Silk-stimulated collagen and claudin-1 expression and silk-stimulated anti-inflammatory effects

Abstract

The present disclosure provides silk fibroin compositions and methods of use thereof for stimulating collagen expression, claudin-1, and / or anti-inflammatory effects in a subject.

Description

[Technical Field]

[0001] Cross-reference to related art This application claims the benefit of U.S. Provisional Patent Application No. 63 / 256,942, filed October 18, 2021, and U.S. Provisional Patent Application No. 63 / 256,896, filed October 18, 2021, both of which are incorporated herein by reference in their entireties.

[0002] The present disclosure is in the field of silk fibroin compositions and methods for stimulating collagen expression. [Background technology]

[0003] Silk is a natural polymer produced by a variety of insects and spiders. Silk contains a filamentous core protein, silk fibroin, and a colloidal coating consisting of a non-filamentous protein, sericin.

[0004] There is a need for stable silk fibroin peptide solutions suitable for collagen stimulation via topical or parenteral administration. Summary of the Invention

[0005] The present disclosure provides a method for treating or preventing a disorder, disease, or condition that is alleviated by stimulating or modulating collagen expression in a subject in need thereof, the method comprising the step of stimulating or modulating collagen expression in a range 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 15 kDa to about 20 kDa, about 14 kDa to about 30 kDa, about 17 kDa to about 39 kDa, about 20 kDa to about 25 kDa, about 25 kDa to about Methods are provided, including administering to a subject a composition comprising silk fibroin fragments having an average weight-average molecular weight selected from 30 kDa, about 30 kDa to about 35 kDa, about 35 kDa to about 40 kDa, about 39 kDa to about 54 kDa, about 39 kDa to about 80 kDa, about 40 kDa to about 45 kDa, about 45 kDa to about 50 kDa, about 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, and a polydispersity of 1 to about 5. In some embodiments, the composition further comprises 0 to 500 ppm lithium bromide. In some embodiments, the composition further comprises 0 to 500 ppm sodium carbonate. In some embodiments, the silk fibroin fragments have a polydispersity of 1 to about 1.5. In some embodiments, the silk fibroin fragments have a polydispersity of about 1.5 to about 2.0. In some embodiments, the silk fibroin fragments have a polydispersity of about 1.5 to about 3.0. In some embodiments, the silk fibroin fragments have a polydispersity of about 2.0 to about 2.5. In some embodiments, the silk fibroin fragments have a polydispersity of about 2.5 to about 3.0. In some embodiments, the silk fibroin fragments are present in the composition at about 0.001% to about 10.0% by weight, based on the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin, based on the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin, based on the total weight of the silk fibroin fragments. In some embodiments, the silk fibroin fragments do not spontaneously or gradually gel or show a visible change in color or turbidity when in aqueous solution for at least 10 days prior to formulation into a composition.In some embodiments, the silk fibroin fragments are present in the composition at about 0.01 wt % to about 10.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 0.01 wt % to about 1.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 1.0 wt % to about 2.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 2.0 wt % to about 3.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 3.0 wt % to about 4.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 4.0 wt % to about 5.0 wt %, based on the total weight of the composition. In some embodiments, the silk fibroin fragments are present in the composition at about 5.0 wt % to about 6.0 wt %, based on the total weight of the composition. In some embodiments, the composition is formulated as an injectable composition or a topical composition. In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a dermatologically acceptable carrier. In some embodiments, the composition further comprises an injectable acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a suspension, emulsion, powder, solution, dispersion, or elixir. In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, jelly, cream, lotion, foam, slurry, ointment, oil, paste, suppository, spray, semi-solid composition, solid composition, stick, or mousse. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of sesame oil, corn oil, cottonseed oil, or peanut oil. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of mannitol or dextrose. In some embodiments, the pharmaceutically acceptable carrier comprises about 0.001% to about 10% (w / v) hyaluronic acid.In some embodiments, the pharmaceutically acceptable carrier comprises about 1% to about 10% (w / v), about 10% to about 25% (w / v), about 25% to about 50% (w / v), or about 50% to about 99.99% (w / v) hyaluronic acid. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of aliphatic oils, fatty alcohols, fatty acids, glycerides, acylglycerols, and phospholipids. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of monoglycerides, diglycerides, or triglycerides. In some embodiments, the pharmaceutically acceptable carrier comprises an aqueous phase. In some embodiments, the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in-oil emulsion. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a hydrocarbon oil, a fatty acid, a fatty acid oil, a fatty acid ester, or a cationic quaternary ammonium salt. In some embodiments, a portion of the pharmaceutically acceptable carrier is a polyepoxy linker, a diepoxy linker, a polyepoxy-PEG, a diepoxy-PEG, a polyglycidyl-PEG, a diglycidyl-PEG, a polyacrylate PEG, a diacrylate PEG, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)butane ... and modified with a crosslinker, crosslink precursor, or activator selected from 1,2,7,8-diepoxyethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic acid dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, and any combination thereof.In some embodiments, the polyepoxy linker is selected from 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether. In some embodiments, the composition is administered parenterally. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is administered by injection. In some embodiments, the composition is administered by subcutaneous, intradermal, transdermal, or subdermal injection. In some embodiments, the composition is administered by intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, or intracavernosal injection. In some embodiments, the composition is administered by depot injection. In some embodiments, the composition is administered by infiltration injection. In some embodiments, the composition is administered by an indwelling catheter. In some embodiments, the composition is administered by a microneedle. In some embodiments, administering the composition reduces expression of one or more metalloproteinases (MMPs) in the subject. In some embodiments, stimulating or modulating collagen expression comprises increasing collagen expression.In some embodiments, collagen expression is reduced by about 1%, 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%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% 9%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75% , about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% increase.In some embodiments, collagen expression is about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, approx. 123%, approx. 124%, approx. 125%, approx. 126%, approx. 127%, approx. 128%, approx. 129%, approx. 130%, approx. 131%, approx. 132%, approx. 133%, approx. 134%, approx. 135%, approx. 136%, approx. 137%, approx. 138%, approx. 139%, approx. 140%, approx. 141%, approx. 142%, approx. 143%, approx. 144%, approx. 145%, approx. 146%, approx. 147%, approx. 148% ,approximately 149%,approximately 150%,approximately 151%,approximately 152%,approximately 153%,approximately 154%,approximately 155%,approximately 156%,approximately 157%,approximately 158%,approximately 159%,approximately 160%,approximately 161%,approximately 162%,approximately 163%,approximately 164%,approximately 165%,approximately 166%,approximately 167%,approximately 168%,approximately 169%,approximately 170%,approximately 171%,approximately 172%,approximately 173%,approximately 174%,approximately 1 75%, about 176%, about 177%, about 178%, about 179%, about 180%, about 181%, about 182%, about 183%, about 184%, about 185%, about 186%, about 187%, about 188%, about 189%, about 190%, about 191%, about 192%, about 193%, about 194%, about 195%, about 196%, about 197%, about 198%, about 199%, or about 200% increase. In some embodiments, administering the composition results in one or more of preventing or improving wrinkles in a subject, preventing or improving age spots in a subject, preventing or improving dry skin in a subject, or improving uneven skin tone in a subject. In some embodiments, administering the composition results in one or more of preventing or improving sagging skin in a subject, preventing or improving skin aging in a subject, preventing or improving reduced skin tensile strength in a subject, preventing or improving photodamaged skin in a subject, or preventing or improving stretch marks in a subject.In some embodiments, the disorder, disease, or condition comprises wrinkles, age spots, dry skin, uneven skin tone, sagging skin, aging skin, reduced skin tensile strength, photodamaged skin, or stretch marks. In some embodiments, the disorder, disease, or condition comprises thyroid hormone-induced myocardial infarction. In some embodiments, the disorder, disease, or condition comprises a rupture, injury, or tear of a tendon. In some embodiments, the tendon is selected from the group consisting of teres minor tendon, infraspinatus tendon, supraspinatus tendon, subscapularis tendon, deltoid tendon, biceps tendon, triceps tendon, brachioradialis tendon, supinator tendon, flexor carpi radialis tendon, flexor carpi ulnaris tendon, extensor carpi radialis longus tendon, extensor carpi radialis brevis tendon, iliopsoas tendon, obturator internus tendon, adductor longus tendon, peroneus or magnus tendon, gluteus maximus or gluteus medius tendon, quadriceps tendon, patellar tendon, popliteus tendon, sartorius tendon, gastrocnemius tendon, Achilles tendon, soleus tendon, tibialis anterior tendon, peroneus longus tendon, flexor digitorum longus tendon, interosseus tendon, flexor digitorum profundus tendon, abductor digitorum minimi tendon, opponens pollicis tendon, flexor pollicis longus tendon, extensor or abductor pollicis tendons), flexor hallucis longus tendon, flexor digitorum brevis tendon, medius tendon, abductor hallucis longus tendon, abductor digitorum minimi tendon, ophthalmic tendon, levator palpebrae tendon, masseter tendon, temporalis tendon, trapezius tendon, sternocleidomastoid tendon, semispinalis capitis or splenius capitis tendon, mylohyoid or thyrohyoid tendon, sternohyoid tendon, rectus abdominis tendon, external oblique tendon, transverse abdominis tendon, latissimus dorsi tendon, and erector spinae tendon. In some embodiments, the disorder, disease, or condition comprises Werner's syndrome. In some embodiments, the disorder, disease, or condition comprises impaired diabetic skin integrity. In some embodiments, the disorder, disease, or condition comprises arthritis. In some embodiments, the disorder, disease, or condition comprises rheumatoid arthritis. In some embodiments, the disorder, disease, or condition comprises tumor progression or tumor growth. In some embodiments, the disorder, disease, or condition comprises impaired cardiac function. In some embodiments, the disorder, disease, or condition comprises Ehlers-Danlos syndrome. In some embodiments, the disorder, disease, or condition comprises abdominal aortic aneurysm. In some embodiments, the disorder, disease, or condition comprises a wound. In some embodiments, the disorder, disease, or condition comprises a disease of the skin or connective tissue. In some embodiments, the disorder, disease, or condition comprises a disease of cartilage. In some embodiments, the disorder, disease, or condition comprises relapsing polychondritis, Tietze's syndrome, cellulitis, Ehlers-Danlos syndrome, keloids (including acne keloids), mucopolysaddaridosis II), necrotizing disorders (including granuloma annulare and necrobiosis lipoidica), osteogenesis imperfecta, cutis laxa, dermatomyositis, Dupuytren's contracture, homocystinuria, lupus erythematosus (including cutaneous, discoid, deep, systemic, and nephritic), Marfan syndrome, mixed connective tissue disease, mucinosis (including follicular), mucopolysaccharidoses (I, II, UU, IV, IV, and VII), myxedema, scleredema adultae, and synovial cysts. In some embodiments, the disorder, disease, or condition is selected from tissue neoplasms, Noonan's syndrome, bone poikilosis, panniculitis including erythema induratum, nodular nonsuppurative and peritoneal, penile sclerosis, pseudoxanthoma elasticum, rheumatic diseases including arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, Kaplan's syndrome, Felty's syndrome, rheumatoid nodules, ankylosing spondylitis, and Still's disease), osteophytosis, polymyalgia rheumatica, localized scleroderma, and systemic sclerosis (CREST syndrome). In some embodiments, the disorder, disease, or condition is selected from angiolymphocytosis with eosinophilia; cicatricialis (including hypertrophic); skin fistula, cutis laxa laxa); dermatitis including acrodermatitis, atopic dermatitis, contact dermatitis (allergic contact, photoallergy, sumac), irritant dermatitis (phototoxicity, diaper rash), occupational dermatitis; exfoliative skin eczema, including inflammation, dermatitis herpetiformis, seborrheic dermatitis, drug eruptions (toxic epidermal necrolysis, erythema nodosum, serum sickness, etc.), dyshidrosis, intertrigo, neurodermatitis, and radiation dermatitis; dermatomyositis; Erythema, including erythematous, polymorphic (Stevens-Johnson syndrome), and nodular (Sweet syndrome); exanthem, including subitum exanthem; facial skin disorders, including acneiform eruptions (keloids, rosacea, rosacea vulgaris, and Fabre-Lacouchot syndrome); skin disorders of the feet, including athlete's foot; skin disorders of the hands; keratoacanthoma; callus, cholesteatoma (including middle ear), ichthyosis (including congenital ichthyosiform erythroderms, epidermolytic hyperkeratosis, lamellar ichthyosis, ichthyosis vulgaris, X-linked ichthyosis, and Sjögren-Larsson syndrome), pyorrhagic keratoderma, palmoplantar keratodermakeratoderms, follicular keratosis, seborrheic keratosis, parakeratosis, and porokeratosis; leg skin diseases, mastocytosis (urticaria pigmentosa), necrobiosis (granuloma annulare and necrobiosis lipoidica), photosensitivity (photoallergic or phototoxic dermatitis, variola vaccinia, sundurn, and xeroderma pigmentosum); argyria, hyperpigmentation, melanosis, aconthosis nigricans, lentigines, Peutz-Jeghers syndrome, hypopigmentation, congenital albinism, pibaldism, vitiligo, incontinentia pigmenti, urticaria pigmentosa, xeroderma pigmentosum, and prurigo; pruritus (including anal and vulvar regions); pyoderma, including ecthyma and pyoderma gangrenosum; scalp diseases (sclap dermatoses; adult scleroderma (sclerodema adultorum); neonatal scleroderma (sclerma neonatorum); skin appendage disorders including hair disorders (alopecia, folliculitis, hirsutism, hypertrichosis, kinky hair syndrome), nail disorders (nail-patella syndrome, ingrown nails or nail dysplasia, onychomycosis, paronychia), sebaceous gland disorders (rhinophyma, neoplasms), sweat gland disorders (hidradenitis suppurativa, hyperhidrosis, hypohidrosis, milia, Fox-Fordyce disease, neoplasms) Hereditary skin diseases including alfinism, cutis laxa, familial benign chronic pemphigus, porphyria, acrodermatitis, ectodermal hypoplasia, Ellis-van Creveld syndrome, focal cutaneous hypoplasia, Ehlers-Danlos syndrome, epidermolysis bullosa, and ichthyosis; infectious skin diseases including dermatomycosis, blastomycosis, candidiasis, chromoblastomycosis, maduramycosis, paracoccidioidomycosis, sporotrichosis, and tinea; cervicofacial actinomycosis, bacilliary angiomatosisbacterial skin diseases including angiomatosis, ecthyma, erysipelas, erythema chronica migrans, erythrasma, granuloma inguinale, hidradenitis suppurativa, maduramycosis, paronychia, pinta, rhinosclerosis, staphylococcal skin infections (furuncolosis, carbuncles, impetigo, scalded skin syndrome), cutaneous syphilis, cutaneous tuberculosis, yaws; parasitic skin diseases including larva migrans, leishmaniasis, pediculosis, and scabies; viral skin diseases including erythema infectiosum, exanthema subitum, herpes simplex, molluscum contagiosum, and warts. [Brief explanation of the drawings]

[0006] Embodiments disclosed herein will be further described with reference to the accompanying drawings, in which the drawings are not necessarily to scale, emphasis instead generally being placed upon illustrating the principles of the embodiments disclosed herein.

[0007] [Figure 1A] Figures 1A-1C show a schematic of collagen synthesis in young and aged skin, as well as the proposed role of silk fibroin in stimulating collagen synthesis. Figure 1A: In healthy young skin, dermal fibroblasts in a dense collagen matrix continually strengthen the matrix by producing new collagen. In young skin, intact collagen within the dermal extracellular matrix (ECM) provides attachment sites and mechanical resistance for fibroblasts. Fibroblasts stretch and produce new collagen (green), promoting the integrity and stability of the ECM. [Figure 1B] Figure 1B: With aging, fibroblasts produce less new collagen and the collagen matrix degrades. With aging, reduced collagen synthesis and increased MMP activity lead to fragmentation of collagen fibrils, resulting in a loss of mechanical tension for fibroblasts and a loss of ECM integrity and stability. [Figure 1C]Figure 1C: Addition of silk fibroin to the matrix stimulates collagen production by fibroblasts and restores the structural integrity of the matrix. The added silk fibroin stimulates fibroblasts to produce collagen, likely through direct interaction with fibroblasts and cross-linking of collagen fragments. This is predicted to promote restoration of ECM integrity and a younger appearance of skin. (Adapted from Varani et al. Am J Pathol. 2006, 168:1861). [Figure 2] Collagen production is shown to depend on silk composition. Intracellular collagen production at various silk concentrations is shown as a function of silk type. Stimulation rate is the increase in collagen formation compared to the negative control. Silk average MW composition: Silk A = low MW (average weight average molecular weight selected from about 14 kDa to about 30 kDa), Silk B = medium MW (average weight average molecular weight selected from about 39 kDa to about 54 kDa). [Figure 3] In vitro model: extracellular matrix production. The timeline shows the chronological order of the experiment and treatment conditions. [Figure 4] In vitro model: collagen production. The positive control treatment was TGF-β (10 ng / mL) + Vitamin C (20 μg / mL). [Figure 5A] Figures 5A and 5B show that treatment of human dermal fibroblasts with silk in the presence of Vit C increases total collagen production. Figure 5A shows Sirius red staining of human dermal fibroblasts with Vit C and co-treatment with TGF-β (serving as a positive control), vehicle control, retinoic acid, medium MW silk, or low MW silk for 5 days. The scale bar represents 650 μm. [Figure 5B] Figure 5B is a spectrophotometric analysis of the Sirius Red stained cells of Figure 5A. n=1. [Figure 6] Medium and low MW silks increase total collagen production. Spectrophotometric analysis of Sirius Red on human dermal fibroblasts after 24 hours of stimulation (n=2). [Figure 7]Medium and low MW silk upregulated COL1A1 gene expression in human dermal fibroblasts. Quantitative PCR for COL1A1 in silk and retinoic acid treated human dermal fibroblasts 8 hours after treatment. n=2 per group. Over 8-fold increase in TGF-b+Vit.C treated human dermal fibroblasts (serving as positive control). [Figure 8A] Figures 8A and 8B show that low MW silk upregulates collagen 1 protein expression. Figure 8A is a representative flow cytometry histogram of collagen 1 expression in retinoic acid-treated, vehicle-treated, and low MW silk-treated human dermal fibroblasts gated on live cells. Data shown represent n=3 per group. [Figure 8B] Figure 8B shows the percent increase in mean fluorescence intensity (MFI) of collagen 1 in retinoic acid-treated and low MW silk-treated cells compared to vehicle controls. Data are summarized as mean + SEM, *p<0.05 by one-way ANOVA followed by post-hoc t-test with Bonferroni correction. n=3 per group: 34.5% increase in collagen 1 MFI in TGF-β + Vitamin C-treated human dermal fibroblasts (serving as a positive control). Representative immunohistochemical staining showing collagen 1 (green staining) and Hoechst (blue) colocalization in retinoic acid-treated and low MW silk-treated human dermal fibroblasts (n=1). [Figure 9] Low MW silk does not alter COL4A1 protein expression. Quantitative analysis of COL4A1+ cell frequency (in viable cells) in retinoic acid-treated, vehicle-treated, and low MW silk-treated human dermal fibroblasts. n=2 per group. [Figure 10] Activated Silk™ molecules exhibit collagen 1 stimulation similar to retinoic acid in human dermal fibroblasts. [Figure 11] Activated Silk™ 33B upregulates COL1A1 gene expression in human dermal fibroblasts. Quantitative PCR for COL1A1 in silk and retinoic acid treated human dermal fibroblasts 8 hours after treatment. n=2 per group. [Figure 12] 1 is a flow chart illustrating various embodiments for producing silk fibroin protein fragments (SPF) of the present disclosure. [Figure 13] 1 is a flow chart illustrating various parameters that can be modified during the extraction and lysis steps during the process of producing a silk protein fragment solution of the present disclosure. [Figure 14A] Figures 14A and 14B show cross sections of EFT-400 tissue exposed to low MW silk (RITC-labeled) for 2 x 5 hours, counterstained with DAPI. The 5x magnification image (Figure 14A) shows the entire tissue thickness, while the 10x magnification image (Figure 14B) focuses on the epidermis. [Figure 14B] Figures 14A and 14B show cross sections of EFT-400 tissue exposed to low MW silk (RITC-labeled) for 2 x 5 hours, counterstained with DAPI. The 5x magnification image (Figure 14A) shows the entire tissue thickness, while the 10x magnification image (Figure 14B) focuses on the epidermis. [Figure 15A] Figures 15A and 15B show cross sections of EFT-400 tissue exposed to medium MW silk (FITC-labeled) for 2 x 5 hours, counterstained with DAPI. The 5x magnification image (Figure 15A) shows the entire tissue thickness, while the 10x magnification image (Figure 15B) focuses on the epidermis. [Figure 15B] Figures 15A and 15B show cross sections of EFT-400 tissue exposed to medium MW silk (FITC-labeled) for 2 x 5 hours, counterstained with DAPI. The 5x magnification image (Figure 15A) shows the entire tissue thickness, while the 10x magnification image (Figure 15B) focuses on the epidermis. [Figure 16] Fluorescence imaging of fluorescently tagged silk fibroin. [Figure 17-1] Figures 17A-D show that silk fibroin described herein restores claudin-1 expression in damaged human skin (N=1, 52-year-old Caucasian female). [Figure 17-2]Figures 17E-H show that silk fibroin described herein restores claudin-1 expression in damaged human skin (N=1, 52-year-old Caucasian female). [Figure 17-3] 17I-N show that silk fibroin described herein restores claudin-1 expression in damaged human skin (N=1, 52-year-old Caucasian female). [Figure 18] We demonstrate that the silk fibroin described herein restores claudin-1 expression in damaged human skin. [Figure 19] We demonstrate how the silk fibroin described herein restores claudin-1 expression and improves the skin barrier. [Figure 20] Figures 20A-21H in Figure 20 show how medium-skid silk increases claudin-1 protein expression in human neonatal epidermal keratinocytes in vitro. (20A-20H) Representative immunohistochemistry images of keratinocyte cells (approximately 80-90% confluence) treated without (20A, 20G) or with (20B-20F, 20H) medium-skid (33B) silk polypeptide (0.5 mg / mL-6 mg / mL = 0.05-0.6% w / v) for 24 hours. Claudin-1 expression (red) increases with the addition of medium-skid silk. Panels G and H were treated with a normal rabbit IgG isotype control antibody to demonstrate nonspecific binding of the target primary antibody. [Figure 21] Figures 21A-21D in Figure 21 show how low-skid silk increases claudin-1 protein expression in human neonatal epidermal keratinocytes in vitro. Representative immunohistochemistry images of keratinocyte cells (approximately 80-90% confluence) treated without (21A) or with (21B-21D) low-skid (27p) silk polypeptide (0.5 mg / mL-7 mg / mL = 0.05-0.7% w / v) for 24 hours. Claudin-1 expression (red) increases with the addition of low-skid silk. [Figure 22A]Figures 22A and 22B show the results of experiments for detecting claudin-1 upregulation in skin biopsies. Punch skin biopsies were obtained from human donors ranging in age from 30 to 60 years. 22A: Skin biopsies were pretreated with acetone as described in Materials and Methods, and then silk or other reagents were added to them as indicated in the figure. [Figure 22B] 22B: When biopsies were first treated with acetone and then with vehicle, claudin-1 (orange staining) disappeared and did not regenerate. However, when mesodermal (33B) silk was applied after acetone treatment, claudin-1 expression was restored. [Figure 23] Experimental results show that low (27P) and medium (33B) skid silk restore claudin-1 expression in human skin. Punch skin biopsies were obtained from human donors ranging in age from 30 to 52 years. Skin biopsies were treated with acetone as described in Materials and Methods, and then silk or other reagents were added to them as indicated in the figures. Skin sections were stained for claudin-1 (orange staining) and cell nuclei (blue staining). Skin biopsies treated with low (27P) and medium (33B) silk polypeptides upregulated claudin-1 expression after treatment with acetone (a representative experiment is shown in this figure). (2 mg / mL = 0.2%, 3 mg / mL = 0.3%, 4 mg / mL = 0.4%) [Figure 24] Figures 24A-24D in Figure 24 show quantification of claudin-1 upregulation. A, B The ratio of claudin-1 (red intensity) to DAPI (cell number) within each experiment was averaged to represent claudin-1 expression per cell. Data were normalized to the untreated sample, and error bars represent the standard deviation of the normalized data. C, D Analysis showing the total area of ​​claudin-1 in human skin samples. Data are expressed as percentage ± SEM, *p<0.05 (see Materials and Methods for further details). (2 mg / mL = 0.2%, 3 mg / mL = 0.3%, 4 mg / mL = 0.4%, 5 mg / mL = 0.5%, 6 mg / mL = 0.6%, 7 mg / mL = 0.7%, 60 mg / mL = 6%). [Figure 25]Low-skid (27P) silk upregulates collagen expression in dermal skin fibroblasts. Human dermal fibroblasts were treated with various concentrations of low-skid (27P) silk polypeptide, and collagen expression was visualized. Collagen expression was upregulated at 2 mg / mL of low-skid (27P) silk polypeptide (0.2%). [Figure 26] Low-skid (27P) silk upregulates collagen expression in dermal skin fibroblasts at 2 mg / mL (0.2% w / v). Human dermal fibroblasts were treated with various concentrations of low-skid (27P) silk polypeptide, and collagen expression was visualized and quantified. Collagen expression was significantly upregulated at 2 mg / mL of low-skid (27P) silk polypeptide (0.25 mg / mL = 0.025%, 0.5 mg / mL = 0.05%, 2 mg / mL = 0.2%, 7 mg / mL = 0.7%). [Figure 27] Low-skid (27P) silk accelerates cell migration in wound closure assays. Human primary keratinocytes were grown in serum- and growth factor-free medium (see Materials and Methods for further details) (negative control). After they formed a layer, a scratch was created that disrupted the continuity of the layer. Cells were allowed to migrate into the resulting "wound" (gap) and the rate at which they filled it was measured. Keratinocytes treated with serum- and growth factor-free medium refilled approximately 20% of the total gap created ("wound closure"). When keratinocytes were treated with medium containing serum and growth factors, wound closure was nearly complete (positive control). Keratinocytes treated with medium and 0.5 mg / mL (0.05%) of low-skid (27P) silk polypeptide also showed nearly complete closure. [Figure 28]CD44 interaction with silk polypeptides. Results of a solid-phase protein-protein interaction assay. Low-skid silk (27P) and medium-skid silk (33B) were immobilized on a high-binding 96-well plate. Human CD44-hFc protein bound to both the low- (27P) and medium- (33B) silk polypeptide compositions (compare lanes 3 with 4 and 7 with 6). Medium-skid (33B) silk had higher nonspecific binding to the secondary antibody (compare lane 6 with 2), but when CD44-hFc was added, the resulting signal was much higher (compare lane 7 with 6). Absorbance values ​​are the average of three technical replicates. Independent experiments showed similar results (not shown). [Figure 29] 1 is a graph showing a summary of expert assessment of fine lines and wrinkles. [Figure 30] 1 is a graph showing a summary of expert assessment of skin firmness. [Figure 31] 1 is a graph showing a summary of expert assessment of redness. [Figure 32] 1 is a graph summarizing TEWL data (measured by Tewameter®). [Figure 33] 1 is a graph summarizing NumWr data (measured by silicone profilometry). [Figure 34] 1 is a graph summarizing bear data (measured by silicone profilometry). [Figure 35] 1 is a graph showing top box responses from the self-perception questionnaire for Study 33B. DETAILED DESCRIPTION OF THE INVENTION

[0008] Methods for producing silk fibroin or silk fibroin fragments 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. Methods for using silk fibroin or silk fibroin fragments in coating applications, including animal hair coating applications, are known and are described, for example, in U.S. Patent Application Publication Nos. 2016 / 0222579 and 2016 / 0281294. Compositions and methods for using silk fibroin or silk fibroin fragments in cosmetic applications are known and are described, for example, in U.S. Patent Application Publication Nos. 2018 / 0280274 and 2018 / 0008522, and WO 2019 / 005848. All publications cited herein are incorporated by reference in their entirety.

[0009] definition Unless otherwise defined, as used in the previous section and throughout the remainder of the specification, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned herein are incorporated herein by reference in their entirety.

[0010] All percentages, parts, and ratios are based on the total weight of the collagen boosting composition of the present invention unless otherwise specified. All such weights relating to listed ingredients are based on the active level and therefore do not include solvents or by-products that may be included in commercially available materials unless otherwise specified. The term "weight percent" may be referred to herein as "% by weight" or % w / w.

[0011] As used herein, the terms "a," "an," or "the" are generally interpreted to encompass both the singular and the plural.

[0012] As used herein, the term "about" generally refers to a particular numerical value that falls within an acceptable range of error as determined by one of ordinary skill in the art, which depends in part on how the numerical value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean a range of ±20%, ±10%, or ±5% of the given numerical value.

[0013] As used herein, the term "dermatologically acceptable carrier" means a carrier that is suitable for use in contact with mammalian keratinous tissue without causing any adverse effects, such as, for example, undue toxicity, incompatibility, instability, allergic response, etc. Dermatologically acceptable carriers may include, without limitation, water, liquid or solid emollients, humectants, solvents, and the like.

[0014] As used herein, the term "hydrophilic-lipophilic balance" (HLB) of a surfactant is a measure of the degree to which it is hydrophilic or hydrophobic, determined by calculating the values ​​of different regions of the molecule, according to Griffin's method: HLB=20*M h / M(where M h HLB values ​​are described by the HLB (where M is the molecular mass of the hydrophilic portion of the surfactant and M is the molecular mass of the entire surfactant molecule) and give results on a scale of 0 to 20. An HLB value of 0 corresponds to a completely lipophilic molecule, and a value of 20 corresponds to a completely hydrophilic molecule. HLB values ​​can be used to predict a molecule's surfactant properties: HLB < 10: lipid soluble (water insoluble), HLB > 10: water soluble (lipid insoluble), HLB = 1-3: antifoaming agent, 3-6: W / O (water-in-oil) emulsifier, 7-9: wetting agent and spreading agent, 8-16: O / W (oil-in-water) emulsifier, 13-16: surfactant, 16-18: solubilizer or hydrotrope.

[0015] As used herein, "average weight average molecular weight" refers to the average of two or more values ​​of weight average molecular weight of silk fibroin or fragments thereof of the same composition, the two or more values ​​being determined by two or more separate experimental readings.

[0016] As used herein, the term "polydispersity (PD)" of a polymer is generally used as a measure of the broadness of the molecular weight distribution of a polymer, and polydispersity

number

[0017] As used herein, the term "substantially homogeneous" can refer to silk fibroin-based protein fragments that are distributed in a normal distribution around an identified molecular weight. As used herein, the term "substantially homogeneous" can refer to, for example, a uniform distribution of components or additives, such as silk fibroin fragments, dermatologically acceptable carriers, etc., throughout the compositions of the present disclosure.

[0018] As used herein, the terms "silk fibroin peptide," "silk fibroin protein fragment," and "silk fibroin fragment" are used interchangeably. Molecular weight or number of amino acid units is defined when molecular size is the critical parameter.

[0019] SPF definition and characteristics As used herein, a "silk protein fragment" (SPF) includes one or more of the following: a "silk fibroin fragment" as defined herein, a "recombinant silk fragment" as defined herein, a "spider silk fragment" as defined herein, a "silk fibroin-like protein fragment" as defined herein, and / or a "chemically modified silk fragment" as defined herein. SPFs can have any molecular weight value or range described herein and any polydispersity value or range described herein. As used herein, in some embodiments, the term "silk protein fragment" also refers to a silk protein comprising or consisting of at least two identical repeating units, each independently selected from a natural silk polypeptide or a variation thereof, the amino acid sequence of a natural silk polypeptide, or a combination of both.

[0020] SPF molecular weight and polydispersityIn one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 1 to about 5 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 5 to about 10 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 10 to about 15 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 15 to about 20 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 14 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 20 to about 25 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 25 to about 30 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 30 to about 35 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 35 to about 40 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 39 to about 54 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 40 to about 45 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 45 to about 50 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 50 to about 55 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 55 to about 60 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 60 to about 65 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 65 to about 70 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 70 to about 75 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 75 to about 80 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 80 to about 85 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 85 to about 90 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 90 to about 95 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 95 to about 100 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 100 to about 105 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 105 to about 110 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 110 to about 115 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 115 to about 120 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 120 to about 125 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 125 to about 130 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 130 to about 135 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 135 to about 140 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 140 to about 145 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 145 to about 150 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 150 to about 155 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 155 to about 160 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 160 to about 165 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 165 to about 170 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 170 to about 175 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 175 to about 180 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 180 to about 185 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 185 to about 190 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 190 to about 195 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 195 to about 200 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 200 to about 205 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 205 to about 210 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 210 to about 215 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 215 to about 220 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 220 to about 225 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 225 to about 230 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 230 to about 235 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 235 to about 240 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 240 to about 245 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 245 to about 250 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 250 to about 255 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 255 to about 260 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 260 to about 265 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 265 to about 270 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 270 to about 275 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 275 to about 280 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 280 to about 285 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 285 to about 290 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 290 to about 295 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 295 to about 300 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 300 to about 305 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 305 to about 310 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 310 to about 315 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 315 to about 320 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 320 to about 325 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 325 to about 330 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 330 to about 335 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 335 to about 340 kDa.In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 340 to about 345 kDa. In one embodiment, the composition of the present disclosure comprises an SPF having an average weight average molecular weight selected from about 345 to about 350 kDa.

[0021] In some embodiments, the compositions of the present disclosure include SPF compositions selected from composition numbers 1001 through 2450, having a weight average molecular weight selected from about 1 kDa to about 145 kDa, and having a polydispersity selected from between 1 and about 5 (including but not limited to a polydispersity of 1), between 1 and about 1.5 (including but not limited to a polydispersity of 1), between about 1.5 and about 2, between about 1.5 and about 3, between about 2 and about 2.5, between about 2.5 and about 3, between about 3 and about 3.5, between about 3.5 and about 4, between about 4 and about 4.5, and between about 4.5 and about 5: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0022] As used herein, "low molecular weight," "low MW," or "low-MW" SPFs can include SPFs having a weight average molecular weight or average weight average molecular weight selected from about 5 kDa to about 38 kDa, about 14 kDa to about 30 kDa, or about 6 kDa to about 17 kDa. In some embodiments, the target low molecular weight of a particular SPF is about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa, about 14 kDa, about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, or about 22 kDa. The weight average molecular weight of the polymer may be about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, or about 38 kDa.

[0023] As used herein, "medium molecular weight," "medium MW," or "mid-MW" SPF can include SPFs having a weight average molecular weight or average weight average molecular weight selected from between about 31 kDa and about 55 kDa, or between about 39 kDa and about 54 kDa. In some embodiments, the target mid-molecular weight for a particular SPF can be a weight average molecular weight of about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, or about 55 kDa.

[0024] As used herein, "high molecular weight," "high MW," or "high-MW" SPF can include SPFs having a weight average molecular weight, or an average weight average molecular weight, selected from between about 55 kDa and about 150 kDa. In some embodiments, the target high molecular weight for a particular SPF can be about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, about 75 kDa, about 76 kDa, about 77 kDa, about 78 kDa, about 79 kDa, or about 80 kDa.

[0025] In some embodiments, the molecular weights described herein (e.g., low molecular weight silk, medium molecular weight silk, high molecular weight silk) can be converted to the approximate number of amino acids contained within each SPF, as would be understood by one skilled in the art. For example, the average weight of an amino acid may be about 110 Daltons (i.e., 110 g / mol). Thus, in some embodiments, dividing the molecular weight of a linear protein by 110 Daltons can be used to approximate the number of amino acid residues contained therein.

[0026] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between 1 and about 5.0, including but not limited to, a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 1.5 and about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between 1 and about 1.5, including but not limited to, a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 1.5 and about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 2.0 and about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 2.5 and about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 3.0 and about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 3.5 and about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from about 4.0 to about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity selected from between about 4.5 to about 5.0.

[0027] In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of 1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 1.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 2.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.6.In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 3.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.0. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.1. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.2. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.3. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.4. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.5. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.6. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.7. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.8. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 4.9. In one embodiment, the SPF in the composition of the present disclosure has a polydispersity of about 5.0.

[0028] 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.

[0029] Silk fibroin fragments Methods for producing silk fibroin or silk fibroin protein fragments and their applications in various fields are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177, 10,287,728, and 10,301,768, all of which are incorporated herein in their entirety. Raw silk from the silkworm Bombyx mori is composed of two major proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin is a fibrous protein with a semi-crystalline structure that provides rigidity and strength. As used herein, the term "silk fibroin" refers to fibers from Bombyx mori cocoons, which have a weight-average molecular weight of approximately 370,000 Da. Crude silkworm fibers consist of double strands of fibroin. The adhesive substance that holds these double strands together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da. Silk fibroin is an amphiphilic polymer with large hydrophobic domains that account for the majority of the polymer's high molecular weight. The hydrophobic regions are interrupted by small hydrophilic spacers, and the N- and C-termini of the chains are also highly hydrophilic. The hydrophobic domains of the H chains contain a repeating hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeats of Gly-Ala / Ser / Tyr dipeptides, which can form stable antiparallel sheet crystallites. The amino acid sequence of the L chain is non-repetitive, making it more hydrophilic and relatively elastic. The hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) chain segments in the silk fibroin molecule are alternately arranged to allow the silk fibroin molecule to self-assemble.

[0030] Provided herein are methods for producing pure, highly scalable silk fibroin-protein fragment mixture solutions that can be used across multiple industries for a variety of applications. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to the fragmentation of any SPF described herein, including, but not limited to, recombinant silk proteins and silk-like or fibroin-like proteins.

[0031] As used herein, the term "fibroin" includes silkworm fibroin and insect or spider silk proteins. In one embodiment, the fibroin is obtained from Bombyx mori. Raw silk from Bombyx mori is composed of two primary proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin is a fibrous protein with a semi-crystalline structure that provides rigidity and strength. As used herein, the term "silk fibroin" refers to Bombyx mori cocoon fibers having a weight-average molecular weight of approximately 370,000 Da. Conversion of these insoluble silk fibroin fibers into water-soluble silk fibroin protein fragments requires the addition of concentrated neutral salt (e.g., 8-10 M lithium bromide), which interferes with the intermolecular and intramolecular ionic and hydrogen bonds that would otherwise render the fibroin protein insoluble in water. Methods for making silk fibroin protein fragments and / or compositions thereof are known and are described, for example, in U.S. Patent Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0032] Raw silk cocoons from the silkworm Bombyx mori were cut into small pieces. The small pieces were treated in an aqueous solution of Na2CO3 at approximately 100°C for approximately 60 minutes to remove sericin (degumming). The volume of water used was approximately 0.4 times the raw silk weight, and the amount of Na2CO3 was approximately 0.848 times the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The volume of rinse water for each cycle was 0.2 L x the weight of the raw silk cocoon pieces. Excess water was removed from the degummed silk cocoon pieces. After the DI water washing step, the wet, degummed silk cocoon pieces were dried at room temperature. Degummed silk cocoon pieces were mixed with a LiBr solution, and the mixture was heated to approximately 100°C. The warmed mixture was placed in a drying oven and heated at approximately 100°C for approximately 60 minutes to achieve complete dissolution of the native silk protein. The resulting silk fibroin solution was filtered and dialyzed for 72 hours using tangential flow filtration (TFF) and a 10 kDa membrane against deionized water. The resulting aqueous silk fibroin solution had a concentration of approximately 8.5% by weight. The 8.5% silk solution was then diluted with water to yield a 1.0% w / v silk solution. TFF can then be used to further concentrate the pure silk solution to a concentration of 20.0% w / w silk in water.

[0033] Dialyzing silk through a series of water changes is a manual, time-intensive process that can be accelerated by varying certain parameters, e.g., diluting the silk solution before dialysis. The dialysis process can be scaled up for production by using semi-automated equipment, e.g., tangential flow filtration systems.

[0034] In some embodiments, silk solutions are prepared under various preparation condition parameters, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3 M LiBr was prepared and left at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in an oven at 60°C. Samples from each set were removed at 4, 6, 8, 12, 24, 168, and 192 hours.

[0035] In some embodiments, silk solutions are prepared under various preparation condition parameters, such as 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, a 9.3 M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5 mL of the hot LiBr solution was added to 1.25 g of silk and placed in a 60°C oven. Samples from each set were removed at 1, 4, and 6 hours.

[0036] In some embodiments, silk solutions are prepared under various preparation condition parameters. For example, four different silk extraction combinations were used: 90°C for 30 minutes, 90°C for 60 minutes, 100°C for 30 minutes, and 100°C for 60 minutes. Briefly, 9.3M LiBr solution was heated to one of four temperatures: 60°C, 80°C, 100°C, or boiling. 5mL of the hot LiBr solution was added to 1.25g of silk and placed in an oven at the same temperature of LiBr. Samples from each set were removed at 1, 4, and 6 hours. 1mL of each sample was added to 7.5mL of 9.3M LiBr and refrigerated for viscosity testing.

[0037] In some embodiments, SPF is achieved by dissolving raw undegummed, partially degummed, or degummed silkworm fiber with a neutral lithium bromide salt. The raw silkworm silk is treated to remove any sericin and to obtain a desired weight average molecular weight (M WThe silk fibroin protein fragments are processed under selected temperatures and other conditions to achieve a desired size and polydispersity (PD). The selection of process parameters can be varied to achieve different final silk protein fragment characteristics, depending on the intended use. The resulting final fragment solution is pure silk fibroin protein fragments and water with undetectable levels of process contaminants, parts per million (ppm), levels acceptable in the pharmaceutical, medical, and consumer eye care markets. The concentration, size, and polydispersity of the SPF can be further varied depending on the desired use and performance requirements.

[0038] FIG. 5 is a flowchart illustrating various embodiments for producing pure silk fibroin protein fragments (SPF) of the present disclosure. It should be understood that not all of the illustrated steps are necessarily required to produce all of the silk solutions of the present disclosure. As illustrated in FIG. 5, Step A, cocoons (heat-treated or non-heat-treated), silk fibers, silk powder, spider silk, or recombinant spider silk can be used as the silk source. When starting with raw silk cocoons from Bombyx mori, the cocoons can be cut into small pieces, e.g., pieces of approximately equal size (Step B1). The raw silk is then extracted and rinsed to remove sericin (Step C1a). This results in raw silk that is substantially free of sericin. In one embodiment, water is heated to a temperature between 84°C and 100°C (ideally to the boiling point), and then Na2CO3 (sodium carbonate) is added to the boiling water until the Na2CO3 is completely dissolved. Raw silk is added to boiling water / Na2CO3 (100°C) and soaked for approximately 15-90 minutes, with longer boiling times resulting in smaller silk protein fragments. In one embodiment, the volume of water is equal to about 0.4 x the weight of raw silk, and the volume of Na2CO3 is equal to about 0.848 x the weight of raw silk. In one embodiment, the volume of water is equal to 0.1 x the weight of raw silk, and the volume of Na2CO3 is maintained at 2.12 g / L.

[0039] The aqueous NaCO solution is then drained, and excess water / NaCO is removed from the silk fibroin fibers (e.g., by ringing out the fibroin extract through manual or mechanical dehydration cycles). The resulting silk fibroin extract is rinsed with warm to hot water, typically at a temperature ranging from about 40°C to about 80°C, through at least one change in water volume (repeated as many times as necessary) to remove any remaining adsorbed sericin or contaminants. The resulting silk fibroin extract is silk fibroin substantially depleted of sericin. In one embodiment, the resulting silk fibroin extract is rinsed with water at a temperature of about 60°C. In one embodiment, the amount of rinse water for each cycle is equal to 0.1 L to 0.2 L times the weight of the raw silk. To maximize the rinsing effect, it may be advantageous to agitate, rotate, or circulate the rinse water. After rinsing, excess water is removed from the extracted silk fibroin fibers (e.g., manually or mechanically ringing out the fibroin extract). Alternatively, methods known to those skilled in the art, such as pressure, temperature, or other reagents, or a combination thereof, can be used for the purpose of sericin extraction. Alternatively, silk glands (100% sericin-free silk protein) can be directly removed from the worm. This can result in sericin-free liquid silk protein without any change in protein structure.

[0040] The extracted fibroin fibers are then completely dried. Once dry, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at ambient to boiling temperatures (step C1b). In one embodiment, the solvent is a solution of lithium bromide (LiBr) (the boiling point for LiBr is 140°C). Alternatively, the extracted fibroin fibers are not dried, but are wet and placed in a solvent; the solvent concentration can then be varied to achieve a concentration similar to that achieved when dry silk is added to the solvent. The final concentration of LiBr in the solvent can range from 0.1 M to 9.3 M. Complete dissolution of the extracted fibroin fibers can be achieved by varying the treatment time and temperature, along with the concentration of the dissolving solvent. Other solvents can be used, including, but not limited to, phosphate phosphoric acid, calcium nitrate, calcium chloride solution, or concentrated aqueous solutions of other inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed in the already heated solvent solution and then maintained at a temperature ranging from about 60° C. to about 140° C. for 1 to 168 hours. In one embodiment, the silk fibers should be fully immersed in the solvent solution and then placed in a drying oven at a temperature of about 100° C. for about 1 hour.

[0041] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an effect on the time required to completely dissolve the fibroin, as well as on the molecular weight and polydispersity of the resulting final SPF mixed solution. In one embodiment, the silk solvent solution concentration is less than or equal to 20% w / v. In addition, stirring during addition or dissolution may be used to promote dissolution at varying temperatures and concentrations. The temperature of the LiBr solution provides control over the molecular weight and polydispersity of the resulting silk protein fragment mixture. In one embodiment, higher temperatures dissolve the silk more quickly, resulting in increased process scalability and mass production of the silk solution. In one embodiment, using a LiBr solution heated to a temperature between 80°C and 140°C reduces the time required in the oven to achieve complete dissolution. Varying the time and temperature of the dissolution solvent at or above 60°C changes and controls the MW and polydispersity of the SPF mixed solution formed from native silk fibroin protein of the original molecular weight.

[0042] Alternatively, whole cocoons can be placed directly into a solvent, such as LiBr, bypassing the extraction (step B2). This requires subsequent filtration of the silkworm particles from the silk and solvent solution and removal of sericin (step C2) using methods known in the art to separate hydrophobic and hydrophilic proteins, such as column separation and / or chromatography, ion exchange, chemical precipitation with salt and / or pH, and / or enzymatic digestion and filtration or extraction (all methods are general examples of standard protein separation methods, but are not limited to these). Non-heat-treated cocoons from which the silkworms have been removed can instead be placed directly into a solvent, such as LiBr, bypassing the extraction. The methods described above can be used for sericin isolation, with the advantage that non-heat-treated cocoons contain significantly less silkworm debris.

[0043] Dialysis can be used to remove the dissolving solvent from the resulting dissolved fibroin protein fragment solution by dialyzing the solution against a volume of water (step E1). Pre-filtration before dialysis is useful to remove any debris (i.e., silkworm remnants) from the silk and LiBr solution (step D). In one example, a 3 μm or 5 μm filter is used at a flow rate of 200-300 mL / min to filter down to a 0.1%-1.0% silk-LiBr solution, if desired, before dialysis and potential concentration. A method disclosed herein, as described above, is to use time and / or temperature to reduce the concentration from 9.3 M LiBr to a range of 0.1 M-9.3 M to facilitate filtration and downstream dialysis, particularly when considering creating a scalable process. Alternatively, the 9.3 M LiBr-silk protein fragment solution can be diluted with water to facilitate filtration and dialysis of debris without additional time or temperature. The result of dissolution, upon filtration for the desired time and temperature, is a translucent, particle-free, room-temperature storage-stable LiBr solution of silk protein fragments of known MW and polydispersity. It is advantageous to periodically change the dialysis water (e.g., change the water after 1 hour, 4 hours, then every 12 hours for a total of six changes) until the solvent is removed. The total number of water volume changes can be varied based on the resulting concentration of the solvent used for dissolving and fragmenting the silk protein. After dialysis, the final silk solution can be further filtered to remove any remaining debris (i.e., silkworm remains).

[0044] Alternatively, tangential flow filtration (TFF), a rapid and efficient method for the separation and purification of biomolecules, can be used to remove the solvent from the resulting dissolved fibroin solution (step E2). TFF provides highly pure aqueous silk protein fragment solutions and allows for process scalability to produce large volumes of solution in a controlled and repeatable manner. The silk and LiBr solution can be diluted (20% to 0.1% silk in either water or LiBr) before TFF. Prefiltration, as described above, before TFF processing can maintain filter efficiency and potentially avoid the formation of a silk gel boundary layer on the surface of the filter as a result of the presence of debris particles. Prefiltration before TFF is also useful to remove any remaining debris (i.e., silkworm remnants) from the silk and LiBr solution, which could cause spontaneous or long-term gelation of the resulting water-only solution (step D). TFF (recirculating or single-pass) can be used to generate water-silk protein fragment solutions of 0.1% to 30.0% silk (more preferably 0.1% to 6.0% silk). Different cutoff size TFF membranes may be required based on the desired concentration, molecular weight, and polydispersity of the silk protein fragment mixture in solution. Membranes ranging from 1 to 100 kDa may be required to vary the molecular weight silk solutions, for example, by varying the length of extraction boiling time or the time and temperature in the dissolving solvent (e.g., LiBr). In one embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution to achieve the final desired silk-to-water ratio. Additionally, TFF single-pass, TFF, and other methods known in the art, such as falling film evaporators, can be used to concentrate the solution after removal of the dissolving solvent (e.g., LiBr) (with a resulting desired concentration ranging from 0.1% to 30% silk). This can be used as an alternative to the standard HFIP concentration method known in the art to generate water-based solutions. Larger pore membranes can also be utilized to filter out small silk protein fragments and create higher molecular weight silk solutions with and / or without tighter polydispersity values.

[0045] Assays for LiBr and Na2CO3 detection can be performed using an HPLC system equipped with an evaporative light scattering detector (ELSD). Calculations were performed by linear regression of the resulting peak areas for the analytes plotted against concentration. Two or more samples of various formulations of the present disclosure were used for sample preparation and analysis. Typically, four samples of different formulations were weighed directly into a 10 mL volumetric flask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and maintained at 2-8 °C with occasional shaking for 2 hours to extract the analytes from the film. After 2 hours, the solution was diluted with 20 mM ammonium formate (pH 3.0). The sample solution from the volumetric flask was transferred into an HPLC vial and injected into the HPLC-ELSD system for estimation of sodium carbonate and lithium bromide.

[0046] The analytical method developed for the quantification of Na2CO3 and LiBr in silk protein preparations was found to be linear in the range of 10-165 μg / mL, with RSDs of 2% and 1% for area and 0.38% and 0.19% for retention time for sodium carbonate and lithium bromide, respectively. This analytical method can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein preparations.

[0047] 6 is a flow chart illustrating various parameters that can be modified during the extraction and dissolution steps of the process to produce a silk protein fragment solution of the present disclosure. Selected method parameters can be altered to achieve distinct final solution characteristics depending on the intended use, e.g., molecular weight and polydispersity. It should be understood that not all of the illustrated steps are necessarily required to produce all silk solutions of the present disclosure.

[0048] In one embodiment, a silk protein fragment solution useful for a wide range of applications is prepared according to the following steps: forming small pieces of silk cocoons from Bombyx mori silkworms; extracting the small pieces in an aqueous Na2CO3 solution at about 100°C for about 60 minutes (the volume of water is equal to about 0.4 x the raw silk weight, and the amount of Na2CO3 is about 0.848 x the weight of the pieces) to form a silk fibroin extract; rinsing the silk fibroin extract three times at about 60°C in a volume of rinse water for about 20 minutes per rinse (the rinse water for each cycle is equal to about 0.2 L x the weight of the pieces), removing excess water from the silk fibroin extract; and extracting the silk fibroin extract. drying, dissolving the dried silk fibroin extract in a LiBr solution (the LiBr solution is first heated to about 100°C to create and maintain a silk and LiBr solution); placing the silk and LiBr solution in a drying oven at about 100°C for about 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into a mixture with the desired molecular weight and polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to result in 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 utilized to purify the silk solution to achieve the final desired silk-to-water ratio. TFF can then be used to further concentrate the silk solution to a concentration of 2.0 wt% silk in water.

[0049] While not wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., temperature of the LiBr solution added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, uniformities, and colors. Also, without wishing to be bound by any particular theory, increasing the temperature for extraction, extending the extraction time, using higher temperature LiBr solutions, both initially and over time, when dissolving the silk, and increasing the time at temperature (e.g., in an oven as shown herein, or alternative heat source) all resulted in solvent and silk solutions with lower viscosity and more uniformity.

[0050] The extraction process can be completed in a larger vessel, such as an industrial washing machine, capable of maintaining temperatures at or between 60°C and 100°C. The rinsing process can also be completed in an industrial washing machine, eliminating manual rinse cycles. Dissolution of silk in a LiBr solution can occur in a vessel other than a convection oven, such as a stirred tank reactor. Dialyzing silk through a series of water changes is a manual, 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 for production by using semi-automated equipment, such as a tangential flow filtration system.

[0051] Varying the extraction (i.e., time and temperature), LiBr (i.e., temperature of the LiBr solution added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, uniformities, and colors. Increasing the temperature for extraction, extending the extraction time, using higher temperature LiBr solutions, both 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 herein) all result in solvent and silk solutions with lower viscosities and more uniformity. While nearly all parameters result in viable silk solutions, methods that achieve complete dissolution in less than 4-6 hours are preferred for process scalability.

[0052] In one embodiment, a solution of silk fibroin protein fragments having a weight average selected from between about 6 kDa and about 17 kDa is prepared according to the following steps: degumming the silk source by adding the silk source to an aqueous solution of boiling (100°C) sodium carbonate for a treatment time of about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract containing non-detectable levels of sericin; draining the solution from the silk fibroin extract; and heating the silk fibroin extract at about 60°C to about 140°C. The method includes dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of 140°C, maintaining the silk fibroin-lithium bromide solution in an oven having a temperature of about 140°C for up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk protein fragments, the aqueous solution comprising fragments having a weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm of sodium carbonate residue as measured using a high-performance liquid chromatography sodium carbonate assay. The aqueous solution of silk fibroin protein fragments may be freeze-dried. In some embodiments, the silk fibroin protein fragment solution may be further processed into various forms, including gels, powders, and nanofibers.

[0053] In one embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from between about 17 kDa and about 39 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes to effect degumming; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C upon placing the silk fibroin extract in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in a drying oven having a temperature in the range of about 60°C to about 100°C for a period of up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk fibroin protein fragments, the aqueous solution of silk fibroin protein fragments comprising about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments comprising about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of silk fibroin protein fragments comprising fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay.

[0054] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight average molecular weight selected from between about 6 kDa and about 17 kDa comprises the following steps: degumming a silk source by adding the silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes and about 60 minutes; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; and removing the silk fibroin extract from the solution containing undetectable levels of sericin. The method includes dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature ranging from 0°C to about 140°C, maintaining the silk fibroin-lithium bromide solution in an oven having a temperature of about 140°C for a period of at least 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk protein fragments, the aqueous solution comprising fragments having an average weight-average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin protein fragments may contain less than 100 ppm of sodium carbonate residue as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin protein fragments may be lyophilized. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin protein fragments.The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding at least one of zinc oxide or titanium dioxide. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The gel may contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

[0055] In some embodiments, a method for preparing an aqueous solution of silk fibroin protein fragments having an average weight average molecular weight selected from between about 17 kDa and about 39 kDa comprises the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes to effect degumming, removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin, draining the solution from the silk fibroin extract, dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C, and then dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 60°C to about 80°C. maintaining the silk fibroin-lithium bromide solution in an oven having a temperature ranging from about 10 ppm to about 100°C for a period of at least 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of pure silk fibroin protein fragments (the aqueous solution of pure silk fibroin protein fragments comprises about 10 ppm to about 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments comprises about 10 ppm to about 100 ppm of sodium carbonate residue, and the aqueous solution of pure silk fibroin protein fragments comprises fragments having an average weight-average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity of 1 to about 5, or about 1.5 to about 3.0). The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of pure silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin protein fragments may contain less than 100 ppm of residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay. The method may further include adding a therapeutic agent to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a molecule selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof.The aqueous solution of pure silk fibroin protein fragments may be freeze-dried. The method may further include adding an alpha hydroxy acid to the aqueous solution of pure silk fibroin protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. The method may further include adding at least one of zinc oxide or titanium dioxide. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel can be made from an aqueous solution of the pure silk fibroin protein fragments produced by the present method. The gel can contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel can have a silk content of at least 2% and a vitamin content of at least 20%.

[0056] In one embodiment, a solution of silk fibroin protein fragments having a weight average molecular weight selected from between about 39 kDa and about 80 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to effect degumming; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; and placing the silk fibroin extract in a lithium bromide solution having a starting temperature in the range of about 80°C to about 140°C. The method includes the steps of dissolving the silk fibroin extract and maintaining the silk fibroin-lithium bromide solution in a drying oven having a temperature in the range of about 60°C to about 100°C for a period of up to 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk fibroin protein fragments, the aqueous solution of silk fibroin protein fragments comprising about 10 ppm to about 300 ppm of lithium bromide residue, about 10 ppm to about 100 ppm of sodium carbonate residue, fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa, and a polydispersity of 1 to about 5, or about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may contain less than 100 ppm sodium carbonate residual as measured using a high performance liquid chromatography sodium carbonate assay. In some embodiments, the method may further comprise adding an active agent (e.g., a therapeutic agent) to the aqueous solution of pure silk fibroin protein fragments. The method may further comprise adding an active agent selected from one of an antioxidant or an enzyme to the aqueous solution of pure silk fibroin protein fragments. The method may further comprise adding a vitamin to the aqueous solution of pure silk fibroin protein fragments. The vitamin may be vitamin C or a derivative thereof. The aqueous solution of pure silk fibroin protein fragments may be lyophilized.The method may further include adding an alpha-hydroxy acid to the aqueous solution of pure silk fibroin protein fragments. The alpha hydroxy acid may be selected from the group consisting of glycolic acid, lactic acid, tartaric acid, and citric acid. The method may further include adding hyaluronic acid or a salt form thereof at a concentration of about 0.5% to about 10.0% to the aqueous solution of pure silk fibroin protein fragments. A film may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The film may contain about 1.0% to about 50.0% by weight of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0% to about 20.0% by weight. The film may contain about 30.0% to about 99.5% by weight of pure silk fibroin protein fragments. A gel may be made from the aqueous solution of pure silk fibroin protein fragments produced by the method. The gel may contain about 0.5% to about 20.0% by weight of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% by weight and a vitamin content of at least 20% by weight.

[0057] The molecular weight of the silk protein fragments can be controlled based on specific parameters utilized during the extraction step, including extraction time and temperature; the dissolution step, including the LiBr temperature during the silk immersion in lithium bromide and the time the solution is maintained at a specific temperature; and the filtration step. By controlling process parameters using the disclosed method, it is possible to create silk fibroin protein fragment solutions with a variety of different molecular weights selected from 5 kDa to 200 kDa or 10 kDa to 80 kDa, and with polydispersities equal to or less than 2.5. By varying process parameters to achieve silk solutions with different molecular weights, it is possible to target a range of final fragment mixture products with desirable polydispersities equal to or less than 2.5 based on desired performance requirements. For example, high-molecular-weight silk films containing ophthalmic drugs may have a controlled, sustained release rate compared to lower-molecular-weight films, making them ideal for use as delivery vehicles in eye care products. Additionally, silk fibroin protein fragment solutions with polydispersities greater than 2.5 can be achieved. Furthermore, two solutions with different average molecular weights and polydispersities can be mixed to create a combined solution. Alternatively, liquid silk glands (100% sericin-free silk protein) directly removed from silkworms can be used in combination with any of the silk fibroin protein fragment solutions disclosed herein. The molecular weight of the pure silk fibroin protein fragment composition was determined using high-pressure liquid chromatography (HPLC) with a refractive index detector (RID). Polydispersity was calculated using Cirrus GPC Online GPC / SEC software version 3.3 (Agilent).

[0058] Differences in processing parameters can result in regenerated silk fibroin that varies in molecular weight and peptide chain size distribution (polydispersity, PD), which in turn affects the performance of the regenerated silk fibroin, including mechanical strength, water solubility, etc.

[0059] Parameters were varied during the processing of raw silk cocoons into silk solution. Varying these parameters affected the MW of the resulting silk solution. The parameters manipulated included (i) extraction time and temperature, (ii) LiBr temperature, (iii) dissolution oven temperature, and (iv) dissolution time. Experiments were conducted to determine the effect of varying the extraction time. Tables 1-7 summarize the results. The summary is as follows: A sericin extraction time of 30 minutes resulted in a larger molecular weight than a sericin extraction time of 60 minutes. The molecular weight decreases over time in the oven. 140°C LiBr and oven resulted in a lower confidence interval below a molecular weight of 9500 Da. The 30 minute extractions at 1st and 4th hours have undigested silk. A 30-minute extraction at hour 1 resulted in a significantly higher molecular weight, with the lower limit of the confidence interval being 35,000 Da. The molecular weight range within the upper limit of the confidence interval was 18,000–216,000 Da (which is important for providing a solution with a specific upper limit). [Table 2] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7]

[0060] Experiments were conducted to determine the effect of varying the extraction temperature. The results are summarized in Table 7. The summary is as follows: The sericin extracted at 90℃ had a higher MW than the sericin extracted at 100℃. Both 90°C and 100°C show a decrease in MW over time in the oven. [Table 8]

[0061] Experiments were conducted to determine the effect of varying the temperature of lithium bromide (LiBr) when added to silk. The results are summarized in Tables 8-9. The summary is as follows: No effect on molecular weight or confidence intervals (all CIs approximately 10500-6500 Da) Tests demonstrate that once LiBr is added and begins to dissolve, the temperature of the LiBr-silk melt drops rapidly below the original LiBr temperature due to the majority of its mass being silk at room temperature. [Table 9] [Table 10]

[0062] Experiments were conducted to determine the effect of oven / melt temperature. The results are summarized in Tables 10-14. The summary is as follows: Oven temperature has less effect on silk extracted for 60 minutes than on silk extracted for 30 minutes. Without wishing to be bound by theory, it is believed that the 30 minute silk is less degraded during extraction and therefore oven temperature has more effect on the larger MW and less degraded portion of the silk. For the 60°C vs. 140°C oven, silk extracted for 30 minutes showed a very significant effect of the lower MW at the higher oven temperature, while silk extracted for 60 minutes had an effect, but it was much less. The 140°C oven yielded a lower limit in the confidence interval at approximately 6000 Da. [Table 11] [Table 12] [Table 13] [Table 14] [Table 15]

[0063] Raw silk cocoons from the silkworm Bombyx mori were cut into small pieces. The raw silk cocoon pieces were boiled in an aqueous solution of Na2CO3 (approximately 100°C) for approximately 30 to 60 minutes to remove sericin (degumming). The volume of water used was approximately 0.4 times the weight of the raw silk, and the amount of Na2CO3 was approximately 0.848 times the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed three times with deionized water at approximately 60°C (20 minutes per rinse). The volume of rinse water for each cycle was 0.2 L times the weight of the raw silk cocoon pieces. Excess water was removed from the degummed silk cocoon pieces. After the DI water washing step, the wet, degummed silk cocoon pieces were allowed to dry at room temperature. Degummed silk cocoon pieces were mixed with a LiBr solution, and the mixture was heated to approximately 100°C. The warmed mixture was placed in a dry oven and heated at a temperature ranging from approximately 60°C to approximately 140°C for approximately 60 minutes to achieve complete dissolution of the native silk proteins. The resulting solution was cooled to room temperature and then dialyzed using a 3,500 Da MWCO membrane to remove the LiBr salt. Multiple exchanges were performed using octane bromide (Br - ) Br as determined in a hydrolyzed fibroin solution lead on a dual-junction ion-selective electrode. - It was carried out in Di water until the ions were below 1 ppm.

[0064] The resulting silk fibroin aqueous solution has a concentration of approximately 8.0% w / v containing pure silk fibroin protein fragments with an average weight-average molecular weight selected from between about 6 kDa to about 16 kDa, about 17 kDa to about 39 kDa, and about 39 kDa to about 80 kDa and a polydispersity of about 1.5 to about 3.0. The 8.0% w / v was diluted with DI water to provide 1.0% w / v, 2.0% w / v, 3.0% w / v, 4.0% w / v, and 5.0% w / v coating solutions.

[0065] Various % silk concentrations have been produced through the use of tangential flow filtration (TFF). In all cases, a 1% silk solution was used as the input feed. A range of 750-18,000 mL of 1% silk solution was used as the starting volume. The solution is diafiltered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, the solution undergoes ultrafiltration to increase concentration through water removal. See example below.

[0066] Six silk solutions were utilized in a standard silk construction with the following results: Solution #1 has a silk concentration of 5.9 wt %, an average MW of 19.8 kDa, and a PDI of 2.2 (made by 60 min boiling extraction, LiBr dissolution at 100° C. for 1 hour).

[0067] Solution #2 is 6.4 wt% silk concentration (made by 30 min boiling extraction, LiBr dissolution at 60° C. for 4 h).

[0068] Solution #3 is 6.17 wt% silk concentration (made by 30 min boiling extraction, LiBr dissolution at 100° C. for 1 hour).

[0069] Solution #4 has a silk concentration of 7.30% by weight: The 7.30% silk solution was produced from a 30-minute extraction batch of 100 g of silk cocoons per batch. The extracted silk fibers were then dissolved in 9.3 M LiBr at 100°C for 1 hour in an oven at 100°C. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 15,500 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of around 1300 mL. 1262 mL of 7.30% silk was then recovered. Water was added to the feed solution to help remove the remaining solution, and 547 mL of 3.91% silk was then recovered.

[0070] Solution #5 has a silk concentration of 6.44 wt%: The 6.44 wt% silk solution was produced by starting with 60-minute extraction batches of a mixture of 25, 33, 50, 75, and 100 g of silk cocoons per batch. The extracted silk fibers were then dissolved in a 100°C oven for 1 hour using 9.3 M LiBr at 100°C. 35, 42, 50, and 71 g of silk fibers per batch were dissolved and combined to create 20% silk in LiBr. The dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 17,000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of around 3000 mL. 1490 mL of 6.44% silk was then recovered. Water was added to the feed solution to help remove any remaining solution, and 1454 mL of 4.88% silk was then recovered.

[0071] Solution #6 has a silk concentration of 2.70% by weight: A 2.70% silk solution was produced starting with a 60-minute extraction batch of 25 g of silk cocoons per batch. The extracted silk fibers were then dissolved in a 100°C oven for 1 hour using 9.3 M LiBr at 100°C. 35.48 g of silk fibers were dissolved per batch to produce 20% silk in LiBr. The silk dissolved in LiBr was then diluted to 1% silk and filtered through a 5 μm filter to remove large debris. 1000 mL of the 1% filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once the LiBr was removed, this solution was ultrafiltered to a volume of around 300 mL. 312 mL of 2.7% silk was then recovered.

[0072] The preparation of silk fibroin solutions with high molecular weights is given in Table 15. [Table 16]

[0073] Silk aqueous coating compositions for application to textiles are given in Tables 16 and 17 below. [Table 17] [Table 18]

[0074] Three silk solutions were utilized in film preparation with the following results: Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and 2.2 PD (made by boiling extraction for 60 minutes, LiBr dissolution at 100° C. for 1 hour).

[0075] Solution #2 is a 6.4% silk concentration (made by boiling extraction for 30 minutes, LiBr dissolution at 60° C. for 4 hours).

[0076] Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes, dissolving in LiBr at 100° C. for 1 hour).

[0077] Films were prepared according to Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published online September 22, 2011, doi:10.1038 / nprot.2011.379). Four milliliters of 1% or 2% (weight / volume) aqueous silk solution was added to a 100 mm Petri dish (the volume of silk can vary for thicker or thinner films and is not critical) and allowed to dry uncovered overnight. The bottom of a vacuum desiccator was filled with water. The dried film was placed in the desiccator, and a vacuum was applied to allow the film to water-anneal for 4 hours before removal from the dish. Films cast from Solution #1 did not yield structurally continuous films. The film cracked into several small pieces. These small pieces dissolved in water despite the water-annealing treatment.

[0078] Silk solutions of various molecular weights and / or combinations of molecular weights can be optimized for gel applications. The following provides an example of this process, but it is not intended to be limiting in application or formulation. Three silk solutions were utilized in gel creation with the following results: Solution #1 has a silk concentration of 5.9%, an average MW of 19.8 kDa, and 2.2 PD (made by boiling extraction for 60 minutes, LiBr dissolution at 100° C. for 1 hour).

[0079] Solution #2 is a 6.4% silk concentration (made by boiling extraction for 30 minutes, LiBr dissolution at 60° C. for 4 hours).

[0080] Solution #3 is a 6.17% silk concentration (made by boiling extraction for 30 minutes, dissolving in LiBr at 100° C. for 1 hour).

[0081] "Egel" is an electrogelation process as described in Rockwood et al. Briefly, 10 ml of aqueous silk solution was added to a 50 ml conical tube, and a pair of platinum wire electrodes was immersed in the silk solution. A potential of 20 volts was applied to the platinum electrodes for 5 minutes, and the power supply was turned off to collect the gel. Solution #1 did not form EGEL over the 5 minutes of applied current.

[0082] Solutions #2 and #3 were gelled according to published horseradish peroxidase (HRP) protocols, and behavior appeared to be typical of published solutions.

[0083] Materials and Methods: The following equipment and materials are used in determining silk molecular weight: Agilent 1100 with chemstation software version 10.01, refractive index detector (RID), analytical balance, volumetric flasks (1000 mL, 10 mL, and 5 mL), HPLC-grade water, ACS-grade sodium chloride, ACS-grade sodium phosphate dibasic heptahydrate, phosphoric acid, dextran MW standards—nominal molecular weights 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa, 50 mL PET or polypropylene disposable centrifuge tubes, graduated pipettes, amber glass HPLC vials with Teflon caps; Phenomenex PolySep GFC P-4000 column (size: 7.8 mm x 300 mm).

[0084] Procedure steps: A) Preparation of 1 L mobile phase (0.1 M sodium chloride solution in 0.0125 M sodium phosphate buffer). Take a clean, dry 250 mL beaker, place it on a balance, and tare it. Add approximately 3.3509 g of sodium phosphate dibasic heptahydrate to the beaker. Note the exact weight of sodium phosphate dibasic. Dissolve the weighed sodium phosphate by adding 100 mL of HPLC water to the beaker. Be careful not to spill the contents of the beaker. Carefully transfer the solution into a clean, dry 1000 mL volumetric flask. Rinse the beaker and transfer the rinse into the volumetric flask. Repeat the rinse 4-5 times. Accurately weigh approximately 5.8440 g of sodium chloride into a separate, clean, dry 250 mL beaker. Dissolve the weighed sodium chloride in 50 mL of water and transfer the solution to the sodium phosphate solution in the volumetric flask. Rinse the beaker and transfer the rinse into the volumetric flask. Adjust the pH of the solution to 7.0 ± 0.2 using phosphoric acid. Bring the volume in the volumetric flask to 1000 mL with HPLC water and stir vigorously to mix the solution uniformly. Filter the solution through a 0.45 μm polyamide membrane filter. Transfer the solution to a clean, dry solvent bottle and label the bottle. The volume of this solution can be adjusted to meet your needs by adjusting the amounts of sodium phosphate dibasic heptahydrate and sodium chloride accordingly.

[0085] B) Preparation of dextran molecular weight standard solution At least five different molecular weight standards are used for each batch of samples run so that the expected values ​​of the samples being tested are bracketed by the values ​​of the standards used. Six 20 mL scintillation glass vials are labeled with each molecular weight standard. Approximately 5 mg of each dextran molecular weight standard is accurately weighed and the weight is recorded. The dextran molecular weight standards are dissolved in 5 mL of mobile phase to make a 1 mg / mL standard solution.

[0086] C) Preparation of sample solution When preparing the sample solution, if the amount of sample available is limited, the preparation may be adjusted as long as the ratio is maintained. Depending on the sample type and silk protein content in the sample, weigh enough sample into a 50 mL disposable centrifuge tube on an analytical balance to create a 1 mg / mL sample solution for analysis. Dissolve the sample in an equal volume of mobile phase to create a 1 mg / mL solution. Cap the tube tightly and mix the sample (in solution). Allow the sample solution to stand at room temperature for 30 minutes. Mix the sample solution gently again for 1 minute and centrifuge at 4000 RPM for 10 minutes.

[0087] D) HPLC analysis of the samples Transfer 1.0 mL of all standard and sample solutions into individual HPLC vials. Inject molecular weight standards (single injection each) and each sample in duplicate. Analyze all standard and sample solutions using the following HPLC conditions: [Table 19]

[0088] E) Data Analysis and Calculations—Calculation of Average Molecular Weight Using Cirrus Software The chromatographic data files of the standards and analytical samples are uploaded into the Cirrus SEC data collection and molecular weight analysis software. The weight average molecular weight (M) for each injection of the sample is calculated. w ), number average molecular weight (M n ), peak average molecular weight (M p ), and polydispersity are calculated.

[0089] Spider silk fragments Spider silk is a natural polymer composed of three domains: a repetitive middle core domain that dominates the protein chain, and nonrepetitive N- and C-terminal domains. The large core domain is organized in a block copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides, alternate. Dragline silk is a protein complex composed of major ampullate gland dragline silk protein 1 (MaSp1) and major ampullate gland dragline silk protein 2 (MaSp2). Both silks are approximately 3500 amino acids long. MaSp1 can be found in the fiber core and periphery, while MaSp2 clusters in a specific core region. The large central domains of MaSp1 and MaSp2 are organized in a block copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides, alternate in the core domain. Specific secondary structures have been assigned to the poly(A) / (GA), GGX, and GPGXX motifs, including β-sheets, β-helices, and β-spirals, respectively. The primary sequence, composition, and secondary structural elements of the repetitive core domain are responsible for the mechanical properties of spider silk; in contrast, the non-repetitive N- and C-terminal domains are essential for the storage of liquid silk dope in the lumen and for fiber formation in the spinning ducts.

[0090] The main difference between MaSp1 and MaSp2 is the presence of proline (P) residues, which account for 15% of the total amino acid content in MaSp2, whereas MaSp1 is proline-free. By calculating the number of proline residues in N. clavipes dragline silk, it is possible to estimate the presence of two proteins in the fiber: 81% MaSp1 and 19% MaSp2. Different spiders have different ratios of MaSp1 and MaSp2. For example, dragline silk fibers from the orb weaver Argiope aurantia contain 41% MaSp1 and 59% MaSp2. Such changes in the ratio of major ampullate gland silk can affect the performance of the silk fiber.

[0091] At least seven different types of silk proteins are known for one orb-weaver spider. Silks differ in primary sequence, physical properties, and function. For example, dragline silk, used to construct frames, radii, and lifelines, is known for its remarkable mechanical properties, including strength, toughness, and elasticity. On an equal weight basis, spider silk has higher toughness than steel and Kevlar®. Flagellate silk, found in the capture spiral, has an extensibility of up to 500%. Minor ampullate silk, found in the accessory spirals and captures of orb webs, has high toughness and strength similar to major ampullate silk, but does not hypercontract in water.

[0092] Spider silk is known for its high tensile strength and toughness. Recombinant silk proteins also impart advantageous properties to cosmetic or dermatological compositions, particularly by improving moisturizing or softening properties, good film-forming properties, and low surface density. The diverse and unique biomechanical properties, along with its biocompatibility and slow degradation rate, make spider silk an excellent candidate for cosmetic products (e.g., nail and hair strengtheners, skin care products), and industrial materials (e.g., nanowires, nanofibers, surface coatings) as a biomaterial for tissue engineering, guided tissue repair, and drug delivery.

[0093] 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 mutants, analogs, derivatives, or the like of natural spider silk proteins. For superior tenacity, the polypeptide may be derived from the major ampullate silk protein produced in the major ampullate gland of spiders. Examples of major ampullate silk proteins include the major ampullate gland spidroins MaSp1 and MaSp2 from Nephila clavipes and ADF3 and ADF4 from Araneus diadematus. Examples of polypeptides derived from major ampullate silk proteins include mutants, analogs, derivatives, or the like of major ampullate silk proteins. Furthermore, the polypeptide may be derived from the flagellate gland silk protein produced in the flagellate gland of spiders. Examples of flagellate gland silk proteins include flagellate gland silk proteins derived from Nephila clavipes.

[0094] Examples of polypeptides derived from major dragline silk proteins include polypeptides containing two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1), preferably polypeptides containing five or more units thereof, and more preferably polypeptides containing ten or more units thereof. Alternatively, polypeptides derived from major dragline silk proteins may be polypeptides containing units of the amino acid sequence represented by Formula 1:REP1-REP2(1), which have at their C-terminus an amino acid sequence represented by any of SEQ ID NOS: 1-3 in U.S. Patent No. 9,051,453 or an amino acid sequence having 90% or greater identity to any of SEQ ID NOS: 1-3 in U.S. Patent No. 9,051,453. In polypeptides derived from major dragline silk proteins, the units of the amino acid sequence represented by Formula 1:REP1-REP2(1) may be identical or different from each other. When recombinant proteins are produced using a microorganism, such as Escherichia coli, as a host, the molecular weight of the polypeptide derived from the major drugline silk protein is, from the viewpoint of productivity, 500 kDa or less, or 300 kDa or less, or 200 kDa or less.

[0095] In formula (1), REP1 represents polyalanine. In REP1, the number of consecutive alanine residues is preferably 2 or more, more preferably 3 or more, even more preferably 4 or more, and particularly preferably 5 or more. Furthermore, in REP1, the number of consecutive alanine residues is preferably 20 or less, more preferably 16 or less, even more preferably 12 or less, and particularly preferably 10 or less. In formula (1), REP2 is an amino acid sequence consisting of 10 to 200 amino acid residues. The total number of glycine, serine, glutamine, and alanine residues contained in the amino acid sequence is 40% or more, preferably 60% or more, and more preferably 70% or more of the total number of amino acid residues contained therein.

[0096] In major dragline silks, REP1 corresponds to the crystalline region in the fiber where crystalline β-sheets are formed, while REP2 corresponds to the amorphous region in the fiber where the majority of the components lack regular organization and are more flexible. Furthermore, [REP1-REP2] corresponds to the repeat region (repeat sequence) composed of crystalline and amorphous regions, which is a characteristic sequence of dragline silk proteins.

[0097] Recombinant silk fragments In some embodiments, recombinant silk protein refers to a recombinant spider silk polypeptide, a recombinant insect silk polypeptide, or a recombinant mussel silk polypeptide. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a recombinant spider silk polypeptide of the Araneidae or Araneoids, or a recombinant insect silk polypeptide of Bombyx mori. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a recombinant spider silk polypeptide of the Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a block copolymer having repeating units derived from a naturally occurring spider silk polypeptide of the Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragments disclosed herein comprise a block copolymer having synthetic repeating units derived from a spider silk polypeptide of the Araneidae or Araneoids, and non-repeating units derived from naturally occurring repeating units of a spider silk polypeptide of the Araneidae or Araneoids.

[0098] Recent advances in genetic engineering have provided a route to producing various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein, "recombinant silk protein" refers to a synthetic protein that is heterologously produced in a prokaryotic or eukaryotic expression system using genetic engineering methods.

[0099] Various methods for synthesizing recombinant silk peptides are known and are described by Ausubel et al., Current Protocols in Molecular Biology § 8 (John Wiley & Sons 1987, (1990)), which is incorporated herein by reference. Escherichia coli, a Gram-negative, rod-shaped bacterium, is a well-established host for industrial-scale production of proteins. Therefore, the majority of recombinant silk has been produced in E. coli. E. coli is easy to manipulate, has a short generation time, is relatively low cost, and can be scaled up to produce large amounts of protein.

[0100] Recombinant silk proteins can be produced by transformed prokaryotic or eukaryotic systems containing cDNAs encoding silk proteins, fragments of these proteins, or analogs of such proteins. Recombinant DNA approaches allow for the production of recombinant silk with programmed sequence, secondary structure, architecture, and precise molecular weight. There are four major steps in this process: (i) design and assembly of a synthetic silk-like gene into a gene "cassette," (ii) insertion of this segment into a DNA recombinant vector, (iii) transformation of this recombinant DNA molecule into a host cell, and (iv) expression and purification of selected clones.

[0101] The term "recombinant vector," as used herein, includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors, such as lambda phage, viral vectors, such as adenovirus or baculovirus vectors, or artificial chromosome vectors, such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and suitable DNA sequences necessary for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, or plants) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific, desired DNA fragments and may lack functional sequences necessary for expression of the desired DNA fragment.

[0102] Prokaryotic systems include gram-negative or gram-positive bacteria. Prokaryotic expression vectors can include an origin of replication that can be recognized by the host organism, a homologous or heterologous promoter that is functional in the host, and a DNA sequence that codes for a spider silk protein, a fragment of this protein, or a similar 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 (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Propionibacterium, Staphylococcus, or Streptomyces cells.

[0103] Eukaryotic systems include yeast and insect, mammalian, or plant cells, in which case the expression vector may contain a yeast plasmid origin of replication or an autonomously replicating sequence, a promoter, a DNA sequence encoding the spider silk protein, a fragment, or a similar protein, a polyadenylation sequence, a transcription termination site, and finally, a selection gene. Non-limiting examples of eukaryotic expression organisms include yeasts such as Saccharomyces cerevisiae, Pichia pastoris, basidiosporogenous, ascosporogenous, etc., filamentous fungi such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum, Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g., Saccharomyces cerevisiae), Schizosaccharomyces, Pichia (e.g., Pichia pastoris), or Yarrowia cells, mammalian cells such as HeLa cells, COS cells, CHO cells, etc., insect cells such as Sf9 cells, MEL cells, etc., and "insect host cells" such as Spodoptera frugiperda or Trichoplusia ni cells, etc. SF9 cells, SF-21 cells, or High-Five cells, where SF-9 and SF-21 are ovary cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia nii, "plant host cells," such as tobacco, potato, or pea cells.

[0104] Various heterologous host systems have been explored to produce different types of recombinant silk. Recombinant partial spidroins and engineered silks have been cloned and expressed in bacteria (Escherichia coli), yeast (Pichia pastoris), insects (Bombyx mori larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT / hamster), and transgenic animals (mouse, goat). Most silk proteins are produced with an N- or C-terminal His tag to simplify purification and produce sufficient protein quantities.

[0105] In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems may include transgenic animals and plants. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include bacteria, yeast, and mammalian cell lines. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include E. coli. In some embodiments, suitable hosts for expressing recombinant spider silk proteins using heterologous systems include transgenic B. mori silkworms generated using genome editing techniques (e.g., CRISPR).

[0106] Recombinant silk proteins in the present disclosure include synthetic proteins based on repeating units of natural silk proteins. In addition to synthetic repetitive silk protein sequences, they can additionally contain one or more naturally occurring non-repetitive silk protein sequences.

[0107] In some embodiments, "recombinant silk protein" refers to recombinant silkworm silk protein or a fragment thereof. Recombinant production of silk fibroin and silk sericin has been reported. Various hosts have been used for production, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas, Rhodopseudomonas, Bacillus, and Strepomyces. See EP0230702, which is incorporated herein by reference in its entirety.

[0108] Also provided herein is the design and biological synthesis of silk fibroin protein-like multiblock polymers containing the GAGAGX (SEQ ID NO: 1) hexapeptide (X is A, Y, V, or S) derived from the repeating domain of the B. mori silk heavy chain (H chain).

[0109] In some embodiments, the present disclosure provides silk protein-like multiblock polymers derived from the repeat domain of B. mori silk heavy chain (H chain) containing GAGAGS (SEQ ID NO: 2) hexapeptide repeat units. GAGAGS (SEQ ID NO: 2) hexapeptide repeat units are the core units of the H chain and play an important role in the formation of crystalline domains. Silk protein-like multiblock polymers containing GAGAGS (SEQ ID NO: 2) hexapeptide repeat units spontaneously aggregate into a β-sheet structure, similar to natural silk fibroin protein, and have any weight-average molecular weight described herein.

[0110] In some embodiments, the present disclosure provides a silk peptide-like multiblock copolymer composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat fragment derived from the heavy chain of B. mori silk and a mammalian elastin VPGVG (SEQ ID NO: 3) motif produced by E. coli. In some embodiments, the present disclosure provides a fusion silk fibroin protein composed of a GAGAGS (SEQ ID NO: 2) hexapeptide repeat fragment derived from the heavy chain of B. mori silk and a GVGVP (SEQ ID NO: 4) motif produced by E. coli, in a silk protein-like multiblock polymer having any weight-average molecular weight described herein.

[0111] In some embodiments, the present disclosure provides (GAGAGS) 16 (SEQ ID NO: 5). In some embodiments, the present disclosure provides a B. mori silkworm recombinant protein composed of a repeat fragment of (GAGAGS) produced by E. coli. 16 (SEQ ID NO: 5) Repeated and non-repeated fragments (GAGAGS) 16 -F-COOH, (GAGAGS) 16 -FF-COOH, (GAGAGS) 16 -FFF-COOH, (GAGAGS) 16 -FFFF-COOH, (GAGAGS) 16 -FFFFFFFF-COOH, (GAGAGS) 16-FFFFFFFFFFFF-COOH, wherein F has the amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG (SEQ ID NO: 6), and is in a silk protein-like multiblock polymer having any weight average molecular weight described herein.

[0112] In some embodiments, "recombinant silk protein" refers to a recombinant spider silk protein or fragment thereof. The production of recombinant spider silk proteins based on partial cDNA clones has been reported. The recombinant spider silk proteins produced as such contain a portion of the repeat sequence derived from the drug-line spider silk protein spidroin 1 from the spider Nephila clavipes. See Xu et al. (Proc. Natl. Acad. Sci. USA, 87:7120-7124 (1990). A cDNA clone encoding a portion of the repeat sequence of spidroin 2, a second fibroin protein from the dragline silk of Nephila clavipes, and its recombinant synthesis are described in J. Biol. Chem., 1992, volume 267, pp. 19320-19324. Recombinant synthesis of spider silk proteins, including Nephila clavipes protein fragments and variants, from transformed Escherichia coli is described in U.S. Patents 5,728,810 and 5,989,894. A cDNA clone encoding a small ampullate gland spider silk protein and its expression are described in U.S. Patents 5,733,771 and 5,756,677. A cDNA clone encoding a flagellate gland silk protein from an orb-web spinning spider is described in U.S. Patents 5,733,771 and 5,756,677. A cDNA clone encoding a flagellate gland silk protein from an orb-web spinning spider is described in U.S. Patents 5,733,771 and 5,756,677. No. 6,268,169 describes the recombinant synthesis of spider silk-like proteins derived from repetitive peptide sequences found in the natural spider drug line of Nephila clavipes using Escherichia coli, Bacillus subtilis, and Pichia pastoris recombinant expression systems.WO 03 / 020916 describes cDNA clones encoding spider silk proteins with repeat sequences derived from Nephila madagascariensis, Nephila senegalensis, Tetragnata kauaiensis, Tetragnata versicolor, Argiope aurantia, Argiope trifasciata, Gasteracantha mammosa, and the large ampullate gland of Latrodectus geometricus, the flagellate gland of Argiope trifasciata, the ampullate gland of Dolomedes tenebrosus, two pairs of silk glands from Plectreulis tristis, and the silk gland of the Mygalomorph Euagrus tissoceus, and their recombinant production. Each of the above references is incorporated herein by reference in its entirety.

[0113] In some embodiments, the recombinant spider silk protein is a hybrid protein of spider silk protein and insect silk protein, spider silk protein and collagen, spider silk protein and resilin, or spider silk protein and keratin. The spider silk repeating unit comprises or consists of the amino acid sequence of a region comprising or consisting of at least one peptide motif that occurs repeatedly within a naturally occurring major ampullate gland polypeptide, such as a dragline spider silk polypeptide, a minor ampullate gland polypeptide, a flagellate gland polypeptide, a condensed gland spider silk polypeptide, a botryoid gland spider silk polypeptide, or a piriform gland spider silk polypeptide.

[0114] In some embodiments, the recombinant spider silk proteins of the present disclosure comprise repeating units of natural spider silk proteins, consensus sequences, and, optionally, synthetic spider silk proteins derived from one or more natural, non-repetitive spider silk protein sequences. The repeating units of natural spider silk polypeptides may comprise Araneida or Araneoid dragonfly spider silk polypeptides or flagellate gland spider silk polypeptides.

[0115] As used herein, a spider silk "repeat unit" comprises or consists of at least one peptide motif that occurs repeatedly within a naturally occurring major ampullate gland polypeptide, such as a dragline spider silk polypeptide, a minor ampullate gland polypeptide, a flagellate gland polypeptide, a condensed gland spider silk polypeptide, a botryoid gland spider silk polypeptide, or a piriform gland spider silk polypeptide. A "repeat unit" refers to a region in amino acid sequence that corresponds to a region comprising or consisting of at least one peptide motif (e.g., AAAAAA) (SEQ ID NO: 21) or GPGQQ (SEQ ID NO: 5)) that occurs repeatedly within a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4, or Flag) (i.e., the same amino acid sequence), or to an amino acid sequence substantially similar thereto (i.e., a variant amino acid sequence). A "repeat unit" having an amino acid sequence that is "substantially similar" to a corresponding amino acid sequence within a naturally occurring silk polypeptide (i.e., a wild-type repeat unit) is also similar in terms of its properties; for example, a silk protein comprising a "substantially similar repeat unit" is still insoluble and retains its insolubility. For example, a "repeating unit" having an amino acid sequence that is "identical" to that of a native silk polypeptide can be a portion of a silk polypeptide that corresponds to one or more peptide motifs of MaSpI, MaSpII, ADF-3, and / or ADF-4. For example, a "repeating unit" having an amino acid sequence that is "substantially similar" to that of a native silk polypeptide can 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.

[0116] As used herein, the term "consensus peptide sequence" refers to an amino acid sequence containing a frequently occurring amino acid at a particular position (e.g., "G"), where other amino acids not yet determined are replaced by the placeholder "X." In some embodiments, the consensus sequence is: (i) GPGXX (SEQ ID NO: 7), where X is an amino acid selected from A, S, G, Y, P, and Q; (ii) GGX, where X is an amino acid selected from Y, P, R, S, A, T, N, and Q, preferably Y, P, and Q; (iii) A x , where x is an integer from 5 to 10.

[0117] The consensus peptide sequences GPGXX (SEQ ID NO: 7) and GGX, i.e., glycine-rich motifs, provide flexibility to silk polypeptides and, therefore, to threads formed from silk proteins containing the motifs. Specifically, the repeated GPGXX (SEQ ID NO: 7) motif forms a turn helix structure, imparting elasticity to silk polypeptides. Both major ampullate and flagellate gland silks contain the GPGXX (SEQ ID NO: 7) motif. The repeated GGX motif is associated with a helical structure with three amino acids per turn and is found in most spider silks. The GGX motif may provide additional elastic properties to silk. The repeated polyalanine Ax (peptide) motif forms a crystalline β-sheet structure that provides strength to silk polypeptides, as described, for example, in WO 03 / 057727.

[0118] In some embodiments, the recombinant spider silk proteins of the present disclosure comprise two identical repeating units, each comprising at least one, and preferably one, amino acid sequence selected from the group consisting of GGRPSDTYG (SEQ ID NO: 8) and GGRPSSSYG (SEQ ID NO: 9) derived from resilin, an elastomeric protein found in most arthropods that provides low stiffness and high strength.

[0119] As used herein, "non-repetitive unit" refers to an amino acid sequence that is "substantially similar" to the corresponding non-repetitive (carboxy-terminal) amino acid sequence in the native dragline polypeptides of Spider Alaneus diadematus (ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2), NR3 (SEQ ID NO: 41), and NR4 (SEQ ID NO: 42), as described in U.S. Patent No. 8,367,803. The C16 peptide (spider silk protein eADF4, molecular weight 47.7 kDa, AMSilk) contains 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 10), an amino acid sequence adapted from the native sequence of ADF4 from A. diadematus. Non-repetitive ADF-4 and its variants exhibit efficient assembly behavior.

[0120] Among the synthetic spider silk proteins, the recombinant silk proteins in the present disclosure include, in some embodiments, the C16 protein having the polypeptide sequence SEQ ID NO: 1 described in U.S. Patent No. 8,288,512. In addition to the polypeptide sequence shown in SEQ ID NO: 1, functional equivalents, functional derivatives, and salts of this sequence, among others, are also included.

[0121] As used herein, "functional equivalents" refers to variants of the above amino acid sequences which, at at least one sequence position, have an amino acid other than the amino acid specifically mentioned.

[0122] In some embodiments, the recombinant spider silk protein of the present disclosure is spidroin major 1, as described by Xu et al., PNAS, USA, 87, 7120, (1990); Hinman and Lewis, J. Biol. Chem., 267,19320, (1922), or a recombinant spider silk protein described in U.S. Patent Application No. 2016 / 0222174, and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, 8,642,734, 8,367,803, 8,097,583, 8,030,024, 7,754,851, 7,148,039, and 7,060,260, or alternatively, a spider silk protein corresponding to a minor spidroin described in Patent Application No. WO95 / 25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spider silk proteins suitable for the recombinant RSPF of the present disclosure include ADF3 and ADF4 from the "major ampullate" gland of Araneus diadematus.

[0123] Recombinant silk is also described in other patents and patent applications, which are incorporated herein by reference: US2004 / 590196, US7,754,851, US2007 / 654470, US7,951,908, US2010 / 785960, US8,034,897, US2009 / 0263430, US2008 / 226854, US2009 / 0123967, US2005 / 712095, US2007 / 991037, US2009 / 0162896, US2008 / 85266, US8,372,436, US2007 / 9899 07, US2009 / 267596, US2010 / 319542, US2009 / 265344, US2012 / 684607, US2004 / 583227, US8,030,024, US2006 / 643569, US7,868,146, US2007 / 99191 6, US8,097,583, US2006 / 643200, US8,729,238, US8,877,903, US2019 / 0062557, US2016 / 0280960, US2011 / 0201783, US2008 / 991916, US2011 / 98666 2, US2012 / 697729, US2015 / 0328363, US9,034,816, US2013 / 0172478, US9,217,017, US2017 / 0202995, US8,721,991, US2008 / 227498, US9,233,067, US8,288,512, US2008 / 161364, US7,148,039, US1999 / 247806, US2001 / 861597, US2004 / 887100, US9,481,719, US8,765,688, US2008 / 80705, US2010 / 809102, US8,367,803, US2010 / 664902, US7,569,660, US1999 / 138833, US2000 / 591632, US2012 / 0065126, US2010 / 0278882, US2008 / 161352, US2010 / 0015070, US2009 / 513709, US2009 / 0194317, US2004 / 559286, US2005 / 89551, US2008 / 187824, US2005 / 0266242, US2005 / 0227322, and US20044418.

[0124] Recombinant silk is also described in other patents and patent applications, which are incorporated herein by reference: US2019 / 0062557, US2015 / 0284565, US2013 / 0225476, US2013 / 0172478, US2013 / 0136779, US2013 / 0109762, US2012 / 0252294, US2011 / 0230911, US2011 / 0201783, US2010 / 029 8877, US10,478,520, US10,253,213, US10,072,152, US9,233,067, US9,217,017, US9,034,816, US8,877,903, US8,729,238, US8,721,991, US8,097,583, US8,034,897, US8,030,024, US7,951,908, US7,868,146, and US7,754,851.

[0125] In some embodiments, the recombinant spider silk proteins of the present disclosure comprise or consist of 2 to 80 repeating units, each of which is selected from the group consisting of GPGXX, GGX, and A, as defined herein. x are independently selected from

[0126] In some embodiments, the recombinant spider silk protein of the present disclosure comprises or consists of repeating units, each independently selected from the group consisting of GPGAS (SEQ ID NO:11), GPGSG (SEQ ID NO:12), GPGGY (SEQ ID NO:13), GPGGP (SEQ ID NO:14), GPGGA (SEQ ID NO:15), GPGQQ (SEQ ID NO:16), GPGGG (SEQ ID NO:17), GPGQG (SEQ ID NO:18), GPGGS (SEQ ID NO:19), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO:20), AAAAAA (SEQ ID NO:21), AAAAAA (SEQ ID NO:22), AAAAAA (SEQ ID NO:23), AAAAAA (SEQ ID NO:24), AAAAAA (SEQ ID NO:25), AAAAAA (SEQ ID NO:26), AAAAAA (SEQ ID NO:27), AAAAAA (SEQ ID NO:28), AAAAAA (SEQ ID NO:29), AAAAAA (SEQ ID NO:30), AAAAAA (SEQ ID NO:31), AAAAAA (SEQ ID NO:32), AAAAAA (SEQ ID NO:33), AAAAAA (SEQ ID NO:34), AAAAAA (SEQ ID NO:35), AAAAAA (SEQ ID NO:36), AAAAAA (SEQ ID NO:37), AAAAAA (SEQ ID NO:38), AAAAAA (SEQ ID NO:39), AAAAAA (SEQ ID NO:40), AAAAAA (SEQ ID NO:41), AAAAAA (SEQ ID NO:42), AAAAAA (SEQ ID NO:43), AAAAAA (SEQ ID NO:44), AAAAAA (SEQ ID NO:45), AAAAAA (SEQ ID NO:46), AAAAAA (SEQ ID NO:47), AAAAAA (SEQ ID NO: No. 21), AAAAAAA (SEQ ID NO: 22), AAAAAAAA (SEQ ID NO: 23), AAAAAAAAA (SEQ ID NO: 24), AAAAAAAAAA (SEQ ID NO: 25), GGRPSDTYG (SEQ ID NO: 26), and GGRPSSSYG (SEQ ID NO: 27), (i) GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 28), (ii) GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 29), (iii) GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 30): (iv) GP GGAGGPYGPGGAGGPYGPGGAGGPY (SEQ ID NO: 31), (v) GGTTIIEDLDITIDGADGPITISEELTI (SEQ ID NO: 32), (vi) PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 33), (vii) SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 34), (viii) GGAGGAGGAGGSGGAGGS (SEQ ID NO: 35), (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGP GGY (SEQ ID NO: 36), (x) GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 37), (xi) GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 38), (xii) GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 39), (xiii) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 40), (xiv) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 40) or U.S. Patent No. 8,877,903, for example, a synthetic spider peptide having a sequential sequence of GPGAS (SEQ ID NO: 11), GGY, GPGSG (SEQ ID NO: 12) in the peptide chain, or a sequential sequence of AAAAAAAA (SEQ ID NO: 23), GPGGY (SEQ ID NO: 13), GPGGP (SEQ ID NO: 14) in the peptide chain, or a sequential sequence of AAAAAAAA (SEQ ID NO: 23), GPGQG (SEQ ID NO: 18), GGR in the peptide chain.

[0127] In some embodiments, the present disclosure provides silk protein-like multi-block peptides that mimic amino acid repeat units derived from natural spider silk proteins, such as spidroin major 1 domain, spidroin major 2 domain, or spidroin minor 1 domain, and profiles of variations between the repeat units without alteration of the three-dimensional structure, wherein these silk protein-like multi-block peptides comprise amino acid repeat units corresponding to one of the following sequences (I), (II), (III), and / or (IV):

[0128] [(XGG) w (XGA)(GXG) x (AGA) y (G) z A.G.] p Formula (I), wherein X corresponds to tyrosine or to glutamine, w is an integer equal to 2 or 3, x is an integer between 1 and 3, y is an integer between 5 and 7, z is an integer equal to 1 or 2, and p is an integer, and has any weight average molecular weight described herein; and / or [(GPG2YGPGQ2) a (X')2S(A) b 〕 p Formula (II), wherein X' corresponds to the amino acid sequence GPS or GPG, a is equal to 2 or 3, b is an integer between 7 and 10, and p is an integer, and has any weight average molecular weight described herein; and / or [(GR)(GA) l (A) m (GGX) n (GA) n (A)m 〕 p Formula (III) and / or [(GGX) n (GA) m (A) l 〕 p Formula (IV), wherein X" corresponds to tyrosine, glutamine, or alanine, l 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.

[0129] In some embodiments, a recombinant spider silk protein or analog of a spider silk protein comprising an amino acid repeat unit of sequence (V): [(Xaa Gly Gly) w (Xaa Gly Ala)(Gly Xaa Gly) x (Ala Gly Ala) y (Gly)zAla 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.

[0130] In some embodiments, the recombinant spider silk protein in the present disclosure is selected from the group consisting of ADF-3 or a mutant thereof, ADF-4 or a mutant thereof, MaSpI (SEQ ID NO: 43) or a mutant thereof, MaSpII (SEQ ID NO: 44) or a mutant thereof, as described in U.S. Pat. No. 8,367,803.

[0131] In some embodiments, the present disclosure provides water-soluble recombinant spider silk proteins produced in mammalian cells.The solubility of spider silk proteins produced in mammalian cells is due to the presence of COOH-terminal amino acids in these proteins, making them more hydrophilic.These COOH-terminal amino acids are not present in spider silk proteins expressed in microbial hosts.

[0132] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a water-soluble recombinant spider silk protein C16 modified at the amino or carboxyl terminus selected from the amino acid sequences consisting of GCGGGGGG (SEQ ID NO: 41), GKGGGGGG (SEQ ID NO: 42), GCGGSGGGGSGGGG (SEQ ID NO: 43), GKGGGGGGSGGGG (SEQ ID NO: 44), and GCGGGGGSGGGG (SEQ ID NO: 45). In some embodiments, the recombinant spider silk protein of the present disclosure comprises a ... such that the molecular weight of the protein is in the ranges described herein. 16 NR4, C 32 NR4, C16, C32, NR4C 16 NR4, NR4C 32 NR4, NR3C 16 NR3 or NR3C 32 Includes NR3.

[0133] In some embodiments, the recombinant spider silk protein of the present disclosure comprises a recombinant spider silk protein having a synthetic repeat peptide segment and amino acid sequence adapted from the native sequence of ADF4 from A. diadematus, as described in U.S. Patent No. 8,877,903. In some embodiments, the RSPF of the present disclosure comprises a recombinant spider silk protein having a repeat peptide unit derived from a native spider silk protein, such as a spidroin major 1 domain, a spidroin major 2 domain, or a spidroin minor 1 domain, where the repeat peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 46) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 47), as described in U.S. Patent No. 8,367,803.

[0134] In some embodiments, the disclosure provides recombinant spider proteins composed of GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 48) repeat fragments and having molecular weights as described herein.

[0135] As used herein, the term "recombinant silk" refers to recombinant spider and / or silkworm silk proteins or fragments thereof. In one embodiment, the spider silk protein is selected from the group consisting of gland silk (grape-shaped gland silk), egg sac silk (cylindrical gland silk), egg case silk (tubular gland silk), non-sticky dragline silk (major ampullate gland silk), attachment silk (pyriform gland silk), adhesive silk core fiber (flap-shaped gland silk), and adhesive silk outer fiber (aggregate gland silk). For example, recombinant spider silk proteins include those described in U.S. Patent Application No. 2016 / 0222174 and U.S. Patent Nos. 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734, as described herein.

[0136] Some organisms produce multiple silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb-weaving spiders have six unique types of glands that produce different silk polypeptide sequences polymerized into fibers tailored to their environment or life cycle niche. Fibers are named after the gland from which they originate, and the polypeptides are labeled with the gland's abbreviation (e.g., "Ma") and "Sp" for spidroin (for spider fibroin). In orb-weavers, these types include the major ampullate gland (MaSp, also called dragline), minor ampullate gland (MiSp), flagellate gland (Flag), botryoid gland (AcSp), tubular gland (TuSp), and piriform gland (PySp). This combination of polypeptide sequences across fiber types, domains, and variation among different genera and species leads to a wide range of potential properties that can be exploited through the commercial production of recombinant fibers. To date, the majority of research using recombinant silk has focused on the major ampullate gland spidroin (MaSp).

[0137] AcSp silks tend to have high toughness, resulting from a combination of moderately high strength and moderately high extensibility. AcSp silks are characterized by large block ("ensemble repeat") sizes, often incorporating polyserine and GPX motifs. TuSp silks, or cylindrical silks, tend to have large diameters, with moderate strength and high extensibility. TuSp silks are characterized by their polyserine and polythreonine content and short tracts of polyalanine. MaSp silks tend to have high strength and moderate extensibility. MaSp silks can be one of two subtypes: MaSp1 and MaSp2. MaSp1 silks are generally less extensible than MaSp2 silks and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silk is characterized by polyalanine, GGX, and GPX motifs. MiSp silk tends to have moderate strength and moderate extensibility. MiSp silk is characterized by GGX, GA, and polyA motifs and often contains a spacer element of approximately 100 amino acids. Flagellate gland (Flag) silk tends to have very high extensibility and moderate strength. Flag silk is usually characterized by GPG, GGX, and a short spacer motif.

[0138] Silk polypeptides are characteristically composed of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repeat domains exhibit a hierarchical structure. They contain a series of blocks (also called repeat units). The blocks are sometimes perfectly repeated, and sometimes imperfectly (forming quasi-repetitive domains) throughout the silk repeat domain. The length and composition of the blocks vary between different silk types and across different species. Table 1 of U.S. Published Application No. 2016 / 0222174, incorporated herein in its entirety, lists examples of block sequences from selected species and silk types; further examples are provided in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pp. 169-184 (2011), and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, the blocks are arranged in regular patterns to form larger macrorepeats that appear multiple times (usually 2-8) within the repeat domain of the silk sequence. Repeated blocks within a repeat domain or macrorepeat, and repeated macrorepeats within a repeat domain, may be separated by spacing elements.

[0139] The structure of certain spider silk block copolymer polypeptides from block and / or macrorepeat domains according to certain embodiments of the present disclosure is illustrated in U.S. Patent Application Publication No. 2016 / 0222174.

[0140] Recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in recombinant prokaryotic or eukaryotic systems can be purified according to methods known in the art. In a preferred embodiment, commercially available expression / secretion systems can be used, whereby the recombinant polypeptide is expressed and then secreted from the host cells and easily purified from the surrounding medium. If an expression / secretion vector is not used, an alternative approach involves purifying the recombinant block copolymer polypeptide from cell lysates (cell remains after disruption of cellular integrity) derived from prokaryotic or eukaryotic cells in which the polypeptide was expressed. Methods for producing such cell lysates are known to those skilled in the art. In some embodiments, the recombinant block copolymer polypeptide is isolated from the cell culture supernatant.

[0141] Recombinant block copolymer polypeptides may be purified by affinity separation, e.g., immunological interaction with an antibody that specifically binds to the recombinant polypeptide or a nickel column for isolation of recombinant polypeptides tagged with 6-8 histidine residues at their N- or C-termini. Alternative tags can include the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by those skilled in the art.

[0142] Solutions of such polypeptides (ie, recombinant silk proteins) may then be prepared and used as described herein.

[0143] In another embodiment, recombinant silk proteins may be prepared according to the methods described in U.S. Pat. No. 8,642,734, which is incorporated herein in its entirety and used as described herein.

[0144] In one embodiment, a recombinant spider silk protein is provided. Spider silk proteins typically consist of 170 to 760 amino acid residues, such as 170 to 600 amino acid residues, preferably 280 to 600 amino acid residues, such as 300 to 400 amino acid residues, and more preferably 340 to 380 amino acid residues. A small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require the use of harsh solvents for solubilization and polymerization. Recombinant spider silk proteins may contain more than 760 residues, particularly when the spider silk protein comprises three or more fragments derived from the N-terminal portion of the spider silk protein, including an N-terminal fragment consisting of at least one fragment (NT) derived from the corresponding portion of the spider silk protein and repeat fragments (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. Spider silk proteins typically comprise a single fragment (NT) derived from the N-terminal portion of the spider silk protein, however, in a preferred embodiment, the N-terminal fragment comprises at least two, such as two fragments (NT) derived from the N-terminal portion of the spider silk protein. Thus, spidroins have the formula NT m -REP, and alternatively NT m Spidroins can be represented schematically by the formula NT2-REP or NT-REP, and alternatively NT2-REP-CT or NT-REP-CT. The protein fragments are typically covalently linked via peptide bonds. In one embodiment, the spider silk protein consists of an NT fragment linked to a REP fragment, which is optionally linked to a CT fragment.

[0145] In one embodiment, the first step of the method for producing isolated spider silk protein polymers involves the expression of a polynucleic acid molecule encoding the spider silk protein in a suitable host, such as E. coli. The protein thus obtained is isolated using standard procedures. Optionally, lipopolysaccharides and other pyrogens are actively removed at this stage.

[0146] In the second step of the method for producing isolated spider silk protein polymers, a solution of spider silk protein in a liquid medium is provided. The terms "soluble" and "in solution" mean that the protein does not visibly aggregate or precipitate from the solvent at 60,000 x g. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, typically a buffered aqueous medium, such as 10-50 mM Tris-HCl buffer or phosphate buffer. The liquid medium has a pH of 6.4 or greater and / or an ionic composition that prevents polymerization of the spider silk protein. That is, the liquid medium has either a pH of 6.4 or greater, an ionic composition that prevents polymerization of the spider silk protein, or both.

[0147] Ionic compositions that prevent spider silk protein polymerization can be easily prepared by those skilled in the art using the methods disclosed herein. Preferred ionic compositions that prevent spider silk protein polymerization have an ionic strength of greater than 300 mM. Specific examples of ionic compositions that prevent spider silk protein polymerization 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.

[0148] The presence of NT fragments improves solution stability and prevents polymer formation under these conditions. This can be advantageous when immediate polymerization would be undesirable, such as during protein purification, large-batch preparation, or when other conditions need to be optimized. The pH of the liquid medium is preferably adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as up to 10.5, to achieve high solubility of the spider silk protein. It can also be advantageous to adjust the pH of the liquid medium to a range of 6.4 to 6.8, which provides sufficient solubility of the spider silk protein but facilitates subsequent pH adjustment to 6.3 or lower.

[0149] In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or less and an ionic composition that permits polymerization. That is, if the liquid medium in which the spider silk protein is dissolved has a pH of 6.4 or more, the pH is reduced to 6.3 or less. Those skilled in the art are well aware of various methods for achieving this, typically including the addition of a strong or weak acid. If the liquid medium in which the spider silk protein is dissolved has an ionic composition that prevents polymerization, the ionic composition is changed to permit polymerization. Those skilled in the art are well aware of various methods for achieving this, such as dilution, dialysis, or gel filtration. If necessary, this step involves reducing the pH of the liquid medium to 6.3 or less and changing the ionic composition to permit polymerization. The pH of the liquid medium is preferably adjusted to 6.2 or less, such as 6.0 or less. In particular, it may be advantageous from a practical standpoint to limit the pH drop from 6.4 or 6.4-6.8 in the previous step to 6.3 or 6.0-6.3, e.g., 6.2, in this step. In a preferred embodiment, the pH of the liquid medium for this step is greater than or equal to 3, such as greater than or equal to 4.2. The resulting pH range, for example, 4.2 to 6.3, promotes rapid polymerization.

[0150] In the fourth step, spider silk proteins are polymerized in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk proteins. The presence of the NT fragments improves the solubility of spider silk proteins at pHs above 6.4 and / or accelerates polymer formation at pHs below 6.3, where the ionic composition allows polymerization of the spider silk proteins. The resulting polymers are preferably solid and macroscopic, and they are formed in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk proteins. In a preferred embodiment, the pH of the liquid medium in this step is 3 or greater, such as 4.2 or greater. The resulting pH range, e.g., 4.2 to 6.3, promotes rapid polymerization. The resulting polymers are provided in the molecular weights described herein and may be prepared in solution form for use as needed for article coating.

[0151] Ionic compositions that allow the polymerization of spider silk proteins can be easily prepared by those skilled in the art using the methods disclosed herein. Preferred ionic compositions that allow the polymerization of spider silk proteins have an ionic strength of less than 300 mM. Specific examples of ionic compositions that allow the polymerization of spider silk proteins include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate, and combinations of these ions that lack a preventive effect on the polymerization of spider silk proteins, such as a combination of 10 mM phosphate or 20 mM phosphate and 150 mM NaCl. The ionic strength of the liquid medium is preferably adjusted to a range of 1 to 250 mM.

[0152] Without wishing to be limited to any particular theory, it is postulated that NT fragments have oppositely charged poles and that environmental changes in pH affect the charge balance on the surface of the protein, leading to polymerization, while salt inhibits the same event.

[0153] At neutral pH, one might expect polymerization to be prevented by the energetic cost of burying the excess negative charge at the acidic pole. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces eventually dominate, explaining the observed salt- and pH-dependent polymerization behavior of NTs and NT-containing minispidroins. In some embodiments, it is proposed that the pH-induced NT polymerization and increased efficiency of NT-minispidroin fibril assembly are due to changes in the surface electrostatic potential, and that clustering of acidic residues at one pole of NTs shifts their charge balance, causing the polymerization transition to occur at pH values ​​below 6.3.

[0154] In a fifth step, the resulting, preferably solid, spider silk protein polymer is isolated from the liquid medium, optionally including actively removing lipopolysaccharides and other pyrogens from the spidroin polymer.

[0155] Without wishing to be limited to any particular theory, it has been observed that the formation of spidroin polymers proceeds via the formation of water-soluble spidroin dimers. The present disclosure also provides a method for producing isolated spider silk protein dimers, the first two method steps being as described above. The spider silk protein exists as a dimer in a liquid medium with a pH of 6.4 or higher and / or an ionic composition that prevents polymerization of the spider silk protein. The third step involves isolating the dimer obtained in the second step, optionally including removal of lipopolysaccharides and other pyrogens. In a preferred embodiment, the spider silk protein polymer of the present disclosure consists of polymerized protein dimers. The present disclosure thus provides a novel use of spider silk proteins, preferably those disclosed herein, for producing spider silk protein dimers.

[0156] According to another aspect, the present disclosure provides a polymer of the spider silk protein disclosed herein. In one embodiment, the protein polymer is obtainable by any one of the methods according to the present disclosure. Thus, the present disclosure provides various uses of recombinant spider silk proteins, preferably those disclosed herein, for producing polymers of spider silk proteins as recombinant silk-based coatings. According to one embodiment, the present disclosure provides a novel use of a spider silk protein dimer, preferably those disclosed herein, for producing polymers of isolated spider silk proteins as recombinant silk-based coatings. In these uses, the polymer is preferably produced in a liquid medium having a pH of 6.3 or less and an ionic composition that allows polymerization of the spider silk protein. In one embodiment, the pH of the liquid medium is 3 or greater, such as 4.2 or greater. The resulting pH range, e.g., 4.2 to 6.3, promotes rapid polymerization.

[0157] Using the methods of the present disclosure, it is possible to control the polymerization process, which allows for the optimization of parameters to obtain silk polymers with desired properties and shapes.

[0158] In one embodiment, the recombinant silk proteins described herein include those described in US Pat. No. 8,642,734, which is incorporated by reference in its entirety.

[0159] In another embodiment, the recombinant silk proteins described herein may be prepared according to the methods described in U.S. Patent No. 9,051,453, which is incorporated herein by reference in its entirety.

[0160] The amino acid sequence represented by SEQ ID NO: 1 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence consisting of 50 amino acid residues of the amino acid sequence of ADF3 at the C-terminus (NCBI accession number: AAC47010, GI: 1263287). The amino acid sequence represented by SEQ ID NO: 2 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 of 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 of U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO: 1 with 29 residues removed from the C-terminus.

[0161] An example of a polypeptide containing a unit of the amino acid sequence represented by Formula 1: REP1-REP2(1) and having at its C-terminus an amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 or an amino acid sequence having 90% or more homology to any one of SEQ ID NOs: 1 to 3 in U.S. Pat. No. 9,051,453 is a polypeptide having the amino acid sequence represented by SEQ ID NO: 8 in U.S. Pat. No. 9,051,453. The polypeptide having the amino acid sequence represented by SEQ ID NO:8 in U.S. Patent No. 9,051,453 was obtained by the following mutations: an amino acid sequence (SEQ ID NO:5 in U.S. Patent No. 9,051,453) consisting of an initiation codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site was added to the N-terminus of the amino acid sequence of ADF3 (NCBI Accession No. AAC47010, GI:1263287), the repeat region from residues 1 to 13 was approximately doubled, and translation terminated at amino acid residue 1154. The C-terminal sequence of the polypeptide having the amino acid sequence represented by SEQ ID NO:8 in U.S. Patent No. 9,051,453 is identical to the amino acid sequence represented by SEQ ID NO:3.

[0162] Furthermore, a polypeptide comprising a unit of the amino acid sequence represented by Formula 1: REP1-REP2(1) and having at its C-terminus an amino acid sequence represented by any one of SEQ ID NOS: 1 to 3 in U.S. Pat. No. 9,051,453 or an amino acid sequence having 90% or more homology to any one of SEQ ID NOS: 1 to 3 in U.S. Pat. No. 9,051,453 may be a protein having an amino acid sequence represented by SEQ ID NOS: 8 in U.S. Pat. No. 9,051,453 in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has a repeat region composed of crystalline and amorphous regions.

[0163] Furthermore, an example of a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1: REP1-REP2(1) is a recombinant protein derived from ADF4 having the amino acid sequence represented by SEQ ID NO: 15 in U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 15 in U.S. Patent No. 9,051,453 is an amino acid sequence obtained by adding an amino acid sequence consisting of an initiation codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site (SEQ ID NO: 5 in U.S. Patent No. 9,051,453) to the N-terminus of a partial amino acid sequence of ADF4 obtained from the NCBI database (NCBI accession number: AAC47011, GI: 1263289). Furthermore, a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1) may be a polypeptide having 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 having regions composed of crystalline and amorphous regions. Further, an example of a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1:REP1-REP2(1) is a recombinant protein derived from MaSp2 having the amino acid sequence represented by SEQ ID NO:17 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO: 17 in U.S. Patent No. 9,051,453 is an amino acid sequence obtained by adding an amino acid sequence (SEQ ID NO: 5 in U.S. Patent No. 9,051,453) consisting of an initiation codon, a His 10 tag, and an HRV3C protease (human rhinovirus 3C protease) recognition site to the N-terminus of a partial sequence of MaSp2 obtained from the NCBI web database (NCBI accession number: AAT75313, GI: 50363147). Furthermore, a polypeptide comprising two or more units of the amino acid sequence represented by Formula 1: REP1-REP2(1) may have the amino acid sequence represented by SEQ ID NO: 17 in U.S. Patent No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has a region composed of a crystalline region and an amorphous region.

[0164] Examples of polypeptides derived from flagellate gland silk proteins include polypeptides containing 10 or more units of the amino acid sequence represented by Formula 2: REP3(2), preferably polypeptides containing 20 or more units thereof, and more preferably polypeptides containing 30 or more units thereof. When recombinant proteins are produced using a microorganism such as Escherichia coli as a host, the molecular weight of the polypeptide derived from flagellate gland silk protein is preferably 500 kDa or less, more preferably 300 kDa or less, and even more preferably 200 kDa or less, from the viewpoint of productivity.

[0165] In formula (2), REP3 represents an amino acid sequence consisting of Gly-Pro-Gly-Gly-X, where X represents an amino acid selected from the group consisting of Ala, Ser, Tyr, and Val.

[0166] A key feature of spider silk is that it does not have crystalline regions, but rather has repeating regions composed of amorphous regions. Because major dragline silks and similar have repeating regions composed of crystalline and amorphous regions, they are expected to have both high stress and stretchability. On the other hand, for flagellate silk, the stress is inferior to that of major dragline silk, but the stretchability is high. The reason for this is thought to be that most of the flagellate silk is composed of amorphous regions.

[0167] An example of a polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2: REP3(2) is a recombinant protein derived from flagellate gland silk protein having the amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9,051,453. The amino acid sequence represented by SEQ ID NO:19 in U.S. Pat. No. 9,051,453 was obtained by combining a partial sequence of the flagellate gland silk protein of Nephila clavipes obtained from the NCBI database (NCBI accession number: AAF36090, GI:7106224), specifically the amino acid sequence from residues 1220 to 1659 from the N-terminus corresponding to the repeat portion and motif (referred to as the PR1 sequence), with a partial sequence of the flagellate gland silk protein of Nephila clavipes obtained from the NCBI database (NCBI accession number: AAC38847, GI:2833649), specifically the C-terminal amino acid sequence from residues 816 to 907 from the C-terminus, and then adding an amino acid sequence consisting of an initiation codon, a His 10 tag, and an HRV3C protease recognition site (SEQ ID NO:5 in U.S. Pat. No. 9,051,453) to the N-terminus of the combined sequence. Furthermore, a polypeptide comprising 10 or more units of the amino acid sequence represented by Formula 2:REP3(2) may be a polypeptide having the amino acid sequence represented by SEQ ID NO: 19 of U.S. Pat. No. 9,051,453, in which one or more amino acids have been substituted, deleted, inserted, and / or added, and which has a repeat region made up of amorphous regions.

[0168] Polypeptides can be produced using a host transformed with an expression vector containing a gene encoding the polypeptide. The method for producing the gene is not particularly limited, but it can be produced by amplifying a gene encoding a natural spider silk protein from cells derived from a spider by cloning it using polymerase chain reaction (PCR) or other methods, or it can be chemically synthesized. The method for chemically synthesizing the gene is also not particularly limited, but it can be synthesized, for example, as follows: based on information on the amino acid sequence of a natural spider silk protein obtained from the NCBI web database, oligonucleotides automatically synthesized using an AKTA OligoPilot Plus 10 / 100 (GE Healthcare Japan Corporation) are ligated by PCR or other methods. At this point, to facilitate protein purification and observation, a gene encoding a protein having the amino acid sequence described above can be synthesized with an amino acid sequence consisting of an initiation codon and a His 10 tag added to the N-terminus.

[0169] Examples of expression vectors include plasmids, phages, viruses, and the like, which are capable of expressing proteins based on DNA sequences. The plasmid-type expression vector is not particularly limited, as long as it can express a target gene in a host cell and amplify itself. For example, when E. coli Rosetta (DE3) is used as a host, pET22b(+) plasmid vector, pCold plasmid vector, and the like can be used. Among these, the pET22b(+) plasmid vector is preferred for protein productivity. Examples of hosts include animal cells, plant cells, microorganisms, etc.

[0170] The polypeptide used in the present disclosure is preferably derived from ADF3, which is one of the two major dragline silk proteins of Araneus diadematus. This polypeptide has the advantages of being essentially high in strength, extensibility, and toughness, and is easily synthesized.

[0171] Accordingly, the recombinant silk proteins (e.g., recombinant spider silk-based proteins) used in accordance with the embodiments, articles, and / or methods described herein may be any of the silk proteins described above or in any of the U.S. Patent Nos. 8,173,772; 8,278,416; 8,618,255; 8,642,734; 8,691,581; 8,729,235; 9,115,204; 9,157,070; 9,309,299; 9,644,012; 9,708,376; 9,051,453; 9,617 ,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332, and U.S. Patent Publication Nos. 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066, and 2014 / 0079674. , 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 014161 No. 8, No. 2015 / 0291673, No. 2015 / 0291674, No. 2015 / 0239587, No. 2015 / 0344 No. 542, No. 2015 / 0361144, No. 2015 / 0374833, No. 2015 / 0376247, No. 2016 / 00 No. 24464, No. 2017 / 0066804, No. 2017 / 0066805, No. 2015 / 0293076, No. 2016 / No. 0222174, No. 2017 / 0283474, No. 2017 / 0088675, No. 2019 / 0135880, No. 201 5 / 0329587, 2019 / 0040109, 2019 / 0135881, 2019 / 0177363, 2 019 / 0225646, 2019 / 0233481, 2019 / 0031842, 2018 / 0355120, No. 2019 / 0186050, No. 2019 / 0002644, No. 2020 / 0031887, No. 2018 / 0273590,Same No. 20191 / 094403, No. 2019 / 0031843, No. 2018 / 0251501, No. 2017 / 0066805, No. 2018 / 0127553, No. 2019 / 032952 No. 6, No. 2020 / 0031886, No. 2018 / 0080147, No. 2019 / 0352349, No. 2020 / 0043085, No. 2019 / 0144819, No. 2019 / 0228 449, 2019 / 0340666, 2020 / 0000091, 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191, which are incorporated herein by reference in their entireties.

[0172] Silk fibroin-like protein fragments The recombinant silk proteins of the present disclosure include synthetic proteins based on the repeating units of natural silk proteins. In addition to synthetic repeating silk protein sequences, they may additionally contain one or more natural non-repetitive silk protein sequences. As used herein, "silk fibroin-like protein fragment" refers to a protein fragment having a molecular weight and polydispersity as defined herein and a degree of homology to a protein selected from natural silk proteins, fibroin heavy chains, fibroin light chains, or any protein containing one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeating units. In some embodiments, the degree of homology is selected from about 99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, about 85%, about 84%, about 83%, about 82%, about 81%, about 80%, about 79%, about 78%, about 77%, about 76%, about 75%, or less than 75%.

[0173] As described herein, proteins, such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contain between about 9% and about 45% glycine, or about 9% glycine, or about 10% glycine, about 43% glycine, about 44% glycine, about 45% glycine, or about 46% glycine. As described herein, proteins, such as natural silk proteins, fibroin heavy chains, fibroin light chains, or any proteins comprising one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contain between about 13% and about 30% alanine, or about 13% alanine, or about 28% alanine, or about 29% alanine, or about 30% alanine, or about 31% alanine. As described herein, a protein, such as a natural silk protein, a fibroin heavy chain, a fibroin light chain, or any protein containing one or more GAGAGS (SEQ ID NO: 2) hexaamino acid repeat units, contains between 9% and about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0174] In some embodiments, the silk fibroin-like proteins described herein are about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about Contains 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, or about 55% glycine. In some embodiments, the silk fibroin-like proteins described herein contain about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, or about 39% alanine. In some embodiments, the silk fibroin-like proteins described herein contain about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, or about 22% serine. In some embodiments, the silk fibroin-like proteins described herein may independently include any amino acid known to be contained in native fibroin. In some embodiments, the silk fibroin-like proteins described herein may independently exclude any amino acid known to be contained in native fibroin. In some embodiments, an average of 2 out of 6 amino acids, 3 out of 6 amino acids, or 4 out of 6 amino acids in the silk fibroin-like proteins described herein are glycines. In some embodiments, an average of 1 in 6 amino acids, 2 in 6 amino acids, or 3 in 6 amino acids in the silk fibroin-like proteins described herein is alanine.In some embodiments, an average of 0 out of 6 amino acids, 1 out of 6 amino acids, or 2 out of 6 amino acids in the silk fibroin-like proteins described herein is serine.

[0175] Other properties of SPF The compositions of the present disclosure are or exhibit "biocompatible," meaning that the compositions are compatible with living tissues or living systems by being non-toxic, non-intoxicating, non-physiologically reactive, and non-immunely rejecting. 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.

[0176] In some embodiments, the compositions described herein may be biocompatible compositions (e.g., biocompatible coatings comprising silk) and may be evaluated to comply with the international standard ISO 10993-1 entitled "Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process." In some embodiments, the compositions described herein may be biocompatible compositions and may be evaluated under ISO 10993-1 for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantability, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation.

[0177] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0178] In one embodiment, the composition of the present disclosure has a stability of about 1 day. In one embodiment, the composition of the present disclosure has a stability of about 2 days. In one embodiment, the composition of the present disclosure has a stability of about 3 days. In one embodiment, the composition of the present disclosure has a stability of about 4 days. In one embodiment, the composition of the present disclosure has a stability of about 5 days. In one embodiment, the composition of the present disclosure has a stability of about 6 days. In one embodiment, the composition of the present disclosure has a stability of about 7 days. In one embodiment, the composition of the present disclosure has a stability of about 8 days. In one embodiment, the composition of the present disclosure has a stability of about 9 days. In one embodiment, the composition of the present disclosure has a stability of about 10 days.

[0179] In one embodiment, the stability of the compositions of the present disclosure is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days.

[0180] In one embodiment, the composition of the present disclosure has a stability of 10 days to 6 months. In one embodiment, the composition of the present disclosure has a stability of 6 months to 12 months. In one embodiment, the composition of the present disclosure has a stability of 12 months to 18 months. In one embodiment, the composition of the present disclosure has a stability of 18 months to 24 months. In one embodiment, the composition of the present disclosure has a stability of 24 months to 30 months. In one embodiment, the composition of the present disclosure has a stability of 30 months to 36 months. In one embodiment, the composition of the present disclosure has a stability of 36 months to 48 months. In one embodiment, the composition of the present disclosure has a stability of 48 months to 60 months.

[0181] In one embodiment, the SPF composition of the present disclosure is not soluble in aqueous solution due to the crystallinity of the protein. In one embodiment, the SPF composition of the present disclosure is soluble in aqueous solution. In one embodiment, the SPF of the composition of the present disclosure comprises about two-thirds crystalline and about one-third amorphous regions. In one embodiment, the SPF of the composition of the present disclosure comprises about half crystalline and about 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 disclosed composition comprises 80% crystalline fraction and 20% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 75% crystalline fraction and 25% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 70% crystalline fraction and 30% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 65% crystalline fraction and 35% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 60% crystalline fraction and 40% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 50% crystalline fraction and 50% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 40% crystalline fraction and 60% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 35% crystalline fraction and 65% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 30% crystalline fraction and 70% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 25% crystalline fraction and 75% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 20% crystalline fraction and 80% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 15% crystalline fraction and 85% amorphous region. In one embodiment, the SPF of the disclosed composition comprises 10% crystalline fraction and 90% amorphous region.In one embodiment, the SPF of the disclosed composition comprises 5% crystalline fraction and 90% amorphous region, hi one embodiment, the SPF of the disclosed composition comprises 1% crystalline fraction and 99% amorphous region.

[0182] As used herein, the term "substantially free of inorganic residue" means that a composition exhibits 0.1% (w / w) or less of a residue. In one embodiment, "substantially free of inorganic residue" refers to a composition exhibiting 0.05% (w / w) or less of a residue. In one embodiment, "substantially free of inorganic residue" refers to a composition exhibiting 0.01% (w / w) or less of a residue. In one embodiment, the amount of inorganic residue is between 0 ppm ("non-detectable" or "ND") and 1000 ppm. In one embodiment, the amount of inorganic residue is ND to about 500 ppm. In one embodiment, the amount of inorganic residue is ND to about 400 ppm. In one embodiment, the amount of inorganic residue is ND to about 300 ppm. In one embodiment, the amount of inorganic residue is ND to about 200 ppm. In one embodiment, the amount of inorganic residue is ND to about 100 ppm. In one embodiment, the amount of inorganic residue is between 10 ppm and 1000 ppm.

[0183] As used herein, the term "substantially free of inorganic residue" means that a composition exhibits 0.1% (w / w) or less residue. In one embodiment, "substantially free of inorganic residue" refers to a composition exhibiting 0.05% (w / w) or less residue. In one embodiment, "substantially free of organic residue" refers to a composition exhibiting 0.01% (w / w) or less residue. In one embodiment, the amount of organic residue is between 0 ppm ("non-detectable" or "ND") and 1000 ppm. In one embodiment, the amount of organic residue is ND to about 500 ppm. In one embodiment, the amount of organic residue is ND to about 400 ppm. In one embodiment, the amount of organic residue is ND to about 300 ppm. In one embodiment, the amount of organic residue is ND to about 200 ppm. In one embodiment, the amount of organic residue is ND to about 100 ppm. In one embodiment, the amount of organic residue is between 10 ppm and 1000 ppm.

[0184] The compositions of the present disclosure are "biocompatible," meaning that the compositions are compatible with living tissues or systems by being non-toxic, non-intoxicating, or physiologically reactive, and by not causing immune rejection. Such biocompatibility can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days, in one embodiment, the extended period is about 14 days, and in one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0185] The compositions of the present disclosure are "hypoallergenic," meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be demonstrated by participants topically applying the compositions of the present disclosure to their skin for an extended period of time. In one embodiment, the extended period is about 3 days. In one embodiment, the extended period is about 7 days. In one embodiment, the extended period is about 14 days. In one embodiment, the extended period is about 21 days. In one embodiment, the extended period is about 30 days. In one embodiment, the extended period is selected from the group consisting of about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, and indefinitely.

[0186] The following are non-limiting examples of suitable ranges for various parameters in and for preparing silk solutions of the present disclosure. Silk solutions of the present disclosure may include one or more (but not necessarily all) of these parameters, but may be prepared using various combinations of such parameter ranges.

[0187] In one embodiment, the SPF (%) in the solution is less than 30.0% by weight. In one embodiment, the SPF (%) in the solution is less than 25.0% by weight. In one embodiment, the SPF (%) in the solution is less than 20.0% by weight. In one embodiment, the SPF (%) in the solution is less than 19.0% by weight. In one embodiment, the SPF (%) in the solution is less than 18.0% by weight. In one embodiment, the SPF (%) in the solution is less than 17.0% by weight. In one embodiment, the SPF (%) in the solution is less than 16.0% by weight. In one embodiment, the SPF (%) in the solution is less than 15.0% by weight. In one embodiment, the SPF (%) in the solution is less than 14.0% by weight. In one embodiment, the SPF (%) in the solution is less than 13.0% by weight. In one embodiment, the SPF (%) in the solution is less than 12.0% by weight. In one embodiment, the SPF (%) in the solution is less than 11.0% by weight. In one embodiment, the SPF (%) in the solution is less than 10.0% by weight. In one embodiment, the SPF (%) in the solution is less than 9.0% by weight. In one embodiment, the SPF (%) in the solution is less than 8.0% by weight. In one embodiment, the SPF (%) in the solution is less than 7.0% by weight. In one embodiment, the SPF (%) in the solution is less than 6.0% by weight. In one embodiment, the SPF (%) in the solution is less than 5.0% by weight. In one embodiment, the SPF (%) in the solution is less than 4.0% by weight. In one embodiment, the SPF (%) in the solution is less than 3.0% by weight. In one embodiment, the SPF (%) in the solution is less than 2.0% by weight. In one embodiment, the SPF (%) in the solution is less than 1.0% by weight. In one embodiment, the SPF (%) in the solution is less than 0.9% by weight. In one embodiment, the SPF (%) in the solution is less than 0.8% by weight. In one embodiment, the SPF (%) in the solution is less than 0.7% by weight. In one embodiment, the SPF (%) in the solution is less than 0.6% by weight. In one embodiment, the SPF (%) in the solution is less than 0.5% by weight. In one embodiment, the SPF (%) in the solution is less than 0.4% by weight. In one embodiment, the SPF (%) in the solution is less than 0.3% by weight. In one embodiment, the SPF (%) in the solution is less than 0.2% by weight.In one embodiment, the SPF (%) in the solution is less than 0.1% by weight.

[0188] In one embodiment, the SPF (%) in the solution is greater than 0.1% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.2% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.3% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.4% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.5% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.6% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.7% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.8% by weight. In one embodiment, the SPF (%) in the solution is greater than 0.9% by weight. In one embodiment, the SPF (%) in the solution is greater than 1.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 2.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 3.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 4.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 5.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 6.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 7.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 8.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 9.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 10.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 11.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 12.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 13.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 14.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 15.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 16.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 17.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 18.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 19.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 20.0% by weight. In one embodiment, the SPF (%) in the solution is greater than 25.0% by weight.

[0189] In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 30.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 25.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 20.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 15.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 10.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 9.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 8.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 7.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 6.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 6.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 5.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 5.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 4.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 4.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 3.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 3.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 2.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 2.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 2.4% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 5.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 4.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 4.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 3.5% by weight.In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 3.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.5% by weight to about 2.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 4.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 3.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 3.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 2.5% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 2.4% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 2.0% by weight.

[0190] In one embodiment, the SPF (%) in the solution is in the range of about 20.0% by weight to about 30.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 10.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 1.0% by weight to about 10.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 2% by weight to about 10.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 0.1% by weight to about 6.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 6.0% by weight to about 10.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 6.0% by weight to about 8.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 6.0% by weight to about 9.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 10.0% to about 20.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 11.0% to about 19.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 12.0% to about 18.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 13.0% to about 17.0% by weight. In one embodiment, the SPF (%) in the solution is in the range of about 14.0% to about 16.0% by weight. In one embodiment, the SPF (%) in the solution is about 1.0% by weight. In one embodiment, the SPF (%) in the solution is about 1.5% by weight. In one embodiment, the SPF (%) in the solution is about 2.0% by weight. In one embodiment, the SPF (%) in the solution is about 2.4% by weight. In one embodiment, the SPF (%) in the solution is about 3.0% by weight. In one embodiment, the SPF (%) in the solution is about 3.5% by weight. In one embodiment, the SPF (%) in the solution is about 4.0% by weight. In one embodiment, the SPF (%) in the solution is about 4.5% by weight. In one embodiment, the SPF (%) in the solution is about 5.0% by weight. In one embodiment, the SPF (%) in the solution is about 5.5% by weight. In one embodiment, the SPF (%) in the solution is about 6.0% by weight. In one embodiment, the SPF (%) in the solution is about 6.5% by weight. In one embodiment, the SPF (%) in the solution is about 7.0% by weight. In one embodiment, the SPF (%) in the solution is about 7.5% by weight.In one embodiment, the SPF (%) in the solution is about 8.0% by weight. In one embodiment, the SPF (%) in the solution is about 8.5% by weight. In one embodiment, the SPF (%) in the solution is about 9.0% by weight. In one embodiment, the SPF (%) in the solution is about 9.5% by weight. In one embodiment, the SPF (%) in the solution is about 10.0% by weight.

[0191] In one embodiment, the sericin percentage in the solution is between undetectable and 25.0% by weight. In one embodiment, the sericin percentage in the solution is between undetectable and 5.0% by weight. In one embodiment, the sericin percentage in the solution is 1.0% by weight. In one embodiment, the sericin percentage in the solution is 2.0% by weight. In one embodiment, the sericin percentage in the solution is 3.0% by weight. In one embodiment, the sericin percentage in the solution is 4.0% by weight. In one embodiment, the sericin percentage in the solution is 5.0% by weight. In one embodiment, the sericin percentage in the solution is 10.0% by weight. In one embodiment, the sericin percentage in the solution is 25.0% by weight.

[0192] In some embodiments, the silk fibroin protein fragments of the present disclosure have a storage stability of 10 days to 3 years, depending on storage conditions, percent SPF, and number of shipments and shipping conditions (they do not slowly or spontaneously gel when stored in aqueous solution, there is no aggregation of the fragments, and therefore no increase in molecular weight over time). Additionally, the pH can be altered to prevent premature folding and aggregation of the silk, thereby extending shelf life and / or assisting shipping conditions. In one embodiment, the stability of the LiBr-silk fragment solution is 0-1 year. In one embodiment, the stability of the LiBr-silk fragment solution is 0-2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0-3 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0-4 years. In one embodiment, the stability of the LiBr-silk fragment solution is 0-5 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1-2 years. In one embodiment, the stability of the LiBr-silk fragment solution is 1-3 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 1 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 3 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 2 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 4 years. In one embodiment, the LiBr-silk fragment solution is stable for 3 to 5 years. In one embodiment, the LiBr-silk fragment solution is stable for 4 to 5 years.

[0193] In one embodiment, the composition of the present disclosure has a stability of 10 days to 6 months. In one embodiment, the composition of the present disclosure has a stability of 6 months to 12 months. In one embodiment, the composition of the present disclosure has a stability of 12 months to 18 months. In one embodiment, the composition of the present disclosure has a stability of 18 months to 24 months. In one embodiment, the composition of the present disclosure has a stability of 24 months to 30 months. In one embodiment, the composition of the present disclosure has a stability of 30 months to 36 months. In one embodiment, the composition of the present disclosure has a stability of 36 months to 48 months. In one embodiment, the composition of the present disclosure has a stability of 48 months to 60 months.

[0194] In one embodiment, compositions of the present disclosure having an SPF have undetectable levels of LiBr residual. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is from 10 ppm to 1000 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is from 10 ppm to 300 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 25 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 50 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 75 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 100 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 200 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 300 ppm. In one embodiment, the amount of LiBr residual in a composition of the present disclosure is less than 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 500 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 600 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 700 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 800 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of LiBr residual in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of LiBr residue in the composition of the present disclosure is between undetectable and 300 ppm. In one embodiment, the amount of LiBr residue in the composition of the present disclosure is between undetectable and 250 ppm. In one embodiment, the amount of LiBr residue in the composition of the present disclosure is between undetectable and 200 ppm. In one embodiment, the amount of LiBr residue in the composition of the present disclosure is between undetectable and 150 ppm.In one embodiment, the composition of the present disclosure contains a LiBr residue in an amount of from undetectable to 100 ppm. In one embodiment, the composition of the present disclosure contains a LiBr residue in an amount of from 100 ppm to 200 ppm. In one embodiment, the composition of the present disclosure contains a LiBr residue in an amount of from 200 ppm to 300 ppm. In one embodiment, the composition of the present disclosure contains a LiBr residue in an amount of from 300 ppm to 400 ppm. In one embodiment, the composition of the present disclosure contains a LiBr residue in an amount of from 400 ppm to 500 ppm.

[0195] In one embodiment, the disclosed compositions having SPF have undetectable levels of Na2CO3 residue. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 100 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 200 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 300 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 400 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 500 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 600 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 700 ppm. In one embodiment, the amount of Na2CO3 residue in the disclosed compositions is less than 800 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is less than 900 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is less than 1000 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 500 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 450 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 400 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 350 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 300 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 250 ppm. In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is undetectable to 200 ppm. In one embodiment, the composition of the present disclosure contains a non-detectable amount of Na2CO3 residue in a range of from 150 ppm to 150 ppm. In one embodiment, the composition of the present disclosure contains a non-detectable amount of Na2CO3 residue in a range of from 100 ppm to 200 ppm. In one embodiment, the composition of the present disclosure contains a non-detectable amount of Na2CO3 residue in a range of from 200 ppm to 300 ppm. In one embodiment, the composition of the present disclosure contains a non-detectable amount of Na2CO3 residue in a range of from 300 ppm to 400 ppm.In one embodiment, the amount of Na2CO3 residue in the composition of the present disclosure is between 400 ppm and 500 ppm.

[0196] A unique feature of the SPF solution compositions of the present disclosure is their storage stability of 10 days to 3 years, depending on storage conditions, percent silk, and number of shipments and shipping conditions (they do not slowly or spontaneously gel when stored in aqueous solution, there is no aggregation of fragments, and therefore no increase in molecular weight over time). Additionally, the pH can be altered to prevent premature folding and aggregation of the silk, thereby extending shelf life and / or supporting shipping conditions. In one embodiment, the SPF solution composition of the present disclosure has storage stability at room temperature (RT) for up to 2 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 4 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 6 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 8 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 10 weeks. In one embodiment, the SPF solution composition of the present disclosure has storage stability at RT for up to 12 weeks. In one embodiment, the SPF solution composition of the present disclosure has a storage stability at RT ranging from about 4 weeks to about 52 weeks.

[0197] Table 18 below shows storage stability test results for embodiments of the SPF compositions of the present disclosure. [Table 20]

[0198] In some embodiments, the water solubility of silk films derived from the silk fibroin protein fragments described herein can be modified by solvent annealing (water annealing or methanol annealing), chemical cross-linking, enzymatic cross-linking, and heat treatment.

[0199] In some embodiments, the annealing process may include inducing beta-sheet formation in a silk fibroin protein fragment solution used as a coating material. Techniques for promoting annealing (e.g., increased crystallinity) or otherwise "molecular packing" of silk fibroin protein-based fragments have been described. In some embodiments, amorphous silk films are annealed to introduce beta-sheets in the presence of a solvent selected from the group consisting of water and organic solvents. In some embodiments, amorphous silk films are annealed to introduce beta-sheets in the presence of water (aqueous annealing process). In some embodiments, amorphous silk fibroin protein fragment films are annealed to introduce beta-sheets in the presence of methanol. In some embodiments, annealing (e.g., beta-sheet formation) is induced by the addition of an organic solvent. Suitable organic solvents include, but are not limited to, methanol, ethanol, acetone, isopropanol, or combinations thereof.

[0200] In some embodiments, annealing is performed by so-called "water annealing" or "water vapor annealing," in which water vapor is used as an intermediate plasticizer or catalyst to promote beta-sheet packing. In some embodiments, the water annealing process can be carried out under vacuum. Suitable such methods are described in Jin HJ et al. (2005), Water-stable Silk Films with Reduced Beta-Sheet Content, Advanced Functional Materials, 15:1241-1247; Xiao H. et al. (2011), Regulation of Silk Material Structure by Temperature-Controlled Water Vapor Annealing, Biomacromolecules, 12(5):1686-1696.

[0201] A key feature of the water annealing process is that it drives the formation of crystalline beta sheets in the silk fibroin protein fragment peptide chains, causing the silk fibroin to self-assemble into a continuous film. In some embodiments, the crystallinity of the silk fibroin protein fragment film is controlled by controlling the temperature of the water vapor and the duration of the annealing. In some embodiments, the annealing is carried out at a temperature ranging from about 65°C to about 110°C. In some embodiments, the temperature of the water is maintained at about 80°C. In some embodiments, the annealing is carried out at a temperature selected from the group consisting of about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, about 90°C, about 95°C, about 100°C, about 105°C, and about 110°C.

[0202] In some embodiments, the annealing process may take from about 1 minute to about 40 minutes, from about 1 minute to about 50 minutes, from about 1 minute to about 60 minutes, from about 1 minute to about 70 minutes, from about 1 minute to about 80 minutes, from about 1 minute to about 90 minutes, from about 1 minute to about 100 minutes, from about 1 minute to about 110 minutes, from about 1 minute to about 120 minutes, from about 1 minute to about 130 minutes, from about 5 minutes to about 40 minutes, from about 5 minutes to about 50 minutes, from about 5 minutes to about 60 minutes, from about 5 minutes to about 70 minutes, from about 5 minutes to about 80 minutes, from about 5 minutes to about 90 minutes, from about 5 minutes to about 100 minutes, from about 5 minutes to about 110 minutes, from about 5 minutes to about 120 minutes, from about 5 minutes to about 130 minutes, from about 10 minutes to about 40 minutes, from about 10 minutes to about 50 minutes, from about 10 minutes to about 60 minutes, from about 10 minutes to about 150 minutes, from about 15 minutes to about 200 minutes, from about 15 minutes to about 250 minutes, from about 15 minutes to about 260 minutes, from about 15 minutes to about 270 minutes, from about 15 minutes to about 280 minutes, from about 15 minutes to about 290 minutes, from about 15 minutes to about 300 minutes, from about 15 minutes to about 310 minutes, from about 15 minutes to about 320 minutes, from about 15 minutes to about 330 minutes, from about 15 minutes to about 340 minutes, from about 15 minutes to about 350 minutes, from about 15 minutes to about 360 minutes, from about 15 minutes to about 370 minutes, from about Approximately 70 minutes, approximately 10 minutes to approximately 80 minutes, approximately 10 minutes to approximately 90 minutes, approximately 10 minutes to approximately 100 minutes, approximately 10 minutes to approximately 110 minutes, approximately 10 minutes to approximately 120 minutes, approximately 10 minutes to approximately 130 minutes, approximately 15 minutes to approximately 40 minutes, approximately 15 minutes to approximately 50 minutes, approximately 15 minutes to approximately 60 minutes, approximately 15 minutes to approximately 70 minutes, approximately 15 minutes to approximately 80 minutes, approximately 15 minutes to Approximately 90 minutes, approximately 15 minutes to approximately 100 minutes, approximately 15 minutes to approximately 110 minutes, approximately 15 minutes to approximately 120 minutes, approximately 15 minutes to approximately 130 minutes, approximately 20 minutes to approximately 40 minutes, approximately 20 minutes to approximately 50 minutes, approximately 20 minutes to approximately 60 minutes, approximately 20 minutes to approximately 70 minutes, approximately 20 minutes to approximately 80 minutes, approximately 20 minutes to approximately 90 minutes, approximately 20 minutes to approximately 100 minutes, approximately 20 minutes ~approx. 110 minutes, ~approx. 20 minutes ~approx. 120 minutes, ~approx. 20 minutes ~approx. 130 minutes, ~approx. 25 minutes ~approx. 40 minutes, ~approx. 25 minutes ~approx. 50 minutes, ~approx. 25 minutes ~approx. 60 minutes, ~approx. 25 minutes ~approx. 70 minutes, ~approx. 25 minutes ~approx. 80 minutes, ~approx. 25 minutes ~approx. 90 minutes, ~approx. 25 minutes ~approx. 100 minutes, ~approx. 25 minutes ~approx. 110 minutes, ~approx. 25 minutes ~approx. 120 minutes, ~approx. 5 minutes to approximately 130 minutes, approximately 30 minutes to approximately 40 minutes, approximately 30 minutes to approximately 50 minutes, approximately 30 minutes to approximately 60 minutes, approximately 30 minutes to approximately 70 minutes, approximately 30 minutes to approximately 80 minutes, approximately 30 minutes to approximately 90 minutes, approximately 30 minutes to approximately 100 minutes, approximately 30 minutes to approximately 110 minutes, approximately 30 minutes to approximately 120 minutes, approximately 30 minutes to approximately 130 minutes, approximately 35 minutes to approximately 40 minutes, approximately 35 minutes to approximately 50 minutes, approximately 35 minutes to approximately 60 minutes, approximately 35 minutes to approximately 70 minutes, approximately 35 minutes to approximately 80 minutes, approximately 35 minutes to approximately 90 minutes, approximately 35 minutes to approximately 100 minutes, approximately 35 minutes to approximately 110 minutes, approximately 35 minutes to approximately 120 minutes, approximately 35 minutes to approximately 130 minutes, approximately 40 minutes to approximately 50 minutes, approximately 40 minutes to approximately 60 minutes, approximately 40 minutes to approximately 70 minutes, approximately 40 minutes to approximately 80 minutes, approximately 40 minutes to approximately 90 minutes, approximately 40 minutes to approximately 100 minutes, approximately 40 minutes to approximately 110 minutes, approximately 40 minutes to approximately 120 minutes, approximately 40 minutes to approximately 130 minutes, approximately 45 minutes to approximately 50 minutes, approximately 45 minutes to approximately 60 minutes, approximately 45 minutes to approximately 70 minutes, approximately 45 minutes to approximately 80 minutes, approximately 45 minutes to approximately 90 minutes, approximately 45 minutes to approximately 100 minutes,The annealing process lasts for a period selected from the group consisting of about 45 minutes to about 110 minutes, about 45 minutes to about 120 minutes, and about 45 minutes to about 130 minutes. In some embodiments, the annealing process lasts for a period ranging from about 1 minute to about 60 minutes. In some embodiments, the annealing process lasts for a period ranging from about 45 minutes to about 60 minutes. Longer water annealing after treatment has been associated with increased crystallinity of the silk fibroin protein fragments.

[0203] In some embodiments, the annealed silk fibroin protein fragment film is soaked in 100% methanol at room temperature for 60 minutes, and the composition of the silk fibroin protein fragment film changes from a predominantly amorphous random coil to a crystalline antiparallel beta sheet structure by methanol annealing.

[0204] In some embodiments, the SPF solution described herein can be used to prepare SPF microparticles by precipitation with methanol. Alternatively, flash drying, fluidized bed drying, spray drying, or vacuum drying can be applied to remove water from the silk solution. The SPF powder can then be stored and handled without refrigeration or other special handling procedures. In some embodiments, the SPF powder comprises low molecular weight silk fibroin protein fragments. In some embodiments, the SPF powder comprises medium molecular weight silk fibroin protein fragments. In some embodiments, the SPF powder comprises a mixture of low molecular weight and medium molecular weight silk fibroin protein fragments.

[0205] Silk protein fragments in collagen boosting compositions and methods thereof The present disclosure provides a method for treating or preventing a disorder, disease, or condition that is alleviated by stimulating or modulating collagen expression in a subject in need thereof, the method comprising administering to a subject a collagen protein having a stimulating or modulating collagen expression level of from about 1 kDa to about 5 kDa, from about 5 kDa to about 10 kDa, from about 6 kDa to about 17 kDa, from about 10 kDa to about 15 kDa, from about 15 kDa to about 20 kDa, from about 14 kDa to about 30 kDa, from about 17 kDa to about 39 kDa, from about 20 kDa to about 25 kDa, from about 25 kDa to about 30 kDa, from about 30 kDa to about 35 kDa, The present invention provides a method for treating a silk fibroin disorder, comprising administering to a subject a composition comprising silk fibroin fragments having an average weight-average molecular weight selected from 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 60 kDa to about 100 kDa, and about 80 kDa to about 144 kDa, and a polydispersity of 1 to about 5, or any other silk protein fragment described herein. Any other molecular weight, molecular weight range, and polydispersity of silk fibroin fragments, or any other silk protein fragment described herein, can be used in the methods and compositions of the present disclosure.

[0206] In some embodiments, the composition further comprises 0-500 ppm lithium bromide. In some embodiments, the composition further comprises 0-500 ppm sodium carbonate. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, have a polydispersity of 1 to about 1.5. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, have a polydispersity of about 1.5 to about 2.0. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, have a polydispersity of about 1.5 to about 3.0. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, have a polydispersity of about 2.0 to about 2.5. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, have a polydispersity of about 2.5 to about 3.0. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 0.001% to about 10.0% by weight, based on the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin, based on the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin, based on the total weight of the composition. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, do not spontaneously or gradually gel or exhibit a visible change in color or turbidity when in aqueous solution for at least 10 days prior to formulation into a composition. In some embodiments, the silk fibroin fragment, or any other silk protein fragment described herein, is present in the composition at about 0.01% to about 10.0% by weight, based on the total weight of the composition.In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 0.01 wt % to about 1.0 wt % relative to the total weight of the composition. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 1.0 wt % to about 2.0 wt % relative to the total weight of the composition. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 2.0 wt % to about 3.0 wt % relative to the total weight of the composition. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 3.0 wt % to about 4.0 wt % relative to the total weight of the composition. In some embodiments, the silk fibroin fragments, or any other silk protein fragments described herein, are present in the composition at about 4.0 wt % to about 5.0 wt % relative to the total weight of the composition. In some embodiments, the silk fibroin fragment, or any other silk protein fragment described herein, is present in the composition at about 5.0% to about 6.0% by weight, based on the total weight of the composition.

[0207] In some embodiments, the composition is formulated as an injectable composition or as a topical composition. In some embodiments, the composition is formulated to improve the appearance and feel of skin by, but not limited to, boosting collagen (for example, but not limited to, stimulating or regulating collagen expression). In some embodiments, the composition is formulated to boost collagen on the skin. In some embodiments, the composition is formulated to boost collagen intradermally. In some embodiments, the composition is formulated to boost collagen on the scalp. In some embodiments, the composition is formulated as a liquid solution for boosting collagen. In some embodiments, the composition is formulated as a film for boosting collagen. In some embodiments, the composition is formulated as a solid for boosting collagen. In some embodiments, the composition is formulated as a powder for boosting collagen. In some embodiments, the composition is formulated as a gel for boosting collagen. In some embodiments, the composition is formulated as a silk gel for boosting collagen. In some embodiments, the composition is formulated as a silk / HA gel with or without lidocaine for boosting collagen. In some embodiments, the composition is formulated as a soap for boosting collagen. In some embodiments, the composition is formulated as a cream for boosting collagen. In some embodiments, the composition is formulated as a lotion for boosting collagen. In some embodiments, the composition is formulated as a shampoo for boosting collagen. In some embodiments, the composition is formulated as a conditioner for boosting collagen. In some embodiments, the composition is formulated as a nutrient for boosting collagen. In some embodiments, the composition is formulated as a mask for boosting collagen. In some embodiments, the composition is formulated as an over-the-counter product for boosting collagen.In some embodiments, the composition is formulated as a medicament for boosting collagen. In some embodiments, the composition is formulated as a therapeutic agent for boosting collagen. In some embodiments, the composition is formulated as a silk-coated woven material for boosting collagen. In some embodiments, the composition is formulated as a silk-coated nonwoven material for boosting collagen.

[0208] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier. In some embodiments, the composition further comprises a dermatologically acceptable carrier. In some embodiments, the composition further comprises an injectable acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a suspension, emulsion, powder, solution, dispersion, or elixir. In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, jelly, cream, lotion, foam, slurry, ointment, oil, paste, suppository, spray, semi-solid composition, solid composition, stick, or mousse. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of sesame oil, corn oil, cottonseed oil, or peanut oil. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of mannitol or dextrose. In some embodiments, the pharmaceutically acceptable carrier comprises about 0.001% to about 10% (w / v) hyaluronic acid. In some embodiments, the pharmaceutically acceptable carrier comprises about 1% to about 10% (w / v), about 10% to about 25% (w / v), about 25% to about 50% (w / v), or about 50% to about 99.99% (w / v) hyaluronic acid. In some embodiments, the HA described herein has a molecular weight of 100,000 daltons or more, 150,000 daltons or more, 1 million daltons or more, or 2 million daltons or more. In some embodiments, the HA described herein has a molecular weight of 100,000 daltons or less, 150,000 daltons or less, 1 million daltons or less, or 2 million daltons or less. In some embodiments, the HA described herein has a high molecular weight (e.g., an HA molecular weight of about 1 MDa to about 4 MDa). In some embodiments, the HA described herein has a low molecular weight (e.g., an HA molecular weight of less than about 1 MDa). In some embodiments, the HA source may be a hyaluronate, such as sodium hyaluronate. In some embodiments, the HA is cross-linked. The cross-linked HA can be formulated into various shapes, such as a membrane, a gel, a semi-gel, a sponge, or a microsphere. In some embodiments, the cross-linked HA is in a fluid gel form, i.e., it takes the shape of its container.The viscosity of the HA gel or semi-gel can be altered by adding unconjugated HA and / or hyaluronate. Viscosity can also be adjusted by varying the degree of SPF-SPF, SPF-HA, and / or HA-HA crosslinking, as described herein. In some embodiments, about 4% to about 12% of the HA can be crosslinked as HA-HA or HA-SPF.

[0209] In some embodiments, the pharmaceutically acceptable carrier comprises one or more of aliphatic oils, fatty alcohols, fatty acids, glycerides, acylglycerols, and phospholipids. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of monoglycerides, diglycerides, or triglycerides. In some embodiments, the pharmaceutically acceptable carrier comprises an aqueous phase. In some embodiments, the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in-oil emulsion. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of hydrocarbon oils, fatty acids, fatty acid oils, fatty acid esters, or cationic quaternary ammonium salts. In some embodiments, a portion of the pharmaceutically acceptable carrier is a polyepoxy linker, a diepoxy linker, a polyepoxy-PEG, a diepoxy-PEG, a polyglycidyl-PEG, a diglycidyl-PEG, a polyacrylate PEG, a diacrylate PEG, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light, glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)butane ... and modified with a crosslinker, crosslink precursor, or activator selected from 1,2,7,8-diepoxyethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic acid dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, and any combination thereof.In some embodiments, the polyepoxy linker is selected from 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether, polyethylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, tri-methylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether.

[0210] In some embodiments, the composition further comprises an anesthetic compound. In some embodiments, the compound is selected from benzocaine, chloroprocaine, cocaine, cyclomethycaine, dimethocaine, piperocaine, propoxycaine, procaine, proparacaine, tetracaine, articaine, bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine, mepivacaine, prilocaine, ropivacaine, and trimecaine. In some embodiments, the composition further comprises lidocaine. In some embodiments, the concentration of lidocaine in the composition is about 0.01% to about 1%, including any increment of 0.01%. In some embodiments, the concentration of lidocaine in the composition is about 0.3%.

[0211] In certain embodiments, the compositions described herein can include one or more anesthetics in an amount effective to ameliorate or reduce pain or discomfort at the site of injection of the composition. Local anesthetics include ambucaine, amoranone, amylocaine, benoxinate, benzocaine, betoxicaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butetamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethicaine, dibucaine, dimethisoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprosin, phenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl-p-aminobenzoate, leuconazole ... The analgesic may be selected from the group consisting of sinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, nepain, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudocaine, pyrrocaine, ropivacaine, salicylic alcohol, tetracaine, tricaine, trimecaine, zolamine, and salts thereof.

[0212] In some embodiments, the compositions described herein are present in an amount by weight of about 0.01% to about 0.02%, or about 0.03% to about 0.04%, or about 0.05% to about 0.06% to about 0.07%, or about 0.08% to about 0.09%, or about 0.1% to about 0.2%, or about 0.3% to about 0.4%, or about 0.5% to about 0.6%, or about 0.7% to about 0.8%, or about 0.9% to about 1.0%, or about 1% to about 1.5%, or about 1.5% to about 2.0%, or about 2.0% to about 2.5%. or about 2.5% to about 3.0%, or about 3.0% to about 3.5%, or about 3.5% to about 4.0%, or about 4.0% to about 4.5%, or about 4.5% to about 5.0%, or about 5.0% to about 5.5%, or about 5.5% to about 6.0%, or about 6.0% to about 6.5%, or about 6.5% to about 7.0%, or about 7.5% to about 8.0%, or about 8.0% to about 8.5%, or about 8.5% to about 9.0%, or about 9.5% to about 10% of lidocaine or other anesthetics listed herein.

[0213] In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as a gel. The gel can be an injectable gel, such as, but not limited to, a tissue filler or a gel for topical administration. Suitable gels are described, for example, in WO 2019 / 005848, which is incorporated herein by reference. In some embodiments, the gel comprises silk fibroin or silk fibroin fragments, or any other SPF described herein, hyaluronic acid (HA), and polyethylene glycol (PEG) and / or polypropylene glycol (PPG). In some embodiments, a portion of the HA is modified or crosslinked with one or more linker moieties comprising one or more of polyethylene glycol (PEG), polypropylene glycol (PPG), and a secondary alcohol, the linker moiety being attached to the HA at one end of the linker. In some embodiments, a portion of the silk fibroin or silk fibroin fragments, or any other SPF described herein, is modified or crosslinked. In some embodiments, a portion of the silk fibroin or silk fibroin fragments, or any other SPF described herein, is free. In some embodiments, a portion of silk fibroin or silk fibroin fragments, or any other SPF described herein, is crosslinked to HA. In some embodiments, a portion of silk fibroin or silk fibroin fragments, or any other SPF described herein, is crosslinked to silk fibroin or silk fibroin fragments, or any other SPF described herein. In some embodiments, the silk fibroin or silk fibroin fragments are substantially devoid of sericin. In some embodiments, the gel has a degree of modification (MoD) of about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%. In some embodiments, the modification or crosslinking is achieved using diepoxy-PEG, polyglycidyl-PEG, diglycidyl-PEG, diepoxy-PPG, polyglycidyl-PPG, diglycidyl-PPG, or any combination thereof as a crosslinker.In some embodiments, the modification or crosslinking is achieved using a polyethylene glycol diglycidyl ether having a MW of about 200 Da, about 500 Da, 1000 Da, about 2,000 Da, or about 6000 Da. In some embodiments, the modification or crosslinking is achieved using a polypropylene glycol diglycidyl ether having a MW of about 380 Da, or about 640 Da. In some embodiments, the gel is a hydrogel. In some embodiments, the gel further comprises water. In some embodiments, the gel is a single phase. In some embodiments, the total concentration of HA and silk in the gel is about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, or about 30 mg / mL. In some embodiments, the ratio of HA to silk fibroin or silk fibroin fragments in the gel is about 92 / 8, about 93 / 7, about 94 / 6, about 95 / 5, about 96 / 4, about 97 / 3, about 18 / 12, about 27 / 3, about 29.4 / 0.6, about 99 / 1, about 92.5 / 7.5, or about 90 / 10. In some embodiments, the gel is a dermal filler. In some embodiments, the gel is biodegradable. In some embodiments, the gel is injectable. In some embodiments, the gel is injectable through a 30G or 27G needle. In some embodiments, the gel has a storage modulus (G') of about 5 Pa to about 500 Pa. In some embodiments, G' is measured using an oscillatory stress of about 1 Hz, about 5 Hz, or about 10 Hz. In some embodiments, the gel has a complex viscosity of about 1 Pa·s to about 10 Pa·s. In some embodiments, the complex viscosity is measured by an oscillatory stress of about 1 Hz, about 5 Hz, or about 10 Hz. In some embodiments, the gel comprises a glycosaminoglycan selected from the group consisting of hyaluronic acid (HA), carboxymethylcellulose (CMC), starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthan gum, chitosan, pectin, agar, carrageenan, and guar gum.

[0214] In some embodiments, the composition is administered parenterally. In some embodiments, the composition is an injectable composition. In some embodiments, the composition is administered by injection. In some embodiments, the composition is administered by subcutaneous, intradermal, transdermal, or subdermal injection. In some embodiments, the composition is administered by intramuscular, intravenous, intraperitoneal, intraosseous, intracardiac, intraarticular, or intracavernosal injection. In some embodiments, the composition is administered by depot injection. In some embodiments, the composition is administered by infiltration injection. In some embodiments, the composition is administered by an indwelling catheter. In some embodiments, the composition, or portions thereof, are biocompatible, biodegradable, bioabsorbable, bioresorbable, or a combination thereof. In some embodiments, the compositions provided herein comprise a fluid component, e.g., a single fluid or a solution substantially comprising one or more fluids. In some embodiments, the composition comprises water or an aqueous solution. In some embodiments, the composition is subcutaneously injectable, implantable, or deliverable by any means known in the art, e.g., after surgical removal of tissue. In some embodiments, the composition is a dermal filler. In some embodiments, the composition is sterile.

[0215] In embodiments, the weight percent water content in the compositions described herein is about 1%, or about 2%, or about 3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, or about 10%, or about 11%, or about 12%, or about 13%, or about 14%, or about 15%, or about 16%, or about 17%, or about 18%, or about 19%, or about 20%, or about 21%, or about 22%, or about 23%, or about 24%, or about 25%, or about 26%, or about 27%, or about 28%, or about 29%, or about 30%, or about 31%, or about 32%, or about 33%, or about 34%, or about 35%, or about 36%, or about 37%, or about 38%, or about 39%, or about 40%, or about 41%, or about 42%, or about 43%, or about 44%, or about 45%, or about 46%, or about 47%, or about 48%, or about 49%, or about 50%, or about 51%, or about 52%, or about 53%, or about 54%, or about 55%, or about 56%, or about 57%, or about 58%, or about 59%, or about 60%, or about 61%, or about 62%, or about 63%, or about 64%, or about 65%, or about 66%, or about 67%, or about 68%, or about 69%, or about 70%, or about 71% %, or about 72%, or about 73%, or about 74%, or about 75%, or about 76%, or about 77%, or about 78%, or about 79%, or about 80%, or about 81%, or about 82%, or about 83%, or about 84%, or about 85%, or about 86%, or about 87%, or about 88%, or about 89%, or about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%.

[0216] In some embodiments, in addition to boosting collagen expression, compositions can be administered into and around soft tissues to provide volume, assist with, or otherwise treat soft tissue defects. The compositions described herein can be administered at multiple levels below the dermis. As used herein, the term "soft tissue" can refer to tissues that connect, support, or surround other structures and organs of the body. For example, soft tissues described herein can include, but are not limited to, skin, dermal tissue, subdermal tissue, cutaneous tissue, subcutaneous tissue, intradural tissue, muscle, tendons, ligaments, fibrous tissue, fat, blood vessels and arteries, nerves, and synovial (intradermal) tissue. In some embodiments, the present disclosure provides a method of treating a soft tissue condition in an individual, comprising administering one or more compositions disclosed herein to the site of the soft tissue condition in the individual, wherein administration of the composition improves the soft tissue condition, thereby treating the soft tissue condition. In some embodiments, the soft tissue condition is a breast tissue condition, a facial tissue condition, a neck condition, a skin condition, an upper arm condition, a lower arm condition, a hand condition, a shoulder condition, a back condition, a torso (including abdomen) condition, a buttocks condition, a thigh condition, a lower leg condition (including a calf condition), a foot condition (including a plantar fat pad condition), an eye condition, a genital condition, or a condition affecting any other body part, region, or area.

[0217] In some embodiments, the present disclosure provides compositions and methods of treatment for regions of the dermis, including, but not limited to, regions of the skin including the epidermal-dermal junction, and the dermis, including the superficial dermis (papillary region) and deep dermis (reticular region). Skin is composed of three primary layers: the epidermis, which provides waterproofing and acts as a barrier against infection; the dermis, which serves as a location for skin appendages; and the hypodermis (subcutaneous fat layer). The epidermis does not contain blood vessels and is nourished by diffusion from the dermis. The main types of cells that make up the epidermis are keratinocytes, melanocytes, Langerhans cells, and Merkel cells.

[0218] In one embodiment, the compositions described herein can be provided in methods for treating one or more conditions in a patient in need thereof, hi some embodiments, a therapeutically effective amount of the composition can be delivered to a tissue of a patient in need thereof to treat a condition or other tissue defect.

[0219] As used herein, the terms "treating," "treat," or "treatment" refer to reducing or eliminating the cosmetic or clinical symptoms of a condition, e.g., a soft tissue condition, in a patient, or delaying or preventing the onset of cosmetic or clinical symptoms of a condition in an individual.

[0220] In some embodiments, the condition treated by the compositions described herein may include a soft tissue condition. Soft tissue conditions include, but are not limited to, augmentation, reconstruction, disease, disorder, defect, or imperfection of a body part, region, or area. In one aspect, soft tissue conditions treated by the disclosed compositions include, but are not limited to, facial augmentation, facial reconstruction, facial disease, facial disorder, facial defect, or facial imperfection. In some embodiments, soft tissue conditions treated by the compositions described herein include, but are not limited to, skin dehydration, lack of skin elasticity, rough skin, lack of skin firmness, stretch lines or marks, pale skin, dermal depressions, sunken cheeks, sunken temples, thin lips, urethral defects, skin imperfections, breast imperfections, retro-orbital defects, facial folds, or wrinkles. In some embodiments, soft tissue conditions treated by the compositions described herein include, but are not limited to, breast imperfections, defects, diseases, and / or disorders, such as defects resulting from breast augmentation, breast reconstruction, mastopexy, micromastia, thoracic hypoplasia, Poland's syndrome, implant complications such as capsular contraction and / or rupture; facial imperfections, defects, diseases, or disorders, such as facial augmentation, facial reconstruction, Parry-Romberg syndrome, lupus erythematosus bumps, skin depressions, sunken cheeks, sunken temples, thin lips, nasal imperfections or defects, retro-orbital imperfections or defects, facial folds, lines, and / or glabellar lines. any wrinkles, nasolabial folds, perioral lines, and / or marionette lines, and / or other facial contour deformations or imperfections; neck imperfections, defects, diseases, or disorders; skin imperfections, defects, diseases, and / or disorders; other soft tissue imperfections, defects, diseases, and / or disorders, such as augmentation or reconfiguration of, or diseases or disorders affecting, the upper arms, lower legs, hands, shoulders, back, trunk including abdomen, buttocks, upper thighs, lower legs including calves, feet including plantar fat pads, eyes, genitalia, or other body parts, regions, or areas; urinary incontinence, fecal incontinence, other types of incontinence; and gastroesophageal reflux disease (GERD).

[0221] In some embodiments, the compositions described herein may be delivered to soft tissues, including, but not limited to, skin, dermal tissue, subdermal tissue, cutaneous tissue, subcutaneous tissue, intradural tissue, muscle, tendons, ligaments, fibrous tissue, fat, blood vessels and arteries, nerves, and synovial (intradermal) tissue.

[0222] In some embodiments, the compositions described herein can be placed directly into a wound to aid healing by providing an artificial biodegradable matrix with cell adhesion, migration, and proliferation signals. In some embodiments, the compositions described herein can be coated onto a biodegradable mesh or other implant material, or can themselves be formed into sheets or other structures or maintained in a hydrated form.

[0223] In some embodiments, the amount of composition used with any of the methods disclosed herein is determined based on the desired change and / or improvement, reduction and / or elimination of symptoms of the desired condition, clinical and / or cosmetic effect desired by the individual and / or physician, and the body part or area to be treated. The effectiveness of composition administration can be expressed by one or more of the following clinical and / or cosmetic measures: changed and / or improved soft tissue shape, changed and / or improved soft tissue size, changed and / or improved soft tissue contour, changed and / or improved tissue function, tissue ingrowth support, and / or new collagen deposition, sustained engraftment of the composition, improved patient satisfaction and / or quality of life, and reduced use of implantable foreign bodies. For example, for breast augmentation procedures, the effectiveness of the compositions and methods can be expressed by one or more of the following clinical and / or cosmetic measures: increased breast size, changed breast shape, changed breast contour, sustained engraftment, reduced risk of capsular contraction, reduced rate of liponecrotic cyst formation, improved patient satisfaction and / or quality of life, and reduced use of breast implants.

[0224] In some embodiments, administering the composition reduces expression of one or more metalloproteinases (MMPs) in the subject. In some embodiments, stimulating or modulating collagen expression comprises increasing collagen expression.

[0225] In some embodiments, collagen expression is reduced by about 1%, 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%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94% 9%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75% , about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% increase.

[0226] In some embodiments, collagen expression is about 101%, about 102%, about 103%, about 104%, about 105%, about 106%, about 107%, about 108%, about 109%, about 110%, about 111%, about 112%, about 113%, about 114%, about 115%, about 116%, about 117%, about 118%, about 119%, about 120%, about 121%, about 122%, approx. 123%, approx. 124%, approx. 125%, approx. 126%, approx. 127%, approx. 128%, approx. 129%, approx. 130%, approx. 131%, approx. 132%, approx. 133%, approx. 134%, approx. 135%, approx. 136%, approx. 137%, approx. 138%, approx. 139%, approx. 140%, approx. 141%, approx. 142%, approx. 143%, approx. 144%, approx. 145%, approx. 146%, approx. 147%, approx. 148% ,approximately 149%,approximately 150%,approximately 151%,approximately 152%,approximately 153%,approximately 154%,approximately 155%,approximately 156%,approximately 157%,approximately 158%,approximately 159%,approximately 160%,approximately 161%,approximately 162%,approximately 163%,approximately 164%,approximately 165%,approximately 166%,approximately 167%,approximately 168%,approximately 169%,approximately 170%,approximately 171%,approximately 172%,approximately 173%,approximately 174%,approximately 1 An increase of 75%, about 176%, about 177%, about 178%, about 179%, about 180%, about 181%, about 182%, about 183%, about 184%, about 185%, about 186%, about 187%, about 188%, about 189%, about 190%, about 191%, about 192%, about 193%, about 194%, about 195%, about 196%, about 197%, about 198%, about 199%, or about 200%.

[0227] In some embodiments, collagen expression is increased by about 225%, or about 250%, or about 275%, or about 300%, or about 325%, or about 350%, or about 375%, or about 400%, or about 425%, or about 450%, or about 475%, or about 500%, or about 525%, or about 550%, or about 575%, or about 600%, or about 625%, or about 650%, or about 675%, or about 700%, or about 725%, or about 750%, or about 775%, or about 800%, or about 825%, or about 850%, or about 875%, or about 900%, or about 925%, or about 950%, or about 975%, or about 1000% relative to basal levels.

[0228] In some embodiments, administering the composition results in one or more of preventing or improving wrinkles in a subject, preventing or improving age spots in a subject, preventing or improving dry skin in a subject, enhancing uneven skin tone in a subject, or improving the appearance and feel of skin. Improving the appearance and feel of skin includes, but is not limited to, improving the appearance and feel of damaged skin, as well as improving the appearance and feel of otherwise visibly undamaged skin. In some embodiments, administering the composition results in one or more of preventing or improving sagging skin in a subject, preventing or improving skin aging in a subject, preventing or improving reduced skin tensile strength in a subject, preventing or improving photodamaged skin in a subject, or preventing or improving striae stretch marks in a subject. In some embodiments, the disorder, disease, or condition comprises wrinkles, age spots, dry skin, uneven skin tone, sagging skin, aging skin, reduced skin tensile strength, photodamaged skin, or stretch marks. In some embodiments, the disorder, disease, or condition comprises a skin condition. In some embodiments, the skin condition can be dehydration of the skin, lack of skin elasticity, rough skin, lack of skin firmness, stretch lines, stretch marks, pale skin, dimples in the dermis, sunken cheeks, sunken temples, thin lips, retro-orbital defects, facial folds, and wrinkles.

[0229] In some embodiments, the disclosed treatment methods include augmentation, reconstruction, treatment of disease, treatment of disorder, treatment of a defect or imperfection in a body part, region, or area, hi some embodiments, the disclosed treatment methods include facial augmentation, facial reconstruction, treatment of facial disease, treatment of facial disorder, treatment of facial defect, or treatment of facial imperfection.

[0230] In some embodiments, the provided methods of treatment include one or more of administering a composition of the present disclosure before, after, or during laser treatment, administering a composition of the present disclosure before, after, or during skin peeling, or administering a composition of the present disclosure before, after, or during radiation treatment. In some embodiments, the provided methods of treatment include one or more of administering a composition of the present disclosure to treat burns, including but not limited to any type of burn (e.g., scald, sunburn, fire burn, hot water burn, radiation burn, chemical burn, etc.). In some embodiments, the provided methods of treatment include one or more of administering a composition of the present disclosure to treat burns, including but not limited to first-degree, second-degree, or third-degree burns. In some embodiments, the provided methods of treatment include one or more of administering a composition of the present disclosure to treat age-related skin conditions.

[0231] In some embodiments, the disorder, disease, or condition comprises thyroid hormone-induced myocardial hypertrophy, hi some embodiments, the disorder, disease, or condition comprises tendon rupture, injury, or tear. In some embodiments, the tendon is selected from the group consisting of teres minor tendon, infraspinatus tendon, supraspinatus tendon, subscapularis tendon, deltoid tendon, biceps tendon, triceps tendon, brachioradialis tendon, supinator tendon, flexor carpi radialis tendon, flexor carpi ulnaris tendon, extensor carpi radialis longus tendon, extensor carpi radialis brevis tendon, iliopsoas tendon, obturator internus tendon, adductor longus tendon, peroneus or magnus tendon, gluteus maximus or gluteus medius tendon, quadriceps tendon, patellar tendon, popliteus tendon, sartorius tendon, gastrocnemius tendon, Achilles tendon, soleus tendon, tibialis anterior tendon, peroneus longus tendon, flexor digitorum longus tendon, interosseus tendon, flexor digitorum profundus tendon, abductor digitorum minimi tendon, opponens pollicis tendon, flexor pollicis longus tendon, extensor or abductor pollicis tendons), flexor hallucis longus tendon, flexor digitorum brevis tendon, medius tendon, abductor hallucis longus tendon, abductor digitorum minimi tendon, ophthalmic tendon, levator palpebrae tendon, masseter tendon, temporalis tendon, trapezius tendon, sternocleidomastoid tendon, semispinalis capitis or splenius capitis tendon, mylohyoid or thyrohyoid tendon, sternohyoid tendon, rectus abdominis tendon, external oblique tendon, transverse abdominis tendon, latissimus dorsi tendon, and erector spinae tendon. In some embodiments, the disorder, disease, or condition comprises Werner's syndrome. In some embodiments, the disorder, disease, or condition comprises impaired diabetic skin integrity. In some embodiments, the disorder, disease, or condition comprises arthritis. In some embodiments, the disorder, disease, or condition comprises rheumatoid arthritis. In some embodiments, the disorder, disease, or condition comprises tumor progression or tumor growth. In some embodiments, the disorder, disease, or condition comprises impaired cardiac function. In some embodiments, the disorder, disease, or condition comprises Ehlers-Danlos syndrome. In some embodiments, the disorder, disease, or condition comprises abdominal aortic aneurysm. In some embodiments, the disorder, disease, or condition comprises a wound. In some embodiments, the disorder, disease, or condition comprises a disease of the skin or connective tissue. In some embodiments, the disorder, disease, or condition comprises a disease of cartilage. In some embodiments, the disorder, disease, or condition comprises relapsing polychondritis, Tietze's syndrome, cellulitis, Ehlers-Danlos syndrome, keloids (including acne keloids), mucopolysaddaridosis II), necrotizing disorders (including granuloma annulare and necrobiosis lipoidica), osteogenesis imperfecta, cutis laxa, dermatomyositis, Dupuytren's contracture, homocystinuria, lupus erythematosus (including cutaneous, discoid, deep, systemic, and nephritic), Marfan syndrome, mixed connective tissue disease, mucinosis (including follicular), mucopolysaccharidoses (I, II, UU, IV, IV, and VII), myxedema, scleredema adultae, and synovial cysts. In some embodiments, the disorder, disease, or condition is selected from tissue neoplasms, Noonan's syndrome, bone poikilosis, panniculitis including erythema induratum, nodular nonsuppurative and peritoneal, penile sclerosis, pseudoxanthoma elasticum, rheumatic diseases including arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, Kaplan's syndrome, Felty's syndrome, rheumatoid nodules, ankylosing spondylitis, and Still's disease), osteophytosis, polymyalgia rheumatica, localized scleroderma, and systemic sclerosis (CREST syndrome). In some embodiments, the disorder, disease, or condition is selected from angiolymphocytosis with eosinophilia; cicatricialis (including hypertrophic); skin fistula, cutis laxa laxa); dermatitis including acrodermatitis, atopic dermatitis, contact dermatitis (allergic contact, photoallergy, sumac), irritant dermatitis (phototoxicity, diaper rash), occupational dermatitis; exfoliative skin eczema, including inflammation, dermatitis herpetiformis, seborrheic dermatitis, drug eruptions (toxic epidermal necrolysis, erythema nodosum, serum sickness, etc.), dyshidrosis, intertrigo, neurodermatitis, and radiation dermatitis; dermatomyositis; Erythema, including erythematous, polymorphic (Stevens-Johnson syndrome), and nodular (Sweet syndrome); exanthem, including subitum exanthem; facial skin disorders, including acneiform eruptions (keloids, rosacea, rosacea vulgaris, and Fabre-Lacouchot syndrome); skin disorders of the feet, including athlete's foot; skin disorders of the hands; keratoacanthoma; callus, cholesteatoma (including middle ear), ichthyosis (including congenital ichthyosiform erythroderms, epidermolytic hyperkeratosis, lamellar ichthyosis, ichthyosis vulgaris, X-linked ichthyosis, and Sjögren-Larsson syndrome), pyorrhagic keratoderma, palmoplantar keratodermakeratoderms, follicular keratosis, seborrheic keratosis, parakeratosis, and porokeratosis; leg skin diseases, mastocytosis (urticaria pigmentosa), necrobiosis (granuloma annulare and necrobiosis lipoidica), photosensitivity (photoallergic or phototoxic dermatitis, variola vaccinia, sundurn, and xeroderma pigmentosum); argyria, hyperpigmentation, melanosis, aconthosis nigricans, lentigines, Peutz-Jeghers syndrome, hypopigmentation, congenital albinism, pibaldism, vitiligo, incontinentia pigmenti, urticaria pigmentosa, xeroderma pigmentosum, and prurigo; pruritus (including anal and vulvar regions); pyoderma, including ecthyma and pyoderma gangrenosum; scalp diseases (sclap dermatoses; adult scleroderma (sclerodema adultorum); neonatal scleroderma (sclerma neonatorum); skin appendage disorders including hair disorders (alopecia, folliculitis, hirsutism, hypertrichosis, kinky hair syndrome), nail disorders (nail-patella syndrome, ingrown nails or nail dysplasia, onychomycosis, paronychia), sebaceous gland disorders (rhinophyma, neoplasms), sweat gland disorders (hidradenitis suppurativa, hyperhidrosis, hypohidrosis, milia, Fox-Fordyce disease, neoplasms) Hereditary skin diseases including alfinism, cutis laxa, familial benign chronic pemphigus, porphyria, acrodermatitis, ectodermal hypoplasia, Ellis-van Creveld syndrome, focal cutaneous hypoplasia, Ehlers-Danlos syndrome, epidermolysis bullosa, and ichthyosis; infectious skin diseases including dermatomycosis, blastomycosis, candidiasis, chromoblastomycosis, maduramycosis, paracoccidioidomycosis, sporotrichosis, and tinea; cervicofacial actinomycosis, bacilliary angiomatosisbacterial skin diseases including angiomatosis, ecthyma, erysipelas, erythema chronica migrans, erythrasma, granuloma inguinale, hidradenitis suppurativa, maduramycosis, paronychia, pinta, rhinosclerosis, staphylococcal skin infections (furuncolosis, carbuncles, impetigo, scalded skin syndrome), cutaneous syphilis, cutaneous tuberculosis, yaws; parasitic skin diseases including larva migrans, leishmaniasis, pediculosis, and scabies; viral skin diseases including erythema infectiosum, exanthema subitum, herpes simplex, molluscum contagiosum, and warts.

[0232] In some embodiments, the amount of a composition used in any of the methods disclosed herein is typically a therapeutically effective amount. As used herein, the term "therapeutically effective amount" is synonymous with "effective amount," "therapeutically effective dose," and / or "effective dose" and refers to the amount of a composition that elicits the expected biological, cosmetic, or clinical response in a patient in need thereof. As a non-limiting example, an effective amount is an amount sufficient to achieve one or more of the clinical and / or cosmetic measures disclosed herein. The appropriate effective amount to be administered for a particular application of the disclosed methods can be determined by one of skill in the art using the guidance provided herein. For example, the effective amount can be estimated from any and all in vitro and in vivo assays described herein. Those skilled in the art will recognize that an individual's condition can be monitored throughout the course of treatment and the effective amount of the compositions disclosed herein administered can be adjusted accordingly.

[0233] In some embodiments, the amount of the composition administered is, but is not limited to, at least 0.001 g, or at least 0.002 g, or at least 0.003 g, or at least 0.004 g, or at least 0.005 g, or at least 0.006 g, or at least 0.007 g, or at least 0.008 g, or at least 0.009 g, or at least 0.01 g, or at least 0.02 g, or at least 0.03 g, or at least 0.04 g, or at least 0.05 g, or at least 0.06 g, or at least 0.07 g, or at least 0.08 g, or at least 0.09 g, or at least 0.1 g, or at least 0.2 g, or at least 0.3 g, or at least 0.4 g, or at least 0.5 g, or at least 0.6 g, or at least 0.7 g, or at least or at least 0.8g, or at least 0.9g, or at least 1g, or at least 2g, or at least 3g, or at least 4g, or at least 5g, or at least 6g, or at least 7g, or at least 8g, or at least 9g, or at least 10g, or at least 11g, or at least 12g, or at least 13g, or at least 14g, or at least 15g, or at least 20g, or at least 25g, or at least 30g, or at least 35g, or at least 40g, or at least 45g, or at least 50g, or at least 55g, or at least 60g, or at least 65g, or at least 70g, or at least 75g, or at least 80g, or at least 85g, or at least 90g, or at least 95g, or at least 100g.

[0234] In some embodiments, the amount of the composition administered is, but is not limited to, up to 0.001g, or up to 0.002g, or up to 0.003g, or up to 0.004g, or up to 0.005g, or up to 0.006g, or up to 0.007g, or up to 0.008g, or up to 0.009g, or up to 0.01g, or up to 0.02g, or up to 0.03g, or up to 0.04g, or up to 0.05g, or up to 0.06g, or up to 0.07g, or up to 0.08g, or up to 0.09g, or up to 0.1g, or up to 0.2g, or up to 0.3g, or up to 0.4g, or up to 0.5g, or up to 0. 6g, or up to 0.7g, or up to 0.8g, or up to 0.9g, or up to 1g, or up to 2g, or up to 3g, or up to 4g, or up to 5g, or up to 6g, or up to 7g, or up to 8g, or up to 9g, or up to 10g, or up to 11g, or up to 12g, or up to 13g, or up to 14g, or up to 15g, or up to 20g, or up to 25g, or up to 30g, or up to 35g, or up to 40g, or up to 45g, or up to 50g, or up to 55g, or up to 60g, or up to 65g, or up to 70g, or up to 75g, or up to 80g, or up to 85g, or up to 90g, or up to 95g, or up to 100g.

[0235] In some embodiments, the amount of the composition administered is, but is not limited to, about 0.001 g, or about 0.002 g, or about 0.003 g, or about 0.004 g, or about 0.005 g, or about 0.006 g, or about 0.007 g, or about 0.008 g, or about 0.009 g, or about 0.01 g, or about 0.02 g, or about 0.03 g, or about 0.04 g, or about 0.05 g, or about 0.06 g, or about 0.07 g, or about 0.08 g, or about 0.09 g, or about 0.1 g, or about 0.2 g, or about 0.3 g, or about 0.4 g, or about 0.5 g, or about 0.6g, or about 0.7g, or about 0.8g, or about 0.9g, or about 1g, or about 2g, or about 3g, or about 4g, or about 5g, or about 6g, or about 7g, or about 8g, or about 9g, or about 10g, or about 11g, or about 12g, or about 13g, or about 14g, or about 15g, or about 20g, or about 25g, or about 30g, or about 35g, or about 40g, or about 45g, or about 50g, or about 55g, or about 60g, or about 65g, or about 70g, or about 75g, or about 80g, or about 85g, or about 90g, or about 95g, or about 100g.

[0236] In some embodiments, the amount of the composition administered is, but is not limited to, 0.001 g to 0.01 g, or 0.01 g to 0.1 g, or 0.1 g to 1 g, or 1 g to 10 g, or 10 g to 20 g, or 20 g to 30 g, or 30 g to 40 g, or 40 g to 50 g, or 50 g to 60 g, or 60 g to 70 g, or 70 g to 80 g, or 80 g to 90 g, or 90 g to 100 g.

[0237] In some embodiments, the volume of the composition administered is, but is not limited to, at least 0.01 mL, or at least 0.02 mL, or at least 0.03 mL, or at least 0.04 mL, or at least 0.05 mL, or at least 0.06 mL, or at least 0.07 mL, or at least 0.08 mL, or at least 0.09 mL, or at least 0.10 mL, or at least 0.15 mL, or at least 0.20 mL, or at least 0.25 mL, or at least 0.30 mL, or at least 0.35 mL, or at least 0.40 mL, or at least 0.45 mL, or at least 0.50 mL, or at least 0.55 mL, or at least 0.60 mL, or at least 0.65 mL, or at least 0.70 mL, or at least 0.75 mL, or at least 0.80 mL, or at least 0.85 mL, or at least 0.90 mL, or at least 0.95mL, or at least 1mL, or at least 2mL, or at least 3mL, or at least 4mL, or at least 5mL, or at least 6mL, or at least 7mL, or at least 8mL, or at least 9mL, or at least 10mL, or at least 15mL, or at least 20mL, or at least 25mL, or at least 30mL, or at least 35mL, or at least 40mL, or at least 45mL, or at least 50mL, or at least 55mL, or at least 60mL, or at least 65mL, or at least 70mL, or at least 75mL, or at least 80mL, or at least 85mL, or at least 90mL, or at least 95mL, or at least 100mL, or at least 110mL, or at least 120mL, or at least 130mL, or at least 140mL, or at least 150mL, or at least 160mL, or at least 170mL, or at least 180mL, or at least 190mL, or at least 200mL, or at least 210mL, or at least 220mL, or at least 230mL, or at least 240mL, or at least 250mL, or at least 260mL, or at least 270mL, or at least 280mL, or at least 290mL, or at least 300mL, or at least 325, 350mL, or at least 375mL, or at least 400mL, or at least 425mL, or at least 450mL, or at least 475mL, or at least 500mL, or at least 525mL, or at least 550mL, Or at least 575 mL, or at least 600 mL, or at least 625 mL, or at least 650 mL, or at least 675 mL, or at least 700 mL, or at least 725 mL, or at least 750 mL, or at least 775 mL, or at least 800 mL, or at least 825 mL, or at least 850 mL, or at least 875 mL, or at least 900 mL, or at least 925 mL, or at least 950 mL, or at least 975 mL, or at least 1000 mL.

[0238] In some embodiments, the volume of the composition administered can be, but is not limited to, up to 0.01 mL, or up to 0.02 mL, or up to 0.03 mL, or up to 0.04 mL, or up to 0.05 mL, or up to 0.06 mL, or up to 0.07 mL, or up to 0.08 mL, or up to 0.09 mL, or up to 0.10 mL, or up to 0.15 mL, or up to 0.20 mL, or up to 0.25 mL, or up to 0.30 mL, or up to 0.35 mL, or up to 0.40 mL, or up to 0.45 mL, or up to 0.50 mL, or up to 0.55 mL, or up to 0.60 mL, or up to 0.65 mL, or up to 0.70 mL, or up to 0.75 mL, or up to 0.80 mL, or up to 0.85 mL, or up to 0.90 mL, or up to 0.95mL, or up to 1mL, or up to 2mL, or up to 3mL, or up to 4mL, or up to 5mL, or up to 6mL, or up to 7mL, or up to 8mL, or up to 9mL, or up to 10mL, or up to 15mL, or up to 20mL, or up to 25mL, or up to 30mL, or up to 35mL, or up to 40mL, or up to 45mL, or up to 50mL, or up to 55mL, or up to 60mL, or up to 65mL, or up to 70mL, or up to 75mL, or up to 80mL, or up to 85mL, or up to 90mL, or up to 95mL, or up to 100mL, or up to 110mL, or up to 120mL, or up to 130mL, or up to 140mL, or up to 150mL, or up to 160mL, or up to 170mL, or up to 180mL, or up to 190mL, or up to 200mL, or up to 210mL, or up to 220mL, or up to 230mL, or up to 240mL, or up to 250mL, or up to 260mL, or up to 270mL, or up to 280mL, or up to 290mL, or up to 300mL, or up to 325, 350mL, or up to 375mL, or up to 400mL, or up to 425mL, or up to 450mL, or up to 475mL, or up to 500mL, or up to 525mL, or up to 550mL, or Up to 575mL, or up to 600mL, or up to 625mL, or up to 650mL, or up to 675mL, or up to 700mL, or up to 725mL, or up to 750mL, or up to 775mL, or up to 800mL, or up to 825mL, or up to 850mL, or up to 875mL, or up to 900mL, or up to 925mL, or up to 950mL, or up to 975mL, or up to 1000mL.

[0239] In some embodiments, the volume of the composition administered is, but is not limited to, about 0.01 mL, or about 0.02 mL, or about 0.03 mL, or about 0.04 mL, or about 0.05 mL, or about 0.06 mL, or about 0.07 mL, or about 0.08 mL, or about 0.09 mL, or about 0.10 mL, or about 0.15 mL, or about 0.20 mL, or about 0.25 mL, or about 0.30 mL, or about 0.35 mL, or about 0.40 mL, or about 0.45 mL, or about 0.50 mL, or about 0.55 mL, or about 0.60 mL, or about 0.65 mL, or about 0. 70 mL, or about 0.75 mL, or about 0.80 mL, or about 0.85 mL, or about 0.90 mL, or about 0.95 mL, or about 1 mL, or about 2 mL, or about 3 mL, or about 4 mL, or about 5 mL, or about 6 mL, or about 7 mL, or about 8 mL, or about 9 mL, or about 10 mL, or about 11 mL, or about 12 mL, or about 13 mL, or about 14 mL, or about 15 mL, or about 16 mL, or about 17 mL, or about 18 mL, or about 19 mL, or about 20 mL, or about 21 mL, or about 22 mL, or about 23 mL, or about 24 mL, or about 25 mL, or about 2 6mL, or about 27mL, or about 28mL, or about 30mL, or about 35mL, or about 36mL, or about 37mL, or about 38mL, or about 39mL, or about 40mL, or about 41mL, or about 42mL, or about 43mL, or about 44mL, or about 45mL, or about 46mL, or about 47mL, or about 48mL, or about 49mL, or about 50mL, or about 51mL, or about 52mL, or about 53mL, or about 54mL, or about 55mL, or about 56mL, or about 57mL, or about 58mL, or about 59mL, or about 60mL, or about 61mL, or about 62m L, or about 63 mL, or about 64 mL, or about 65 mL, or about 66 mL, or about 67 mL, or about 68 mL, or about 69 mL, or about 70 mL, or about 71 mL, or about 72 mL, or about 73 mL, or about 74 mL, or about 75 mL, or about 76 mL, or about 77 mL, or about 78 mL, or about 79 mL, or about 80 mL, or about 81 mL, or about 82 mL, or about 83 mL, or about 84 mL, or about 85 mL, or about 86 mL, or about 87 mL, or about 88 mL, or about 89 mL, or about 90 mL, or about 91 mL, or about 92 mL, or about 93 mL,or about 94 mL, or about 95 mL, or about 96 mL, or about 97 mL, or about 98 mL, or about 99 mL, or about 100 mL, or about 110 mL, or about 120 mL, or about 130 mL, or about 140 mL, or about 150 mL, or about 160 mL, or about 170 mL, or about 180 mL, or about 190 mL, or about 200 mL, or about 210 mL, or about 220 mL, or about 230 mL, or about 240 mL, or about 250 mL, or about 260 mL, or about 270 mL, or about 280 mL L, or about 290 mL, or about 300 mL, or about 310 mL, or about 320 mL, or about 330 mL, or about 340 mL, or about 350 mL, or about 360 mL, or about 370 mL, or about 380 mL, or about 390 mL, or about 400 mL, or about 410 mL, or about 420 mL, or about 430 mL, or about 440 mL, or about 450 mL, or about 460 mL, or about 470 mL, or about 480 mL, or about 490 mL, or about 500 mL, or about 510 mL, or about 520 mL, or about 530 mL, or about 540 mL, or about 550 mL, or about 560 mL, or about 570 mL, or about 580 mL, or about 590 mL, or about 600 mL, or about 610 mL, or about 620 mL, or about 630 mL, or about 640 mL, or about 650 mL, or about 660 mL, or about 670 mL, or about 680 mL, or about 690 mL, or about 700 mL, or about 710 mL, or about 720 mL, or about 730 mL, or about 740 mL, or about 750 mL, or about 760 mL, or About 770 mL, or about 780 mL, or about 790 mL, or about 800 mL, or about 810 mL, or about 820 mL, or about 830 mL, or about 840 mL, or about 850 mL, or about 860 mL, or about 870 mL, or about 880 mL, or about 890 mL, or about 900 mL, or about 910 mL, or about 920 mL, or about 930 mL, or about 940 mL, or about 950 mL, or about 960 mL, or about 970 mL, or about 980 mL, or about 990 mL, or about 1000 mL.

[0240] In some embodiments, the volume of the composition administered may be, but is not limited to, 0.01 mL to 0.10 mL, or 0.10 mL to 1 mL, or 1 mL to 10 mL, or 10 mL to 100 mL, or 50 mL to 100 mL, or 100 mL to 150 mL, or 150 mL to 200 mL, or 200 mL to 250 mL, or 250 mL to 300 mL, or 300 mL to 350 mL, or 350 mL to 400 mL, or 400 mL to 450 mL, or 450 mL to 500 mL, or 500 mL to 550 mL, or 550 mL to 600 mL, or 600 mL. L to 650mL, or 650mL to 700mL, or 700mL to 750mL, or 750mL to 800mL, or 800mL to 850mL, or 850mL to 900mL, or 900mL to 950mL, or 950mL to 1000mL, or 1mL to 25mL, or 1mL to 50mL, or 1mL to 75mL, or 1mL to 100mL, or 10mL to 25mL, or 10mL to 50mL, or 10mL to 75mL, or 100mL to 250mL, or 100mL to 500mL, or 100mL to 750mL, or 100mL to 1000mL.

[0241] Silk fibroin protein fragments as collagen-stimulating compositions Raw silk from the silkworm, Bombyx mori, is composed of two major proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin is a fibrous protein with a semi-crystalline structure that provides rigidity and strength. As used herein, the term "silk fibroin" refers to the fiber of Bombyx mori cocoons, which has a weight-average molecular weight of approximately 370,000 Da. Crude silkworm fiber is composed of double strands of fibroin. The adhesive substance that holds these double fibers together is sericin. Silk fibroin is composed of heavy chains (H chains) with a weight-average molecular weight of approximately 350,000 Da and light chains (L chains) with a weight-average molecular weight of approximately 25,000 Da.

[0242] Conversion of these fibrillar silk fibroins into water-soluble silk fibroin protein fragments requires the addition of concentrated heavy salts (e.g., 8-10 M lithium bromide), which interfere with the inter- and intramolecular ionic and hydrogen bonds that would otherwise render the fibroin protein insoluble in water. Methods for making silk fibroin or silk 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.

[0243] Provided herein is a silk protein fragment (SPF) mixed solution obtained by dissolving raw unrefined, partially refined, or refined silkworm fiber in a neutral lithium bromide salt. The raw silkworm fiber is treated to remove sericin, and the desired weight average molecular weight (M W The silk protein fragments are processed under selected temperatures and other conditions to achieve a desired concentration (P / D) and polydispersity (PD). The selected process parameters can be modified to achieve different final silk protein fragment properties depending on the intended use. The resulting final fragment solution is silk protein fragments and water with PPM to undetectable levels of process contaminants, levels acceptable for the pharmaceutical, medical, and consumer cosmetic markets. The concentration, size, and polydispersity of the silk protein fragments in the solution can be further modified depending on the desired application and performance requirements.

[0244] In one embodiment, a silk protein fragment solution useful for application in collagen stimulating compositions and methods of making and using the same is prepared according to the following steps: forming small pieces of silk cocoons from Bombyx mori silkworms; extracting the pieces in aqueous NaCO solution at about 100°C for about 60 minutes (the volume of water is equal to about 0.4 x the raw silk weight, and the amount of NaCO is about 0.848 x the weight of the pieces) to form a silk fibroin extract; rinsing the silk fibroin extract three times at about 60°C for about 20 minutes per rinse in a volume of rinse water (the rinse water for each cycle is equal to about 0.2 L x the weight of the pieces); removing excess water from the silk fibroin extract; drying the silk fibroin extract. the steps of drying the silk fibroin extract, dissolving the dried silk fibroin extract in a LiBr solution (where the LiBr solution is first heated to and maintained at approximately 100°C to create a silk and LiBr solution), placing the silk and LiBr solution in a drying oven at approximately 100°C for approximately 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into a mixture with the desired molecular weight and polydispersity, filtering the solution to remove any remaining debris from the silkworm, diluting the solution with water to result in 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 utilized to purify the silk solution to achieve the final desired silk-to-water ratio. TFF can then be used to further concentrate the silk solution to a concentration of 2.0 wt% silk in water.

[0245] While not wishing to be bound by any particular theory, varying the extraction (i.e., time and temperature), LiBr (i.e., temperature of the LiBr solution added to the silk fibroin extract or vice versa), and dissolution (i.e., time and temperature) parameters resulted in solvent and silk solutions with different viscosities, uniformities, and colors. Also, without wishing to be bound by any particular theory, increasing the temperature for extraction, extending the extraction time, using higher temperature LiBr solutions when dissolving the silk (e.g., in an oven as shown herein or with an alternative heat source), both initially and over a longer period of time, all resulted in lower viscosity, more uniform solvent and silk solutions.

[0246] In one embodiment, a solution of silk fibroin-based protein fragments having a weight average selected from about 6 kDa to about 17 kDa is prepared according to the following steps: degumming the silk source by adding the silk source to an aqueous solution of boiling (100°C) sodium carbonate for a treatment time of about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract containing non-detectable levels of sericin; draining the solution from the silk fibroin extract; and heating the silk fibroin extract to a temperature ranging from about 60°C to about 140°C using lithium bromide. The method includes dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature upon placement in the lithium bromide solution; maintaining the silk fibroin-lithium bromide solution in an oven having a temperature of about 140°C for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk protein fragments, wherein the aqueous solution comprises fragments having a weight-average molecular weight selected from about 6 kDa to about 17 kDa, and the aqueous solution of silk fibroin-based protein fragments comprises a polydispersity of about 1.5 to about 3.0. The method may further include drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin-based protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin-based protein fragments may contain less than 100 ppm of sodium carbonate residue as measured using a high-performance liquid chromatography sodium carbonate assay. The aqueous solution of silk fibroin-based protein fragments may be freeze-dried.

[0247] In one embodiment, a solution of silk fibroin-based protein fragments having a weight average molecular weight selected from about 17 kDa to about 39 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to about 60 minutes to effect degumming, removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin, draining the solution from the silk fibroin extract, dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature ranging between about 80°C to about 140°C, and then dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature ranging between about 60°C to about 100°C. maintaining the silk fibroin-lithium bromide solution in an oven at a temperature for a period of at least 1 hour, removing the lithium bromide from the silk fibroin extract, and producing an aqueous solution of silk fibroin-based protein fragments, wherein the aqueous solution of silk fibroin-based protein fragments comprises about 10 ppm to 300 ppm of lithium bromide residue, the aqueous solution of silk protein fragments comprises about 10 ppm to 100 ppm of sodium carbonate residue, the aqueous solution of silk fibroin-based protein fragments comprises fragments having a weight-average molecular weight selected from about 17 kDa to about 39 kDa, and the aqueous solution of silk fibroin-based protein fragments comprises a polydispersity of about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin-based protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. An aqueous solution of the silk fibroin-based protein fragment may contain less than 100 ppm residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay.

[0248] In one embodiment, a solution of silk fibroin-based protein fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa is prepared according to the following steps: adding a silk source to a boiling (100°C) aqueous solution of sodium carbonate for a treatment time of about 30 minutes to effect degumming; removing sericin from the solution to produce a silk fibroin extract containing undetectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a lithium bromide solution having a starting temperature ranging from about 80°C to about 140°C upon placing the silk fibroin extract in the lithium bromide solution. maintaining the silk fibroin-lithium bromide solution in an oven having a temperature ranging from about 60°C to about 100°C for a period of at least 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk fibroin-based protein fragments, wherein the aqueous solution of silk fibroin-based protein fragments comprises about 10 ppm to 300 ppm of lithium bromide residue, about 10 ppm to 100 ppm of sodium carbonate residue, fragments having a weight average molecular weight selected from about 39 kDa to about 80 kDa, and the aqueous solution of silk fibroin-based protein fragments comprises a polydispersity of about 1.5 to about 3.0. The method may further comprise drying the silk fibroin extract prior to the dissolving step. The aqueous solution of silk fibroin-based protein fragments may contain less than 300 ppm of lithium bromide residue as measured using a high-performance liquid chromatography lithium bromide assay. An aqueous solution of the silk fibroin-based protein fragment may contain less than 100 ppm residual sodium carbonate as measured using a high performance liquid chromatography sodium carbonate assay.

[0249] In one embodiment, the silk fibroin-based protein fragments in solution are substantially devoid of sericin and have a weight average molecular weight selected from about 6 kDa to about 17 kDa and a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the silk fibroin-based protein fragments in solution are substantially devoid of sericin and have a weight average molecular weight selected from about 17 kDa to about 39 kDa and a polydispersity selected from about 1.5 to about 3.0. In one embodiment, the silk fibroin-based protein fragments in solution are substantially devoid of sericin and have a weight average molecular weight selected from about 39 kDa to about 80 kDa and a polydispersity selected from about 1.5 to about 3.0.

[0250] As used herein, the terms "substantially free of sericin" or "substantially devoid of sericin" refer to silk fibers from which the majority of the sericin protein has been removed. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 10.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 9.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 8.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 7.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 6.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.01% to about 5.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.05% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.1% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 0.5% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 1.0% to about 4.0% by weight of sericin. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 1.5% to about 4.0% by weight of sericin, and in one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 2.0% to about 4.0% by weight of sericin.In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having about 2.5% to about 4.0% sericin by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content of about 0.01% to about 0.1% by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.1% by weight. In one embodiment, silk fibroin substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.05% by weight. In one embodiment, when a silk source is added to a boiling (100°C) aqueous solut...

Claims

1. i) stimulation or modulation of collagen expression, and / or ii) stimulation or modulation of claudin-1 expression, and / or iii) stimulation or modulation of expression of a gene selected from ADA2, CD209, CD30E, CR1, F13A1, KL, PDGFRA and TLR4; 1. A composition for treating or preventing a disorder, disease, or condition alleviated by 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 15 kDa to about 20 kDa, about 17 kDa Da ~ about 39kDa, about 14kDa - about 30kDa, about 20kDa - about 25kDa, about 25kDa - about 30kDa, about 30kDa - about 35kDa, about 35kD and a polydispersity of 1 to about 5; A composition characterized in that the concentration of silk fibroin fragments in the composition is from about 0.001% w / v to about 10% w / v.

2. The composition described in claim 1, further comprising 0 to 500 ppm of lithium bromide.

3. The composition of claim 1, further comprising 0 to 500 ppm sodium carbonate.

4. 10. The composition of claim 1, wherein the silk fibroin fragments have a polydispersity of 1 to about 1.

5.

5. 10. The composition of claim 1, wherein the silk fibroin fragments have a polydispersity of about 1.5 to about 2.

0.

6. 10. The composition of claim 1, wherein the silk fibroin fragments have a polydispersity of about 1.5 to about 3.

0.

7. 10. The composition of claim 1, wherein the silk fibroin fragments have a polydispersity of about 2.0 to about 2.

5.

8. 10. The composition of claim 1, wherein the silk fibroin fragments have a polydispersity of about 2.5 to about 3.

0.

9. 9. The composition of any one of claims 1 to 8, wherein the silk fibroin fragments do not spontaneously or gradually gel or undergo a visible change in color or turbidity when in aqueous solution for at least 10 days prior to formulation into the composition.

10. The composition of any one of claims 1 to 8, wherein the silk fibroin fragments are present in the composition at about 0.001% w / v to about 1% w / v.

11. The composition of any one of claims 1 to 8, wherein the silk fibroin fragments are present in the composition at about 0.01% w / v to about 1% w / v.

12. The composition of any one of claims 1 to 8, wherein the silk fibroin fragments are present in the composition at about 0.025% w / v to about 1% w / v.

13. The composition of any one of claims 1 to 8, wherein the silk fibroin fragments are present in the composition at about 0.05% w / v to about 0.7% w / v.

14. A composition according to any one of claims 1 to 8, formulated as an injectable composition or a topical composition.

15. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.

16. The composition of claim 15 , wherein the pharmaceutically acceptable carrier comprises an aqueous phase.

17. 17. The composition of claim 15 or 16, wherein the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in-oil emulsion.

18. The composition of claim 1 formulated for administration to an epithelial surface.

19. 20. The composition of claim 18, wherein the epithelial surface is a superficial epidermal region, a stratum corneum, an ocular surface, or an intestinal surface.

20. A composition described in any one of claims 1 to 8, formulated to reduce transepidermal water loss.

21. A composition according to any one of claims 1 to 8 formulated as a barrier preparation.

22. A composition described in any one of claims 1 to 8 formulated as a wound closure preparation.

23. The composition of any one of claims 1 to 8, formulated to prevent or improve wrinkles in a subject, prevent or improve age spots in a subject, prevent or improve dry skin in a subject, or prevent or improve uneven skin tone in a subject.

24. A composition according to any one of claims 1 to 8, formulated to prevent or improve sagging skin in a subject, prevent or improve skin aging in a subject, prevent or improve reduced tensile strength of skin in a subject, prevent or improve photodamaged skin in a subject, or prevent or improve stretch marks in a subject.

25. 9. The composition of any one of claims 1 to 8, wherein the disease or condition comprises wrinkles, age spots, dry skin, uneven skin tone, sagging skin, aging skin, reduced skin tensile strength, photodamaged skin, or stretch marks.

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