Composite polymeric materials and products and methods of making same

Abstract

The invention provides a leather product and a method for manufacturing the leather product. The leather article comprises a substrate and a coating comprising a cellulose derivative with improved color fastness to rubbing, the cellulose derivative may be methylcellulose, ethylcellulose, ethylmethylcellulose, carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methylcellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose, or carboxymethyl cellulose. The cellulose acetate-cellulose acetate composite material is prepared from ethyl hydroxyethyl cellulose, cellulose acetate, cellulose triacetate, cellulose propionate, cellulose acetate propionate, cellulose acetate butyrate, microcrystalline cellulose, cellulose nitrate or cellulose sulfate.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] 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. Technical Field

[0003] The present disclosure relates to composite polymeric materials comprising, in part, a cellulose derivative coating composition, optionally including silk fibroin or fragments thereof, and various additives, for coating various substrates. Background Art

[0004] Silk is a natural polymer produced by various insects and spiders and consists of a filament core protein, fibroin, and a gelatinous coating composed of a non-filamentous protein, sericin. Silk fibers are lightweight, breathable, and hypoallergenic. Summary of the Invention

[0005] The present disclosure provides an article comprising one or more surfaces coated with: a fibroin or a fragment thereof having a fibroin mass 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 40 kDa, about 50 kDa to about 60 kDa, about 60 kDa to about 70 kDa, about 70 kDa to about 80 kDa, about 80 kDa to about 90 kDa, about 90 kDa to about 100 kDa, about 100 kDa to about 150 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 an average weight average molecular weight of about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 50 kDa to about 55 kDa, about 55 kDa to about 60 kDa, about 60 kDa to about 100 kDa, or about 80 kDa to about 114 kDa, and a polydispersity ranging from 1 to about 5; and one or more components selected from cellulose derivatives, plasticizers, and cross-linking agents. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, 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, polyacridines, polyureas, glutaraldehyde, and starch dialdehyde. In some embodiments, the silk fibroin 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 or fragment thereof. In some embodiments, the fibroin or fragment thereof does not spontaneously or gradually gel and does not undergo a visible change in color or turbidity in aqueous solution for at least 10 days before being added to the article. In some embodiments, a portion of the fibroin or fragment thereof is coated on the surface of a leather substrate. In some embodiments, a portion of the fibroin or fragment thereof is injected into a layer of the leather substrate. In some embodiments, a portion of the fibroin or fragment thereof is in a recessed portion of 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 gellan gum with a low acyl content.In some embodiments, the w / w ratio between the fibroin or fragment thereof and the polysaccharide is selected from the group consisting of 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, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:6 0, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75, 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 15:85, 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, 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, close to 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 fibroin or fragment thereof and the polysaccharide is selected from 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, close to 10.9:1, close to 10.8:1, close to 10.7:1, close to 10.6:1, close to 10.5:1, close to 10.4:1, close to 10.3:1, close to 10.2:1, close to 10.1:1, close to 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 preparation further comprises one or more polyols, and / or one or more polyethers. In some embodiments, the polyol comprises one or more of ethylene 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 or fragments thereof and the one or more polyols and / or one or more polyethers is selected from the group consisting of 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, about 0.1:1, about 1:0.1, about 1:0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, 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 silicone, dye, pigment, and 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 further comprises one or more of the following: isocyanates, isocyanate adducts and / or isocyanate reactive derivatives; polydiisocyanates, polydiisocyanate adducts and / or polydiisocyanate reactive derivatives; aziridines, aziridine adducts and / or aziridine reactive derivatives; carbodiimides, carbodiimide adducts and / or carbodiimide reactive derivatives; aldehydes, aldehyde adducts and / or aldehyde reactive derivatives; polyisocyanates, polyisocyanate adducts and / or polyisocyanate reactive derivatives; polyaziridines, polyaziridine adducts and / or polyaziridine reactive derivatives. and / or polyaziridine reaction derivatives; polycarbodiimide, polycarbodiimide adduct and / or polycarbodiimide reaction derivatives; polyaldehyde, polyaldehyde adduct and / or polyaldehyde reaction derivatives; polyurethane, polyurethane adduct and / or polyurethane reaction derivatives; polyacrylate, polyacrylate adduct and / or polyacrylate reaction derivatives; polyester, polyester adduct and / or polyester reaction derivatives; wax, wax adduct and / or wax reaction derivatives; protein, protein adduct and / or protein reaction derivatives; or alcohol, alcohol adduct and / or alcohol reaction derivative.

[0006] In some embodiments, one or more surfaces of the article have a higher colorfastness to rubbing than one or more surfaces of a similar article that has not been similarly coated. In some embodiments, the article comprises leather.

[0007] The present disclosure also provides a method for coating one or more surfaces of an article, the method comprising: applying a first composition comprising a fibroin or a fragment thereof having a thickness 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 39 kDa, about 40 kDa to about 45 kDa, about 50 kDa to about 50 kDa, about 50 kDa to about 5 ... kDa to about 40kDa, about 39kDa to about 54kDa, about 39kDa to about 80kDa, about 40kDa to about 45kDa, about 45kDa to about 50kDa, about 50kDa to about 55kDa, about 55kDa to about 60kDa, about 60kDa to about 100kDa, or about 80kDa to about 114kDa, and a polydispersity ranging from 1 to about 5; and applying a second composition comprising one or more components selected from cellulose derivatives and plasticizers. In some embodiments, the first composition further comprises a crosslinking agent. In some embodiments, the crosslinking agent is selected from polyisocyanates, polycarbodiimides, polyacridines, polyureas, glutaraldehyde, and starch dialdehyde. In some embodiments, the cellulose derivative is selected from methylcellulose, ethylcellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, 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 fibroin or its fragment 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 composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the fibroin or its fragment. In some embodiments, the fibroin or its fragment does not spontaneously or gradually gel and does not undergo a visible change in color or turbidity in aqueous solution for at least 10 days before being formulated into a composition and applied to one or more surfaces of the article. In some embodiments, the article comprises leather. In some embodiments, a portion of the silk preparation is coated on the surface of the leather substrate, and / or a portion of the silk preparation is injected into a layer of the leather substrate, and / or a portion of the silk preparation enters a recessed portion of the leather substrate. In some embodiments, the silk preparation 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, alginates, pectins, chitin, chitosan, carrageenan, 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 fibroin or fragment thereof and the rheology modifier in the silk preparation 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, close to 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 fibroin or fragment thereof and the rheology modifier in the silk preparation is selected from 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, close to 10.9:1, close to 10.8:1, close to 10.7:1, close to 10.6:1, close to 10.5:1, close to 10.4:1, close to 10.3:1, close to 10.2:1, close to 10.1:1, close to 10:1, about 9.9:1, about 9.8:1, about 9.7:1, about 9.6:1 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 In some embodiments, the rheology modifier has a w / v concentration of about 0.01% to about 5%, or about 0.1% to about 1%. In some embodiments, the filament 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 ethylene glycol, glycerol, sorbitol, D-sorbitol, glucose, sucrose, mannitol, mannitol, D-mannitol and dextrose. In some embodiments, the polyether is one or more polyethylene glycols (PEG). In some embodiments, the w / w ratio between the fibroin or its fragment and the plasticizer in the silk preparation is selected from 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, about 0.1:1, about 1:0.1, about 1: 0.2, about 1:0.3, about 1:0.4, about 1:0.5, about 1:0.6, about 1:0.7, about 1:0.8, about 1:0.9, about 1:1.1, about 1:1.2, about 1:1.3, about 1:1.4, about 1:1.5, about 1:1.6, about 1:1.7, about 1:1.8, about 1:1.9, about 1:2, about 1:2.1, about 1:2.2, about 1:2.3, 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 w / v concentration of the plasticizer in the silk preparation is about 0.01% to about 10%. In some embodiments, the silk preparation further comprises a defoaming agent at a concentration of about 0.001% to about 1%. In some embodiments, the defoaming agent comprises 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 preparation is a liquid, gel, paste, wax, or cream. In some embodiments, the silk preparation comprises one or more sub-formulations to be applied simultaneously or at different times. In some embodiments, the concentration of fibroin or its fragments in the silk preparation is from about 0.1% w / v to about 15% w / v. In some embodiments, the concentration of fibroin or its fragments in the silk preparation is from about 0.5% w / v to about 12% w / v. In some embodiments, the concentration of fibroin or its fragments in the silk preparation 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 fibroin or its fragments in the silk preparation is about 3% w / v, about 3.25% w / v, about 3.5% w / v, about or about 10% w / v.In some embodiments, the concentration of fibroin or its fragments in the silk preparation is from about 5 mg / mL to about 125 mg / mL. In some embodiments, the concentration of fibroin or its fragments in the silk preparation 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 g / 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, 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 the group consisting of dyeing, drying, water annealing, mechanical stretching, trimming, polishing, applying pigments, applying colorants, applying acrylic formulations, applying polyurethane formulations, chemically fixing, stamping, applying silicone finishes, providing a Uniflex treatment, and / or providing a Finiflex treatment, wherein the step of applying the silk formulation to the leather surface is performed before, during, or after the one or more additional steps. In some embodiments, treating the leather substrate with the silk formulation results in one or more of the following: increased gloss, increased color saturation, enhanced color, increased color fixation, reduced dye usage, and / or improved color fastness. In some embodiments, the improvement is over a leather substrate that has not been similarly treated with the silk formulation formulated into a composition and applied to one or more surfaces of the article. In some embodiments, the article comprises leather.

[0008] Disclosed herein are silk-coated leather products and methods for their preparation. Silk and silk protein fragments and silk and silk protein fragment (SPF) compositions as described herein can be used to replace or supplement any chemicals used in any chemical processing step, as a surface treatment, to lock in color, and thereby change the appearance, feel, texture, and / or quality of leather.

[0009] 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 change the sheen or luster of the leather and / or to obtain finishing effects such as matte, glossy, mirrored, embossed, etc.

[0010] 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 leather goods, such as follicle defects or other mechanical defects, whether surface or internal to the leather or leather goods.

[0011] 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, leather goods, and / or leather products, or to change the grade of leather or leather goods, thereby expanding the applicable market area for a given leather type.

[0012] In some embodiments, silk and silk protein fragments and silk and silk protein fragment compositions as described herein can be used to improve the hand of leather, such as described for its feel or softness.

[0013] 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 during the finishing stage or in any other suitable process step to lock in color, adjust the final coloration, or change the chemical properties of the pigment or improve the delivery of the colorant.

[0014] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions as described herein can be used before or after any mechanical processing steps typical of leather processing, including but not limited to Uniflex processing, Finiflex processing, hot stamping processing, polishing processing, epidermis trimming or drying before or after. In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions as 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 as 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 as described herein can be used before any embossing process described herein.

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

[0016] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions described herein can be used by stamping on leather.

[0017] 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.

[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 leather processing steps, such as the finishing process, to replace any chemical substances used to stabilize, alter the sheen, luster, color, darkness, hue, finish, feel, weight, etc.

[0019] 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 leather processing steps, such as the finishing process, in addition to any chemical used to stabilize, change the sheen, luster, color, darkness, hue, finish, feel, etc.

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

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

[0022] In some embodiments, silk and silk protein fragments and silk and silk protein fragment compositions as described herein can be used to perform one or more functions during the tanning stage through the dyeing stage of leather processing.

[0023] 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 leather processing steps, such as the finishing process, to change the contact angle of solvents applied to semi-finished or finished leather.

[0024] 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 leather processing steps, such as the finishing process, as defect fillers for pre-dyed or post-dyed skins. In some embodiments, such uses include in combination with pigments, dyes, blending agents, softeners, rheology modifiers, and the like.

[0025] 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 any process described herein and can be used for any purpose described herein, and such use can be enhanced by the additional use of one or more physical and chemical processing treatments, including but not limited to O2 plasma, the use of crosslinkers, photocrosslinkers or ultraviolet treatment.

[0026] In some embodiments, the silk and silk protein fragments and silk and silk protein fragment compositions described herein can be mixed with or replaced with material categories including, but not limited to, water-based paints, waxes, oils, proteins or other binders, fillers, feel modifiers, leveling agents, solvent varnishes, water-based varnishes, penetrants, acrylic resins, butadiene resins, dense resins, hybrid resins, impregnating resins, rheology modifiers, solvent passivators, solvent urethanes, water-based passivators, water-based topcoats, chrome, dye dispersants, acid dyes, basic dyes, chrome-based or other dyes and / or colorants.

[0027] In some embodiments, the leather preparation process may include treating the leather with the silk and / or SPF composition described herein. In some embodiments, the silk and / or SPF composition may include one or more chemical agents (e.g., silicone, polyurethane, etc.) as described below.

[0028] In one embodiment, the present disclosure provides a method for treating leather with the silk and / or SPF composition described herein, wherein the method may include the following steps: dyeing the leather; mechanically stretching the leather; trimming the leather; polishing the leather; applying (optionally by spraying) a pigment and / or acrylic coating to the leather; chemically fixing the leather, stamping the leather, applying silicone or other finishes to the leather; providing the leather with a Uniflex treatment; and / or filling defects on the surface of the leather or inside the leather with silk or an SPF composition; wherein one or more of the foregoing steps includes applying the silk and / or SPF composition to the leather before, during, or after the steps.

[0029] In one embodiment, the present disclosure provides a method for treating leather with silk and / or an SPF composition as described herein, wherein the method may include the following steps: dyeing the leather; mechanically stretching the leather; trimming the leather; performing a first polishing of the leather; applying a colorant and / or an acrylic coating to the leather (optionally by spraying); performing a second polishing of the leather; providing the leather with a Finiflex treatment; and / or filling defects on the surface of the leather or inside the leather with silk or an SPF composition; wherein one or more of the foregoing steps includes applying the silk composition to the leather before, during, or after said steps.

[0030] In some embodiments, the silk and / or SPF composition described herein can be applied to leather or leather products by any of the methods described herein, but can also be applied by hand spraying, spraying using a mechanical spraying device, by brushing, bath coating, rubbing, wet mixing, washing, drumming, dipping, squeezing, injecting, plastering, roller coating, and / or filling.

[0031] In some embodiments, the silk and / or SPF composition described herein can be applied alone, mixed with one or more chemical substances (e.g., chemical agents), 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 composition described herein can be applied to finished leather or leather products, mechanically treated leather or leather products, or drum-finished leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied to defects in finished leather or leather products, mechanically treated leather or leather products, or drum-finished leather or leather products.

[0032] In some embodiments, the silk and / or SPF composition described herein can be applied to leather or leather products as a defect filler before dyeing and before finishing. In some embodiments, the silk and / or SPF composition described herein can be applied to leather or leather products as a defect filler after dyeing and before finishing. In some embodiments, the silk and / or SPF composition described herein can be applied to leather or leather products as a defect filler after dyeing and after finishing.

[0033] In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by hand. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by fingers. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a brush-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a marker-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a pen-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a pipette-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a syringe-type applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by 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 products as a blemish filler by using a heated die device applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a sponge applicator. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a roller coater. In some embodiments, the silk and / or SPF compositions described herein can be applied to leather or leather products as a blemish filler by using a "glue gun" like applicator.

[0034] In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to cowhide leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to sheepskin leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to lambskin leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to horsehide leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to crocodile leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to alligator leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to birdhide leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to animalhide leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to split leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to suede leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to wet chrome tanned leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to modified leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to aniline leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to bonded leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to brushed leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to tumbled leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to buffed leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to bycast leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a defect filler to suede leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to plongé leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to chrome-tanned leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to combination-tanned leather or leather products.In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to cordovan leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to nubuck leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to crockproof leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to drum-finished leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to embossed leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to reinforced grain leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to grain leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to metallized leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to bare leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to natural grain leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to nubuck leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to patent leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to pearlescent leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to pressed leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to printed leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to protected leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to pure aniline leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied as a flaw filler to tanned / retanned leather or leather products. In some embodiments, the silk and / or SPF compositions described herein can be applied to round hand leather or leather products as a defect filler. In some embodiments, the silk and / or SPF compositions described herein can be applied to saddle leather or leather products as a defect filler.In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to semi-aniline leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to reduced leather or leather products. In some embodiments, the silk and / or SPF composition described herein can be applied as a defect filler to split leather or leather products.

[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 before or after the liming 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 before or after the deliming and / or bating steps. 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 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 before or after the tanning 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 before or after the neutralization, dyeing, and / or fat liquoring steps. 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 any drying steps. 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 step. 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 step. 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.

[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 the liming 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 the deliming and / or bating steps. 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 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 the tanning 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 the neutralization, dyeing, and / or esterification steps. 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 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 finishing step. 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 step.

[0037] 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 in a process that includes one or more steps, such as one or more dyeing steps. 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 in 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 described herein (with or without one or more chemical agents) can be used to treat leather in 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 described herein (with or without one or more chemical agents) can be used to treat leather in 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 the polishing step. Mechanical steps include, but are not limited to, drying, polishing, stamping, Uniflex and / or Finiflex, stretching, and / or finishing. 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 in 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 the 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 in a process that includes one or more steps, such as one or more chemical treatment steps. In some embodiments, the silk and / or SPF composition can be used before, during, or after the chemical treatment steps. 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 fixing treatment steps.

[0038] In one embodiment, a method for processing leather with fibroin, which may include silk-based proteins or fragments thereof, and / or SPF, is provided to provide leather processed with fibroin. In some embodiments, the method may include preparing a fibroin solution or other composition that may contain one or more of low molecular weight fibroin, medium molecular weight fibroin, and high molecular weight 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 may include preparing a fibroin solution or other composition that may include a concentration 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), or less than about 12% by weight (w / w). 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% by weight (w / w), or less than about 27% by weight (w / w), or less than about 28% by weight (w / w), or less than about 29% by weight (w / w), or less than about 30% by weight (w / w), or less than about 31% by weight (w / w), or less than about 32% by weight (w / w), or less than about 33% by weight (w / w), or less than about 34% by weight (w / w), or less than about 35% by weight (w / w), or less than about 36% by weight (w / w), or less than about 37% by weight (w / w), or less than about 38% by weight (w / w), or less than about 39% by weight (w / w), w / w), or 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) of one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin. In some embodiments, the method may include treating the surface of the leather material with the silk fibroin solution or composition before, during, or after any processing step.In some embodiments, the method may 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 may include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after color lock. In some embodiments, the method may 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 may 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 may include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after colorant delivery enhancement. In some embodiments, the method may 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 may include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after Finiflex treatment. In some embodiments, the method may include treating the surface of the leather material with a silk fibroin solution or composition before, during, or after hot stamping treatment. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the polishing process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the cuticle trimming. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the finishing process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the tanning process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the dyeing process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the stretching process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the drying process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the trimming process. In some embodiments, the method may include treating the surface of the leather material with a fibroin solution or composition before, during, or after the polishing process. .

[0039] In one embodiment, a method is provided for coating leather with silk fibroin, which may include a silk-based protein or fragment thereof, and / or SPF to provide silk fibroin-coated leather. In some embodiments, the method 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 may include preparing a fibroin solution or other composition that may include a concentration 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), or less than about 12% by weight (w / w). 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% by weight (w / w), or less than about 27% by weight (w / w), or less than about 28% by weight (w / w), or less than about 29% by weight (w / w), or less than about 30% by weight (w / w), or less than about 31% by weight (w / w), or less than about 32% by weight (w / w), or less than about 33% by weight (w / w), or less than about 34% by weight (w / w), or less than about 35% by weight (w / w), or less than about 36% by weight (w / w), or less than about 37% by weight (w / w), or less than about 38% by weight (w / w), or less than about 39% by weight (w / w) In some embodiments, the method may comprise coating the surface of the leather material with a silk fibroin solution before, during, or after any processing step. In some embodiments, the method may comprise coating the surface of the leather material with a silk fibroin solution before, during, or after the pigment is delivered. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after color locking.In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after final color adjustment. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after pigment chemical modification. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after colorant delivery enhancement. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after Uniflex treatment. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after Finiflex treatment. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after hot stamping treatment. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after polishing treatment. In some embodiments, the method may include coating the surface of the leather material with the fibroin solution before, during, or after skin trimming. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after the finishing process. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after tanning. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after dyeing. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after stretching. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after drying. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after trimming. In some embodiments, the method may include coating the surface of the leather material with a fibroin solution before, during, or after polishing.

[0040] In some embodiments, the method may include filling and / or repairing defects on the surface of leather materials with a silk fibroin composition, such as a silk fibroin glue, paste, gel, wax, putty, etc. In one embodiment, a method for repairing leather with silk fibroin and / or SPF that may include silk-based proteins or fragments thereof to provide leather repaired with silk fibroin is provided. In some embodiments, the method may include preparing a silk fibroin solution or other composition that may include 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) of one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin.In some embodiments, the method may include preparing a fibroin solution or other composition that may include a concentration 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), or less than about 12% by weight (w / w), or less than about 13% by weight (w / w). 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% by weight (w / w), or less than about 27% by weight (w / w), or less than about 28% by weight (w / w), or less than about 29% by weight (w / w), or less than about 30% by weight (w / w), or less than about 31% by weight (w / w), or less than about 32% by weight (w / w), or less than about 33% by weight (w / w), or less than about 34% by weight (w / w), or less than about 35% by weight (w / w), or less than about 36% by weight (w / w), or less than about 37% by weight (w / w), or less than about 38% by weight (w / w), or less than about 39% by weight (w / w), or 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 less than about 28% by weight (w / w), or less than about 29% by weight (w / w), or less than about 30% by weight (w / w), or less than about 31% by weight (w / w), or less than about 32% by weight (w / w), or less than about 33% by weight (w / w), or less than about 34% by weight (w / w), or less than about 35% by weight (w / w), or less than about 36% by weight (w / w), or less than about 37% by weight (w / w), or less than about 38% by weight (w / w), or less than about 39% by weight (w / w), or 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) of one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin. In some embodiments, the method may 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 may 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 may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after color locking.In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the final color adjustment. In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the pigment chemical change. In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the colorant delivery improvement. In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the Uniflex treatment. In some embodiments, the method may 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 may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the hot stamping treatment. In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after the polishing treatment. In some embodiments, the method may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after cuticle trimming. In some embodiments, the method may 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 may 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 may 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 may 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 may 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 may 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 may include repairing the surface and / or defects of the leather material with a silk fibroin solution or composition before, during, or after polishing.

[0041] In one embodiment, a method is provided for coating leather with fibroin, which may include a silk-based protein or fragment thereof, and / or SPF to provide fibroin-coated leather, wherein the fibroin coated on the fibroin-coated leather may be heat-resistant to a selected temperature. In some embodiments, the method may include preparing a fibroin solution or other composition that may include one or more of low molecular weight fibroin, medium molecular weight fibroin, and high molecular weight 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 may include preparing a fibroin solution or other composition that may include a concentration 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), 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% by weight (w / w), or less than about 2 7% by weight (w / w), or less than about 28% by weight (w / w), or less than about 29% by weight (w / w), or less than about 30% by weight (w / w), or less than about 31% by weight (w / w), or less than about 32% by weight (w / w), or less than about 33% by weight (w / w), or less than about 34% by weight (w / w), or less than about 35% by weight (w / w), or less than about 36% by weight (w / w), or less than about 37% by weight (w / w), or less than about 38% by weight (w / w), or less than about 39% by weight (w / w), or 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 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), or less than about 51% by weight (w / w), or less than about 52% by weight (w / w), or less than about 53% by weight (w / w), or less than about 54% by weight (w / w), or less than about 55% by weight (w / w), or less than about 56% by weight (w / w), or less than about 57% by weight (w / w), or less than about 58% by weight (w / w), or less than about 59% by weight (w / w), or less than about % weight (w / w), or less than about 42% weight (w / w), or less than about 43% weight (w / w), or less than about 44% weight (w / w), or less than about 45% weight (w / w), or less than about 46% weight (w / w), or less than about 47% weight (w / w), or less than about 48% weight (w / w), or less than about 49% weight (w / w), or less than about 50% weight (w / w) of one or more of low molecular weight silk fibroin, medium molecular weight silk fibroin, and high molecular weight silk fibroin. In some embodiments, the method may include coating the surface of the leather material with the silk fibroin solution. In some embodiments, the method may include drying the surface of the leather material that has been coated with the silk fibroin solution or composition to provide a silk fibroin-coated leather material, wherein drying the surface of the leather material includes heating the surface of the material without significantly reducing the silk fibroin coating properties. In some embodiments, the method may include filling defects on the surface of the leather material with a fibroin composition, such as a fibroin glue, paste, gel, wax, putty, or the like.

[0042] In one embodiment, the silk fibroin-processed leather material of the present disclosure may be processed with one or more of low molecular weight silk, medium molecular weight silk, and high molecular weight silk to provide a resulting coated leather material having enhanced hydrophobicity or hydrophilicity. In one embodiment, the silk fibroin-coated leather material of the present disclosure may be coated with one or more of low molecular weight silk, medium molecular weight silk, and high molecular weight silk to provide a resulting coated leather material having enhanced hydrophobicity or hydrophilicity. In one embodiment, the silk fibroin-repaired leather material of the present disclosure may have one or more defects repaired, masked, or hidden with one or more of low molecular weight silk, medium molecular weight silk, and high molecular weight silk to provide a resulting leather material having enhanced properties, including an enhanced quality grade.

[0043] In one embodiment, the leather material processed with the silk fibroin of the present disclosure can be processed with a composition comprising low molecular weight silk and medium molecular weight silk. In one embodiment, the leather material coated with the silk fibroin of the present disclosure can be coated with a composition comprising low molecular weight silk and medium molecular weight silk. In one embodiment, the leather material with silk fibroin defects repaired with the silk fibroin 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 from about 99:1 to about 1:99, from about 95:5 to about 5:95, from about 90:10 to about 10:90, from about 75:25 to about 25:75, from about 65:35 to about 35:65, or from about 55:45 to about 45:55.In some embodiments, the w / w ratio between the low molecular weight filaments and the medium molecular weight filaments 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 one embodiment, the w / w ratio between the low molecular weight filaments and the medium molecular weight filaments 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, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:4 0, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69 , about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75, 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 15:85, 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 one embodiment, the leather material processed by the silk fibroin of the present disclosure can be processed with a composition comprising low molecular weight silk and high molecular weight silk. In one embodiment, the leather material coated with the silk fibroin of the present disclosure can be coated with a composition comprising low molecular weight silk and high molecular weight silk. In one embodiment, the leather material repaired by the silk fibroin defect 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 filaments and the high molecular weight filaments 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 one embodiment, the w / w ratio between the low molecular weight filaments and the high molecular weight filaments 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, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:4 0, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69 , about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75, 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 15:85, 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 one embodiment, the leather material processed with the silk fibroin of the present disclosure can be processed with a composition comprising medium molecular weight silk and high molecular weight silk. In one embodiment, the leather material coated with the silk fibroin of the present disclosure can be coated with a composition comprising medium molecular weight silk and high molecular weight silk. In one embodiment, the leather material with silk fibroin defects repaired with the silk fibroin 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 from about 99:1 to about 1:99, from about 95:5 to about 5:95, from about 90:10 to about 10:90, from about 75:25 to about 25:75, from about 65:35 to about 35:65, or from about 55:45 to about 45:55. In some embodiments, the w / w ratio between the medium molecular weight filaments and the high molecular weight filaments 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 one embodiment, the w / w ratio between the medium molecular weight filaments and the high molecular weight filaments 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 78:24, about 79:25, about 79:26, about 79:27, about 79:28, about 79:29, about 80:21 : 23, about 76:24, about 75:25, about 74:26, about 73:27, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about 52: 48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about 27:7 3. about 26:74, about 25:75, 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 15:85, 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.

[0046] In one embodiment, the leather material processed with the silk fibroin 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 one embodiment, the leather material coated with the silk fibroin 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 one embodiment, the leather material defect-repaired with the silk fibroin 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 one embodiment, the w / w ratio between the low molecular weight filaments, medium molecular weight filaments, and high molecular weight filaments 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.

[0047] In one embodiment, the present disclosure provides a silk and / or SPF treated leather product, wherein the treatment comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a silk and / or SPF coated leather product, wherein the coating comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a silk and / or SPF defect-repaired leather product, wherein the defect filling comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa.

[0048] In one embodiment, the present disclosure provides a silk and / or SPF processed leather product, wherein the processing comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a silk and / or SPF coated leather product, wherein the coating comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a silk and / or SPF defect repaired leather product, wherein the defect filling comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa.

[0049] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or a fragment thereof, wherein the average number of amino acid residues of the silk-based protein or the fragment thereof is about 1 to 400 residues, or 1 to 300 residues, or 1 to 200 residues, or 1 to 100 residues, or 1 to 50 residues, or 5 to 25 residues, or 10 to 20 residues. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof, wherein the average number of amino acid residues is about 1 to 400 residues, or 1 to 300 residues, or 1 to 200 residues, or 1 to 100 residues, or 1 to 50 residues, or 5 to 25 residues, or 10 to 20 residues. In one embodiment, the present disclosure provides a leather product comprising one or more leather defect-filling portions, wherein the composition comprises a silk-based protein or fragment thereof having an average number of amino acid residues of about 1 to 400 residues, or 1 to 300 residues, or 1 to 200 residues, or 1 to 100 residues, or 1 to 50 residues, or 5 to 25 residues, or 10 to 20 residues.

[0050] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa.

[0051] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product having a coating, 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 one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa.

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

[0053] In one embodiment, the present disclosure provides a leather product processed with a silk protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or the fragment thereof comprises a fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) sericin. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or the fragment thereof comprises a fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) sericin. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition and a coating, wherein the composition comprises a silk-based protein or fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof comprises a fibroin-based protein or protein fragment having about 0.01% (w / w) to about 10% (w / w) sericin.

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

[0055] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof. In one embodiment, the present disclosure provides a leather product with a coating, wherein the coating comprises a silk-based protein or fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof.

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

[0057] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: a natural silk-based protein or a fragment thereof, a recombinant silk-based protein or a fragment thereof, and a combination thereof, wherein the silk-based protein or a fragment thereof is a natural silk-based protein or a fragment thereof, and the natural silk-based protein or a fragment thereof is selected from the group consisting of: a spider silk-based protein or a fragment thereof, a silkworm silk-based protein or a fragment thereof, and a combination thereof. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: a natural silk-based protein or a fragment thereof, a recombinant silk-based protein or a fragment thereof, and a combination thereof, wherein the silk-based protein or a fragment thereof is a natural silk-based protein or a fragment thereof, and the natural silk-based protein or a fragment thereof is selected from the group consisting of: a spider silk-based protein or a fragment thereof, a silkworm silk-based protein or a fragment thereof, and a combination thereof. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof, and the natural silk-based protein or fragment thereof is selected from the group consisting of: spider silk-based proteins or fragments thereof, silkworm silk-based proteins or fragments thereof, and combinations thereof.

[0058] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or a fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof, and the natural silk-based protein or fragment thereof is selected from the group consisting of: spider silk-based proteins or fragments thereof, silk-based proteins or fragments thereof, and combinations thereof, wherein the natural silk-based protein or fragment thereof is a silk-based protein or fragment thereof, and the silk-based protein or fragment thereof is a Bombyx mori silk-based protein or fragment thereof. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having a weight average molecular weight ranging from 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, the natural silk-based protein or fragment thereof is 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, wherein the natural silk-based protein or fragment thereof is a silkworm silk-based protein or fragment thereof, and the silk-based protein or fragment thereof is a Bombyx mori silk-based protein or fragment thereof. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or fragment thereof having a weight average molecular weight ranging from 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, and wherein the natural silk-based protein or fragment thereof is a silkworm silk-based protein or fragment thereof. In one embodiment, the present disclosure provides a leather product having a leather defect filling composition, wherein the composition comprises a silk-based protein or a fragment thereof having a weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof, and the natural silk-based protein or fragment thereof is selected from the group consisting of: spider silk-based proteins or fragments thereof, silk-based proteins or fragments thereof, and combinations thereof, wherein the natural silk-based protein or fragment thereof is a silk-based protein or fragment thereof, and the silk-based protein or fragment thereof is a Bombyx mori silk-based protein or fragment thereof.

[0059] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: a natural silk-based protein or a fragment thereof, a recombinant silk-based protein or a fragment thereof, and a combination thereof, wherein the silk-based protein or a fragment thereof is a natural silk-based protein or a fragment thereof, and the natural silk-based protein or a fragment thereof is selected from the group consisting of: a spider silk-based protein or a fragment thereof, a silk-based protein or a fragment thereof, and a combination thereof, wherein the natural silk-based protein or a fragment thereof is a silk-based protein or a fragment thereof, and the silk-based protein or a fragment thereof is a Bombyx mori silk-based protein or a fragment thereof. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: a natural silk-based protein or a fragment thereof, a recombinant silk-based protein or a fragment thereof, and a combination thereof, wherein the silk-based protein or a fragment thereof is a natural silk-based protein or a fragment thereof, and the natural silk-based protein or a fragment thereof is selected from the group consisting of: a spider silk-based protein or a fragment thereof, a silkworm silk-based protein or a fragment thereof, and a combination thereof, wherein the natural silk-based protein or a fragment thereof is a silkworm silk-based protein or a fragment thereof, and the silk-based protein or a fragment thereof is a Bombyx mori silk-based protein or a fragment thereof. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or a fragment thereof having an average weight average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: a natural silk-based protein or a fragment thereof, a recombinant silk-based protein or a fragment thereof, and a combination thereof, wherein the silk-based protein or a fragment thereof is a natural silk-based protein or a fragment thereof, and wherein the natural silk-based protein or a fragment thereof is a silkworm silk-based protein or a fragment thereof. In one embodiment, the present disclosure provides a leather product having a leather defect filling composition, wherein the composition comprises a silk-based protein or a fragment thereof having an average weight-average molecular weight ranging from about 5 kDa to about 144 kDa, wherein the silk-based protein or a fragment thereof is selected from the group consisting of: natural silk-based proteins or fragments thereof, recombinant silk-based proteins or fragments thereof, and combinations thereof, wherein the silk-based protein or fragment thereof is a natural silk-based protein or fragment thereof, and the natural silk-based protein or fragment thereof is selected from the group consisting of: spider silk-based proteins or fragments thereof, silk-based proteins or fragments thereof, and combinations thereof, wherein the natural silk-based protein or fragment thereof is a silk-based protein or fragment thereof, and the silk-based protein or fragment thereof is a Bombyx mori silk-based protein or fragment thereof.

[0060] In one embodiment, the present disclosure provides a leather product processed 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 ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product 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 ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product 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 ranging from about 5 kDa to about 144 kDa.

[0061] In one embodiment, the present disclosure provides a leather product processed 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 one embodiment, the present disclosure provides a leather product 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 one embodiment, the present disclosure provides a leather product 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.

[0062] In one embodiment, the present disclosure provides a leather product processed 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 ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product 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 ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product 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 ranging from about 5 kDa to about 144 kDa.

[0063] In one embodiment, the present disclosure provides a leather product processed 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 one embodiment, the present disclosure provides a leather product 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 one embodiment, the present disclosure provides a leather product 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.

[0064] In one embodiment, the present disclosure provides a leather product 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 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 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof has a polydispersity between about 1.5 and about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel prior to processing the leather product and does not undergo a visible change in color or turbidity when in solution for at least 10 days. In one embodiment, the present disclosure provides a leather article having a coating, 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, wherein the silk-based protein or fragment thereof has an average weight average molecular weight range selected from the group consisting of: about 5 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 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or fragment thereof has a polydispersity between about 1.5 and about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel prior to coating the leather article and does not undergo a visible change in color or turbidity when in solution for at least 10 days. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or a fragment thereof having a weight average molecular weight ranging from 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 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 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk-based protein or protein fragment thereof has a polydispersity between about 1.5 and about 3.0, and wherein the protein or protein fragment does not spontaneously or gradually gel prior to repairing the leather product and does not undergo a visible change in color or turbidity for at least 10 days in solution.

[0065] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product having a coating, wherein the coating comprises a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or fragment thereof having a weight-average molecular weight ranging from about 5 kDa to about 144 kDa.

[0066] In one embodiment, the present disclosure provides a leather product processed with a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa. In one embodiment, the present disclosure provides a leather product having a coating, 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 one embodiment, the present disclosure provides a leather product comprising a leather defect filling composition, wherein the composition comprises a silk-based protein or fragment thereof having an average weight average molecular weight of about 5 kDa to about 144 kDa. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] The presently disclosed embodiments will be further explained with reference to the accompanying drawings. The drawings shown are not necessarily to scale, with emphasis generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0068] Figure 1 Is a flow chart showing various embodiments for producing pure fibroin-based protein fragments (SPF) of the present disclosure.

[0069] Figure 2 is a flow chart showing various parameters that may be modified during the extraction and solubilization steps in producing the SPF of the present disclosure.

[0070] Figure 3 The general steps used in leather processing are shown.

[0071] Figure 4 is a photograph of a felt pad (and associated leather sample) after 600 consecutive wet Veslic rubbing cycles, comparing leather samples treated with a fibroin fragment composition (bottom sample - entry B2) versus leather samples treated with polyurethane (top 2 samples). Note the damage to the polyurethane sample and dye bleed from the leather to the felt after 600 cycles.

[0072] Figure 5 Photographs of the felt pad after 10 wet Veslic rubbing cycles on leather samples treated with items A1, A2, B1, and B2 (Table 1).

[0073] Figure 6 The following is a photograph of a water droplet placed on a sample treated with either a silk fibroin fragment or a cross-linked polyurethane coating system after wet Veslic rubbing. In the case of the silk fibroin fragment (entry B2), the sample was subjected to 600 rubbing cycles, while the polyurethane sample was subjected to only 10 cycles. The photograph was taken after the water droplet had been placed for 5 minutes. Note that when using a commercial reference system designed as a topcoat, water penetrated into the leather matrix.

[0074] Figures 7A-7B is a graphical analysis showing the results of Water Vapor Transmission Test #1 on coated leather (8A) and uncoated leather (8B).

[0075] Figures 8A-8B is a graphical analysis showing the results of Water Vapor Transmission Test #2 on coated leather (9A) and uncoated leather (9B).

[0076] Figures 9A-9B is a graphical analysis showing the results of Water Vapor Transmission Test #3 on coated leather (10A) and uncoated leather (10B).

[0077] Figures 10A-10B This is a photo of uncoated ordinary leather.

[0078] Figures 11A-11B FTIR analysis of uncoated plain leather is shown.

[0079] Figures 12A-12B is a photograph of leather treated with an adhesive coating of the coating system disclosed herein.

[0080] Figures 13A-13B Shown is an FTIR analysis of leather treated with an adhesive coating of the coating system disclosed herein.

[0081] Figures 14A-14B is a photograph of treated leather finished with a topcoat of the coating system disclosed herein.

[0082] Figures 14C-14D Shown is an FTIR analysis of treated leather finished with a topcoat of the coating system disclosed herein.

[0083] Figure 15A Is an IR spectrum of a leather sample treated with the coating system disclosed herein using a LN-MCT detector.

[0084] Figure 15B Macro ATR imaging of a leather sample treated with an adhesive primer of the coating system disclosed herein is shown.

[0085] Figure 15CMacro ATR imaging of a leather sample treated with a topcoat of the coating system disclosed herein is shown.

[0086] Figures 16A-16H

[0014] Figures 16A and 16B show soil release test results for various soil sources on leather treated with the coating system disclosed herein. 16A: mud, 16B: water, 16C: mustard, 16D: corn oil, 16E: wine, 16F: ketchup, 16G: French dressing, 16H: coffee.

[0087] Figures 17A-17C Is a photograph of leather samples treated with the coating system disclosed herein used in industrial trials.

[0088] Figures 18A-18I is a photograph of a felt pad (and related leather samples treated with the coating system disclosed herein) after 600 consecutive wet Veslic rub cycles (Note: Figure 18H Only 360 cycles were performed).

[0089] Figures 19A-19D Is a photograph showing the results of Bally flexural testing of various leather samples treated with the coating system disclosed herein.

[0090] Figures 20A-20I are photographs showing the results of adhesive tape testing conducted on various leather samples treated with the adhesive coating system.

[0091] Figure 21 Is a photograph showing the difference between leather samples treated with the adhesive coating system disclosed herein before and after milling.

[0092] Figures 22A-22I are photographs showing the results of adhesive tape testing conducted on various leather samples treated with the adhesive coating system disclosed herein.

[0093] Figure 23 are photographs showing the difference in leather samples treated with the adhesive coating system disclosed herein before and after abrasion.

[0094] Figures 24A-24B are photographs showing the difference in adhesive tape testing performed on leather samples treated with the adhesive coating system disclosed herein before and after abrasion.

[0095] Figures 25A-25C is a microscopic cross-sectional image of a leather surface treated with the coating system disclosed herein.

[0096] Figures 26A-26C Is a microscopic top view image of a leather surface treated with the coating system disclosed herein.

[0097] Figures 27A-27C 27A: Side view, 27B: Top grain view, 27C: Flesh view of leather.

[0098] Figures 28A-28C 28A: Top view, 28B: Side view, 28C: Back view.

[0099] Figures 29A-29C 29A: Top view, 29B: Side view, 29C: Back view.

[0100] Figures 30A-30C 30A: Top view, 30B: Side view, 30C: Back view.

[0101] Figure 31 A picture showing the tensile testing process of AS-104+2% glycerol+50 mM magnesium sulfate film.

[0102] Figure 32 Proposed formulation mechanisms containing varying concentrations of AS-104, 2% glycerol, and salt are shown.

[0103] Figure 33A The elongation at break for AS-104, 2% glycerol, and guanidine hydrochloride (5, 10, 25, and 50 mM) is shown.

[0104] Figure 33B The ultimate tensile strength of AS-104, 2% glycerol, and guanidine hydrochloride (5, 10, 25, and 50 mM) is shown.

[0105] Figure 34A The elongation at break is shown for AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM).

[0106] Figure 34B The ultimate tensile strength of AS-104, 2% glycerol, and sodium chloride (5, 10, 25, and 50 mM) is shown.

[0107] Figure 35A The elongation at break of AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM) is shown.

[0108] Figure 35B The ultimate tensile strength of AS-104, 2% glycerol, and urea (5, 10, 25, and 50 mM) is shown.

[0109] Figure 36A The elongation at break for AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM) is shown.

[0110] Figure 36B The ultimate tensile strength of AS-104, 2% glycerol, and L-arginine hydrochloride (5, 10, 25, and 50 mM) is shown.

[0111] Figure 37A The elongation at break for AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM) is shown.

[0112] Figure 37B The ultimate tensile strength of AS-104, 2% glycerol, and magnesium sulfate heptahydrate (5, 10, 25, and 50 mM) is shown.

[0113] Figure 38A The elongation at break for AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM) is shown.

[0114] Figure 38B The ultimate tensile strength of AS-104, 2% glycerol, and ammonium sulfate (5, 10, 25, and 50 mM) is shown.

[0115] Figure 39A The elongation at break for AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM) is shown.

[0116] Figure 39B The ultimate tensile strength of AS-104, 2% glycerol, and calcium chloride (5, 10, 25, and 50 mM) is shown.

[0117] Figure 40A The elongation at break for AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM) is shown.

[0118] Figure 40B The ultimate tensile strength of AS-104, 2% glycerol, and magnesium chloride (5, 10, 25, and 50 mM) is shown.

[0119] Figure 41A The elongation at break for AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM) is shown.

[0120] Figure 41B The ultimate tensile strength of AS-104, 2% glycerol, and calcium sulfate dihydrate (5, 10, 25, and 50 mM) is shown.

[0121] Figure 42AThe elongation at break for AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM) is shown.

[0122] Figure 42B The ultimate tensile strength of AS-104, 2% glycerol, and calcium lactobionate (5, 10, 25, and 50 mM) is shown.

[0123] Figure 43 All data on elongation at break are summarized.

[0124] Figure 44 All data are summarized for ultimate tensile strength.

[0125] Figure 45 Shows the results of Veslic wet and dry tests on 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-9S11-25mML-arginine hydrochloride.

[0126] Figure 46 Shows the results of Veslic wet and dry tests on Bodin Brown 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-9S11-25mML-arginine hydrochloride.

[0127] Figure 47 Shown are 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.

[0128] Figure 48Shown are 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.

[0129] Figure 49A and Figure 49B Shown is the coating with silk + 0.5% wt. GG by point filling before ( Figure 23 A) and after ( Figure 23 B) Topographic traces of a leather sample before and after coating with GG-filament. The traces were captured using a Taylor Hobson CCI HD optical profilometer.

[0130] As noted in the discussion, although the above-identified drawings illustrate embodiments of the present disclosure, other embodiments are also contemplated. This disclosure presents exemplary embodiments by way of illustration and not limitation. Numerous other modifications 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

[0131] In some embodiments, the present disclosure provides a composition comprising a coating comprising two components. In some embodiments, the second component is impregnated on the first component. In some embodiments, the second component undergoes a phase change (such as, but not limited to, Tg, polymerization, etc.). The first coating described herein may include, but is not limited to, a polymer disclosed herein or any protein, such as biodegradable polyurethane, silk protein, collagen, casein, elastin, etc. The second coating described herein may include, but is not limited to, a cellulose derivative disclosed herein. The order of the first coating and the second coating should not be limited, as any coating disclosed herein is interchangeable with any other coating disclosed herein. Although ethyl cellulose may generally be brittle and may crack, in some embodiments, the present disclosure provides a flexible ethyl cellulose coating. The present disclosure provides coatings for any surface, not limited to, for example, protective coatings for leather, fabrics, wood, and food (fruits, vegetables, etc.). In some embodiments, the coatings disclosed herein are made from two or more thin films (perhaps starting with a single film made from two polymers), distributed as a single layer, and applied to a substrate. As disclosed herein, the composite materials and / or coatings disclosed herein can be based on (but not limited to) molecular entanglement, where the EC is crosslinker-free. In some embodiments, all layers are held together by molecular interactions. In some embodiments, all molecular interactions are solidified, coagulated, or polymerized. In some embodiments, the molecular interactions between the two layers solidify the film and the molecules form a larger polymer structure. In some embodiments, the outer layer described herein comprises 1% to 100% EC on the surface. In some embodiments, the first layer (when applied relative to the surface to be coated): participates in molecular entanglement, such as the first and second layers becoming sticky; the first layer can adhere to uneven surfaces; the first layer is thermoplastic, self-assembles, and is soluble in the solvent used for the second layer; the first layer polymerizes by crosslinking, self-assembly, and in some embodiments, the first layer is soluble and can be solidified. In some embodiments, polymers or proteins, such as, but not limited to, silk fibroin, play a role in the first layer. In some embodiments, the second layer (deposited over the first and outer layers) is made of ethylcellulose (EC) or a biomaterial or polymer in a molecular dispersion; in some embodiments, this layer contains approximately 1-5 g / L (volume) of EC in a solvent. In some embodiments, this layer can provide dyes, silk, or other molecules to modulate optical, tactile, and mechanical properties. In some embodiments, the EC acts as a protective barrier that enhances 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 be attached to a dynamic first layer substrate. In some embodiments, the EC can be attached to uneven first layer surfaces. In some embodiments, the majority of the EC faces outward, facing the external environment / forces.In some embodiments, proteins or polymers, such as but not limited to silk, function in the second layer.

[0132] Silk-coated leather articles and methods of making the same are described in WO 2020 / 018821 and WO 2021 / 146654, each of which is incorporated herein by reference in its entirety.

[0133] Leather is a material produced by treating the skin removed from animals using a series of physical, mechanical, and chemical methods, followed by tanning. Leather is composed of woven collagen fiber bundles, with trace amounts of elastic and reticular fibers, of which collagen accounts for 95% to 98%. The natural weave structure of collagen fibers in natural leather is such that thicker fiber bundles are sometimes separated into several finer fiber bundles, and these finer fiber bundles are sometimes combined with other fiber bundles to form another larger fiber bundle.

[0134] Leather in its natural state is a nonwoven material in which the fibrils of the fibers grow together. The silk fibroin and collagen fibers in leather are natural proteins composed of 22 proteinogenic amino acids. Silk fibroin has a high affinity for leather fibers (collagen fibers) due to the presence of hydrophilic amino acid residues in silk fibroin (e.g., due to the physical entanglement of silk fibroin fragments with leather fibers through hydrogen bonding), such as the -OH group from serine, the guanidine group from arginine, the free amine group from lysine, and the -COOH group from aspartic acid and glutamic acid.

[0135] In some embodiments, the fibroin-based protein fragments and solutions described herein can be used as color performance enhancers for leather or leather products. In some embodiments, the present disclosure provides silk-treated leather or leather products that exhibit good dyeability, excellent color fastness, and enhanced color saturation.

[0136] Leather and leather products are treated with fibroin-based protein fragments and solutions, enhancing the quality and aesthetic properties of natural leather using non-toxic, sustainable, natural silk-based compositions. The silk treatment process disclosed herein develops leather products while respecting the tradition and craftsmanship of leather tanning and design without disrupting the leather tanning and design process.

[0137] SPF definition and properties

[0138] As used herein, "silk protein fragment" (SPF) includes, but is not limited to, one or more of the following: a "fibroin fragment" as defined herein; a "recombinant silk fragment" as defined herein; a "spider silk fragment" as defined herein; a "fibroin-like protein fragment" as defined herein; a "chemically modified silk fragment" as defined herein; and / or a "sericin or sericin fragment" as defined herein. The SPF 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 a variant thereof, an amino acid sequence of a naturally occurring silk polypeptide, or a combination of both.

[0139] SPF molecular weight and polydispersity

[0140] In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 to about 70 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 70 to about 75 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 75 to about 80 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 80 to about 85 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 85 to about 90 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 90 to about 95 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 95 to about 100 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 to about 170 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 170 to about 175 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 175 to about 180 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 180 to about 185 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 185 to about 190 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 190 to about 195 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 195 to about 200 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 200 to about 205 kDa.In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 205 to about 210 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 210 to about 215 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 215 to about 220 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 220 to about 225 kDa. In one embodiment, the compositions of the present disclosure comprise an SPF having an average weight average molecular weight selected from about 225 to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 to about 255 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 to about 260 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 to about 270 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 270 to about 275 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 275 to about 280 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 280 to about 285 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 285 to about 290 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 to about 300 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 to about 310 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 to about 330 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 330 to about 335 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 335 to about 340 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 340 to about 345 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 345 to about 350 kDa.

[0141] In some embodiments, the compositions of the present disclosure comprise an SPF composition selected from compositions #1001 to #2450 having a weight average molecular weight selected from about 1 kDa to about 145 kDa and a polydispersity selected from the group consisting of: 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:

[0142]

[0143]

[0144]

[0145]

[0146] As used herein, "low molecular weight," "low MW," or "low-MW" SPF may include an SPF 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 certain SPFs can be a weight average molecular weight of about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, 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.

[0147] As used herein, "medium molecular weight," "medium MW," or "mid-MW" SPFs may 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 medium molecular weight for certain SPFs may 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.

[0148] As used herein, "high molecular weight," "high MW," or "high-MW" SPFs may include SPFs 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 certain SPFs may 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.

[0149] In some embodiments, the molecular weights described herein (e.g., low molecular weight, medium molecular weight, high molecular weight) can be converted to the approximate number of amino acids contained in each SPF, as understood by those of ordinary skill in the art. For example, the average weight of amino acids can be approximately 110 Daltons (i.e., 110 g / mol). Therefore, in some embodiments, the molecular weight of a linear protein divided by 110 Daltons can be used to approximately calculate the number of amino acid residues contained therein.

[0150] In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from 1 to about 5.0, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from 1 to about 1.5, including but not limited to a polydispersity of 1. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 1.5 to about 2.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 2.0 to about 2.5. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 2.5 to about 3.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 3.0 to about 3.5. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 3.5 to about 4.0. In one embodiment, the SPF in the compositions of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.5 to about 5.0.

[0151] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of 1.

[0152] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.1.

[0153] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.2.

[0154] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.3.

[0155] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.4.

[0156] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.5.

[0157] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.6.

[0158] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.7.

[0159] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.8.

[0160] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.9.

[0161] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.0.

[0162] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.1.

[0163] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.2.

[0164] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.3.

[0165] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.4.

[0166] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.5.

[0167] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.6.

[0168] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.7.

[0169] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.8.

[0170] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.9.

[0171] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.0.

[0172] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.1.

[0173] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.2.

[0174] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.3.

[0175] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.4.

[0176] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.5.

[0177] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.6.

[0178] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.7.

[0179] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.8.

[0180] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.9.

[0181] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.0.

[0182] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.1.

[0183] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.2.

[0184] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.3.

[0185] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.4.

[0186] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.5.

[0187] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.6.

[0188] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.7.

[0189] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.8.

[0190] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.9.

[0191] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 5.0.

[0192] In some embodiments, in compositions described herein having a combination of low, medium, and / or high molecular weight SPFs, such low, medium, and / or high molecular weight SPFs may have the same or different polydispersities.

[0193] Silk fibroin fragments

[0194] Methods for preparing silk fibroin or silk fibroin fragments and their use in various fields are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, 10,166,177, 10,287,728, and 10,301,768, all of which are incorporated herein in their entireties. Raw silk from the silkworm is composed of two major proteins: fibroin (about 75%) and sericin (about 25%). Fiber fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. As used herein, the term "fibroin" refers to fibers from the cocoon of the silkworm Bombyx mori having a weight-average molecular weight of about 370,000 Da. Crude silkworm fiber consists of double strands of fibroin. The adhesive holding these twin fibers together is sericin. Silk fibroin consists of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with large hydrophobic domains (of high molecular weight) that constitute the majority of the polymer. These hydrophobic regions are interrupted by small hydrophilic spacers, and the N- and C-termini of the chains are also highly hydrophilic. The hydrophobic domain of the H chain contains a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeating dipeptides of Gly-Ala / Ser / Tyr, which form stable antiparallel-sheet crystallites. The L chain's amino acid sequence is non-repeating, making it more hydrophilic and relatively elastic. The alternating arrangement of hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) segments within the silk fibroin molecule allows for its self-assembly.

[0195] Provided herein are methods for producing pure and highly scalable silk fibroin fragment mixture solutions that can be used for a variety of applications across multiple industries. 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 proteins or silk fibroin-like proteins.

[0196] As used herein, the term "fibroin" includes both silk fibroin and insect or spider silk proteins. In one embodiment, the silk fibroin is obtained from the silkworm, Bombyx mori. Raw silk from the silkworm is composed of two major proteins: fibroin (about 75%) and sericin (about 25%). Fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. As used herein, the term "fibroin" refers to fibers from the cocoon of the silkworm, Bombyx mori, having a weight-average molecular weight of about 370,000 Da. Converting these insoluble silk fibroin fibrils into water-soluble silk fibroin fragments requires the addition of concentrated neutral salts (e.g., 8-10 M lithium bromide), which interfere with the inter- and intramolecular ionic and hydrogen bonding that originally makes silk fibroin insoluble in water. Methods of preparing fibroin fragments and / or compositions thereof are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0197] Raw silk cocoons from silkworms are cut into fragments. The silk cocoon fragments are treated in an aqueous solution of Na2CO3 at about 100°C for about 60 minutes to remove sericin (degumming). The volume of water used is equal to about 0.4x the weight of raw silk, and the amount of Na2CO3 is about 0.848x the weight of the raw silk cocoon fragments. The resulting degummed silk cocoon fragments are rinsed three times with deionized water at about 60°C (each rinse for 20 minutes). The volume of rinse water for each cycle is 0.2L x the weight of the raw silk cocoon fragments. Excess water is removed from the degummed silk cocoon fragments. After the deionized water washing step, the wet degummed silk cocoon fragments are dried at room temperature. The degummed silk cocoon fragments are mixed with a LiBr solution, and the mixture is heated to about 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 fibroin. The resulting fibroin solution was filtered and dialyzed against deionized water using tangential flow filtration (TFF) using a 10 kDa membrane for 72 hours. The resulting aqueous fibroin solution had a concentration of approximately 8.5% by weight. The 8.5% silk solution was then diluted with water to produce a 1.0% w / v silk solution. TFF was then used to further concentrate the pure silk solution to a concentration of 20.0% w / w silk / water.

[0198] Silk dialysis using a series of water changes is a manual and time-intensive process that can be accelerated by modifying certain parameters, such as diluting the silk solution prior to dialysis. The dialysis process can be scaled up using semi-automated equipment, such as a tangential flow filtration system.

[0199] In some embodiments, silk solutions were prepared under various preparation conditions, such as 90°C for 30 min, 90°C for 60 min, 100°C for 30 min, and 100°C for 60 min. 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 a 60°C oven. Samples were removed from each group at 4, 6, 8, 12, 24, 168, and 192 hours.

[0200] In some embodiments, silk solutions were prepared under various preparation conditions, 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 were removed from each group at 1, 4, and 6 hours.

[0201] In some embodiments, silk solutions were prepared under various preparation conditions, such as using four different silk extraction combinations: 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3M 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 grams of silk and placed in an oven at the same temperature as the LiBr solution. Samples were removed from each group at 1, 4, and 6 hours. 1 mL of each sample was added to 7.5 mL of 9.3M LiBr and refrigerated for viscosity testing.

[0202] In some embodiments, SPF is obtained by dissolving raw, undegummed, partially degummed, or degummed silkworm fibers with a neutral lithium bromide salt. The raw silk is treated at a selected temperature and other conditions to remove any sericin and achieve the desired weight-average molecular weight (Mw) and polydispersity (PD) of the fragment mixture. The selection of process parameters can be varied to achieve different final silk protein fragment properties depending on the intended use. The resulting final fragment solution is a fibroin fragment and water with process contaminants ranging from parts per million (ppm) to undetectable levels, which is an acceptable level in the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further varied depending on the desired use and performance requirements.

[0203] Figure 1 Flowcharts showing various embodiments for producing pure silk fibroin fragments (SPF) of the present disclosure. It should be understood that not all steps shown are necessary to prepare all silk solutions of the present disclosure. Figure 1, as shown in step A, cocoons (heat-treated or not), silk fibers, silk powder, spider silk or recombinant spider silk can be used as the silk source. If starting with raw silk cocoons from silkworms, the cocoons can be cut into small pieces, for example pieces of roughly equal size, step B1. The raw silk is then extracted and rinsed to remove any sericin, step C1a. This produces raw silk that is essentially free of sericin. In one 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 boiling water / Na2CO3 (100°C) and immersed for about 15-90 minutes, where boiling for a longer time produces smaller silk protein fragments. In one embodiment, the water volume is equal to about 0.4x the weight of the raw silk, and the Na2CO3 volume is equal to about 0.848x the weight of the raw silk. In one embodiment, the water volume is equal to 0.1x the weight of the raw silk, and the Na2CO3 volume is maintained at 2.12g / L.

[0204] Subsequently, the aqueous NaCO solution is drained and excess water / NaCO is removed from the fibroin fibers (e.g., by manually squeezing out the fibroin extract, using a rotary cycle, etc.). The resulting fibroin extract is rinsed with warm to hot water, typically at a temperature ranging from about 40°C to about 80°C, to remove any residual adsorbed sericin or contaminants, with the water volume changed at least once (repeated as needed). The resulting fibroin extract is substantially sericin-free fibroin. In one embodiment, the resulting fibroin extract is rinsed with water at a temperature of about 60°C. In one embodiment, the volume of rinse water per cycle is equal to 0.1 L to 0.2 L x the weight of the raw silk. It may be advantageous to stir, tumble, or circulate the rinse water to maximize the rinsing effect. After rinsing, excess water is removed from the extracted fibroin fibers (e.g., by manually or by machine squeezing out the fibroin extract). Alternatively, methods known to those skilled in the art, such as pressure, temperature, or other agents, or a combination thereof, can be used for sericin extraction. Alternatively, the silk glands can be directly removed from the insect (100% sericin-free silk). This will result in liquid silk without sericin, without any change in the protein structure.

[0205] The extracted silk 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 and boiling temperature (step C1b). In one embodiment, the solvent is a lithium bromide (LiBr) solution (LiBr has a boiling point of 140°C). Alternatively, the extracted silk fibroin fibers are not dried, but rather wet and placed in the solvent; the solvent concentration can then be varied to achieve a concentration similar to that achieved when the dried silk fibers are added to the solvent. The final concentration of the LiBr solvent can range from 0.1 M to 9.3 M. Complete dissolution of the extracted silk fibroin fibers can be achieved by varying the treatment time and temperature, as well as the concentration of the dissolving solvent. Other solvents can be used, including but not limited to phosphate phosphate, calcium nitrate, calcium chloride solutions, or other concentrated aqueous inorganic salt solutions. To ensure complete dissolution, the silk fibers should be completely immersed in the heated solvent solution and then maintained at a temperature of about 60°C to about 140°C for 1-168 hours. In one 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.

[0206] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an impact on the time required to completely dissolve the silk fibroin and the resulting molecular weight and polydispersity of the final SPF mixture solution. In one embodiment, the silk solvent solution concentration is less than or equal to 20% w / v. In addition, stirring during introduction or dissolution can be used to promote dissolution at different temperatures and concentrations. The temperature of the LiBr solution will provide control over the molecular weight and polydispersity of the resulting silk protein fragment mixture. In one embodiment, higher temperatures will dissolve the silk faster to provide enhanced process scalability and large-scale production of silk solutions. In one embodiment, using a LiBr solution heated to a temperature of 80°C to 140°C reduces the time required to achieve complete dissolution in an oven. Changing the time and dissolving the solvent at a temperature of 60°C or above will change and control the MW and polydispersity of the SPF mixture solution formed from native silk fibroin of original molecular weight.

[0207] Alternatively, extraction can be bypassed by placing the entire cocoon directly into a solvent, such as LiBr (step B2). This requires subsequently filtering the silkworm particles from the silk and solvent solution and removing the sericin using methods known in the art for separating hydrophobic and hydrophilic proteins (e.g., 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 common examples of standard protein separation methods and are not limiting (step C2). Alternatively, extraction can be bypassed by placing the unheated cocoon, from which the silkworms have been removed, into a solvent, such as LiBr. This method can be used for sericin isolation, with the advantage that the unheated cocoon will contain significantly less insect debris.

[0208] Dialysis can be used to remove the dissolving solvent from the resulting dissolved silk fibroin fragment solution by dialyzing the solution against a volume of water, step E1. Prefiltration prior to dialysis helps remove any debris (i.e., silkworm residue) from the silk and LiBr solution, step D. In one example, a 0.1% to 1.0% silk-LiBr solution is filtered using a 3 μm or 5 μm filter at a flow rate of 200-300 mL / min prior to dialysis and, if desired, concentration. The methods disclosed herein, as described above, utilize time and / or temperature to reduce the concentration from 9.3 M LiBr to a range of 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering establishing a scalable process. Alternatively, without using additional time or temperature, the 9.3 M LiBr-silk fibroin fragment solution can be diluted with water to facilitate debris filtration and dialysis. The result of dissolution under filtration at the desired time and temperature is a translucent, particle-free, room temperature storage-stable silk fibroin fragment-LiBr solution of known MW and polydispersity. It is advantageous to periodically change the dialysis water until the solvent has been removed (e.g., change the water after 1 hour, 4 hours, and then every 12 hours, for a total of 6 changes of water). The total number of water volume changes can be varied based on the resulting concentration of the solvent used for silk protein solubilization and fragmentation. After dialysis, the final silk solution can be further filtered to remove any remaining debris (i.e., silkworm residue).

[0209] Alternatively, tangential flow filtration (TFF), which is a rapid and efficient method for separating and purifying biomolecules, can be used to remove the solvent from the resulting dissolved silk fibroin solution, step E2. TFF provides a highly pure aqueous solution of silk fibroin fragments and ensures the scalability of the process to produce large quantities of solution in a controlled and reproducible manner. The silk-LiBr solution can be diluted (from 20% to 0.1% silk in water or LiBr) before TFF. Prefiltration as described above before TFF processing can maintain filtration efficiency and potentially avoid the creation of a silk gel boundary layer on the filter surface due to the presence of debris particles. Prefiltration before TFF also helps to remove any residual debris (i.e., silkworm residues) from the silk-LiBr solution that may cause spontaneous or long-term gelation of the resulting water-only solution, step D. Recirculating or single-pass TFF can be used to produce water-silk protein fragment solutions ranging from 0.1% silk to 30.0% silk (more preferably, 0.1%-6.0% silk). TFF membranes of varying cutoff sizes may be required based on the desired concentration, molecular weight, and polydispersity of the silk protein fragment mixture in the solution. Membranes ranging from 1 to 100 kDa may be required for silk solutions of varying molecular weights, for example, by varying the length of the extraction boiling time or the time and temperature in the dissolving solvent (e.g., LiBr). In one embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution and produce the desired final silk:water ratio. Following removal of the dissolving solvent (e.g., LiBr), the solution can also be concentrated using single-pass TFF, TFF, and other methods known in the art, such as falling film evaporation (to achieve desired concentrations ranging from 0.1% to 30% silk). This can be used as an alternative to standard HFIP concentration methods known in the art for preparing water-based solutions. Larger pore membranes can also be used to filter out small silk protein fragments and produce solutions of higher molecular weight silk with or without narrower polydispersity values.

[0210] The assay for LiBr and Na2CO3 detection can be performed using an HPLC system equipped with an evaporative light scattering detector (ELSD). Calculations are performed by linear regression of the resulting peak areas of the analytes plotted against concentration. More than one sample of many formulations disclosed herein is used for sample preparation and analysis. Typically, four samples of different formulations are weighed directly into a 10 mL volumetric flask. The sample is suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and kept at 2-8°C for 2 hours with occasional shaking to extract the analytes from the membrane. After 2 hours, the solution is diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask is transferred to an HPLC vial and injected into the HPLC-ELSD system to estimate sodium carbonate and lithium bromide.

[0211] The analytical method developed for the quantification of Na2CO3 and LiBr in silk fibroin preparations was found to be linear over the range of 10-165 μg / mL, with injection precision RSDs of 2% and 1% by area for sodium carbonate and 0.38% and 0.19% by retention time for lithium bromide, respectively. The analytical method can be used for the quantitative determination of sodium carbonate and lithium bromide in silk fibroin preparations.

[0212] Figure 2 is a flow chart illustrating various parameters that can be modified during the extraction and solubilization steps in the process of producing a silk protein fragment solution of the present disclosure. The selected process parameters can be varied to achieve different final solution properties, such as molecular weight and polydispersity, depending on the intended application. It should be understood that not all illustrated steps are required to prepare all silk solutions of the present disclosure.

[0213] In one embodiment, a silk protein fragment solution that can be used for a variety of applications is prepared according to the following steps: forming silk cocoon fragments from silkworms; extracting the fragments in a Na2CO3 aqueous solution at about 100°C for about 60 minutes, wherein the water volume is equal to about 0.4× the weight of the raw silk and the Na2CO3 amount is about 0.848× the weight of the fragments to form a fibroin extract; rinsing the fibroin extract in a certain volume of rinse water at about 60°C for three times, each rinse for about 20 minutes, wherein the rinse water in each cycle is equal to about 0.2 L× the weight of the fragments; removing the excess fibroin from the fibroin extract; The method comprises the following steps: measuring water; drying the silk fibroin extract; dissolving the dried silk fibroin extract in a LiBr solution, wherein the LiBr solution is first heated to about 100°C to produce a silk and LiBr solution and maintained; placing the silk and LiBr solution in a drying oven at about 100°C for about 60 minutes to achieve complete dissolution of the native silk protein structure and further fragmentation into a mixture with the desired molecular weight and polydispersity; filtering the solution to remove any residual debris from the silkworm; diluting the solution with water to produce a 1.0 wt% silk solution; and removing the solvent from the solution using tangential flow filtration (TFF). In one embodiment, a 10 kDa membrane is used to purify the silk solution and establish the final desired silk: water ratio. TFF can then be used to further concentrate the silk solution to a concentration of 2.0 wt% silk in water.

[0214] Without wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution when added to the fibroin extract (or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent-silk solutions with varying viscosities, uniformities, and colors. Also without wishing to be bound by any particular theory, increasing the extraction temperature, extending the extraction time, using a higher temperature LiBr solution initially and over time when dissolving the silk, and increasing the time at temperature (e.g., in an oven or alternative heat source as shown here) all resulted in solvent-silk solutions with lower viscosities and more uniformity.

[0215] The extraction step can be completed in a larger vessel, such as an industrial washing machine that can be maintained at a temperature of 60°C to 100°C or therebetween. The rinsing step can also be completed in an industrial washing machine to eliminate manual rinse cycles. The dissolution of silk in LiBr solution can be carried out in a vessel other than a convection oven, such as a stirred tank reactor. Silk dialysis by a series of water changes is a manual and time intensive process that can be accelerated by changing certain parameters, such as diluting the silk solution before dialysis. The dialysis process can be scaled up by using semi-automated equipment (e.g., a tangential flow filtration system).

[0216] Varying the extraction (i.e., time and temperature), LiBr (i.e., the temperature of the LiBr solution when added to the fibroin extract (or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent-silk solutions with varying viscosities, homogeneities, and colors. Increasing the extraction temperature, extending the extraction time, using higher-temperature LiBr solutions initially and over time when dissolving the silk, and increasing the time at temperature (e.g., in an oven or alternative heat source, as shown here) all resulted in solvent-silk solutions with lower viscosities and more homogeneous properties. While nearly all parameters resulted in viable silk solutions, methods that achieved complete dissolution in less than 4 to 6 hours were preferred for process scalability.

[0217] In one embodiment, a solution of silk fibroin fragments having a weight average molecular weight of about 6 kDa to about 17 kDa is prepared according to the following steps: degumming a 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 comprising undetectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having an initial temperature of 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; and preparing an aqueous solution of silk fibroin fragments, the aqueous solution comprising fragments having a weight average molecular weight of 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 the silk fibroin fragments may contain less than 300 ppm of residual lithium bromide, as measured using a high-performance liquid chromatography (HPLC) lithium bromide assay. The aqueous solution of the silk fibroin fragments may contain less than 100 ppm of residual sodium carbonate, as measured using a HPLC sodium carbonate assay. The aqueous solution of the silk fibroin fragments may be freeze-dried. In some embodiments, the silk fibroin fragment solution may be further processed into various forms, including gels, powders, and nanofibers.

[0218] In one embodiment, a solution of fibroin fragments having a weight average molecular weight of about 17 kDa to about 39 kDa is prepared according to 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 about 60 minutes to cause degumming; removing sericin from the solution to produce a fibroin extract containing undetectable levels of sericin; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C; and The method further comprises the steps of: providing a fibroin extract having a starting temperature; maintaining the fibroin-lithium bromide solution in a drying oven at a temperature of about 60° C. to about 100° C. for up to 1 hour; removing lithium bromide from the fibroin extract; and preparing an aqueous solution of fibroin fragments, wherein the aqueous solution of fibroin fragments comprises from about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of fibroin fragments comprises from about 10 ppm to about 100 ppm of sodium carbonate residues, wherein the aqueous solution of fibroin fragments comprises fragments having a weight average molecular weight of from about 17 kDa to about 39 kDa and a polydispersity of from 1 to about 5 or from about 1.5 to about 3.0. The method further comprises drying the fibroin extract prior to the dissolving step. The aqueous solution of fibroin fragments may comprise less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of fibroin fragments may comprise less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay.

[0219] In some embodiments, a method for preparing an aqueous solution of silk fibroin fragments having an average weight average molecular weight of about 6 kDa to about 17 kDa comprises the following steps: degumming a 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 comprising an undetectable level of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having an initial temperature of 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 at least 1 hour; removing the lithium bromide from the silk fibroin extract; and preparing an aqueous solution of silk fibroin fragments comprising fragments having an average weight average molecular weight of 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 comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin fragments may contain less than 300 ppm of residual lithium bromide, as measured using a high-performance liquid chromatography (HPLC) lithium bromide assay. The aqueous solution of pure silk fibroin fragments may contain less than 100 ppm of residual sodium carbonate, as measured using a HPLC sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin fragments. The method may further include adding a molecule selected from the group consisting of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin 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 thereof to the aqueous solution of pure silk fibroin fragments at a concentration of about 0.5% to about 10.0%. The method may further include adding at least one of zinc oxide or titanium dioxide. A film can be made from an aqueous solution of pure silk fibroin fragments produced by this method. The film can contain from about 1.0% to about 50.0% by weight of vitamin C or its derivatives. The film can have a water content ranging from about 2.0% to about 20.0% by weight. The film can contain from about 30.0% to about 99.5% by weight of pure silk fibroin fragments. A gel can be made from an aqueous solution of pure silk fibroin fragments produced by this method. The gel can contain from about 0.5% to about 20.0% by weight of vitamin C or its derivatives. The gel can have a silk content of at least 2% and a vitamin content of at least 20%.

[0220] In some embodiments, a method for preparing an aqueous solution of fibroin fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa comprises 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 about 60 minutes to cause degumming; removing sericin from the solution to produce a fibroin extract comprising an undetectable level of sericin; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a temperature in the range of about 80° C. to about 140° C.; and The method further comprises: preparing an aqueous solution of pure fibroin fragments, wherein the aqueous solution of pure fibroin fragments comprises about 10 ppm to about 300 ppm of lithium bromide residues, wherein the aqueous solution of fibroin fragments comprises about 10 ppm to about 100 ppm of sodium carbonate residues, and wherein the aqueous solution of pure fibroin fragments comprises fragments having an average weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5 or about 1.5 to about 3.0. The method further comprises drying the fibroin extract before the dissolving step. The aqueous solution of pure silk fibroin fragments may contain less than 300 ppm of residual lithium bromide, as measured using a high-performance liquid chromatography (HPLC) lithium bromide assay. The aqueous solution of pure silk fibroin fragments may contain less than 100 ppm of residual sodium carbonate, as measured using a HPLC sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin fragments. The method may further include adding a molecule selected from one of an antioxidant and an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin 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 thereof to the aqueous solution of pure silk fibroin fragments at a concentration of about 0.5% to about 10.0%. The method may further include adding at least one of zinc oxide or titanium dioxide. A film can be made from an aqueous solution of pure silk fibroin fragments produced by this method. The film can contain from about 1.0% to about 50.0% by weight of vitamin C or its derivatives. The film can have a water content ranging from about 2.0% to about 20.0% by weight. The film can contain from about 30.0% to about 99.5% by weight of pure silk fibroin fragments. A gel can be made from an aqueous solution of pure silk fibroin fragments produced by this method. The gel can contain from about 0.5% to about 20.0% by weight of vitamin C or its derivatives.The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

[0221] In one embodiment, a solution of fibroin fragments having a weight average molecular weight selected from the group consisting of about 39 kDa to about 80 kDa is prepared according to 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 cause degumming; removing sericin from the solution to produce a fibroin extract comprising undetectable levels of sericin; draining the solution from the fibroin extract; dissolving the fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C when the fibroin extract is placed in the lithium bromide solution; maintaining the fibroin-lithium bromide solution in a drying oven at a temperature in the range of about 60°C to about 100°C for up to 1 hour; removing the lithium bromide from the fibroin extract; and preparing an aqueous solution of fibroin fragments, wherein the aqueous solution of fibroin fragments comprises about 1 0 ppm to about 300 ppm of lithium bromide residues, about 10 ppm to about 100 ppm of sodium carbonate residues, 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 also include drying the silk fibroin extract before the dissolving step. The aqueous solution of the silk fibroin fragments may contain less than 300 ppm of lithium bromide residues as measured using a high performance liquid chromatography lithium bromide assay. The aqueous solution of the silk fibroin fragments may contain less than 100 ppm of sodium carbonate residues as measured using a high performance liquid chromatography sodium carbonate assay. In some embodiments, the method may also include adding an active agent (e.g., a therapeutic agent) to the aqueous solution of pure silk fibroin fragments. The method may also include adding an active agent selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin fragments may be freeze-dried. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin 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 thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin fragments prepared by this method. A film may be prepared from the aqueous solution of pure silk fibroin fragments prepared by this method. The film may contain about 1.0% to about 50.0% vitamin C or a derivative thereof by weight. 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% pure silk fibroin fragments by weight. A gel may be prepared from the aqueous solution of pure silk fibroin fragments prepared by this method. The gel may comprise from about 0.5 wt % to about 20.0 wt % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2 wt % and a vitamin content of at least 20 wt %.

[0222] The molecular weight of the silk protein fragments can be controlled based on specific parameters used during the extraction step, including extraction time and temperature; specific parameters used during the dissolution step, including the LiBr temperature when the silk is immersed in lithium bromide and the time the solution is held at a specific temperature; and specific parameters used during the filtration step. By controlling the process parameters using the disclosed method, silk fibroin fragment solutions with a polydispersity of 2.5 or less can be produced at various molecular weights selected from 5 kDa to 200 kDa, or 10 kDa to 80 kDa. By varying the process parameters to obtain silk solutions with varying molecular weights, a final product fragment mixture with a desired polydispersity of 2.5 or less can be targeted based on desired performance requirements. For example, a higher molecular weight silk film containing an ophthalmic drug can have a controlled, slower release rate compared to a lower molecular weight film, making it an ideal delivery vehicle for use in eye care products. Additionally, silk fibroin fragment solutions with a polydispersity greater than 2.5 can be obtained. Furthermore, two solutions with different average molecular weights and polydispersities can be mixed to produce a combined solution. Alternatively, liquid silk glands directly removed from insects (100% sericin-free silk) can be used in combination with any of the fibroin fraction solutions disclosed herein. The molecular weight of the pure fibroin fraction compositions was determined using high-pressure liquid chromatography (HPLC) with a refractive index detector (RID). Polydispersity was calculated using Cirrus GPCOnline GPC / SEC software version 3.3 (Agilent).

[0223] Differences in processing parameters can produce regenerated silk fibroin with different molecular weights and peptide chain size distributions (polydispersity, PD), which in turn affect the properties of regenerated silk fibroin, including mechanical strength and water solubility.

[0224] Parameters were varied during the processing of raw silk cocoons into silk solution. These parameters affected the MW of the resulting silk solution. The manipulated parameters included (i) extraction time and temperature, (ii) LiBr temperature, (iii) dissolution oven temperature, and (iv) dissolution time. Experiments were conducted to determine the effects of varying extraction time. Tables AG summarize the results. The following is a summary:

[0225] - 30 minutes of sericin extraction time resulted in a higher molecular weight than 60 minutes of sericin extraction time

[0226] -Molecular weight decreases with time in the oven

[0227] -140℃ LiBr and oven drying result in the lower limit of the confidence interval being below the molecular weight of 9500Da

[0228] -30 min extraction with undigested silk at 1 hour and 4 hours time points

[0229] -30 min extraction resulted in a significantly higher molecular weight at the 1 h time point, with the lower limit of the confidence interval being 35,000 Da

[0230] - The molecular weights achieved at the upper limit of the confidence interval range from 18000 to 216000 Da (important to provide a solution with the specified upper limit).

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] Experiments were conducted to determine the effects of varying the extraction temperature. Table G summarizes the results. Here is a summary:

[0238] -Sericin extraction at 90°C resulted in higher MW than sericin extraction at 100°C

[0239] Both -90°C and 100°C showed a decrease in MW with time in the oven.

[0240]

[0241] Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) as it was added to the silk. Table HI summarizes the results. Here is a summary:

[0242] - No effect on molecular weight or confidence intervals (all CIs approximately 10,500-6,500 Da)

[0243] -The study showed that since most of the material is silk at room temperature, as LiBr is added and begins to dissolve, the temperature of the LiBr-silk solution drops rapidly to below the initial LiBr temperature.

[0244]

[0245]

[0246] Experiments were conducted to determine the effect of varying the oven / dissolution temperature. Table JN summarizes the results.

[0247] Here's the summary:

[0248] - Oven temperature has less effect on silk extracted for 60 minutes than on silk extracted for 30 minutes. Without wishing to be bound by theory, it is believed that the 30-minute silk is less degraded during the extraction process, so the oven temperature has a greater effect on the larger MW, less degraded silk fraction.

[0249] - For the 60°C vs. 140°C ovens, the 30 minute extracted silk showed a very clear effect of lower MW at the higher oven temperature, while the 60 minute extracted silk had an effect but was much smaller

[0250] The -140°C oven resulted in a lower limit of the confidence interval of approximately 6000 Da.

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257] Raw silk cocoons from silkworms are cut into pieces. The raw silk cocoon pieces are boiled in an aqueous solution of Na2CO3 (approximately 100°C) for a period of about 30 to about 60 minutes to remove sericin (degumming). The volume of water used is equal to about 0.4 times the weight of the raw silk, and the amount of Na2CO3 is about 0.848 times the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces are rinsed three times with deionized water at about 60°C (20 minutes per rinse). The volume of rinse water for each cycle is 0.2 L x the weight of the raw silk cocoon pieces. Excess water is removed from the degummed silk cocoon pieces. After the deionized water washing step, the wet degummed silk cocoon pieces are dried at room temperature. The degummed silk cocoon pieces are mixed with a LiBr solution, and the mixture is heated to about 100°C. The heated mixture is placed in a drying oven and heated at a temperature of about 60°C to about 140°C for about 60 minutes to achieve complete dissolution of the natural silk protein. The resulting solution was cooled to room temperature and then dialyzed using a 3,500 Da MWCO membrane to remove the LiBr salt. Multiple exchanges were performed in deionized water until the solution was as described in Oakton Bromide (Br - ) The Br ion measured in the hydrolyzed silk fibroin solution read on a double-junction ion selective electrode was less than 1 ppm.

[0258] The resulting silk fibroin aqueous solution has a concentration of about 8.0% w / v of pure silk fibroin fragments having 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 was diluted with deionized water to provide 1.0% w / v, 2.0% w / v, 3.0% w / v, 4.0% w / v, 5.0% w / v on a coating solution basis.

[0259] Various % silk concentrations were prepared using tangential flow filtration (TFF). In all cases, a 1% silk solution was used as the input feed. Starting volumes of 1% silk solution ranged from 750-18,000 mL. The solution was diafiltered in TFF to remove lithium bromide. Once below a specified residual LiBr level, the solution was subjected to ultrafiltration to increase the concentration by removing water. See the examples below.

[0260] Six (6) silk solutions were used in the standard silk structure and the results were as follows:

[0261] Solution #1 was 5.9 wt% silk concentration, average MW of 19.8 kDa, and 2.2 PDI (prepared using 60 min boiling extraction, 100°C LiBr dissolution for 1 hour).

[0262] Solution #2 was 6.4 wt% silk concentration (prepared using a 30 minute boiling extraction and a 4 hour LiBr dissolution at 60°C).

[0263] Solution #3 was 6.17 wt% silk concentration (prepared using a 30 minute boiling extraction and 100°C LiBr dissolution for 1 hour).

[0264] Solution #4 is a 7.30% silk concentration by weight: A 7.30% silk solution was produced starting with 30-minute extraction batches of 100g silk cocoons. The extracted silk fibers were then dissolved in a 100°C oven using 9.3M LiBr at 100°C for 1 hour. 100g of silk fibers were dissolved in each batch to produce a 20% silk in LiBr solution. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5μm filter to remove large debris. 15,500mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for the TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 1300mL. 1262mL of 7.30% silk was then collected. Water was added to the feed to aid in removing the remaining solution, and 547mL of 3.91% silk was collected.

[0265] Solution #5 is a 6.44 wt% silk concentration: A 6.44 wt% silk solution was produced starting with mixed 60-minute extraction batches of 25, 33, 50, 75, and 100 g of silk cocoons. The extracted silk fibers were then dissolved in a 100°C oven using 9.3 M LiBr at 100°C for 1 hour. 35, 42, 50, and 71 g of silk fibers were dissolved in each batch to produce a 20% silk in LiBr solution and combined. The silk solution in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 17,000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for the TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 3000 mL. 1490 mL of 6.44% silk was then collected. Water was added to the feed to aid in removing the remaining solution, and 1454 mL of 4.88% silk was then collected.

[0266] Solution #6 is a 2.70% silk concentration by weight: 2.70% silk solutions were produced starting with 60-minute extraction batches of 25 g of silk cocoons. The extracted silk fibers were then dissolved in a 100°C oven using 9.3 M LiBr at 100°C for 1 hour. 35.48 g of silk fibers were dissolved per batch to produce a 20% silk solution in LiBr. The silk solution in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 1000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for the TFF. Once the LiBr was removed, the solution was ultrafiltered to a volume of approximately 300 mL. 312 mL of 2.7% silk was then collected.

[0267] The preparation of fibroin solutions with higher molecular weight is given in Table O.

[0268] Table O. Preparation and properties of silk fibroin solution.

[0269]

[0270] The silk aqueous coating compositions for application to the fabric are given in Tables P and Q below.

[0271]

[0272]

[0273]

[0274] Three (3) silk solutions were used in membrane fabrication with the following results:

[0275] Solution #1 was 5.9% silk concentration, average MW of 19.8 kDa, and 2.2 PD (prepared using 60 min boiling extraction and 100 °C LiBr dissolution for 1 h).

[0276] Solution #2 was a 6.4% silk concentration (prepared using a 30-minute boiling extraction and a 4-hour LiBr dissolution at 60°C).

[0277] Solution #3 was 6.17% silk concentration (prepared using a 30 minute boiling extraction and 100°C LiBr dissolution for 1 hour).

[0278] Membranes were prepared according to Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published online September 22, 2011; doi:10.1038 / nprot.2011.379). 4 mL of a 1% or 2% (wt / vol) silk solution in water was added to a 100 mm Petri dish (the silk volume can be varied for thicker or thinner membranes and is not critical) and left to dry overnight. The bottom of a vacuum desiccator was filled with water. The dried membrane was placed in the desiccator and vacuum was applied to water anneal the membrane for 4 hours before removal from the dish. Membranes cast from solution #1 did not yield structurally continuous membranes; the membranes broke into several pieces. Despite water annealing, these membrane fragments dissolved in water.

[0279] Silk solutions of various molecular weights and / or molecular weight combinations can be optimized for gel applications. An example of this approach is provided below, but is not intended to be limiting in application or formulation. Three (3) silk solutions were used in gel fabrication, with the following results:

[0280] Solution #1 was 5.9% silk concentration, average MW of 19.8 kDa, and 2.2 PD (prepared using 60 min boiling extraction, 100°C LiBr dissolution for 1 hour).

[0281] Solution #2 was 6.4% silk concentration (prepared using a 30 minute boiling extraction and a 4 hour LiBr dissolution at 60°C).

[0282] Solution #3 was 6.17% silk concentration (prepared using a 30 minute boiling extraction and 100°C LiBr dissolution for 1 hour).

[0283] "Egel" refers to the electrogelation method described by Rockwood et al. Briefly, 10 ml of a silk solution in water was added to a 50 ml conical tube, and a pair of platinum wire electrodes were immersed in the silk solution. A 20 volt potential 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 Egel during the 5-minute application of current.

[0284] Solutions #2 and #3 were gelled according to the published horseradish peroxidase (HRP) procedure. The behavior appeared typical for the published solutions.

[0285] Materials and Methods: The following equipment and materials were used in the determination of silk molecular weight: Agilent 1100 with ChemStation software version 10.01; refractive index detector (RID); analytical balance; volumetric flasks (1000 mL, 10 mL, and 5 mL); HPLC-grade water; ACS-grade sodium chloride; ACS-grade sodium phosphate dibasic heptahydrate; phosphoric acid; dextran MW standards (nominal molecular weights of 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa); 50 mL PET or polypropylene disposable centrifuge tubes; graduated pipettes; amber glass HPLC vials with Teflon caps; and a Phenomenex PolySep GFC P-4000 column (dimensions: 7.8 mm x 300 mm).

[0286] Procedure:

[0287] A) Preparation of 1 L of mobile phase (0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer)

[0288] Take a clean, dry 250mL beaker, place it on a scale, and tar it. Add approximately 3.3509g of sodium phosphate dibasic heptahydrate to the beaker. Record the exact weight of the sodium phosphate dibasic heptahydrate. Dissolve the weighed sodium phosphate by adding 100mL of HPLC water to the beaker. Be careful not to spill any of the contents of the beaker. Carefully transfer the solution to a clean, dry 1000mL volumetric flask. Rinse the beaker and transfer the rinse solution to the volumetric flask. Repeat the rinse 4-5 times. In a separate, clean, dry 250mL beaker, accurately weigh approximately 5.8440g of sodium chloride. Dissolve the weighed sodium chloride in 50mL of water and transfer the solution to the sodium phosphate solution in the volumetric flask. Rinse the beaker and transfer the rinse solution to the volumetric flask. Adjust the pH of the solution to 7.0 ± 0.2 with phosphoric acid. Bring the volume of the volumetric flask to 1000mL with HPLC water and shake vigorously to mix the solution evenly. Filter the solution through a 0.45μm 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 required by varying the amounts of disodium hydrogen phosphate heptahydrate and sodium chloride accordingly.

[0289] B) Preparation of Dextran Molecular Weight Standard Solution

[0290] Use at least five different molecular weight standards for each sample run so that the expected values ​​for the test samples are encompassed by the values ​​of the standards used. Label six 20 mL scintillation glass vials as molecular weight standards. Accurately weigh approximately 5 mg of each dextran molecular weight standard and record the weight. Dissolve the dextran molecular weight standard in 5 mL of mobile phase to prepare a 1 mg / mL standard solution.

[0291] C) Preparation of sample solution

[0292] When preparing the sample solution, if there are limitations on the amount of sample available, the preparation can be scaled up, as long as the ratio is maintained. Depending on the sample type and the silk protein content in the sample, weigh enough sample to prepare a 1 mg / mL sample solution for analysis into a 50 mL disposable centrifuge tube on an analytical balance. Dissolve the sample in an equal volume of mobile phase to prepare a 1 mg / mL solution. Tightly cap the tubes and mix the sample (in solution). Let the sample solution stand at room temperature for 30 minutes. Gently mix the sample solution for another minute and centrifuge it at 4000 RPM for 10 minutes.

[0293] D) HPLC analysis of samples

[0294] Transfer 1.0 mL of all standard and sample solutions to separate HPLC vials. Inject the molecular weight standards (one injection each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions:

[0295] column PolySep GFC P-4000(7.8x 300mm) Column temperature 25℃ detector Refractive index detector (temperature 35°C) Injection volume 25.0μL Mobile phase 0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer flow 1.0mL / min Runtime 20.0min

[0296] E) Data Analysis and Calculation - Average Molecular Weight Calculation using Cirrus Software

[0297] Upload the chromatographic data files of the standard and analytical samples to the Cirrus SEC data collection and molecular weight analysis software. Calculate the weight average molecular weight (M) of each injected sample. w ), number average molecular weight (M n ), peak average molecular weight (M p ) and polydispersity.

[0298] spider silk fragments

[0299] Spider silk is a natural polymer composed of three domains: a repetitive central core domain that dominates the protein chain, and non-repetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, with two basic sequences alternating between a crystalline polypeptide (poly(A) or poly(GA)) and a less crystalline polypeptide (GGX or GPGXX). Dragline silk is a protein complex composed of major ampullate dragline protein 1 (MaSp1) and major ampullate dragline protein 2 (MaSp2). Both silks are approximately 3,500 amino acids long. MaSp1 is found in the fiber core and periphery, while MaSp2 forms clusters in certain core regions. The large central domain of MaSp1 and MaSp2 is organized in a block copolymer-like arrangement, with two basic sequences—crystalline polypeptides (poly(A) or poly(GA)) and less crystalline polypeptides (GGX or GPGXX)—alternating within the core domain. Specific secondary structures have been assigned to poly(A) / (GA), GGX, and GPGXX motifs, including β-sheets, α-helices, and β-helices, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain determine the mechanical properties of spider silk; whereas the non-repetitive N- and C-terminal domains are crucial for storing the liquid silk dope in the lumen and for fiber formation in the spinning duct.

[0300] The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which make up 15% of the total amino acid content in MaSp2, while MaSp1 contains no prolines. By counting the number of proline residues in N. clavipes dragline silk, it was possible to estimate the presence of both proteins in the fiber: 81% MaSp1 and 19% MaSp2. Different spiders have varying ratios of MaSp1 and MaSp2. For example, dragline silk fibers from the orb weaver Argiope aurantia contain 41% MaSp1 and 59% MaSp2. This variation in the ratio of large ampullate silk can determine the properties of the silk fiber.

[0301] At least seven different types of silk proteins are known for a single species of Theridiidae spider. The silks differ in their primary sequence, physical properties, and function. For example, dragline silks, used to construct the framework, radii, and lifelines, are known for their exceptional mechanical properties, including strength, toughness, and elasticity. Weight for weight, spider silk is tougher than steel and Kevlar. Flageliform silks, found in capture spirals, have extensibility of up to 500%. Small ampullate silks, found in the auxiliary spirals of orb-webs and prey wrapping, have high toughness and strength nearly similar to large ampullate silks, but do not supercontract in water.

[0302] Spider silks are known for their high tensile strength and toughness. Recombinant silk proteins also impart advantageous properties to cosmetic or dermatological compositions, particularly improved hydration or softening, good film-forming properties, and low surface density. These diverse and unique biomechanical properties, combined with biocompatibility and slow degradation rates, make spider silk an excellent candidate as a biomaterial for tissue engineering, guided tissue repair, and drug delivery, as well as for cosmetic products (e.g., nail and hair strengtheners, skin care products), and as industrial materials (e.g., nanowires, nanofibers, surface coatings).

[0303] In one embodiment, 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 natural spider silk proteins and recombinant spider silk proteins, such as variants, analogs, and derivatives of natural spider silk proteins. For excellent toughness, the polypeptide may be derived from major dragline silk proteins produced in the spider's ampulla gland. Examples of major dragline silk proteins include the major ampulla gland spider silk proteins MaSp1 and MaSp2 from Nephila clavipes, and ADF3 and ADF4 from Araneus diadematus. Examples of polypeptides derived from major dragline silk proteins include variants, analogs, and derivatives of major dragline silk proteins. Furthermore, the polypeptide may be derived from whip silk proteins produced in the spider's whip gland. Examples of whip silk proteins include whip silk proteins derived from Nephila clavipes.

[0304] Examples of polypeptides derived from the major dragline protein include polypeptides containing two or more units of the amino acid sequence represented by Formula 1: REP1-REP2 (1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, the polypeptide derived from the major dragline protein may be a polypeptide containing units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453 or an amino acid sequence having 90% or more homology to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453 at the C-terminus. In the polypeptide derived from the major dragline protein, the units of the amino acid sequence represented by Formula 1: REP1-REP2 (1) may be the same as or different from each other. In the case of using a microorganism such as Escherichia coli as a host to produce a recombinant protein, the molecular weight of the polypeptide derived from the main dragline protein is 500 kDa or less, or 300 kDa or less, or 200 kDa or less, taking productivity into consideration.

[0305] In formula (1), REP1 refers to polyalanine. In REP1, the number of consecutively arranged alanine residues is preferably 2 or greater, more preferably 3 or greater, further preferably 4 or greater, and particularly preferably 5 or greater. Furthermore, in REP1, the number of consecutively arranged alanine residues is preferably 20 or less, more preferably 16 or less, further 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 greater, preferably 60% or greater, and more preferably 70% or greater relative to the total number of amino acid residues contained therein.

[0306] In the primary dragline, REP1 corresponds to the crystalline region of the fiber, where crystalline β-sheets are formed, while REP2 corresponds to the amorphous region of the fiber, where most of the strands lack a regular structure and exhibit greater flexibility. Furthermore, [REP1-REP2] corresponds to the repeating region (repeating sequence) composed of the crystalline and amorphous regions, a characteristic sequence of dragline proteins.

[0307] Recombinant silk fragment

[0308] In some embodiments, the recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide, or a recombinant spidroin polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids, or recombinant insect silk polypeptides of the silkworm (Bombyx mori). In some embodiments, the recombinant silk protein fragments disclosed herein include recombinant spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having repeating units derived from natural spider silk polypeptides of the family Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein include block copolymers having synthetic repeating units derived from spider silk polypeptides of the family Araneidae or Araneoids and non-repeating units derived from natural repeating units of spider silk polypeptides of the family Araneidae or Araneoids.

[0309] Recent advances in genetic engineering have provided routes for producing various types of recombinant silk proteins. Recombinant DNA technology has been used to provide more practical sources of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein produced heterologously in a prokaryotic or eukaryotic expression system using genetic engineering methods.

[0310] Various methods for synthesizing recombinant silk peptides are known and 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, rod-shaped bacterium Escherichia coli (E. coli) is a well-established host for industrial-scale protein production. Consequently, most recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively inexpensive, and can be scaled up for larger protein production quantities.

[0311] Recombinant silk proteins can be produced by transforming eukaryotic or prokaryotic systems containing cDNA encoding a silk protein, a fragment of such a protein, or an analog of such a protein. The recombinant DNA approach enables the production of recombinant silk with a programmed sequence, secondary structure, architecture, and precise molecular weight. There are four main steps in the method: (i) design and assembly of synthetic silk-like genes into gene "cassettes", (ii) insertion of such fragments into recombinant DNA vectors, (iii) transformation of such recombinant DNA molecules into host cells, and (iv) expression and purification of selected clones.

[0312] As used herein, the term "recombinant vector" includes any vector known to the skilled artisan, 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 (BAC), yeast artificial chromosomes (YAC) or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the desired coding sequence and the appropriate DNA sequences necessary for expressing the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast or plants) or in an in vitro expression system. Cloning vectors are generally used to engineer and amplify a specific desired DNA fragment and may lack the functional sequences required for expression of the desired DNA fragment.

[0313] Prokaryotic systems include Gram-negative or Gram-positive bacteria. Prokaryotic expression vectors can include an origin of replication recognized by the host organism, a homologous or heterologous promoter functional in the host, and a DNA sequence encoding a spidroin protein, a fragment of such a protein, or an analogous protein. Non-limiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Pseudomonas, Paracoccus, Bacillus (e.g., Bacillus subtilis), Brevibacterium, Corynebacterium, Rhizobium (Sinorrhiza rhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Propionibacterium, Staphylococcus, or Streptomyces cells.

[0314] Eukaryotic systems include yeast and insect, mammalian or plant cells. In this case, the expression vector may include a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding a spider silk protein, a fragment or an analogous protein, a polyadenylation sequence, a transcription termination site and finally, a selection gene. Non-limiting examples of eukaryotic expression organisms include yeast, such as Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous yeasts, ascosporogenous yeasts, filamentous fungi, such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium acremonium, and the like. chrysogenum), Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia (e.g., Pichia pastoris) or Yarrowia cells, etc., mammalian cells, such as HeLa cells, COS cells, CHO cells, etc., insect cells, such as Sf9 cells, MEL cells, etc., “insect host cells”, such as Spodoptera frugiperda or Trichoplusia ni cells, SF9 cells, SF-21 cells or High-Five cells, wherein SF-9 and SF-21 are ovary cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia ni, “plant host cells”, such as tobacco, potato or pea cells.

[0315] A variety of heterologous host systems have been developed for the production of different types of recombinant silk. Recombinant partial spider silk proteins (apidroins) and engineered silk 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 of the silk proteins produced have N- or C-terminal His tags to facilitate purification and produce sufficient protein.

[0316] In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems may include transgenic animals and plants. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include Escherichia coli. In some embodiments, hosts suitable for expressing recombinant spider silk proteins using heterologous systems include transgenic B. mori silkworms generated using genome editing technology (e.g., CRISPR).

[0317] The recombinant silk proteins of the present disclosure comprise synthetic proteins based on the repeating units of natural silk proteins. In addition to the synthetic repetitive silk protein sequences, these may additionally comprise one or more natural non-repetitive silk protein sequences.

[0318] In some embodiments, "recombinant silk protein" refers to recombinant silk protein or fragments thereof. Recombinant production of silk protein and sericin has been reported. Various hosts have been used for production, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas, Rhodopseudomonas, Bacillus, and Streptomyces. See EP 0230702, which is incorporated herein by reference in its entirety.

[0319] Also provided herein is the design and biosynthesis of fibroin-like multiblock polymers comprising the GAGAGX hexapeptide (X is A, Y, V, or S) derived from the repeating domain of the silk heavy chain (H chain).

[0320] In some embodiments, the present disclosure provides a silk protein-like multi-block polymer derived from a repeating domain of a silk heavy chain (H chain) comprising a GAGAGS hexapeptide repeating unit. The GAGAGS hexapeptide is the core unit of the H chain and plays an important role in the formation of the crystalline domain. The silk protein-like multi-block polymer containing the GAGAGS hexapeptide repeating unit spontaneously aggregates into a β-pleated structure similar to natural silk fibroin, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0321] In some embodiments, the present disclosure provides a silk peptide-like multi-block copolymer composed of a GAGAGS hexapeptide repeat derived from the H chain of a silk heavy chain and a mammalian elastin VPGVG motif produced by Escherichia coli. In some embodiments, the present disclosure provides a fusion silk fibroin composed of a GAGAGS hexapeptide repeat derived from the H chain of a silk heavy chain and GVGVP produced by Escherichia coli, wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0322] In some embodiments, the present disclosure provides a mixture consisting of (GAGAGS) 16 In some embodiments, the present disclosure provides a B. mori silkworm recombinant protein consisting of (GA GAGS) 16 Repeated fragments and non-repeated fragments produced by E. coli (GAGAGS) 16 -F-COOH, (GAGAGS) 16 -FF-COOH, (GAGAGS) 16 -FFF-COOH, (GAGAGS) 16 -FFFF-COOH, (GAGAGS) 16 -FFFFFFFF-COOH, (GAGA GS) 16-FFFFFFFFFFFF-COOH, wherein F has the following amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG, and wherein the silk protein-like multi-block polymer has any weight average molecular weight described herein.

[0323] 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 cloning has been reported. The recombinant spider silk proteins thus produced comprise a portion of the repeating sequence of spidroin 1, a dragline silk protein from the spider Nephila clavipes. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87: 7120-7124 (1990)). A cDNA clone encoding a portion of the repeating sequence of spidroin 2, the second core protein of the dragline silk from Nephila clavipes, and its recombinant synthesis are described in J. Biol. Chem., 1992, Vol. 267, pp. 19320-19324. Recombinant synthesis of spider silk proteins comprising Nephila clavipes protein fragments and variants by transformed Escherichia coli is described in U.S. Patents 5,728,810 and 5,989,894. The cloning and expression of cDNA encoding ampullate silk proteins are described in U.S. Patents 5,733,771 and 5,756,677. The cloning of cDNA encoding whip silk proteins from orb-web spinning spiders is described in U.S. Patent 5,994,099. U.S. Patent 6,268,169 describes the recombinant synthesis of a spider silk-like protein derived from a repetitive peptide sequence present in natural spider dragline silk from Nephila clavipes using Escherichia coli, Bacillus subtilis, and Pichia pastoris recombinant expression systems. WO 03 / 020916 describes cDNA clones encoding for spider silk proteins having repetitive sequences derived from the large ampullate glands of the golden orb spider Nephila madagascariensis, Nephilase negalensis, Tetragnatha kauaiensis, Tetragnatha versicolor, Argiope aurantia, Argiope trifasciata, Gasteracantha mammosa, and Latrodectus geometricus, the whip glands of Argiope trifasciata, the ampullate glands of Dolomedes tenebrosus, the two sets of silk glands of Plectreurys tristis, and the silk glands of the mygalomorph Euagrus chisoseus. Each of the above references is incorporated herein by reference in its entirety.

[0324] In some embodiments, the recombinant spidroin protein is a hybrid protein of a spidroin protein and an insect silk protein, a spidroin protein and collagen, a spidroin protein and elastic protein, or a spidroin protein and keratin. The spider silk repeating unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that is repeated in a naturally occurring large ampullate polypeptide, such as a dragline silk polypeptide, a small ampullate polypeptide, a whip-like polypeptide, an aggregated silk polypeptide, a grape-like silk polypeptide, or a pyriform silk polypeptide.

[0325] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a synthetic spider silk protein comprising repeating units derived from a natural spider silk protein, a consensus sequence, and optionally one or more natural non-repetitive spider silk protein sequences. The repeating units of the natural spider silk polypeptide may include a dragline spider silk polypeptide or a whip gland spider silk polypeptide of the family Araneidae or Araneoids.

[0326] As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that is repeated in a naturally occurring macroampullar gland polypeptide, such as a dragline silk polypeptide, a microampullar gland polypeptide, a whip-like gland polypeptide, a polymorphic gland silk polypeptide, a grape-like gland silk polypeptide, or a piriform gland silk polypeptide. A "repeat unit" refers to a region in the amino acid sequence that corresponds to or consists of at least one peptide motif (e.g., AAAAAA or GPGQQ) that is repeated in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., the same amino acid sequence) or a region that corresponds to an amino acid sequence that is substantially similar thereto (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 its properties, for example, a silk protein comprising a "substantially similar repeat unit" remains insoluble and maintains its insolubility. A "repeat unit" having an amino acid sequence that is "identical" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3 and / or ADF-4. A "repeat unit" having an amino acid sequence that is "substantially similar" to the amino acid sequence of a naturally occurring silk polypeptide can, for example, be a portion of a silk polypeptide that corresponds to one or more peptide motifs of MaSpI, MaSpII, ADF-3 and / or ADF-4 but has one or more amino acid substitutions at specific amino acid positions.

[0327] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing an amino acid that frequently occurs at a certain position (e.g., "G") and in which other amino acids that are not further identified are replaced by the placeholder "X." In some embodiments, the consensus sequence is at least one of the following: (i) GPGXX, wherein X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, wherein 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.

[0328] The consensus peptide sequences of GPGXX and GGX, i.e., glycine-rich motifs, provide flexibility to silk polypeptides and, consequently, to threads formed from silk proteins containing these motifs. Specifically, iterative GPGXX motifs form a twisted helical structure that imparts elasticity to silk polypeptides. Both major ampullate and flagellar silks possess GPGXX motifs. Iterative GGX motifs are associated with a helical structure with three amino acids per turn and are present in most spider silks. GGX motifs may provide additional elasticity to silk. Iterative polyalanine Ax (peptide) motifs form a crystalline β-sheet structure to provide strength to silk polypeptides, as described, for example, in WO 03 / 057727.

[0329] In some embodiments, the recombinant spider silk protein of the present disclosure comprises two identical repeating units, each of which comprises at least one, preferably one, amino acid sequence selected from the group consisting of: GGRPSDTYG and GGRPSSSYG derived from resilin. Resilin is an elastomeric protein found in most arthropods that provides low stiffness and high strength.

[0330] As used herein, "non-repeating unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repeating (carboxyl terminal) amino acid sequence in a naturally occurring dragline silk polypeptide (i.e., a wild-type non-repeating (carboxyl 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 as described in U.S. Patent 8,367,803, a C16 peptide comprising 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk), and an amino acid sequence engineered from the native sequence of ADF4 from A. diadematus. Non-repeating ADF-4 and its variants exhibit efficient assembly behavior.

[0331] Among synthetic spider silk proteins, the recombinant silk proteins disclosed herein, in some embodiments, comprise a C16 protein having the polypeptide sequence SEQ ID NO: 1 as described in U.S. Patent 8288512. In addition to the polypeptide sequence shown in SEQ ID NO: 1, functional equivalents, functional derivatives, and salts of this sequence are also specifically included.

[0332] As used herein, "functional equivalents" refer to mutants which have an amino acid different from the specifically mentioned amino acid in at least one sequence position of the above-mentioned amino acid sequences.

[0333] In some embodiments, the recombinant spidroin proteins of the present disclosure comprise an effective amount of at least one natural or recombinant silk protein, including spidroin proteins corresponding to Spidroin major 1 described by Xu et al., PNAS, USA, 87, 7120, (1990), Spidroin major 2 described by Hinman and Lewis, J. Biol., Chem., 267, 19320, (1922), recombinant spidroin proteins as described in U.S. patent application 2016 / 0222174 and U.S. Patents 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,8038,097,583, 8,030,024, 7,754,851, 7,148,039, 7,060,260, or patent application WO 95 / 25165. The above-cited references are each incorporated herein by reference in their entirety. Additional recombinant spider silk proteins suitable for use in the recombinant RSPF of the present disclosure include ADF3 and ADF4 from the "major ampulla" of Araneus diadematus.

[0334] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US 2004590196, US 7,754,851, US 2007654470, US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US 200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542, US 2009265344, US 2012684607, US 2004583227, US 8,030,024, US 2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US 20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US 2012697729, US 20150328363, US 9,034,816, US 20130172478, US 9,217,017, US 20170202995, US 8,721,991, US 2008227498, US 9,233,067, US 8,288,512, US 2008161364, US 7,148,039, US 1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902, US7,569,660, US 1999138833、US 2000591632, US 20120065126, US 20100278882, US2008161352, US 20100015070, US 2009513709, US 20090194317, US 2004559286, US200589551, US 2008187824, US 20050266242, US 20050227322 and US 20044418.

[0335] Recombinant silk is also described in other patents and patent applications incorporated herein by reference: US 20190062557, US 20150284565, US 20130225476, US 20130172478, US 20130136779, US 20130109762, US20120252294, US 20110230911, US 20110201783, US 20100298877, US 10,478,520, US 10,253,213, US 10,072,152, US 9,233,067, US 9,217,017, US 9,034,816, US 8,877,903, US 8,729,238, US 8,721,991, US 8,097,583, US 8,034,897, US 8,030,024, US 7,951,908, US 7,868,146 and US 7,754,851.

[0336] In some embodiments, the recombinant spidroin protein of the present disclosure comprises or consists of 2 to 80 repeating units, each of which is independently selected from GPGXX, GGX, and Ax as defined herein.

[0337] In some embodiments, the recombinant spidroin protein of the present disclosure comprises or consists of repeating units, each of which is independently selected from the group consisting of GPGAS, GPGSG, GPGGY, GPGGP, GPGGA, GPGQQ, GPGGG, GPGQG, GPGGS, GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA, AAAAAA, AAAAAAA, AAAAAAAAA, AAAAAAAAAA, GGRPSDTYG, and GGRPSSSYG, (i) GPYGPGASAAAAAAGGYGPGSGQQ, (ii) GS SAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP, (iii) GPGQQGPGQQGPGQQGPGQQ: (iv) GPGGAGGPYGPGGAGGPYGPGGAGGPY, (v) GGTTIIEDLDITIDGADGPITIS EELTI, (vi)PGSSAAAAAAAAASGPGQGQGQGQGQGGRPSDTYG, (vii)SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG, (viii)GGAGGAGGAGGSGGAGGS(SEQ ID NO: 27), (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY, (x) GPYGPGASAAAAAAGGYGPGCGQQ, (xi) GPYGPGASAAAAAAGGYGPGKGQQ, (xii) GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP, (xiii) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP, (xiv) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP, or variants thereof as described in U.S. Pat. No. 8,877,903, such as a synthetic spider peptide having the sequence of GPGAS, GGY, GPGSG in the peptide chain, or the sequence of AAAAAAAA, GPGGY, GPGGP in the peptide chain, or the sequence of AAAAAAAA, GPGQG, GGR in the peptide chain.

[0338] In some embodiments, the present disclosure provides silk-like multi-block peptides that mimic repeating units of amino acids derived from natural spider silk proteins, such as Spidroin major 1 domain, Spidroin major 2 domain, or Spidroin minor 1 domain, and a profile of variation between repeating units without changing their three-dimensional conformation, wherein these silk-like multi-block peptides comprise repeating units of amino acids corresponding to one of the following sequences (I), (II), (III), and / or (IV).

[0339] [(XGG) w (XGA)(GXG) x (AGA) y (G) z AG] p Formula (I), in which: X corresponds to tyrosine or to 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, and has any weight average molecular weight described herein, and / or

[0340] [(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 from 7 to 10, p is an integer, and has any weight average molecular weight described herein, and / or

[0341] [(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), wherein: X" corresponds to tyrosine, glutamine or alanine, 1 is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer.

[0342] In some embodiments, the recombinant spidroin protein or spidroin protein analog comprises amino acid repeating units of sequence (V):

[0343] [(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.

[0344] In some embodiments, the recombinant spider silk protein in 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.

[0345] In some embodiments, the present disclosure provides water-soluble recombinant spider silk proteins produced in mammalian cells. The solubility of spider silk proteins produced in mammalian cells can be attributed to the presence of COOH-terminal amino acids in these proteins, which render them more hydrophilic. These COOH-terminal amino acids are absent in spider silk proteins expressed in microbial hosts.

[0346] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a water-soluble recombinant spider silk protein C16 modified with an amino or carboxyl terminal group selected from the group consisting of the amino acid sequences: GCGGGGGG, GKGGGGGG, GCGGSGGGGSGGGG, GKGGGGGGSGGGG, and GCGGGGGGSGGGG. 16 NR4, C 32 NR4, C16, C32, NR4C 16 NR4, NR4C 32 NR4, NR3C 16 NR3 or NR3C 32 NR3 so that the molecular weight range of the protein is as described in this article.

[0347] In some embodiments, the recombinant spider silk protein of the present disclosure includes a recombinant spider silk protein having a synthetic repeating peptide segment and an amino acid sequence modified 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 includes a recombinant spider silk protein having a repeating peptide unit derived from a natural spider silk protein, such as a Spidroin major 1 domain, a Spidroin major 2 domain, or a Spidroin minor 1 domain as described in U.S. Patent No. 8,367,803, wherein the repeating peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG.

[0348] In some embodiments, the present disclosure provides a recombinant spidroin protein consisting of GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY repeating segments and having a molecular weight as described herein.

[0349] As used herein, the term "recombinant silk" refers to recombinant spider silk and / or silk proteins or fragments thereof. In one embodiment, the spider silk proteins are selected from the group consisting of swathing silk (Achniform glandular silk), egg sac silk (Cylindriform glandular silk), egg case silk (Tubuliform silk), non-viscous dragline silk (Ampullate glandular silk), attaching thread silk (Piriform glandular silk), sticky core silk fiber (Flagelliform glandular silk) and sticky outer silk fiber (Polygonal glandular silk). For example, the recombinant spidroin proteins described herein include 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, and 8,642,734.

[0350] Some organisms produce a variety of silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb-weaving spiders possess six distinct types of glands that produce different silk polypeptide sequences, which are polymerized into fibers adapted to their environment or lifecycle niche. These fibers are named after the gland from which they originate, and the polypeptides are labeled with glandular abbreviations (e.g., "Ma") and "Sp," short for spider silk core protein. In orb-weaving spiders, these types include major ampullate gland (MaSp, also known as dragline silk), minor ampullate gland (MiSp), flagellum gland (Flag), grape-shaped gland (AcSp), tubular gland (TuSp), and piriform gland (PySp). This combination of fiber types, structural domains, and variations in polypeptide sequences across different genera and species presents a wide range of potential properties that can be manipulated through the commercial production of recombinant fibers. To date, the vast majority of work on recombinant silk has focused on major ampullate gland spidroins (MaSp).

[0351] Acetic (AcSp) filaments tend to have high toughness, which is the result of a combination of medium-to-high strength and medium-to-high ductility. AcSp filaments are characterized by large block ("ensemble repeat") size, which typically contains motifs of polyserine and GPX. Tubular (TuSp or Cylindrical) filaments tend to have large diameters, as well as moderate strength and high ductility. TuSp filaments are characterized by their polyserine and polythreonine content, and short segments of polyalanine. Large ampullate (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be one of two subtypes: MaSp1 and MaSp2. MaSp1 filaments are generally less ductile than MaSp2 filaments and are characterized by polyalanine, GX and GGX motifs. MaSp2 filaments are characterized by polyalanine, GGX and GPX motifs. Small ampullate (MiSp) filaments tend to have moderate strength and moderate ductility. MiSp filaments are characterized by GGX, GA, and poly A motifs and typically contain spacer units of approximately 100 amino acids. Flag filaments tend to have extremely high extensibility and moderate strength. Flag filaments are typically characterized by GPG, GGX, and a short spacer motif.

[0352] Silk polypeptides are uniquely composed of a repeat domain (REP) and non-repeat regions (e.g., C-terminal and N-terminal domains) on either side thereof. In one embodiment, the C-terminal and N-terminal domains are both 75-350 amino acids in length. The repeat domain exhibits a hierarchical structure. The repeat domain comprises a series of blocks (also referred to as repeat units). These blocks repeat in the silk repeat domain, sometimes perfectly, sometimes imperfectly (constituting quasi-repeat domains). The length and composition of the blocks vary between different silk types and between different species. Table 1 of U.S. Published Application No. 2016 / 0222174 (incorporated herein in its entirety) lists examples of block sequences from selected species and silk types, with further examples given 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, pg 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg.2603-2605 (2001). In some cases, blocks can be arranged in a regular pattern to form larger macro-repeats that occur multiple times (typically 2-8 times) in a repeat domain of a silk sequence. Repeat blocks within a repeat domain or macro-repeats and repeat macro-repeats within a repeat domain can be separated by spacer units.

[0353] Construction of certain spider silk block copolymer polypeptides from these block and / or large repeat domains according to certain embodiments of the present disclosure is described in U.S. Published Patent Application No. 2016 / 0222174.

[0354] The recombinant block copolymer polypeptide based on spider silk sequence made by gene expression in recombinant prokaryotic or eukaryotic system can be purified according to methods known in the art. In a preferred embodiment, commercially available expression / secretion system can be used to express the recombinant polypeptide thus, and thereafter secreted from host cell to easily purify from surrounding medium. If expression / secretion vector is not used, alternative method relates to purifying the recombinant block copolymer polypeptide from the cell lysate (cell residue after cell integrity destruction) of the prokaryotic or eukaryotic cell derived from the expressed polypeptide. The method for generating such cell lysate is well known to those skilled in the art. In some embodiments, the recombinant block copolymer polypeptide is separated from the cell culture supernatant.

[0355] Recombinant block copolymer polypeptides can be purified by affinity separation, such as by immunological interaction with antibodies that specifically bind to the recombinant polypeptide or by nickel columns for isolating recombinant polypeptides tagged with 6-8 histidine residues at their N- or C-termini. Alternative tags may include FLAG epitopes or hemagglutinin epitopes. Such methods are commonly used by skilled practitioners.

[0356] Solutions of such polypeptides (ie, recombinant silk proteins) can then be prepared and used as described herein.

[0357] 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.

[0358] In one embodiment, a recombinant spider silk protein is provided. The spider silk protein is generally composed of 170 to 760 amino acid residues, such as 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, such as 300 to 400 amino acid residues, and more preferably 340 to 380 amino acid residues. Small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require the use of harsh solvents for dissolution and polymerization. Recombinant spider silk protein may contain more than 760 residues, particularly in the case where the spider silk protein contains more than two fragments derived from the N-terminal portion of the spider silk protein. The spider silk protein comprises an N-terminal fragment composed of at least one fragment (NT) derived from the corresponding portion of the spider silk protein, and a repeating fragment (REP) derived from the corresponding internal fragment of the spider silk protein. Optionally, the spider silk protein comprises a C-terminal fragment (CT) derived from the corresponding fragment of the spider silk protein. The spidroin protein typically comprises a single fragment (NT) derived from the N-terminal portion of the spidroin protein, but in a preferred embodiment, the N-terminal fragment comprises at least two, such as two, fragments (NT) derived from the N-terminal portion of the spidroin protein. Thus, the spidroin protein can be schematically represented by the formula NT m -REP or NT mThe spidroin protein is represented by the formula NT2-REP or NT-REP, or NT2-REP-CT or NT-REP-CT, wherein m is 1 or higher, such as 2 or higher, and preferably an integer in the range of 1-2, 1-4, 1-6, 2-4, or 2-6. Preferred spidroin proteins can be schematically represented by the formula NT2-REP or NT-REP, or NT2-REP-CT or NT-REP-CT. Protein fragments are typically covalently coupled via peptide bonds. In one embodiment, the spidroin protein consists of one or more NT fragments coupled to a REP fragment, which is optionally coupled to a CT fragment.

[0359] 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 Escherichia coli. The protein thus obtained is isolated using standard procedures. Optionally, lipopolysaccharide and other pyrogens are actively removed at this stage.

[0360] In the second step of the method for producing isolated spidroin protein polymers, a solution of the spidroin protein in a liquid medium is provided. The terms "soluble" and "in solution" mean that the protein does not appreciably aggregate at 60,000 × g and does not precipitate from the solvent. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium such as a 10-50 mM Tris-HCl buffer or a phosphate buffer. The liquid medium has a pH of 6.4 or higher and / or an ionic composition that prevents the polymerization of the spidroin protein. In other words, the liquid medium has a pH of 6.4 or higher, an ionic composition that prevents the polymerization of the spidroin protein, or both.

[0361] A skilled artisan can readily prepare an ionic composition that prevents the polymerization of spider silk proteins using the methods disclosed herein. Preferred ionic compositions that prevent the polymerization of spider silk proteins have an ionic strength greater than 300 mM. Specific examples of ionic compositions that prevent the polymerization of spider silk proteins include greater than 300 mM NaCl, 100 mM phosphate, and combinations of these ions that have the desired preventive effect on spider silk protein polymerization, such as a combination of 10 mM phosphate and 300 mM NaCl.

[0362] The presence of the NT fragment improves the stability of the solution and prevents polymer formation under these conditions. This is advantageous when immediate polymerization may not be ideal, such as during protein purification, in large-scale production, or when other conditions need to be optimized. Preferably, the pH of the liquid medium is adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as up to 10.5, to achieve high solubility of the spider silk protein. It is also advantageous to adjust the pH of the liquid medium to a range of 6.4-6.8, which provides sufficient solubility of the spider silk protein, but it is advantageous to subsequently adjust the pH to 6.3 or lower.

[0363] In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or lower and an ionic composition that permits polymerization. That is, if the liquid medium in which the spidroin protein is dissolved has a pH of 6.4 or higher, the pH is lowered to 6.3 or lower. A skilled artisan is familiar with various ways to achieve this, typically involving the addition of a strong or weak acid. If the liquid medium in which the spidroin protein is dissolved has an ionic composition that prevents polymerization, the ionic composition is altered to permit polymerization. A skilled artisan is familiar with various ways to achieve this, such as dilution, dialysis, or gel filtration. If necessary, this step involves lowering the pH of the liquid medium to 6.3 or lower and altering the ionic composition to permit polymerization. Preferably, the pH of the liquid medium is adjusted to 6.2 or lower, such as 6.0 or lower. In particular, from a practical perspective, it may be advantageous to limit the pH drop from 6.4 or 6.4-6.8 in the previous step to 6.3 or 6.0-6.3, such as 6.2, in this step. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range, eg, 4.2-6.3 promotes rapid polymerization.

[0364] In the fourth step, spidroin is polymerized in a liquid medium having a pH of 6.3 or lower and an ionic composition that permits spidroin polymerization. Although the presence of the NT fragment improves the solubility of spidroin at a pH of 6.4 or higher and / or an ionic composition that prevents spidroin polymerization, it accelerates polymer formation at a pH of 6.3 or lower when the ionic composition permits spidroin polymerization. The resulting polymers are preferably solid and macroscopic, and are formed in a liquid medium having a pH of 6.3 or lower and an ionic composition that permits spidroin polymerization. In a preferred embodiment, the pH of the liquid medium in this step is 3 or higher, such as 4.2 or higher. The resulting pH range, e.g., 4.2-6.3, promotes rapid polymerization. The resulting polymers can be provided at the molecular weights described herein and prepared in solution form, which can be used for coating articles, if desired.

[0365] A skilled artisan can readily prepare an ionic composition that allows spidroin polymerization using the methods disclosed herein. Preferred ionic compositions that allow spidroin polymerization have an ionic strength of less than 300 mM. Specific examples of ionic compositions that allow spidroin polymerization include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack a preventive effect on spidroin polymerization, such as a combination of 10 mM phosphate or 20 mM phosphate and 150 mM NaCl. The ionic strength of the liquid medium is preferably adjusted to a range of 1-250 mM.

[0366] Without wishing to be bound by any particular theory, it is believed that the NT fragments have oppositely charged poles and that changes in environmental pH affect the charge balance on the protein surface and subsequently polymerization, whereas salt inhibits the same event.

[0367] At neutral pH, the energetic cost of burying the excess negative charge of the acidic pole is expected to prevent polymerization. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces eventually dominate, explaining the observed salt- and pH-dependent polymerization behavior of NT and NT-containing minispidroins. It is proposed that, in some embodiments, pH-induced NT polymerization and the enhanced fiber assembly efficiency of NT-minispidroins are attributed to changes in surface electrostatic potential, and that clusters of acidic residues at one pole of NT change their charge balance, resulting in a polymerization transition at pH values ​​of 6.3 or lower.

[0368] In a fifth step, the resulting, preferably solid, spider silk protein polymer is separated from the liquid medium. Optionally, this step involves the active removal of lipopolysaccharides and other pyrogens from the spider silk protein polymer.

[0369] Without wishing to be bound by any particular theory, it has been observed that the formation of spider silk protein polymers proceeds via the formation of water-soluble spider silk protein dimers. The present disclosure therefore also provides a method for producing isolated spider silk protein dimers, wherein the first two method steps are as described above. The spider silk protein is present as a dimer in a liquid medium having 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 polymers of the present disclosure consist of polymerized protein dimers. The present disclosure therefore provides novel uses of spider silk proteins, preferably those disclosed herein, for producing spider silk protein dimers.

[0370] According to another aspect, the present disclosure provides polymers of spider silk proteins as disclosed herein. In one embodiment, polymers of such proteins are obtainable by any of the methods for use therewith according to the present disclosure. Thus, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing spider silk protein polymers for recombinant silk-based coatings. According to one embodiment, the present disclosure provides novel uses of spider silk protein dimers, preferably those disclosed herein, for producing isolated spider silk protein polymers for recombinant silk-based coatings. In these uses, it is preferred that the polymers are prepared in a liquid medium having a pH of 6.3 or lower and an ionic composition that allows polymerization of the spider silk protein. In one embodiment, the pH of the liquid medium is 3 or higher, such as 4.2 or higher. The resulting pH range, for example 4.2-6.3, promotes rapid polymerization.

[0371] Using one or more methods of the present disclosure, the polymerization process can be controlled, and this enables optimization of parameters to obtain silk polymers with desired properties and shapes.

[0372] In one embodiment, the recombinant silk proteins described herein include those described in US Patent No. 8,642,734, which is incorporated herein by reference in its entirety.

[0373] In another embodiment, the recombinant silk proteins described herein can be prepared according to the method described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety.

[0374] The amino acid sequence represented by SEQ ID NO: 1 in U.S. Patent No. 9,051,453 is equivalent 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 equivalent 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 equivalent to the amino acid sequence represented by SEQ ID NO: 1 with 29 residues removed from the C-terminus.

[0375] An example of a polypeptide containing a unit of the amino acid sequence represented by formula 1: REP1-REP2 (1) and having at the C-terminus an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 or an amino acid sequence having 90% or greater homology to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453 is a polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453. A polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453 was obtained by the following mutations: In the amino acid sequence of ADF3 (NCBI Accession No. AAC47010, GI: 1263287), an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) was added to its N-terminus, the first to 13 repeat regions were approximately doubled, and translation ended at amino acid residue 1154. In the polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 3.

[0376] In addition, a polypeptide containing a unit of the amino acid sequence represented by Formula 1: REP1-REP2 (1) and having at the C-terminus an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453 or an amino acid sequence having 90% or greater homology to the amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453 can be a protein having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and / or added and has a repeating region consisting of a crystalline region and a non-crystalline region.

[0377] 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 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051,453 is an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of a partial amino acid sequence of ADF4 (NCBI accession number: AAC47011, GI: 1263289) obtained from the NCBI database. In addition, a polypeptide containing 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 has a repeat region consisting of a crystalline region and a non-crystalline region. In addition, 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 MaSp2 having an 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 of U.S. Patent No. 9,051,453 is an amino acid sequence obtained by adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of a partial sequence of MaSp2 (NCBI Accession No.: AAT75313, GI: 50363147) obtained from the NCBI online database. Furthermore, the 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: 17 of U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted and / or added and has a repeating region consisting of a crystalline region and an amorphous region.

[0378] Examples of polypeptides derived from whip silk protein include polypeptides containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2), preferably polypeptides containing 20 or more units thereof, and more preferably polypeptides containing 30 or more units thereof. In the case of producing recombinant proteins using microorganisms such as Escherichia coli as hosts, the molecular weight of the polypeptide derived from whip silk protein is preferably 500 kDa or less, more preferably 300 kDa or less, and further preferably 200 kDa or less, in view of productivity.

[0379] In formula (2), REP 3 refers to an amino acid sequence consisting of Gly-Pro-Gly-Gly-X, wherein X refers to an amino acid selected from the group consisting of Ala, Ser, Tyr and Val.

[0380] A key characteristic of spider silk is that whip silk lacks crystalline regions but instead contains repeating regions composed of amorphous regions. Since main dragline silk and other silks have repeating regions composed of crystalline and amorphous regions, they are expected to possess high stress and stretchability. Meanwhile, whip silk, while not as stress-resistant as main dragline silk, exhibits high stretchability. This is believed to be because most whip silk consists of amorphous regions.

[0381] 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 whipworm silk protein having the amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9,051,453 is an amino acid sequence obtained by combining a partial sequence of the whip silk protein of Nephila clavipes obtained from the NCBI database (NCBI Accession No.: AAF36090, GI: 7106224), specifically the amino acid sequence from residues 1220 to 1659 of the N-terminus (corresponding to the repeat region and motif) (referred to as the PR1 sequence), with a partial sequence of the whip silk protein of Nephila clavipes obtained from the NCBI database (NCBI Accession No.: AAC38847, GI: 2833649), specifically the C-terminal amino acid sequence from residues 816 to 907 of the C-terminus, and thereafter adding an amino acid sequence consisting of a start codon, a His 10 tag, and an HRV3C protease recognition site (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) to the N-terminus of the combined sequence. Furthermore, the polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2: REP3(2) may be a polypeptide having the amino acid sequence represented by SEQ 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 has a repeating region consisting of an amorphous region.

[0382] The polypeptide can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, and it can be prepared by amplifying a gene encoding a natural spider silk protein from a spider-derived cell by polymerase chain reaction (PCR) or the like and cloning it, or it can be chemically synthesized. The method for chemically synthesizing the gene is also not particularly limited, and it can be synthesized as follows, for example, based on information on the amino acid sequence of the natural spider silk protein obtained from the NCBI online database, by ligating oligonucleotides automatically synthesized using AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Corporation) by PCR. At this time, in order to facilitate the purification and observation of the protein, a gene encoding a protein having an amino acid sequence having an amino acid sequence consisting of a start codon and a His 10 tag added to its N-terminus can be synthesized.

[0383] Examples of expression vectors include plasmids, phages, and viruses that can express proteins based on DNA sequences. Plasmid-type expression vectors are not particularly limited, as long as they allow expression of the target gene in host cells and can be amplified. For example, when using Escherichia coli Rosetta (DE3) as a host, pET22b(+) plasmid vectors and pCold plasmid vectors can be used. Of these, pET22b(+) plasmid vectors are preferred in view of protein productivity. Examples of hosts include animal cells, plant cells, and microorganisms.

[0384] The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, one of the two major dragline proteins of Araneus diadematus. This polypeptide has the advantages of generally having high strength-elongation and toughness and being easily synthesized.

[0385] 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 include one or more 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, 9,15 7,070, 9,309,299, 9,644,012, 9,708,376, 9,051,453, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069 and 10,329,332;and U.S. Patent Publication Nos. 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066, 2014 / 0079674, 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 0141618, 2015 / 0291673, 2015 / 0291674, 2015 / 0239 587, 2015 / 0344542, 2015 / 0361144, 2015 / 0374833, 2015 / 0376247, 2016 / 0024464, 2017 / 0066804, 2017 / 0066805, 2015 / 0293076, 2016 / 0222174, 2017 / 0283474, 2017 / 0088675, 2019 / 0135880, 2015 / 0329587, 2019 / 0040109, 2019 / 0135881, 2 019 / 0177363, 2019 / 0225646, 2019 / 0233481, 2019 / 0031842, 2018 / 0355120, 2019 / 0186050, 2019 / 0002644, 2020 / 0031887, 2018 / 0273590, 20191 / 094403, 2019 / 0031843, 2018 / 0251501, 2017 / 0066805, 2018 / 0127553, 2019 / 0329526, 2020 / 0031886, 2018 / 0080147, 2019 / 0352349, 2020 / 0043085, 2019 / 0144819, 2019 / 0228449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191 (each of which is incorporated herein by reference in its entirety).

[0386] fibroin-like protein fragments

[0387] The recombinant silk protein in the present disclosure comprises a synthetic protein based on the repeating unit of natural silk protein. In addition to the synthetic repeating silk protein sequence, these can additionally comprise one or more natural non-repeating silk protein sequences. As used herein, "fibroin-like protein fragment" refers to a protein fragment having a molecular weight and polydispersity as defined herein and a certain degree of homology with a protein selected from natural silk protein, fibroin heavy chain, fibroin light chain or any protein comprising one or more GAGAGS six amino 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%.

[0388] As described herein, proteins such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS six amino acid repeating units comprise from 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, proteins such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS six amino acid repeating units comprise from 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, proteins such as native silk proteins, fibroin heavy chains, fibroin light chains, or any protein comprising one or more GAGAGS six-amino acid repeating units comprise 9% to about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0389] In some embodiments, the fibroin-like protein described herein comprises 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% or about 55% glycine. In some embodiments, the fibroin-like protein described herein comprises 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 fibroin-like proteins described herein comprise 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 fibroin-like proteins described herein may independently comprise any amino acid known to be contained in natural fibroin. In some embodiments, the fibroin-like proteins described herein may independently not comprise any amino acid known to be contained in natural fibroin. In some embodiments, an average of 2 / 6 amino acids, 3 / 6 amino acids, or 4 / 6 amino acids in the fibroin-like proteins described herein are glycine. In some embodiments, an average of 1 / 6 amino acids, 2 / 6 amino acids, or 3 / 6 amino acids in the fibroin-like proteins described herein are alanine. In some embodiments, an average of 0 / 6 amino acids, 1 / 6 amino acids, or 2 / 6 amino acids in the fibroin-like proteins described herein are serine.

[0390] Sericin or sericin fragments

[0391] Raw silk is primarily composed of fibroin fibers coated with the adhesive substance sericin. Sericin is a gelatinous silk protein that coats the surface of silk threads. In addition to glycine and alanine, it also contains chemically reactive, bulky amino acids such as serine, threonine, and aspartic acid. Throughout the silk production process, sericin plays a crucial role in controlling silk solubility and producing high-quality silk. Furthermore, it serves as an adhesive protein, playing a crucial role. When silk fibers are used as clothing, much of the sericin coating is removed and discarded, making it a valuable, unused resource.

[0392] In some embodiments, the silk protein fragments described herein include sericin or sericin fragments. Methods for preparing sericin or sericin fragments and their applications in various fields are known and described herein, and are also described 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 entirety.

[0393] In some embodiments, sericin removed from raw silk cocoons (e.g., in a degumming step) can be collected and used in the methods described herein.Sericin can also be reconstituted from a powder and used in the compositions and methods of the present disclosure.

[0394] Other properties of SPF

[0395] The compositions of the present disclosure are "biocompatible" or exhibit "biocompatibility," meaning that the compositions are compatible with living tissues or living systems by being non-toxic, non-harmful, or non-physiologically reactive and not causing immune rejection or an inflammatory response. Such biocompatibility can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of: about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely. For example, in some embodiments, the coatings described herein are biocompatible coatings.

[0396] In some embodiments, the compositions described herein (which can be biocompatible compositions) (e.g., biocompatible coatings comprising silk) can be evaluated and conform to the International Standard ISO 10993-1 entitled "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein (which can be biocompatible compositions) can be evaluated for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation according to ISO 106993-1.

[0397] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants applying the compositions of the present disclosure topically to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0398] In one embodiment, the stability of the composition of the present disclosure is about 1 day. In one embodiment, the stability of the composition of the present disclosure is about 2 days. In one embodiment, the stability of the composition of the present disclosure is about 3 days. In one embodiment, the stability of the composition of the present disclosure is about 4 days. In one embodiment, the stability of the composition of the present disclosure is about 5 days. In one embodiment, the stability of the composition of the present disclosure is about 6 days. In one embodiment, the stability of the composition of the present disclosure is about 7 days. In one embodiment, the stability of the composition of the present disclosure is about 8 days. In one embodiment, the stability of the composition of the present disclosure is about 9 days. In one embodiment, the stability of the composition of the present disclosure is about 10 days.

[0399] In one embodiment, the stability of the composition 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.

[0400] In one embodiment, the stability of the composition of the present disclosure is from 10 days to 6 months. In one embodiment, the stability of the composition of the present disclosure is from 6 months to 12 months. In one embodiment, the stability of the composition of the present disclosure is from 12 months to 18 months. In one embodiment, the stability of the composition of the present disclosure is from 18 months to 24 months. In one embodiment, the stability of the composition of the present disclosure is from 24 months to 30 months. In one embodiment, the stability of the composition of the present disclosure is from 30 months to 36 months. In one embodiment, the stability of the composition of the present disclosure is from 36 months to 48 months. In one embodiment, the stability of the composition of the present disclosure is from 48 months to 60 months.

[0401] In one embodiment, the SPF composition of the present disclosure is insoluble in aqueous solution due to the crystallinity of the protein. In one embodiment, the SPF composition of the present disclosure is soluble in aqueous solution. In one embodiment, the SPF of the composition of the present disclosure comprises approximately 2 / 3 crystalline and approximately 1 / 3 amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises approximately half crystalline and approximately half amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 99% crystalline and 1% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 95% crystalline and 5% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 90% crystalline and 10% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 85% crystalline and 15% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 80% crystalline and 20% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 75% crystalline and 25% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 70% crystalline and 30% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 65% crystalline and 35% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 60% crystalline and 40% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 50% crystalline and 50% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 40% crystalline and 60% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 35% crystalline and 65% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 30% crystalline and 70% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 25% crystalline and 75% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 20% crystalline and 80% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 15% crystalline and 85% amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises 10% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 5% crystalline portion and 90% amorphous region. In one embodiment, the SPF of the composition of the present disclosure comprises 1% crystalline portion and 99% amorphous region.

[0402] As used herein, the term "substantially free of inorganic residues" refers to a composition that exhibits 0.1% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of inorganic residues refers to a composition that exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of inorganic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of inorganic residues is from ND to about 500 ppm. In one embodiment, the amount of inorganic residues is from ND to about 400 ppm. In one embodiment, the amount of inorganic residues is from ND to about 300 ppm. In one embodiment, the amount of inorganic residues is from ND to about 200 ppm. In one embodiment, the amount of inorganic residues is from ND to about 100 ppm. In one embodiment, the amount of inorganic residues is from 10 ppm to 1000 ppm.

[0403] As used herein, the term "substantially free of organic residues" refers to a composition that exhibits 0.1% (w / w) or less residues. In one embodiment, substantially free of organic residues refers to a composition that exhibits 0.05% (w / w) or less residues. In one embodiment, substantially free of organic residues refers to a composition that exhibits 0.01% (w / w) or less residues. In one embodiment, the amount of organic residues is from 0 ppm ("not detectable" or "ND") to 1000 ppm. In one embodiment, the amount of organic residues is from ND to about 500 ppm. In one embodiment, the amount of organic residues is from ND to about 400 ppm. In one embodiment, the amount of organic residues is from ND to about 300 ppm. In one embodiment, the amount of organic residues is from ND to about 200 ppm. In one embodiment, the amount of organic residues is from ND to about 100 ppm. In one embodiment, the amount of organic residues is from 10 ppm to 1000 ppm.

[0404] The compositions of the present disclosure exhibit "biocompatibility," meaning that the compositions are compatible with living tissues or living systems because they are non-toxic, harmless, or non-physiologically reactive and do not cause immune rejection. Such biocompatibility can be demonstrated by participants applying the compositions of the present disclosure topically on their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days, in one embodiment, the extended period is about 14 days, in one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0405] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants applying the compositions of the present disclosure topically to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0406] As used herein, in some embodiments, the terms "leather" and / or "leather substrate" refer to natural leather, and may be derived from cowhide, sheepskin, lambskin, horsehide, crocodile, alligator, birdhide, or another known animal hide as will be understood in the art, or processed leather. Unprocessed, processed, coated, and / or repaired leather may include, but is not limited to, modified leather, aniline leather, bonded leather, brushed leather, tumbled leather, bycast leather, suede leather, chrome-tanned leather, combination-tanned leather, cordovan leather, nubuck leather, crockproof leather, drum-finished leather, embossed leather, reinforced grain leather, grain leather, metallized leather, bare leather, natural grain leather, nubuck leather, patent leather, pearlized leather, pressed leather, printed leather, protected leather, pure aniline leather, tanned / retanned leather, round-hand leather, saddle leather, semi-aniline leather, shrunken leather, split leather, split leather, suede leather, and wet-blue leather. In some embodiments, the term "leather" may refer to synthetic or reconstructed leather, including but not limited to leather constructed partially / completely of cellulose, mushroom-based materials, synthetic materials such as vinyl, synthetic materials such as polyamide or polyester.

[0407] As used herein, the term "hand" refers to the feel of a material, which can be further described as soft, crisp, dry, silky, smooth, and combinations thereof. The hand of a material is also referred to as "drape." Materials with a hard hand feel are coarse and rough, and the wearer generally feels less comfortable. Materials with a soft hand feel are smooth and slippery, and the wearer generally feels more comfortable. The hand of a material can be determined by comparing a collection of material samples, or by methods such as the Kawabata Evaluation System (KES) or the Fabric Assurance by Simple Testing (FAST) method. Behera and Hari, Ind. J. Fibre & Textile Res., 1994, 19, 168-71. In some embodiments, and as described herein, silk can change the hand of leather, as can be assessed by the SynTouch Touch-Scale method or another method described herein.

[0408] As used herein, "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 leather products. In some embodiments, a portion of the coating may at least partially penetrate into the substrate. In some embodiments, the coating may at least partially penetrate into the voids of the substrate. In some embodiments, the coating may be injected into the substrate surface so that the application or coating method of the coating may include injecting at least one coating component at least partially (at the melting temperature of the substrate) into the substrate surface. Coating can be applied to the substrate by one or more methods described herein.

[0409] In the described embodiments in which the coating can be injected into the surface of a substrate, the coating can be co-dissolved in the surface of the substrate so that the components of the coating can be mixed 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 injected into the surface of a substrate wherein the substrate comprises leather or leather products.

[0410] As used herein, the term "bath coating" encompasses 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 introduced in US Patent No. 4,521,458, the entire contents of which are incorporated herein by reference.

[0411] As used herein, and unless more specifically described, the term "drying" may refer to drying a coating material as described herein at a temperature above room temperature (ie, 20°C).

[0412] The following are non-limiting examples of suitable ranges for various parameters in and for the preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges for such parameters.

[0413] In one embodiment, the SPF percentage in the solution is less than 30.0% by weight. In one embodiment, the SPF percentage in the solution is less than 25.0% by weight. In one embodiment, the SPF percentage in the solution is less than 20.0% by weight. In one embodiment, the SPF percentage in the solution is less than 19.0% by weight. In one embodiment, the SPF percentage in the solution is less than 18.0% by weight. In one embodiment, the SPF percentage in the solution is less than 17.0% by weight. In one embodiment, the SPF percentage in the solution is less than 16.0% by weight. In one embodiment, the SPF percentage in the solution is less than 15.0% by weight. In one embodiment, the SPF percentage in the solution is less than 14.0% by weight. In one embodiment, the SPF percentage in the solution is less than 13.0% by weight. In one embodiment, the SPF percentage in the solution is less than 12.0% by weight. In one embodiment, the SPF percentage in the solution is less than 11.0% by weight. In one embodiment, the SPF percentage in the solution is less than 10.0% by weight. In one embodiment, the SPF percentage in the solution is less than 9.0% by weight. In one embodiment, the SPF percentage in the solution is less than 8.0% by weight. In one embodiment, the SPF percentage in the solution is less than 7.0% by weight. In one embodiment, the SPF percentage in the solution is less than 6.0% by weight. In one embodiment, the SPF percentage in the solution is less than 5.0% by weight. In one embodiment, the SPF percentage in the solution is less than 4.0% by weight. In one embodiment, the SPF percentage in the solution is less than 3.0% by weight. In one embodiment, the SPF percentage in the solution is less than 2.0% by weight. In one embodiment, the SPF percentage in the solution is less than 1.0% by weight. In one embodiment, the SPF percentage in the solution is less than 0.9% by weight. In one embodiment, the SPF percentage in the solution is less than 0.8% by weight. In one embodiment, the SPF percentage in the solution is less than 0.7% by weight. In one embodiment, the SPF percentage in the solution is less than 0.6% by weight. In one embodiment, the SPF percentage in the solution is less than 0.5% by weight. In one embodiment, the SPF percentage in the solution is less than 0.4% by weight. In one embodiment, the SPF percentage in the solution is less than 0.3% by weight. In one embodiment, the SPF percentage in the solution is less than 0.2% by weight. In one embodiment, the SPF percentage in the solution is less than 0.1% by weight.

[0414] In one embodiment, the SPF percentage in the solution is greater than 0.1% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.2% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.3% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.4% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.5% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.6% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.7% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.8% by weight. In one embodiment, the SPF percentage in the solution is greater than 0.9% by weight. In one embodiment, the SPF percentage in the solution is greater than 1.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 2.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 3.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 4.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 5.0% by weight. In one embodiment, the SPF percentage in the solution is greater than 6.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 7.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 8.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 9.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 10.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 11.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 12.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 13.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 14.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 15.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 16.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 17.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 18.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 19.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 20.0% by weight. In one embodiment, the SPF percentage of the solution is greater than 25.0% by weight.

[0415] In one embodiment, the SPF percentage in the solution is from about 0.1% to about 30.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 25.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 20.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 15.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 9.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 8.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 7.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 6.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 6.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 5.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 2.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 5.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 4.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 4.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.5% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 4.0% by weight.In one embodiment, the SPF percentage in the solution is from about 1.0% to about 3.5% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 3.0% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 2.5% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 2.4% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 2.0% by weight.

[0416] In one embodiment, the SPF percentage in the solution is from about 20.0% to about 30.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution is from about 1.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution is from about 2% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution is from about 0.1% to about 6.0% by weight. In one embodiment, the SPF percentage in the solution is from about 6.0% to about 10.0% by weight. In one embodiment, the SPF percentage in the solution is from about 6.0% to about 8.0% by weight. In one embodiment, the SPF percentage in the solution is from about 6.0% to about 9.0% by weight. In one embodiment, the SPF percentage in the solution is from about 10.0% to about 20.0% by weight. In one embodiment, the SPF percentage in the solution is from about 11.0% to about 19.0% by weight. In one embodiment, the SPF percentage in the solution is from about 12.0% to about 18.0% by weight. In one embodiment, the SPF percentage in the solution is from about 13.0% to about 17.0% by weight. In one embodiment, the SPF percentage in the solution is from about 14.0% to about 16.0% by weight. In one embodiment, the SPF percentage in the solution is about 1.0% by weight. In one embodiment, the SPF percentage in the solution is about 1.5% by weight. In one embodiment, the SPF percentage in the solution is about 2.0% by weight. In one embodiment, the SPF percentage in the solution is about 2.4% by weight. In one embodiment, the SPF percentage in the solution is 3.0% by weight. In one embodiment, the SPF percentage in the solution is 3.5% by weight. In one embodiment, the SPF percentage in the solution is about 4.0% by weight. In one embodiment, the SPF percentage in the solution is about 4.5% by weight. In one embodiment, the SPF percentage in the solution is about 5.0% by weight. In one embodiment, the SPF percentage in the solution is about 5.5% by weight. In one embodiment, the SPF percentage of the solution is about 6.0% by weight. In one embodiment, the SPF percentage of the solution is about 6.5% by weight. In one embodiment, the SPF percentage of the solution is about 7.0% by weight. In one embodiment, the SPF percentage of the solution is about 7.5% by weight. In one embodiment, the SPF percentage of the solution is about 8.0% by weight. In one embodiment, the SPF percentage of the solution is about 8.5% by weight.In one embodiment, the SPF percentage in the solution is about 9.0% by weight. In one embodiment, the SPF percentage in the solution is about 9.5% by weight. In one embodiment, the SPF percentage in the solution is about 10.0% by weight.

[0417] In one embodiment, the percentage of sericin in the solution is between undetectable and 25.0% by weight. In one embodiment, the percentage of sericin in the solution is between undetectable and 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 1.0% by weight. In one embodiment, the percentage of sericin in the solution is 2.0% by weight. In one embodiment, the percentage of sericin in the solution is 3.0% by weight. In one embodiment, the percentage of sericin in the solution is 4.0% by weight. In one embodiment, the percentage of sericin in the solution is 5.0% by weight. In one embodiment, the percentage of sericin in the solution is 10.0% by weight. In one embodiment, the percentage of sericin in the solution is 25.0% by weight.

[0418] In some embodiments, the silk fibroin fragments of the present disclosure are shelf stable (they do not slowly or spontaneously gel when st...

Claims

1. A composite material, the composite material comprising a first polymeric macromolecular substance or a polymer and a second polymeric macromolecular substance or a polymer.

2. The composite material of claim 1, wherein a portion of the first polymeric macromolecular substance or polymer and a portion of the second polymeric macromolecular substance or polymer are physically and / or chemically entangled.

3. The composite material of claim 1, wherein a portion of the first polymeric macromolecular substance or polymer is physically and / or chemically cross-linked.

4. The composite material of claim 1, wherein a portion of the second polymeric macromolecular species or polymer is physically and / or chemically cross-linked.

5. The composite material of claim 1, wherein a portion of the first polymeric macromolecular substance or polymer is chemically and / or physically integrated into a portion of the second polymeric macromolecular substance or polymer.

6. The composite material of claim 1, wherein a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer are inseparable.

7. The composite material according to claim 1, wherein a portion of the first polymeric macromolecular substance or polymer and / or a portion of the second polymeric macromolecular substance or polymer is cross-linked.

8. The composite material of claim 1, wherein a portion of the first polymeric macromolecular species or polymer and / or a portion of the second polymeric macromolecular species or polymer are partially ordered and / or crystalline.

9. The composite material according to claim 1, wherein the first polymerized macromolecular substance or a portion of the polymer and the second polymerized macromolecular substance or a portion of the polymer cannot be delaminated.

10. The composite material of claim 1, wherein a portion of the first polymeric macromolecular species or polymer and a portion of the second polymeric macromolecular species or polymer are self-assembled.

11. The composite material according to any one of claims 1 to 10, wherein a portion of the first polymeric macromolecular substance or polymer in the composite material has a second structure different from the first structure of the first polymeric macromolecular substance or polymer.

12. The composite material of any one of claims 1 to 10, wherein a portion of the second polymeric macromolecular substance or polymer in the composite material has a second structure different from the first structure of the second polymeric macromolecular substance or polymer.

13. The composite material according to any one of claims 1 to 10, wherein a portion of the first polymeric macromolecular substance or polymer in the composite material has a second structure different from the first structure of the first polymeric macromolecular substance or polymer, and a portion of the second polymeric macromolecular substance or polymer in the composite material has a second structure different from the first structure of the second polymeric macromolecular substance or polymer.

14. The composite material according to any one of claims 1 to 13, wherein the first polymeric macromolecular substance or polymer comprises a protein component.

15. The composite material of claim 14, wherein the protein component comprises one or more of silk fibroin or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein and / or whey.

16. The composite material of any one of claims 1 to 13, wherein the first polymeric macromolecular substance or polymer comprises a biodegradable polymer.

17. The composite material of any one of claims 1 to 13, wherein the first polymeric macromolecular substance or polymer comprises one or more polyurethane components.

18. The composite material of any one of claims 1 to 13, wherein the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component, a poly(lactic-co-glycolic acid) (PLGA) component, or both.

19. The composite material according to any one of claims 1 to 18, wherein the second polymeric macromolecular substance or polymer comprises a cellulose and / or cellulose derivative component.

20. The composite material of claim 19, wherein the cellulose derivative is selected from the group consisting of 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.

21. The composite material of claim 19, wherein the cellulose derivative is ethyl cellulose.

22. The composite material of claim 21, wherein the ethoxy content of the ethylcellulose 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%.

23. The composite material of claim 21, 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.

24. The composite material of any one of claims 19 to 23, wherein the second structure of the cellulose derivative comprises less than 100% crystallinity.

25. The composite material of any one of claims 19 to 23, wherein the second structure of the cellulose derivative comprises a crystallinity of from about 5% to less than about 100%.

26. The composite material of any one of claims 19 to 23, wherein the second structure of the cellulose derivative comprises a crystallinity of about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, about 90% to about 99%, or about 90% to about 100%.

27. The composite material of any one of claims 19 to 23, wherein the second structure of the cellulose derivative comprises a crystallinity of less than about 99%, less than about 98%, less than about 97%, less than about 96%, less than about 95%, less than about 94%, less than about 93%, less than about 92%, less than about 91%, less than about 90%, less than about 89%, less than about 88%, less than about 87%, less than about 86%, less than about 85%, less than about 84%, less than about 83%, less than about 82%, less than about 81%, less than about 80%, less than about 79%, less than about 78%, less than about 77%, less than about 76%, less than about 75%, less than about 74%, less than about 73%, less than about 72%, less than about 71%, less than about 70%, less than about 69%, less than about 68%, less than about 67%, less than about 66%, less than about 65%, less than about 64%, less than about 63%, less than about 62%, less than about 61%, less than about 60%, less than about 59%, less than about 5 less than about 8%, less than about 57%, less than about 56%, less than about 55%, less than about 54%, less than about 53%, less than about 52%, less than about 51%, less than about 50%, less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, or less than about 10%.

28. The composite material of any one of claims 1 to 27, wherein the w / w ratio between the first polymeric macromolecular substance or polymer and the second polymeric macromolecular substance polymer in the composite material is from about 1:100 to about 100:

1.

29. The composite material of any one of claims 1 to 27, wherein the w / w ratio between the first polymeric macromolecular species or polymer and the second polymeric macromolecular species polymer in the composite material 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 82:1 8. 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, about 72:28, about 71:29, about 70:30, about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42, about 57:43, about 56:44, about 55 :45, about 54:46, about 53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75, 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 15:85, 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.

30. The composite material of any one of claims 1 to 27, wherein the w / w ratio between the first polymeric macromolecular species or polymer and the second polymeric macromolecular species polymer in the composite material is about 10:1, about 10:2, about 10:3, about 10:4, about 10:5, about 10:6, about 10:7, about 10:8, about 10:9, or about 10:

10.

31. The composite material of any one of claims 1 to 30, wherein the first polymeric macromolecular substance or polymer is isotropically distributed in a cross section of the composite material.

32. The composite material of any one of claims 1 to 30, wherein the first polymeric macromolecular species or polymer is anisotropically distributed across a cross section of the composite material.

33. The composite material of claim 32, wherein a concentration of the first polymerized macromolecular species or polymer closer to a first surface of the composite material is higher than a concentration of the first polymerized macromolecular species or polymer closer to a second surface of the composite material.

34. The composite material of claim 32 or 33, wherein the first polymerized macromolecular substance or polymer is substantially undetectable at the second surface of the composite material.

35. The composite material of any one of claims 1 to 34, wherein the second polymeric macromolecular species or polymer is isotropically distributed across a cross section of the composite material.

36. The composite material of any one of claims 1 to 34, wherein the second polymeric macromolecular substance or polymer is anisotropically distributed in a cross section of the composite material.

37. The composite material of claim 36, wherein a concentration of the second polymerized macromolecular species or polymer closer to the second surface of the composite material is higher than a concentration of the second polymerized macromolecular species or polymer closer to the first surface of the composite material.

38. A composite material as claimed in claim 36 or 37, wherein the second polymerized macromolecular substance or polymer is substantially undetectable at the first surface of the composite substrate-coating interface.

39. The composite material of any one of claims 1 to 38, wherein the first surface of the composite material is an adhesive.

40. The composite material of any one of claims 1 to 38, wherein the second surface of the composite material is an adhesive.

41. The composite material of any one of claims 1 to 38, wherein the first surface of the composite material is an adhesive and the second surface of the composite material is an adhesive.

42. The composite material of any one of claims 1 to 38, wherein a first surface of the composite material is adhesive and a second surface of the composite material is non-adhesive.

43. The composite material of any one of claims 1 to 42, wherein the composite material has increased water resistance compared to one of the following materials: i) a non-composite material comprising the first polymeric macromolecular species or polymer but not the second polymeric macromolecular species or polymer, ii) a non-composite material comprising the second polymeric macromolecular species or polymer but not 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 free of physical and / or chemical molecular entanglements.

44. The composite material of any one of claims 1 to 42, wherein the composite material has increased water vapor permeability compared to one of the following materials: i) a non-composite material comprising the first polymeric macromolecular species or polymer but not the second polymeric macromolecular species or polymer, ii) a non-composite material comprising the second polymeric macromolecular species or polymer but not 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 free of physical and / or chemical molecular entanglements.

45. An article comprising a substrate and a coating, the coating comprising the composite material of any one of claims 1 to 44.

46. ​​The article of claim 45, wherein the substrate comprises an irregular surface.

47. The article of claim 45, wherein the coating has a thickness of about 10 μm to about 1000 μm.

48. The article of any one of claims 45 to 47, wherein the coating amount on the substrate is about 0.01 g / ft 2 About 25g / ft 2 .

49. The article of any one of claims 45 to 48, wherein the amount of first polymeric macromolecular species or polymer in the coating on the substrate is about 0.001 g / ft 2 About 20g / ft 2 .

50. The article of any one of claims 45 to 49, wherein the amount of second polymeric macromolecular species or polymer in the coating on the substrate is about 0.001 g / ft 2 About 15g / ft 2 .

51. The article of any one of claims 45 to 50, wherein the substrate comprises a substantially flexible material.

52. The article of any one of claims 45 to 51, wherein the substrate comprises a leather material or a textile material.

53. The article of any one of claims 45 to 52, wherein the substrate comprises one or more of collagen, cellulose and / or lignin.

54. A method for coating a substrate, the method comprising applying to the surface of the substrate a first composition comprising a first polymeric macromolecular substance or a polymer, and a second composition comprising a second polymeric macromolecular substance or a polymer.

55. The method of claim 54, wherein the first composition comprises an unstructured first polymeric macromolecular substance or polymer, or a first structure of the first polymeric macromolecular substance or polymer.

56. The method of claim 54 or 55, wherein the first polymeric macromolecular species or polymer comprises a protein component.

57. A method as claimed in any one of claims 54 to 56, wherein the protein component comprises one or more of fibroin or fragments, collagen, elastin, gelatin, zein, wheat gluten, pectin, chitin, casein and / or whey.

58. The method of claim 54 or 55, wherein the first polymeric macromolecular substance or polymer comprises a biodegradable polymer.

59. The method of any one of claims 54 to 58, wherein the first polymeric macromolecular species or polymer comprises one or more polyurethane components.

60. The method of any one of claims 54 to 58, wherein the first polymeric macromolecular species or polymer comprises a polylactic acid (PLA) component, a poly(lactic-co-glycolic acid) (PLGA) component, or both.

61. The method of any one of claims 54 to 60, wherein the second composition comprises an unstructured second polymeric macromolecular substance or polymer, or the second polymeric macromolecular substance or polymer of a first structure.

62. The method of any one of claims 54 to 60, wherein the second polymeric macromolecular species or polymer comprises a cellulose and / or cellulose derivative component.

63. The method of claim 62, wherein the cellulose derivative is selected from the group consisting of methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxyethylmethylcellulose, hydroxypropylmethylcellulose, ethylhydroxyethylcellulose, cellulose triacetate, cellulose propionate, cellulose nitrate, cellulose sulfate, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, cellulose acetate, cellulose acetate propionate, cellulose acetate butyrate, and microcrystalline cellulose.

64. The method of claim 62, wherein the cellulose derivative is ethyl cellulose.

65. The method of claim 64, wherein the ethoxy content of the ethylcellulose 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%.

66. The method of claim 64, wherein the degree of substitution of the ethyl cellulose is from 0.5 to 1, from 1 to 1.5, from 1.5 to 2, from 2 to 2.5, or from 2.5 to 3.

67. The method of any one of claims 62 to 66, wherein the cellulose derivative comprises a first structure of the cellulose derivative having a lower crystallinity than a second structure of the cellulose derivative, the second structure of the cellulose derivative having a crystallinity of from about 5% to less than about 100%.

68. The method of any one of claims 54 to 67, wherein the second composition comprising a second polymeric macromolecular substance or a polymer further comprises a solvent component.

69. The method of claim 68, wherein the solvent component comprises an alcohol and / or an alcohol derivative.

70. The method of claim 68 or 69, wherein the solvent component comprises one or more of an alcohol, an ether, a ketone, an aldehyde and / or a ketal.

71. The method of any one of claims 68 to 70, wherein the solvent component comprises from about 75% w / w to about 99% w / w of the composition, from about 80% w / w to about 98% w / w of the composition, from about 85% w / w to about 97.5% w / w of the composition, or from about 85% w / w to about 95% w / w of the composition.

72. The method of any one of claims 68 to 71, wherein the solvent component comprises one or more of methanol, ethanol, n-propanol, 2-propanol, n-butanol, 2-butanol, pentanol, hexanol, acetone, butanone, methoxypropanol, diisopropyl glycerol, 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane, 2,2-dimethyl-1,3-dioxolane-4-methanol, or any combination thereof.

73. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular substance or a polymer further comprises one or more of a polyethylene glycol (PEG) component, a polypropylene glycol (PPG) component and / or a polyether component.

74. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular species or a polymer further comprises a fatty acid or a fatty acid derived amide, and / or one or more of a monoglyceride, a diglyceride and / or a triglyceride.

75. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular substance or a polymer further comprises one or more of a triethylene glycol monomethyl ether component, a diethylene glycol butyl ether component, a diethylene glycol ethyl ether component, a tetradecanedioic acid dimethyl ester component, an erucamide component, and / or a stearic acid glyceryl ester component.

76. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular species or polymer comprises one or more of an isocyanate component, a polyol component, a blocked isocyanate component, and / or a blocked polyol component.

77. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular species or polymer comprises a partially polymerized, partially cross-linked and / or partially cured polyurethane component.

78. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular substance or a polymer further comprises a polyurethane prepolymer component.

79. The method of any one of claims 54 to 72, wherein the first composition comprising a first polymeric macromolecular substance or a polymer further comprises water.

80. The method of any one of claims 54 to 72, wherein the surface of the substrate is first coated with a first composition comprising a first polymeric macromolecular species or a polymer and then coated with a second composition comprising a second polymeric macromolecular species or a polymer.

81. The method of claim 80, further comprising a drying or partial drying step between two coating steps.

82. The method of claim 80 or 81, wherein the first composition comprising a first polymeric macromolecular species or polymer is only partially polymerized, partially dried and / or partially cured prior to applying the second composition comprising a second polymeric macromolecular species or polymer.

83. The method of any one of claims 54 to 82, wherein the second composition comprising a second polymeric macromolecular substance or polymer has a glass transition temperature (Tg) higher than that of the first polymeric macromolecular substance or polymer. g ) is applied at a temperature of 84. The method of any one of claims 54 to 82, wherein the second composition comprising a second polymeric macromolecular substance or polymer has a temperature above the glass transition temperature (T g ) is applied at a temperature of 85. The method of any one of claims 54 to 84, wherein the first composition comprising a first polymeric macromolecular species or a polymer is heated at about 0.5 mL / ft 2 To about 5mL / ft 2 Apply one or more times at a rate.

86. The method of any one of claims 54 to 85, wherein the second composition comprising a second polymeric macromolecular species or a polymer is concentrated at about 0.5 mL / ft 2 To about 5mL / ft 2 Apply one or more times at a rate.

87. An article comprising a substrate and a coating, the article being made by the method of any one of claims 54 to 86.

88. The article of claim 87, wherein the first polymeric macromolecular substance or polymer is isotropically distributed in a cross-section of the coating from the substrate-coating interface to the outer surface of the coating.

89. The article of claim 87, wherein the first polymeric macromolecular substance or polymer is anisotropically distributed in a cross section of the coating from the substrate-coating interface to the outer surface of the coating.

90. The article of claim 87, wherein the concentration of the first polymerized macromolecular species or polymer is higher nearer the substrate-coating interface than nearer the outer surface of the coating.

91. The article of claim 87, wherein the first polymerized macromolecular species or polymer is substantially undetectable at the outer surface of the coating.

92. The article of any one of claims 87 to 91, wherein the second polymeric macromolecular species or polymer is isotropically distributed in a cross-section of the coating from the substrate-coating interface to the outer surface of the coating.

93. The article of any one of claims 87 to 91, wherein the second polymeric macromolecular species or polymer is anisotropically distributed in a cross-section of the coating from the substrate-coating interface to the outer surface of the coating.

94. The article of any one of claims 87 to 91, wherein the concentration of the second polymerized macromolecular species or polymer closer to the substrate-coating interface is lower than the concentration of the second polymerized macromolecular species or polymer closer to the outer surface of the coating.

95. The article of any one of claims 87 to 91, wherein the second polymeric macromolecular species or polymer is substantially undetectable at the substrate-coating interface.

Images