of the cells has been destroyed) 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.

[0202] Recombinant block copolymer polypeptides can be purified by affinity separation, for example, by immunological interaction with an antibody that specifically binds the recombinant polypeptide, or by nickel columns to isolate recombinant polypeptides tagged with 6-8 histidine residues at the N- or C-terminus. Alternative tags may include the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by those skilled in the art.

[0203] Solutions of such polypeptides (i.e., recombinant silk proteins) can then be prepared and used as described herein.

[0204] In another embodiment, recombinant silk proteins can be prepared according to the methods described in U.S. Pat. No. 8,642,734, which is incorporated herein by reference in its entirety, and used as described herein.

[0205] In one embodiment, a recombinant spider silk protein is provided. Spider silk proteins typically consist of 170 to 760 amino acid residues, e.g., 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, e.g., 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 that require harsh solvents for solubilization and polymerization. The recombinant spider silk protein can contain more than 760 residues, particularly when the spider silk protein contains more than two fragments derived from the N-terminal portion of the spider silk protein. The spider silk protein contains an N-terminal fragment (NT) consisting of at least one fragment derived from a corresponding portion of the spider silk protein and a repeat fragment (REP) derived from a corresponding internal fragment of the spider silk protein. Optionally, the spider silk protein contains a C-terminal fragment (CT) derived from a 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, for example two, fragments (NT) derived from the N-terminal portion of the spider silk protein. Thus, spidroins have the formula NT m -REP, or NT m -REP-CT, where m is an integer of 1 or more, e.g., 2 or more, and preferably in the range of 1 to 2, 1 to 4, 1 to 6, 2 to 4, or 2 to 6. Preferred spidroins can be generally represented by the formula NT2-REP or NT-REP, or 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 NT fragment(s) linked to a REP fragment, which is optionally linked to a CT fragment.

[0206] In one embodiment, the first step of the method for producing isolated spider silk protein polymers involves expressing a polynucleic acid molecule encoding the spider silk protein in a suitable host, such as E. coli. The protein thus obtained is isolated using standard procedures. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage.

[0207] The second step in the method for producing isolated spider silk protein polymers involves providing a solution of spider silk protein in a liquid medium. 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 can 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 higher and / or an ionic composition that prevents polymerization of spider silk protein. That is, the liquid medium has either a pH of 6.4 or higher, an ionic composition that prevents polymerization of spider silk protein, or both.

[0208] The ionic composition that prevents the polymerization of spider silk protein can be easily prepared by those skilled in the art using the method disclosed herein.The preferred ionic composition that prevents the polymerization of spider silk protein has an ionic strength of more than 300mM.Specific examples of the ionic composition that prevents the polymerization of spider silk protein include NaCl of more than 300mM, phosphate of 100mM, and the combination of these ions that have the desired inhibitory effect on the polymerization of spider silk protein, such as the combination of 10mM phosphate and 300mM NaCl.

[0209] The presence of NT fragments improves solution stability and prevents polymer formation under these conditions. This can be advantageous when immediate polymerization is undesirable, such as during protein purification, when preparing large batches, or when other conditions need to be optimized. To achieve high solubility of spider silk proteins, it is preferable to adjust the pH of the liquid medium to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as 10.5. 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 spider silk proteins but makes it easier to subsequently adjust the pH to 6.3 or lower.

[0210] In the third step, the liquid medium is adjusted to a pH of 6.3 or less and an ionic composition that allows 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 familiar with 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 allow polymerization. Those skilled in the art are familiar with 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 allow polymerization. It is preferable to adjust the pH of the liquid medium to 6.2 or less, e.g., 6.0 or less. In particular, it may be advantageous from a practical standpoint to limit the pH reduction from 6.4 or 6.4-6.8 in the preceding 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, for example greater than or equal to 4.2. The resulting pH range, for example 4.2 to 6.3, promotes rapid polymerization.

[0211] In the fourth step, spider silk proteins are polymerized in a liquid medium with a pH of 6.3 or less and an ionic composition that allows for the polymerization of spider silk proteins. The presence of NT fragments improves the solubility of spider silk proteins at pHs above 6.4 and / or ionic compositions that prevent the polymerization of spider silk proteins, but accelerates polymer formation at pHs below 6.3 when the ionic composition allows the polymerization of spider silk proteins. The resulting polymers are preferably solid and macroscopic and are formed in a liquid medium with a pH of 6.3 or less and an ionic composition that allows the polymerization of spider silk proteins. In a preferred embodiment, the pH of the liquid medium in this step is 3 or greater, e.g., 4.2 or greater. The resulting pH range, e.g., 4.2 to 6.3, promotes rapid polymerization. The resulting polymers may be provided at the molecular weights described herein or prepared in solution form for use in article coatings as needed.

[0212] Ion compositions that allow spider silk protein polymerization can be easily prepared by those skilled in the art using the methods disclosed herein. Preferred ion compositions that allow spider silk protein polymerization have an ionic strength of less than 300 mM. Specific examples of ion compositions that allow spider silk protein polymerization include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack an inhibitory effect on spider silk protein polymerization, such as a combination of 10 mM phosphate or 20 mM phosphate with 150 mM NaCl. The ionic strength of this liquid medium is preferably adjusted to a range of 1 to 250 mM.

[0213] 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, which then allows polymerization to occur, whereas salt inhibits the same event.

[0214] At neutral pH, the energetic cost of burying the excess negative charge at the acidic pole would be expected to prevent polymerization. However, as the dimer approaches its isoelectric point at lower pH, electrostatic attraction eventually becomes dominant, explaining the observed salt- and pH-dependent polymerization behavior of NTs and NT-containing minispidroins. In some embodiments, we propose that the pH-induced NT polymerization and enhanced efficiency of NT-minispidroin fiber assembly are due to changes in the surface electrostatic potential, whereby the clustering of acidic residues at one pole of the NTs shifts their charge balance such that the polymerization transition occurs at pH values ​​below 6.3.

[0215] In a fifth step, the resulting, preferably solid, spider silk protein polymer is isolated from the liquid medium, optionally including the active removal of lipopolysaccharides and other pyrogens from the spidroin polymer.

[0216] 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 an isolated spider silk protein dimer, 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. Therefore, the present disclosure provides a novel use of spider silk proteins, preferably those disclosed herein, for producing spider silk protein dimers.

[0217] In another aspect, the present disclosure provides a polymer of the spider silk protein disclosed herein. In embodiments, the protein polymer can be obtained by any one of the methods therefor according to the present disclosure. Accordingly, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing spider silk protein polymers as recombinant silk-based coatings. In one embodiment, the present disclosure provides a novel use of spider silk protein dimers, preferably those disclosed herein, for producing isolated spider silk protein polymers as recombinant silk-based coatings. In these uses, the polymers are 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 embodiments, the liquid medium has a pH of 3 or greater, e.g., 4.2 or greater. The resulting pH range, e.g., 4.2 to 6.3, promotes rapid polymerization.

[0218] Using the method(s) of the present disclosure, it is possible to control the polymerization process, thereby optimizing the parameters to obtain silk polymers with desired properties and shapes.

[0219] In embodiments, the recombinant silk proteins described herein include those described in U.S. Patent No. 8,642,734, which is incorporated by reference in its entirety.

[0220] In another embodiment, the recombinant silk proteins described herein can be prepared according to the methods described in U.S. Pat. No. 9,051,453, which is incorporated herein by reference in its entirety.

[0221] The amino acid sequence represented by SEQ ID NO: 1 in U.S. Patent No. 9,051,453 is identical to the amino acid sequence consisting of the C-terminal 50 amino acid residues of the amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI: 1263287). The amino acid sequence represented by SEQ ID NO: 2 in U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 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: 3 in U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 with 29 residues removed from the C-terminus.

[0222] An example of a polypeptide comprising a unit of the amino acid sequence represented by Formula 1: REP1-REP2(1) and having an amino acid sequence at its C-terminus represented by any one of SEQ ID NOS: 1 to 3, or an amino acid sequence having 90% or more identity to any one of SEQ ID NOS: 1 to 3 in U.S. Pat. No. 9,051,453, is a polypeptide having the amino acid sequence represented by SEQ ID NOS: 8 in U.S. Pat. No. 9,051,453. The polypeptide having the amino acid sequence represented by SEQ ID NO:8 in U.S. Patent No. 9,051,453 is 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 to the N-terminus of the amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI:1263287) (SEQ ID NO:5 in U.S. Patent No. 9,051,453), and by modifying the sequence such that the repeat regions from positions 1 to 13 are approximately doubled and translation terminates at amino acid residue 1154. The C-terminal sequence of the polypeptide having the amino acid sequence represented by SEQ ID NO:8 in U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO:3.

[0223] 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: 1 to 3 in U.S. Pat. No. 9,051,453 or an amino acid sequence having 90% or more identity to any one of SEQ ID NOS: 1 to 3 in U.S. Pat. No. 9,051,453 may be a protein having an amino acid sequence represented by SEQ ID NOS: 8 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 consisting of a crystalline region and an amorphous region.

[0224] 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: 15 in U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 15 in U.S. Patent No. 9,051,453 is an amino acid sequence in which 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: 5 in U.S. Patent No. 9,051,453) has been added to the N-terminus of the 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 the amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has a region composed of crystalline and amorphous regions. Further, 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 the amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 17 in U.S. Patent No. 9,051,453 is an amino acid sequence in which 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: 5 in U.S. Patent No. 9,051,453) has been added 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 the amino acid sequence represented by Sequence ID No. 17 of U.S. Pat. No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has regions composed of crystalline and amorphous regions.

[0225] Examples of polypeptides derived from flagelliform 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, and more preferably polypeptides containing 30 or more units. When recombinant proteins are produced using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from flagelliform 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.

[0226] In formula (2), REP3 represents an amino acid sequence consisting of Gly-Pro-Gly-Gly-X, where X represents an amino acid selected from the group consisting of Ala, Ser, Tyr and Val.

[0227] The main characteristic of spider silk is that flagellate silk does not have crystalline regions but has repeating regions consisting of amorphous regions. Large dragline silk, for example, has repeating regions consisting of crystalline and amorphous regions, and is therefore expected to have both high stress and stretchability. On the other hand, flagellate silk has lower stress than large dragline silk, but high stretchability. This is thought to be because flagellate silk is mostly composed of amorphous regions.

[0228] An example of a polypeptide containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2) is a recombinant protein derived from a flagelliform silk protein having the amino acid sequence represented by Sequence ID No. 19 in U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 19 in U.S. Pat. No. 9,051,453 was obtained by combining a partial sequence of the flagellate 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, which corresponds to the repeat portion and motif, respectively (referred to as the PR1 sequence), with a partial sequence of the flagellate 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 to the N-terminus of the combined sequence an amino acid sequence consisting of an initiation codon, a His10 tag, and an HRV3C protease recognition site (SEQ ID NO: 5 in U.S. Pat. No. 9,051,453). Furthermore, a polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2:REP3(1) may be a polypeptide having the amino acid sequence represented by Sequence ID No. 19 of U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and in which the polypeptide has regions comprised of amorphous regions.

[0229] Polypeptides can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for gene production is not particularly limited; it can be produced by amplifying and cloning a gene encoding a natural spider silk protein from spider cells using techniques such as polymerase chain reaction (PCR), or by chemical synthesis. The method for chemically synthesizing the gene is also not particularly limited. For example, it can be synthesized by linking oligonucleotides synthesized automatically using AKTA oligopilot plus 10 / 100 (GE Healthcare Japan) based on the amino acid sequence of a natural spider silk protein obtained from the NCBI web database, using PCR or other methods. To facilitate protein purification and observation, it is also possible to synthesize a gene encoding a protein having an amino acid sequence in which an amino acid sequence consisting of an initiation codon and a His10 tag is added to the N-terminus of the amino acid sequence.

[0230] Expression vectors include plasmids, phages, viruses, and the like that can express proteins based on DNA sequences. Plasmid-type expression vectors are not particularly limited as long as they can express a target gene in host cells and amplify the target gene itself. For example, when Escherichia coli Rosetta (DE3) is used as the 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.

[0231] The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, one of the two major dragline silk proteins of Araneus diadematus, which has the advantages of essentially high strength and toughness, and being easy to synthesize.

[0232] 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 proteins described above or in U.S. Patent Nos. 8,173,772, 8,278,416, 8,618,255, 8,642,734, 8,691,581, 8,729,235, 9,115,204, 9,157,070, 9,309,299, 9,644,012, 9,708,376, 9,051,453, 9,617,311, 9,617,312, 9,617,313, 9,617,314, 9,617,315, 9,617,316, 9,617,317, 9,617,318, 9,617,319 ... Nos. 5, 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, and 2014 / 0079674. , 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 014161 No. 8, No. 2015 / 0291673, No. 2015 / 0291674, No. 2015 / 0239587, No. 2015 / 0344 No. 542, No. 2015 / 0361144, No. 2015 / 0374833, No. 2015 / 0376247, No. 2016 / 00 No. 24464, No. 2017 / 0066804, No. 2017 / 0066805, No. 2015 / 0293076, No. 2016 / No. 0222174, No. 2017 / 0283474, No. 2017 / 0088675, No. 2019 / 0135880, No. 201 5 / 0329587, 2019 / 0040109, 2019 / 0135881, 2019 / 0177363, 2 019 / 0225646, 2019 / 0233481, 2019 / 0031842, 2018 / 0355120, No. 2019 / 0186050, No. 2019 / 0002644, No. 2020 / 0031887, No. 2018 / 0273590,Same No. 20191 / 094403, No. 2019 / 0031843, No. 2018 / 0251501, No. 2017 / 0066805, No. 2018 / 0127553, No. 2019 / 032952 No. 6, No. 2020 / 0031886, No. 2018 / 0080147, No. 2019 / 0352349, No. 2020 / 0043085, No. 2019 / 0144819, No. 2019 / 022 The recombinant silk protein may include one or more of the recombinant silk proteins described in US Pat. Nos. 8449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191, all of which are incorporated by reference herein in their entireties.

[0233] Silk fibroin-like protein fragments Recombinant silk proteins in the present disclosure include synthetic proteins based on repeating units of natural silk proteins. In addition to synthetic repeating silk protein sequences, they can also contain one or more non-repeating natural 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%.

[0234] As described herein, a protein such as a natural silk protein, a fibroin heavy chain, a fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units comprises about 9% to 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, a protein such as a natural silk protein, a fibroin heavy chain, a fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units comprises about 13% to 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 9% to about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0235] 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 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%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 100%, about 101%, about 102%, about 103%, about Contains 8%, 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 comprise 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, on average, two out of six amino acids, three out of six amino acids, or four out of six amino acids in the silk fibroin-like proteins described herein are glycines. In some embodiments, on average, 1 out of 6 amino acids, 2 out of 6 amino acids, or 3 out of 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.

[0236] Sericin or sericin fragments Raw silk consists primarily of silk fibroin fibers, which are coated with the adhesive substance silk sericin. Sericin is a colloidal silk protein that covers the surface of silk threads. It is composed of bulky, chemically reactive amino acids such as serine, threonine, and aspartic acid in addition to glycine and alanine. In the various processes used to produce silk from raw silk, sericin is important for controlling the solubility of silk and producing high-quality silk. It also plays an extremely important role as an adhesive protein. When silk fibers are used in clothing, most of the silk sericin covering the silk threads is removed and discarded, making sericin a valuable, unused resource.

[0237] 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.

[0238] In some embodiments, sericin removed from raw silk cocoons, such as by a degumming process, can be recovered 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.

[0239] Other properties of SPF Compositions of the present disclosure are "biocompatible" or otherwise exhibit "biocompatibility," meaning that they are compatible with living tissues or biological systems by not being toxic, injurious, or physiologically reactive and not causing immune rejection or an inflammatory response. Such biocompatibility can be demonstrated by a participant topically applying a composition of the present disclosure to the skin for an extended period of time. In embodiments, the extended period is about 3 days. In embodiments, the extended period is about 7 days. In embodiments, the extended period is about 14 days. In embodiments, the extended period is about 21 days. In embodiments, the extended period is about 30 days. In embodiments, 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.

[0240] In some embodiments, the compositions described herein may be biocompatible compositions (e.g., biocompatible coatings including silk) and may be evaluated to comply with 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 may be biocompatible compositions and 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.

[0241] 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 having a participant topically apply a composition of the present disclosure to the skin for an extended period of time. In embodiments, the extended period is about 3 days. In embodiments, the extended period is about 7 days. In embodiments, the extended period is about 14 days. In embodiments, the extended period is about 21 days. In embodiments, the extended period is about 30 days. In embodiments, 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.

[0242] In embodiments, the compositions of the present disclosure have a stability of about 1 day. In embodiments, the compositions of the present disclosure have a stability of about 2 days. In embodiments, the compositions of the present disclosure have a stability of about 3 days. In embodiments, the compositions of the present disclosure have a stability of about 4 days. In embodiments, the compositions of the present disclosure have a stability of about 5 days. In embodiments, the compositions of the present disclosure have a stability of about 6 days. In embodiments, the compositions of the present disclosure have a stability of about 7 days. In embodiments, the compositions of the present disclosure have a stability of about 8 days. In embodiments, the compositions of the present disclosure have a stability of about 9 days. In embodiments, the compositions of the present disclosure have a stability of about 10 days.

[0243] In embodiments, 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.

[0244] In embodiments, the compositions of the present disclosure have a stability of 10 days to 6 months. In embodiments, the compositions of the present disclosure have a stability of 6 months to 12 months. In embodiments, the compositions of the present disclosure have a stability of 12 months to 18 months. In embodiments, the compositions of the present disclosure have a stability of 18 months to 24 months. In embodiments, the compositions of the present disclosure have a stability of 24 months to 30 months. In embodiments, the compositions of the present disclosure have a stability of 30 months to 36 months. In embodiments, the compositions of the present disclosure have a stability of 36 months to 48 months. In embodiments, the compositions of the present disclosure have a stability of 48 months to 60 months.

[0245] In embodiments, the SPF compositions of the present disclosure are insoluble in aqueous solutions due to the crystallinity of the protein. In embodiments, the SPF compositions of the present disclosure are soluble in aqueous solutions. In embodiments, the SPF of the compositions of the present disclosure comprises about two-thirds crystalline fraction and about one-third amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises about half crystalline fraction and about half amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises 99% crystalline fraction and 1% amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises 95% crystalline fraction and 5% amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises 90% crystalline fraction and 10% amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises 85% crystalline fraction and 15% amorphous region. In embodiments, the SPF of the compositions of the present disclosure comprises 80% crystalline fraction and 20% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 75% crystalline fraction and 25% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 70% crystalline fraction and 30% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 65% crystalline fraction and 35% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 60% crystalline fraction and 40% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 50% crystalline fraction and 50% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 40% crystalline fraction and 60% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 35% crystalline fraction and 65% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 30% crystalline fraction and 70% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 25% crystalline fraction and 75% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 20% crystalline fraction and 80% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 15% crystalline fraction and 85% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 10% crystalline fraction and 90% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 5% crystalline fraction and 90% amorphous region. In embodiments, the SPF of the disclosed compositions comprises 1% crystalline fraction and 99% amorphous region.

[0246] As used herein, the term "substantially free of inorganic residue" means that the composition exhibits 0.1% (w / w) or less of a residue. In embodiments, substantially free of inorganic residue refers to a composition exhibiting 0.05% (w / w) or less of a residue. In embodiments, substantially free of inorganic residue refers to a composition exhibiting 0.01% (w / w) or less of a residue. In embodiments, the amount of inorganic residue is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In embodiments, the amount of inorganic residue is from ND to about 500 ppm. In embodiments, the amount of inorganic residue is from ND to about 400 ppm. In embodiments, the amount of inorganic residue is from ND to about 300 ppm. In embodiments, the amount of inorganic residue is from ND to about 200 ppm. In embodiments, the amount of inorganic residue is from ND to about 100 ppm. In embodiments, the amount of inorganic residue is from 10 ppm to 1000 ppm.

[0247] As used herein, the term "substantially free of organic residue" means that a composition exhibits 0.1% (w / w) or less of residue, and in embodiments, substantially free of organic residue refers to a composition exhibiting 0.05% (w / w) or less of residue. In embodiments, substantially free of organic residue refers to a composition exhibiting 0.01% (w / w) or less of residue. In embodiments, the amount of organic residue is 0 ppm ("not detectable" or "ND") to 1000 ppm. In embodiments, the amount of organic residue is ND to about 500 ppm. In embodiments, the amount of organic residue is ND to about 400 ppm. In embodiments, the amount of organic residue is ND to about 300 ppm. In embodiments, the amount of organic residue is ND to about 200 ppm. In embodiments, the amount of organic residue is ND to about 100 ppm. In embodiments, the amount of organic residue is 10 ppm to 1000 ppm.

[0248] The compositions of the present disclosure are "biocompatible," meaning that they are compatible with living tissues or systems by not being toxic, injurious, or physiologically reactive and not causing immune rejection. Such biocompatibility can be demonstrated by a participant topically applying a composition of the present disclosure to the skin for an extended period of time. In embodiments, the extended period is about 3 days. In embodiments, the extended period is about 7 days, in embodiments, the extended period is about 14 days, in embodiments, the extended period is about 21 days. In embodiments, the extended period is about 30 days. In embodiments, 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.

[0249] 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 having a participant topically apply a composition of the present disclosure to the skin for an extended period of time. In embodiments, the extended period is about 3 days. In embodiments, the extended period is about 7 days. In embodiments, the extended period is about 14 days. In embodiments, the extended period is about 21 days. In embodiments, the extended period is about 30 days. In embodiments, 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.

[0250] In some embodiments, the terms "leather" and / or "leather substrate" as used herein refer to natural or processed leather derived from bovine skin, ovine skin, lambskin, equine skin, crocodile skin, alligator skin, avian skin, or another known animal skin recognized in the art. Raw, processed, coated, and / or restored leathers include, but are not limited to, altered leather, aniline leather, bonded leather, brushed leather, buffed leather, bycast leather, chamois leather, chrome-tanned leather, mixed-tanned leather, cordovan leather, collected grain leather, crack-proof leather, drummed leather, embossed leather, enhanced grain leather, grained leather, metallized leather, naked leather, natural grain leather, nubuck leather, patent leather, pearlized leather, plated leather, printed leather, protected leather, pure aniline leather, tanned / retanned leather, roundhand leather, saddle leather, semi-aniline leather, shrunken grain leather, side leather, split leather, suede leather, and wet blue. In some embodiments, the term "leather" may refer to synthetic or reconstituted leathers, including, but not limited to, leathers composed partially or completely of synthetic materials such as cellulose, mushroom-based materials, vinyl, polyamide, or polyester.

[0251] As used herein, the term "hand" refers to the feel of a material and can be further described as softness, crispness, dryness, silkiness, smoothness, and combinations thereof. Material hand is also referred to as "drape." A stiff-hand material is rough, abrasive, and generally uncomfortable to the wearer. A soft-hand material is fluid, smooth, and generally more comfortable to the wearer. Material hand can be determined by comparison with a collection of material samples or by using methods such as the Kawabata Evaluation System (KES) or Fabric Assurance by Simple Testing (FAST) method (Behera and Hari, Ind. J. Fibre & Textile Res., 1994, 19, 168-71). In some embodiments, as described herein, silk can alter the hand feel of leather, as can be assessed by the SynTouch Touch-Scale methodology or another methodology described herein.

[0252] As used herein, the term "coating" refers to a material or combination of materials that forms a substantially continuous layer or film on the outer surface of a substrate, such as leather or a leather article. In some embodiments, portions of the coating can at least partially penetrate the substrate. In some embodiments, the coating can at least partially penetrate crevices in the substrate. In some embodiments, the coating can be soaked into the surface of the substrate, such that application of the coating, i.e., the coating process, can include soaking at least one coating component at least partially into the surface of the substrate (at the melting point of the substrate). The coating can be applied to the substrate by one or more of the processes described herein.

[0253] In embodiments described where the coating can be soaked into the surface of a substrate, the coating can be codissolved into the surface of the substrate such that the components of the coating can be intermixed in the surface of the substrate to a depth of at least about 1 nm, or at least about 2 nm, or at least about 3 nm, or at least about 4 nm, or at least about 5 nm, or at least about 6 nm, or at least about 7 nm, or at least about 8 nm, or at least about 9 nm, or at least about 10 nm, or at least about 20 nm, or at least about 30 nm, or at least about 40 nm, or at least about 50 nm, or at least about 60 nm, or at least about 70 nm, or at least about 80 nm, or at least about 90 nm, or at least about 100 nm. In some embodiments, the coating can be soaked into the surface of a substrate, and the substrate comprises leather or a leather article.

[0254] As used herein, the term "bath coating" includes coating a material in a bath, immersing a material in a bath, and submerging a material in a bath. The concept of bath coating is described in U.S. Pat. No. 4,521,458, which is incorporated by reference in its entirety.

[0255] As used herein, the term "drying" may refer to drying the coated materials described herein at temperatures above room temperature (i.e., 20°C), unless otherwise specified.

[0256] The following are non-limiting examples of suitable ranges for various parameters in and for the preparation of silk solutions of the present disclosure: Silk solutions of the present disclosure can be prepared using various combinations of such parameter ranges, which can include one or more, but not necessarily all, of these parameters.

[0257] In embodiments, the percent SPF in the solution is less than 30.0% by weight. In embodiments, the percent SPF in the solution is less than 25.0% by weight. In embodiments, the percent SPF in the solution is less than 20.0% by weight. In embodiments, the percent SPF in the solution is less than 19.0% by weight. In embodiments, the percent SPF in the solution is less than 18.0% by weight. In embodiments, the percent SPF in the solution is less than 17.0% by weight. In embodiments, the percent SPF in the solution is less than 16.0% by weight. In embodiments, the percent SPF in the solution is less than 15.0% by weight. In embodiments, the percent SPF in the solution is less than 14.0% by weight. In embodiments, the percent SPF in the solution is less than 13.0% by weight. In embodiments, the percent SPF in the solution is less than 12.0% by weight. In embodiments, the percent SPF in the solution is less than 11.0% by weight. In embodiments, the percent SPF in the solution is less than 10.0% by weight. In embodiments, the percent SPF in the solution is less than 9.0% by weight. In embodiments, the percent SPF in the solution is less than 8.0% by weight. In embodiments, the percent SPF in the solution is less than 7.0% by weight. In embodiments, the percent SPF in the solution is less than 6.0% by weight. In embodiments, the percent SPF in the solution is less than 5.0% by weight. In embodiments, the percent SPF in the solution is less than 4.0% by weight. In embodiments, the percent SPF in the solution is less than 3.0% by weight. In embodiments, the percent SPF in the solution is less than 2.0% by weight. In embodiments, the percent SPF in the solution is less than 1.0% by weight. In embodiments, the percent SPF in the solution is less than 0.9% by weight. In embodiments, the percent SPF in the solution is less than 0.8% by weight. In embodiments, the percent SPF in the solution is less than 0.7% by weight. In embodiments, the percent of SPF in the solution is less than 0.6% by weight. In embodiments, the percent of SPF in the solution is less than 0.5% by weight. In embodiments, the percent of SPF in the solution is less than 0.4% by weight. In embodiments, the percent of SPF in the solution is less than 0.3% by weight.In an embodiment, the percent of SPF in the solution is less than 0.2% by weight. In an embodiment, the percent of SPF in the solution is less than 0.1% by weight.

[0258] In embodiments, the percent of SPF in the solution is greater than 0.1% by weight. In embodiments, the percent of SPF in the solution is greater than 0.2% by weight. In embodiments, the percent of SPF in the solution is greater than 0.3% by weight. In embodiments, the percent of SPF in the solution is greater than 0.4% by weight. In embodiments, the percent of SPF in the solution is greater than 0.5% by weight. In embodiments, the percent of SPF in the solution is greater than 0.6% by weight. In embodiments, the percent of SPF in the solution is greater than 0.7% by weight. In embodiments, the percent of SPF in the solution is greater than 0.8% by weight. In embodiments, the percent of SPF in the solution is greater than 0.9% by weight. In embodiments, the percent of SPF in the solution is greater than 1.0% by weight. In embodiments, the percent of SPF in the solution is greater than 2.0% by weight. In embodiments, the percent of SPF in the solution is greater than 3.0% by weight. In embodiments, the percent of SPF in the solution is greater than 4.0% by weight. In embodiments, the percent SPF in the solution is greater than 5.0% by weight. In embodiments, the percent SPF in the solution is greater than 6.0% by weight. In embodiments, the percent SPF in the solution is greater than 7.0% by weight. In embodiments, the percent SPF in the solution is greater than 8.0% by weight. In embodiments, the percent SPF in the solution is greater than 9.0% by weight. In embodiments, the percent SPF in the solution is greater than 10.0% by weight. In embodiments, the percent SPF in the solution is greater than 11.0% by weight. In embodiments, the percent SPF in the solution is greater than 12.0% by weight. In embodiments, the percent SPF in the solution is greater than 13.0% by weight. In embodiments, the percent SPF in the solution is greater than 14.0% by weight. In embodiments, the percent SPF in the solution is greater than 15.0% by weigh...

Claims

1. 1. A conjugate comprising a first polymeric macromolecular species or polymer and a second polymeric macromolecular species or polymer, the first polymeric macromolecular species or polymer portion and the second polymeric macromolecular species or polymer portion are physically and / or chemically entangled; the first polymeric macromolecular species or polymer portion is physically and / or chemically crosslinked; the second polymeric macromolecular species or polymer portion is physically and / or chemically crosslinked; the first polymeric macromolecular species or polymer portion is chemically and / or physically incorporated into the second polymeric macromolecular species or polymer portion; the first polymeric macromolecular species or polymer portion and the second polymeric macromolecular species or polymer portion are not separable; the first polymeric macromolecular species or polymer portion and / or the second polymeric macromolecular species or polymer portion are crosslinked; the first polymeric macromolecular species or polymer portion and / or the second polymeric macromolecular species or polymer portion are partially organized and / or crystallized; the first polymeric macromolecular species or polymer portion and the second polymeric macromolecular species or polymer portion are incapable of exfoliation; and the first polymeric macromolecular species or polymer portion and the second polymeric macromolecular species or polymer portion self-assemble; A complex having at least one characteristic selected from the following: a portion of the first polymeric macromolecular species or polymer in the complex having a second structure that is different from the first structure of the first polymeric macromolecular species or polymer; and / or 10. The conjugate of claim 1, wherein a portion of the second polymeric macromolecular species or polymer in the conjugate has a second structure that is different from the first structure of the second polymeric macromolecular species or polymer.

3. The complex described in claim 1, wherein the first polymeric macromolecular species or polymer comprises a protein component.

4. The complex of claim 3, wherein the protein component comprises one or more of silk fibroin protein or fragment, collagen, elastin, gelatin, corn zein, wheat gluten, pectin, chitin, casein, and / or whey.

5. The complex described in claim 1, wherein the first polymeric macromolecular species or polymer comprises a biodegradable polymer.

6. The composite of claim 1, wherein the first polymeric macromolecular species or polymer comprises one or more polyurethane components.

7. The complex of claim 1, wherein the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component, a poly(lactic-co-glycolic acid) (PLGA) component, or both.

8. The complex of claim 1, wherein the second polymeric macromolecular species or polymer comprises cellulose and / or a cellulose derivative as a component.

9. The complex described in claim 8, wherein the cellulose derivative is selected from methyl cellulose, ethyl cellulose, ethyl methyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, ethyl hydroxyethyl cellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose.

10. The composite of claim 8, wherein the cellulose derivative is ethyl cellulose.

11. The complex described in claim 10, wherein the ethoxyl content in the ethyl cellulose is 45.0% to 49.5%, 45.0% to 46.0%, 45.0% to 47.0%, 47.0% to 48.0%, or 48.0% to 49.5%.

12. The composite of claim 10, wherein the degree of substitution of the ethyl cellulose is 0.5 to 1, 1 to 1.5, 1.5 to 2, 2 to 2.5, or 2.5 to 3.

13. The complex described in claim 8, wherein the second structure of the cellulose derivative comprises a crystallinity of less than 100%.

14. The complex described in claim 8, wherein the second structure of the cellulose derivative comprises a crystallinity of from about 5% to less than about 100%.

15. The complex of claim 1, wherein the concentration of the first polymeric macromolecular species or polymer near a first surface of the complex is higher than the concentration of the first polymeric macromolecular species or polymer near a second surface of the complex.

16. The complex of claim 1, wherein the concentration of the second polymeric macromolecular species or polymer near the second surface of the complex is higher than the concentration of the second polymeric macromolecular species or polymer near the first surface of the complex.

17. The composite of claim 1, wherein the composite has increased water resistance and / or increased moisture permeability compared to one of: i) a non-composite material comprising the first polymeric macromolecular species or polymer but excluding the second polymeric macromolecular species or polymer; ii) a non-composite material comprising the second polymeric macromolecular species or polymer but excluding the first polymeric macromolecular species or polymer; or iii) a non-composite material comprising the first polymeric macromolecular species or polymer and the second polymeric macromolecular species or polymer, wherein the polymeric macromolecular species or polymers are not physically and / or chemically molecularly entangled.

18. An article comprising a substrate and a coating, wherein the coating comprises the composite of claim 1.

19. The article of claim 18, wherein the coating has a thickness of about 10 μm to about 1000 μm.

20. The amount of coating on the substrate is from about 0.01 g / ft 2 to about 25 g / ft 2 ; and / or the amount of first polymeric macromolecular species or polymer in the coating on the substrate is from about 0.001 g / ft 2 to about 20 g / ft 2 ; and / or 20. The article of claim 18, wherein the amount of second polymeric macromolecular species or polymer in the coating on the substrate is from about 0.001 g / ft 2 to about 15 g / ft 2 .

21. The article of claim 18, wherein the substrate comprises a leather material or a woven material.