Modified silk fibroin 27p and KDF-β and medical uses thereof

Abstract

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

Description

[0001] MODIFIED SILK FIBROIN 27P AND KDF-P AND MEDICAL USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 749,319 filed January' 24, 2025, and U.S. Provisional Application No.

[0004] 63 / 749.327 filed January 24. 2025.

[0005] REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0006] This application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety7. The XML file, named 032272-5042 -WO.xml, was created on January 26, 2026 and is 69.6 kilobytes in size.

[0007] FIELD

[0008] This disclosure is in the field of silk fibroin compositions and methods for use thereof.

[0009] BACKGROUND

[0010] Silk is a natural polymer produced by a variety' of insects and spiders. Silk comprises a filament core protein, silk fibroin, and a glue-like coating consisting of a nonfilamentous protein, sericin.

[0011] There exists a need for stable silk fibroin peptide solution suitable for medical uses, e.g., collagen stimulation, by topical or parenteral administration.

[0012] SUMMARY

[0013] The disclosure provides peptide compositions, e g., silk fibroin derived peptide compositions.

[0014] Embodiments of the present disclosure provide a method of treatment or prevention of a disorder, disease, or condition alleviated by stimulating or modulating

[0015] 1

[0016] DBl / 165497080.5collagen expression in a subject in need thereof; and / or stimulating or modulating claudin-1 expression in a subject in need thereof; and / or stimulating or modulating one more anti-inflammatory genes in a subject in need thereof, the method comprising administering to the subject a composition comprising fibroin heavy chain protein fragments, fibroin light chain protein fragments, and fibrohexamerin protein fragments, wherein one or more fibroin heavy chain protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 49 selected from Q58E, M64X, N68D, N70D, N77D, M80X, N93D, M103X. Q125E, N132D. Q139E, Q275E, N4191D, Q5216E, and N5262D; one or more fibroin light chain protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 50 selected fromN23D, Q24E, N28D, M69X, N105D, N108D, N118D, N136D, N138D, Q149E. N186D, N200D, Q202E. N204D, N240D. N248D, and Q255E; and one or more fibrohexamerin protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 51 selected from Q62E, N93D, M120X, N149D, N172D, N174D, and N202D, wherein X is methionine oxide. In some embodiments, the composition is formulated as an injectable composition or as a topical composition further comprising a pharmaceutically acceptable carrier, wherein one or more of the following apply: the pharmaceutically acceptable carrier comprises an aqueous phase; the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in-oil emulsion; the pharmaceutically acceptable carrier comprises or is formulated as one or more of a cosmeceutical, a suspension, an emulsion, a powder, a solution, a dispersion, or an elixir; the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, a jelly, a cream, a lotion, a foam, a slurry', an ointment, an oil, a paste, a suppository, a spray, a semisolid composition, a solid composition, a stick, or a mousse; the pharmaceutically acceptable carrier comprises one or more of aliphatic oil, a fatty alcohol, a fatty acid, a glyceride, an acylglycerol, and a phospholipid; the pharmaceutically acceptable carrier comprises one or more of a monoglyceride, a diglyceride, or a triglyceride; and / or the pharmaceutically

[0017] 2

[0018] DBl / 165497080.5acceptable carrier comprises one or more of a hydrocarbon oil, a fatty acid, a fatty oil, a fatty acid ester, or a cationic quaternary ammonium salt. In some embodiments, the composition is formulated for one or more of the following: for administration to an epithelial surface selected from a superficial epidermal area, a stratum comeum, an eye surface, or an intestinal surface; for reducing trans-epidermal water loss; as a barrier formulation; as a wound-closure formulation; for preventing or reversing wrinkles in the subject, preventing or reversing age spots in the subject, preventing or reversing dry skin in the subject, or preventing or reversing uneven skin tone in the subject; for preventing or reversing skin sagging in the subject, preventing or reversing skin aging in the subject, preventing or reversing reduced skin tensile strength in the subject, preventing or reversing photodamaged skin in the subject, or preventing or reversing striae distensae (stretch marks) in the subject. In some embodiments, the disorder, disease, or condition comprises wrinkles, age spots, dry skin, uneven skin tone, skin sagging, skin aging, reduced skin tensile strength, photodamaged skin, or striae distensae (stretch marks). In some embodiments, the disease, disorder or condition is selected from Atopic Dermatitis (Eczema), Psoriasis (e.g., plaque psoriasis), Contact Dermatitis, rosacea, acne (e.g., cystic acne), Ichthyosis (e.g., ichthyosis vulgaris), inflammation, Netherton Syndrome (a genetic disorder that lead to chronic skin inflammation and increased susceptibility to an infection), Xerosis (Dry Skin), Aging Skin, impaired wound healing, Diabetic Dermopathy, Pruritus, Scleroderma, Multiple Sclerosis (MS), Bacterial and fungal Infection, abnormal scarring condition, melanoma, dysbiosis, autoimmune condition like lupus, Systemic Lupus Erythematosus, and rheumatoid arthritis. In some embodiments, the disorder, disease, or condition is an inflammatory disease, disorder or condition selected from Rheumatoid Arthritis, Inflammatory Bowel Disease, Systemic Lupus Erythematosus, Multiple Sclerosis (MS), Psoriasis. Asthma, allergic rhinitis, asthma, food allergy, and another autoimmune condition. In some embodiments, the disease, disorder or condition is selected from inflammatory bowel disease (IBD), Intestinal Permeability, Crohn’s disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), NASH, cirrhosis, metabolic disorders, and 3

[0019] DBl / 165497080.5another disease, a disorder or condition involving regulation of a gut epithelial cell. In some embodiments, the disease, disorder or condition is a neurodegenerative condition selected from Alzheimer’s Disease (AD), Multiple Sclerosis (MS), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the disease, disorder or condition is a metabolic condition selected from obesity and type 2 Diabetes, and non-alcoholic fatty liver disease (NAFLD). In some embodiments, the disease, disorder or condition is a cancer. In some embodiments, the disease, disorder or condition is associated with dysregulation in a gut barrier and / or in a tight junction of the gut; the disease, disorder, or condition is alleviated by decreasing the secretion of TNF-a and IL-6, and / or increasing secretion of IL-10; the disease, disorder, or condition is alleviated by modulating Annexin A2’s role in tumor growth, immune function, neuronal protection, and / or metabolic processes; the disorder, disease, or condition is alleviated by stimulating or modulating a skin barrier-specific protein in a subject in need thereof, wherein the skin barrier-specific protein comprises at least one of desmoplakin (DSP), cystatin A (CSTA), tumor-associated calcium signal transducer 2 (TACSTD2), biglycan (BGN), decorin (DCN) and / or epiplakin 1 (EPPK1), wherein at least one of desmoplakin (DSP), cystatin A (CSTA) and / or tumor-associated calcium signal transducer 2 (TACSTD2) is upregulated post-administration, and / or wherein at least one of biglycan (BGN), decorin (DCN) and / or epiplakin 1 (EPPK1) is down regulated postadministration, and / or wherein at least one of the skin barrier-specific protein is a tight junction protein; and / or administering the composition induces secretion of pro- CollAl, Col3A, and / or a-SMA. In some embodiments, the protein fragments have a weight average molecular weight selected from between about 1 kDa to about 250 kDa, In some embodiments, the protein fragments have a w eight average molecular weight selected from between about 1 kDa and about 20 kDa. from between about 20 kDa and about 40 kDa, from between about 40 kDa and about 60 kDa, from between about 60 kDa and about 80 kDa, from between about 80 kDa and about 100 kDa, from betw een about 100 kDa and about 120 kDa, from betw een about 120 kDa and about 140 kDa. or from between about 140 kDa and about 160 kDa, from between 4

[0020] DBl / 165497080.5about 160 kDa and about 180 kDa, from between about 180 kDa and about 200 kDa, or from between about 200 kDa and about 250 kDa. In some embodiments, the protein fragments have a weight average molecular weight selected from between about 10 kDa and about 20 kDa, from between about 20 kDa and about 40 kDa, from between about 40 kDa and about 60 kDa, from between about 60 kDa and about 80 kDa, from between about 80 kDa and about 100 kDa, from between about 100 kDa and about 120 kDa, from between about 120 kDa and about 140 kDa, from between about 140 kDa and about 160 kDa, or from between about 160 kDa and about 180 kDa. In some embodiments, the protein fragments have a weight average molecular weight selected from between about 21 kDa and about 41 kDa. In some embodiments, the protein fragments have a weight average molecular weight selected from between about 12 kDa and about 20 kDa. In some embodiments, the protein fragments have a poly dispersity between 1 and 3.4. In some embodiments, the protein fragments have a poly dispersity between 1 and about 1.5, between about 1.5 and about 2.0, between about 2.0 and about 2.5, or between about 2.5 and about 3.0. In some embodiments, the protein fragments have a polydispersity between 1 and about 1.7. In some embodiments, the protein fragments have a poly dispersity between 1 and about 1.1, between about 1.1 and about 1.2, between about 1.2 and about 1.3, between about 1.3 and about 1.4, or between about 1.4 and about 1.5. In some embodiments, each modification is independently ranging in the composition between about 1% to about 99%. In some embodiments, a percent modification is defined as (number of protein fragments comprising a modification at a specific position, divided by the total number of protein fragments which include the specific position, whether comprising a modification or not) x 100. In some embodiments, a percentage of amino acid modification in the fibroin heavy chain protein fragments at Q58E is from about 1% to about 10%, at M64X is from about 20% to about 90%, at N68D is from about 30% to 100%, at N70D is from about 20% to 100%, at N77D is from about 80% to 100%, at M80X is from about 10% to about 50%, at N93D is from about 1% to about 40%, at M103X is from about 10% to about 40%, at Q125E is from about 1% to about 30%, at N132D is from about 10% to about 90%, at Q139E is from about 1% to about 5

[0021] DBl / 165497080.530%, at Q275E is from about 1% to about 10%, at N4191D is from about 10% to 100%, at Q5216E is from about 1% to about 20%, and at atN5262D is from about 1% to about 50%. In some embodiments, a percentage of amino acid modification in the fibroin light chain protein fragments at N23D is from about 70% to 100%, at Q24E is from about 1% to about 10%, at N28D is from about 1% to about 40%, at M69X is from about 20X% to about 80%, at N105D is from about 40% to about 80%, at N108D is from about 10% to about 50%, at N118D is from about 20% to about 60%, at N136D is from about 10% to about 60%, at N138D is from about 1% to about 10%. at Q149E is from about 1% to about 20%. at N186D is from about 1% to about 40%, at N200D is from about 40% to 100%, at Q202E is from about 1 % to about 50%, at N204D is from about 40% to 100%, at N240D is from about 20% to 100%, at N248D is from about 10% to about 80%, and at Q255E is from about 1% to about 10%. In some embodiments, a percentage of amino acid modification in the fibrohexamerin protein fragments at Q62E is from about 1% to about 90%, at N93D is from about 1% to about 15%, at M120X is from about 1% to about 70%, at N149D is from about 20% to about 70%, at N172D is from about 40% to about 80%, at N174D is from about 1% to about 80%, and at N202D is from about 1% to about 30%. In some embodiments, the composition comprises a fibroin fragment subpopulation having an isoelectric point selected from between about 4.4 to about 9.6; the composition comprises a fibroin fragment subpopulation having a selfassembly rate factor selected from between 0 to about 5; the composition comprises a fibroin fragment subpopulation having a self-assembly rate factor selected from between about 0.6 to about 4; the composition comprises a fibroin fragment subpopulation each having a self-assembly factor selected from between about 0.5 to about 0.9; the composition comprises a fibroin fragment subpopulation having a self assembly rate factor selected from between about 5 to about 11; the composition comprises a fibroin fragment subpopulation having an Amax selected from between 0 to about 0.7; the composition comprises a fibroin fragment subpopulation having an Amax selected from between about 0.5 to about 0.8; the composition comprises a fibroin fragment subpopulation having a t0.5(min) selected from between 0 to about 6

[0022] DBl / 165497080.51500; and / or the composition comprises a fibroin fragment subpopulation having a t0.5(min) selected from between 0 to about 1000. In some embodiments, the composition comprises a formulation comprising water and one or more of: sodium benzoate (0.50-1.50% w / w), aspen bark (0.50-3.50% w / w), glycerin (0.50-3.50% w / w), fibroin glycoside (Pl -alpha hydrogel, 0.10-1.00% w / w), medium-chain triglyceride oil (MCT, 10-35% w / w), and an acidic agent (0.10-0.50% w / w). In some embodiments, the acidic agent is citric acid (50% Solution). In some embodiments, the protein fragments concentration in the formulation is between about 1% and about 15% w / w.

[0023] BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The presently disclosed embodiments will be further explained with reference to the attached drawings. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the presently disclosed embodiments.

[0025] FIG. 1 depicts Ion Exchange Fractionation Schemes for the isolation of the populations that constitute Low and Mid Skid silk / modified polypeptide compositions. Low and Mid Skid silk / modified polypeptide compositions contains silk / modified polypeptides that are negatively, positively charged, or neutral. Using Q anion exchange chromatography (A) these populations were isolated.

[0026] FIG. 2 depicts chromatogram of Low Skid silk / modified polypeptide composition loaded in a Q-Sepharose HP column (Cytiva). The flow through contains the silk / modified polypeptides that do not get captured in the column and are the depleted in negatively charged amino acids. After the column is loaded with Low or Mid Skid silk / modified polypeptide compositions and the flow through is collected, the column is washed until the UV-280 absorbance becomes less than 200 AU. The captured negatively charged silk / modified polypeptides are eluted with high salt concentration (IM NaCl) and constitute ASH and AS22. The chromatography is performed in Tris-containing buffers but the flow through and the Q-elution were finally dialyzed in water.

[0027] 7

[0028] DBl / 165497080.5FIG. 3 depicts Analytical Size Exclusion Chromatography of Low, Mid Skid silk / modified silk compositions and their constituent AS compositions. Average molecular weight in kDa and poly dispersity measurements are shown.

[0029] FIG. 4A and FIG. 4B depict Analytical Size Exclusion Chromatography of the Low and Mid skid silk / modified peptide compositions and their components (see table 1 for more details). FIG. 4A, Molecular weight of the various Activated Silk new compositions described in this study. FIG. 4B, Poly dispersity (PDI) of the various Activated Silk new compositions described in this study. AS24 reconstitutes the average molecular weight and poly dispersity of the Low' skid silk / modified peptide composition and it consists of 50% AS12 and 50% AS22 (see Table 1 for details). AS6 reconstitutes the average molecular weight and poly dispersity of the Mid skid silk / modified peptide composition and it consists of 50% AS1 and 50% ASH (see Table 1 for details).

[0030] FIG. 5 depicts Isoelectric Focusing Electrophoresis of Low Skid silk / modified polypeptide compositions. Lanes 2, 7; Low7Skid silk different amounts loaded. Lanes 3, 5, 8, 10; AS 12 silk, different preparations different amounts loaded. Lanes 4, 6, 9, 11; AS22 silk, different preparations different amounts loaded.

[0031] FIG. 6A and FIG. 6B depict self-assembly reactions of the of the Low and Mid skid silk / modified peptide compositions and their components (see Table 1 for more details). Both graphs depict the kinetic parameters of gel formation during selfassembly of silk. On graph FIG. 1A calculation of the three self-assembly kinetic parameters is shown. t0.5, Amax and SARF.

[0032] FIGS. 7A-C depict self-assembly kinetics of the Low and Mid skid silk / modified peptide compositions and their components (see table 1 for more details). FIG. 7A, the Self-assembly Rate Factor shows how fast the self-assembly reaction proceeds once it is initiated and the self-assembly nuclei are organized. FIG.

[0033] 7B, Maximum Gel Yield shows how7dense the silk gel is after self-assembly is complete. FIG 7C, Time required for the self-assembly reaction to produce half of the maximum gel amount.

[0034] 8

[0035] DBl / 165497080.5FIG. 8 depict the Low and Mid skid silk / modified peptide compositions and their components (see Table 1 for more details). The Self Assembly Factor reflects the average propensity of silk to self- assemble and form gels.

[0036] FIG. 9 is a graph of molecular weight plotted as a function of time for solubilized fibroin in 9.3 M LiBr at 100 °C - 103 °C

[0037] FIG. 10 is a graph of molecular weight plotted as a function of time for solubilized fibroin in 9.3 M LiBr at 122 °C - 125 °C

[0038] FIG. 11 is a graph illustrating percentage of amino acid modification in silk. FIGS. 12A-12C are graphs illustrating percentage of amino acid modifications in Low and Mid Skid silk. FIG. 12A illustrates heavy chain modifications, FIG. 12B illustrates light chain modifications, and FIG. 12C illustrates fibrohexamerin modifications. N are Asparagines that become aspartic acid and Q are Glutamines that become deamidated. M corresponds to Methionies that become oxidized.

[0039] FIG. 13 A and FIG. 13B are graphs illustrating percentage of amino acid modifications in Low and Mid Skid silk produced and lyophilized. FIG. 13A illustrates heavy chain modifications and FIG. 13B illustrates light chain modifications. N are Asparagines that become aspartic acid and Q are Glutamines that become deamidated. M corresponds to Methionies that become oxidized.

[0040] FIG. 14A and FIG. 14B are graphs illustrating percentage of amino acid modifications in Low Skid silk produced in two different facilities. FIG. 14A illustrates heavy chain modifications and FIG. 14B illustrates light chain modifications. N are Asparagines that become aspartic acid and Q are Glutamines that become deamidated. M corresponds to Methionies that become oxidized.

[0041] FIGS. 15A-15D are graphs illustrating percentage of amino acid modifications in Mid silk produced in Skid and Benchtop scale. N are Asparagines that become aspartic acid and Q are Glutamines that become deamidated. M corresponds to Methionies that become oxidized.

[0042] 9

[0043] DBl / 165497080.5FIG. 16 is a schematic explanation of the method used to calculate percentage ratios of modified amino acids at specific locations along the sequence of each peptide.

[0044] FIG. 17 illustrates an Anion exchange chromatography and size exclusion chromatography scheme of the isolation of Low Skid silk / modified peptide compositions. Low Skid silk / modified polypeptide compositions is composed of a variety of peptide populations, in a wide range of sizes and charge. Using Q- Sepharose anion exchange chromatography as a first step, and HiLoad Superdex 200 size exclusion chromatography as a second purification step, distinct populations of Low Skid silk / modified polypeptide compositions were separated. The Q-Sepharose eluate was loaded onto HiLoad Superdex 200 size exclusion chromatography, which resulted in negatively charged silk compositions / modified peptides fractionated by size.

[0045] FIG. 18 A and FIG. 18B are chromatograms of the anion exchange chromatography and the following size exclusion chromatography of the eluate (Q- eluate) of Low Skid silk / modified polypeptide compositions. FIG. 18A: Anion exchange chromatography was performed with a Q-Sepharose column (Cytiva). Low Skid silk / modified peptide compositions were separated to uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk compositions (eluate - light pink background) by anion exchange chromatography. Light yellow background indicates column wash with 50 mM Tris pH=8.0 before eluting the charged peptide population. FIG. 18B: The negatively charged eluate was loaded onto the Superdex 200 column and was flowed through the column with 50 mM Tris, 200 mM CaC12, pH=8.0. When the UV-280 absorbance started to increase fractions were collected to separate the Low Skid silk / modified peptide compositions by size. The relative elution volume of silk compositions AS77 and AS81 are indicated on the chromatogram.

[0046] FIG. 19A and FIG. 19B illustrates the Analytical Size Exclusion Chromatography of Low7Skid silk / modified silk compositions and their constituent 10

[0047] DBl / 165497080.5AS compositions. FIG. 19A. Average molecular weight in kDa of Low Skid silk (LS) and AS77-AS81 are shown. FIG. 19B. Poly dispersity (PDI) measurements are shown. The numerical data is presented in Table F.

[0048] FIG. 20 is a SDS polyacrylamide gel electrophoresis of Low Skid silk / modified polypeptide compositions. Lanes are indicated by fraction number, at the order of elution from the Superdex 200 column, and their respective silk composition: fraction 6 is AS77, fraction 7 is AS78, fraction 8 is AS79, fraction 9 is AS80, and fraction 10 is AS81.

[0049] FIG. 21 A and FIG. 21B are graphs illustrating self-assembly reactions of the of the Low7Skid silk / modified peptide compositions. Mid Skid Silk reaction was used as a positive control. FIG. 21 A. Illustrate kinetic parameters of gel formation during self-assembly of silk. Self-Assembly parameters of Mid Skid silk: Amax is 0.6780 (Abs), SARF is 8.676, T0.5 is 3.668 h, and the FSAF is 3.08 (Abs / min). FIG. 21B. Is a snapshot of a later time point of the same self-assembly assay, 12 days after setting the assay. None of the tested fraction has self-assembled over time.

[0050] FIG. 22A and FIG. 22B illustrate the characterization of Low7Skid silk compositions by Dynamic Light Scattering. Low skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by the Zetasizer Pro to estimate the diameter particle size of each silk composition. FIG.

[0051] 22A. Illustrates intensity diameter particle size distribution measured for silk compositions AS77, AS78, AS79, AS80, and AS81. FIG. 22B. Illustrate correlogram functions of silk compositions AS77, AS78. AS79, AS80, AS81.

[0052] FIG. 23 illustrates size exclusion chromatography scheme of the isolation of Low Skid silk / modified peptide compositions. Low Skid silk / modified polypeptide compositions is composed of a variety of peptide populations, in a wide range of sizes, using HiLoad Superdex 200 size exclusion chromatography, distinct populations of Low7Skid silk / modified polypeptide compositions were separated.

[0053] FIG. 24 is a chromatogram of Low Skid silk / modified polypeptide compositions loaded onto a Superdex 200 gel filtration column. Low Skid

[0054] 11

[0055] DBl / 165497080.5silk / modified peptide compositions were loaded onto the Superdex 200 column and were flowed through the column with 50 mM Tris, 200 mM CaC12, pH=8.0. When the UV-280 absorbance started to increase fractions were collected to separate the Low Skid silk / modified peptide compositions by size. The relative elution volume of silk compositions AS82, AS86, and AS87 are indicated on the chromatogram.

[0056] FIG. 25A and FIG. 25B illustrate Analytical Size Exclusion Chromatography of Low Skid silk / modified silk compositions and their constituent AS compositions. Fig. QA. Illustrates average molecular weight in kDa of Low Skid silk (LS) and AS82-AS89 are shown. FIG. 25B. Polydispersity (PDI) measurements are shown. The numerical data is presented in Table H.

[0057] FIG. 26 is an SDS polyacrylamide gel electrophoresis of Low Skid silk / modified polypeptide compositions. Lanes are indicated by fraction number, at the order of elution from the Superdex 200 column, and their respective silk composition: fraction 6 is AS82, fraction 7 is AS83, fraction 8 is AS84, fraction 9 is AS85, and fraction 10 is AS86.

[0058] FIG. 27A and FIG. 27B are graphs illustrating self-assembly reactions of the of the Low Skid silk / modified peptide compositions. Mid Skid Silk reaction was used as a positive control. FIG. 27A. Illustrates kinetic parameters of gel formation during self-assembly of silk. Self-Assembly parameters of Mid Skid silk: Amax is 0.6978 (Abs), SARF is 8.591, T0.5 is 3.361 h, and the FSAF is 3.46 (Abs / min). FIG. 27B. Is a snapshot of a later time point of the same self-assembly assay, 18 days after setting the assay. AS87, AS88, and AS89 demonstrate gel formation at this time point, that was already observed five days post assay (LS, Low Skid silk; MS, Mid Skid silk).

[0059] FIGS. 28A-28C are graphs showing characterization of Low Skid silk compositions by Dynamic Light Scattering. Low skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by the Zetasizer Pro to estimate particle size of each silk composition. FIG. 28A. Shows intensity particle size distribution measured for silk compositions AS82, AS83. AS84, AS85, AS86, AS87, AS88, and AS89. FIG. 28B. Shows intensity particle size

[0060] 12

[0061] DBl / 165497080.5distribution measured for silk compositions AS82, Low Skid silk / modified peptide compositions (LS), and Mid Skid silk / modified peptide compositions (MS). FIG. 28C. Shows correlogram functions of silk compositions AS82, AS83, AS84, AS85, AS86, AS87. AS88, AS89, Low Skid silk / modified peptide compositions (LS). Mid Skid silk / modified peptide compositions (MS).

[0062] FIG. 29 illustrates anion exchange chromatography (Q). hydrophobic interaction chromatography (HIC), and size exclusion chromatography (SEC) scheme of the isolation of Low Skid silk / modified peptide compositions. Low Skid silk / modified polypeptide compositions is composed of a variety of peptide populations, in a wide range of sizes and charge. Using Q-Sepharose anion exchange chromatography as a first step. Butyl ImpRes Hydrophobic interactions resin as a second step, and HiLoad Superdex 200 size exclusion chromatography as a third purification step, distinct populations of Low Skid silk / modified polypeptide compositions were isolated. The Q-Sepharose eluate was loaded onto a Butyl ImpRes (HIC) column, and the HIC-eluate was loaded onto a HiLoad Superdex 200 size exclusion chromatography, which resulted in fractionation of negatively charged silk compositions / modified peptides with hydrophobicity characteristics fractionated by size. The Q-Sepharose eluate contained negatively charged peptides in all sizes. Resolving these peptides by Butyl ImpRes column resulted in elution of high- molecular-weight, negatively charged, somewhat hydrophobic silk compositions / modified peptides. The smaller negatively charged peptides were washed as flowthrough and did not bind the Butyl ImpRes column. The Q- HlC(elution) was loaded into Superdex 200 and was separated by size.

[0063] FIGS. 30A-30E are chromatograms of anion exchange chromatography, hydrophobic interactions chromatography, and the following size exclusion chromatography of Low Skid silk / modified polypeptide compositions. FIG. 30A. illustrates anion exchange chromatography was performed with a Q-Sepharose column. Low Skid silk / modified peptide compositions were separated to uncharged peptide population (flowthrough - light blue background) and eluted negatively

[0064] 13

[0065] DBl / 165497080.5charged silk compositions (eluate - light pink background) by anion exchange chromatography. Light yellow background indicates column wash with 50 mM Tris pH=8.0 before eluting the charged peptide population. FIG. 30B. illustrates the negatively charged eluate (Q-elution) was loaded onto a Butyl ImpRes column, in the presence of 300 mM ammonium sulfate [(NH4)2SO4J, to expose hydrophobic domains of the silk peptides, which allows binding to the column. The highly charged peptide population did not bind the column (flowthrough), highlighted in light blue. The column was washed until OD280 was reduced to -100 units (light yellow). Then, the bound silk peptides (Q-HIC(elution)) were eluted by using 50 mM Tris, pH=8.0 without ammonium sulfate (light pink). FIG. 30C. illustrates the Q-HIC(elution) was further fractionated by size exclusion chromatography (SEC), using the gel filtration column Superdex 200. The Q-HIC(elution) fraction was flowed through the column with 50 mM Tris, 200 mM CaC12. pH=8.0. When the UV-280 absorbance started to increase fractions were collected to separate the Low Skid silk / modified peptide compositions by size. The relative elution volume of silk compositions AS 90 and AS94 are indicated on the chromatogram. FIG. 30D. the Q-HIC(flowthrough) fraction was further fractionated by SEC. using the Superdex 200 column, at the same procedure as in (VC). The relative elution volume of silk compositions AS 95 and AS 100 are indicated on the chromatogram. FIG. 30E. illustrates the superimposition of chromatograms (FIG. 30C) and (FIG. 30D). the Q-HIC(elution) fraction has a higher-molecular-weight range compared to the Q-HIC(flowthrough) fractions, which elutes later in SEC, and has lower-molecular-weight range.

[0066] FIG. 31A and FIG. 3 IB are graphs showing analytical Size Exclusion Chromatography of Low Skid silk / modified silk compositions and their constituent AS compositions. FIG. 31 A. Average molecular weight in kDa of Low Skid silk (LS) and AS90-AS100 are shown. FIG. 3 IB. Poly dispersity (PDI) measurements are shown. The numerical data is presented in Table 11.

[0067] FIG. 32A and FIG. 32B are SDS polyacrylamide gel electrophoresis of Low' Skid silk / modified polypeptide compositions. FIG. 32A. Q-HIC(elution) SEC

[0068] 14

[0069] DBl / 165497080.5fractions. FIG. 32B. Q-HIC(flowthrough) SEC fractions. Lanes are indicated by fraction number, at the order of elution from the Superdex 200 column, and their respective silk composition: in FIG. 32A, fraction 6 is AS90, fraction 7 is AS91, fraction 8 is AS92, fraction 9 is AS93. and fraction 10 is AS94. In FIG. 32B, fraction 8 is AS95, fraction 9 is AS96, fraction 10 is AS97, fraction 11 is AS98, fraction 12 is AS99, and fraction 13 is AS 100.

[0070] FIG. 33 illustrates self-assembly reactions of the of the Low Skid silk / modified peptide compositions. Mid Skid Silk reaction was used as a positive control, kinetic parameters of gel formation during self-assembly of silk. Q- HlC(elution) is the elution fraction that was eluted from the Butyl ImpRes column, prior to SEC purification; LS, Low Skid silk; MS, Mid Skid silk. Self-Assembly parameters of Mid Skid silk: Amax is 0.6974 (Abs), SARF is 8.661, T0.5 is 3.834 h, and the FSAF is 3.03 (Abs / min).

[0071] FIGS. 34A-34F illustrate the characterization of Low Skid silk compositions by Dynamic Light Scattering. Low and Mid skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL. filtered, and analyzed by the Zetasizer Pro to estimate the diameter particle size of each silk composition. In FIG. 34A, intensity diameter particle size distribution measured for silk compositions AS90, Q- HlC(elution) fraction (prior to fractionation by SEC), Low Skid silk (LS), and Mid Skid silk (MS). FIG. 34B: Correlogram functions of silk compositions presented in (FIG. 34A). FIG. 34C: Intensity diameter particle size distribution measured for silk compositions AS90-AS94, derived from Q-HIC(elution)-SEC fractionation process. FIG. 34D: Correlogram functions of silk compositions presented in (FIG. 34C). FIG.

[0072] 34E: Intensity diameter particle size distribution measured for silk compositions AS95-AS100, derived from Q-HIC(flowthrough)-SEC fractionation process. FIG.

[0073] 34F: Correlogram functions of silk compositions presented in (FIG. 34E).

[0074] FIG. 35 illustrates size exclusion chromatography scheme of the isolation of Mid Skid silk / modified peptide compositions. Mid Skid silk / modified polypeptide compositions is composed of a variety' of peptide populations, in a wide range of 15

[0075] DBl / 165497080.5sizes, using HiLoad Superdex 200 size exclusion chromatography, distinct populations of Mid Skid silk / modified polypeptide compositions were able to be separated.

[0076] FIG. 36 is a chromatogram of Mid Skid silk / modified polypeptide compositions loaded onto a Superdex 200 gel filtration column. Mid Skid silk / modified peptide compositions were loaded onto the Superdex 200 column and were flowed through the column with 50 mM Tris, 200 mM CaC12, pH=8.0. When the UV-280 absorbance started to increase fractions were collected to separate the Mid Skid silk / modified peptide compositions by size. The relative elution volume of silk compositions AS 107 and ASH 1 are indicated on the chromatogram.

[0077] FIG. 37 and FIG. 37B illustrates analytical Size Exclusion Chromatography of Mid Skid silk / modified silk compositions and their constituent AS compositions. In FIG. 37 A average molecular weight in kDa of Mid Skid silk (MS) and AS106-AS111 are shown. In FIG. 37B poly dispersity (PDI) measurements are shown. The numerical data is presented in Table 13.

[0078] FIG. 38 is a SDS polyacrylamide gel electrophoresis of Mid Skid silk / modified polypeptide compositions. Lanes are indicated by fraction number, at the order of elution from the Superdex 200 column, and their respective silk composition: fraction 6 is AS107, fraction 7 is AS108, fraction 8 is AS109, fraction 9 is AS 110, and fraction 10 is AS 111.

[0079] FIG. 39 illustrates self-assembly reactions of the of the Mid Skid silk / modified peptide compositions, kinetic parameters of gel formation during self-assembly of silk. Dashed red lines show how the self-assembly parameters Amax, SARF, and TO.5 were calculated for unfractionated Mid Skid silk (MS). Numerical parameters of silk compositions AS 106- AS 111 can be found in Tables 12-14. Low Skid silk (LS) was used as a negative control. LS, Low Skid silk; MS, Mid Skid silk.

[0080] FIG. 40A and FIG. 40B illustrates characterization of Mid Skid silk compositions by Dynamic Light Scattering. Mid skid silk / modified peptide compositions ere diluted to a concentration of 1 mg / rnL, filtered, and analyzed by 16

[0081] DBl / 165497080.5the Zetasizer Pro to estimate particle size of each silk composition. FIG. 40A shows intensity particle size distribution measured for silk compositions AS 106, AS 107, AS108, AS109, AS110, AS111, and Mid Skid (MS). FIG. 40B showscorrelation functions of silk compositions presented in (FIG. 40A).

[0082] FIG. 41 illustrates anion exchange chromatography and size exclusion chromatography scheme of the isolation of Mid Skid silk / modified peptide compositions. Mid Skid silk / modified polypeptide compositions is composed of a variety of peptide populations, in a wide range of sizes and charge. Using Q- Sepharose anion exchange chromatography as a first step, and HiLoad Superdex 200 size exclusion chromatography as a second purification step, distinct populations of Mid Skid silk / modified polypeptide compositions were separated. The Q-Sepharose eluate was loaded onto HiLoad Superdex 200 size exclusion chromatography, which resulted in negatively charged silk compositions / modified peptides fractionated by size.

[0083] FIG. 42A and FIG. 42B are chromatograms of the anion exchange chromatography and the following size exclusion chromatography of the eluate (Q- eluate) of Mid Skid silk / modified polypeptide compositions. FIG. 42A. Anion exchange chromatography was performed with a Q-Sepharose column (Cytiva). Mid Skid silk / modified peptide compositions were separated to uncharged peptide population (flowthrough - light blue background) and eluted negatively charged silk compositions (eluate - light pink background) by anion exchange chromatography. Light yellow background indicates column wash with 50 mM Tris pH=8.0 before eluting the charged peptide population. FIG. 42B. The negatively charged eluate (Coelution) was loaded onto the Superdex 200 column and was flowed through the column with 50 mM Tris, 200 mM CaC12, pH=8.0. When the UV-280 absorbance started to increase fractions were collected to separate the Mid Skid silk / modified peptide compositions by size. The relative elution volume of silk compositions AS 101 and AS 105 are indicated on the chromatogram.

[0084] 17

[0085] DBl / 165497080.5FIG. 43A and FIG.43B illustrate analytical Size Exclusion Chromatography of Mid Skid silk / modified silk compositions and their constituent AS compositions. In FIG. 43A, average molecular weight in kDa of Mid Skid silk (MS) and AS101-AS105 are shown. In FIG. 43B, polydispersity (PDI) measurements are show n. The numerical data is presented in Table 16.

[0086] FIG. 44A is a SDS polyacrylamide gel electrophoresis of Mid Skid silk / modified polypeptide compositions. Lanes are indicated by fraction number, at the order of elution from the Superdex 200 column, and their respective silk composition: fraction 6 is AS101, fraction 7 is AS102, fraction 8 is AS103, fraction 9 is AS 104, and fraction 10 is AS 105.

[0087] FIG. 44B illustrates self-assembly reactions of the of the Mid Skid silk / modified peptide compositions. Low Skid Silk reaction was used as a negative control. Kinetic parameters of gel formation during self-assembly of silk are shown. Red dotted lines are shown to clarify the calculations of Amax, SARF (Self-Assembly Rate Factor), and T0.5 parameters in Table 17.

[0088] FIGS. 45A-45C are graphs illustrating characterization of Mid Skid silk compositions by Dynamic Light Scattering. Mid skid silk / modified peptide compositions were diluted to a concentration of 1 mg / mL, filtered, and analyzed by the Zetasizer Pro (Malvern) to estimate the diameter particle size of each silk composition. FIG. 45 A: Intensify diameter particle size distribution by intensify measured for silk compositions AS101, AS102, AS103, AS104, and AS105. FIG. 45B: Intensify diameter particle size distribution by intensify measured for silk compositions AS 101, AS105, and Mid Skid silk (MS), to emphasize the size difference between AS101 and AS105. FIG. 45C: Correlogram functions of silk compositions AS101, AS102, AS103, AS104, AS105, and Mid Skid silk (MS).

[0089] FIG. 46 is an illustration of the values for three molar mass moments (Mn, Mw, and Mz) as it relates to molar mass and the number of molecules at each molar mass. This example is applicable to a poly disperse sample; for a monodisperse sample, Mn = Mw = Mz.

[0090] 18

[0091] DBl / 165497080.5FIG. 47A and FIG. 47B are analytical SEC-MALS of Low, Mid and High Molecular Weight Silk. FIG. 47 A. Weight Average Molecular Weight in kDa of Low, Mid, and High Molecular Weight Silk. FIG. 47B: Polydispersity Index (PDI) measurements of Low. Mid, and High Molecular Weight Silk are shown.

[0092] FIG. 48A and FIG. 48B are analytical SEC-MALS of Low, Mid and High Molecular Weight Silk organized by Silk Type. Individual data points are show and the mean is represented by the heigh of the box. The bars encompass one standard deviation. FIG. 48A. Weight- Average Molecular Weight Ranges for Low, Mid, and High Molecular Weight Silk. FIG. 48B. PDI Ranges for Low, Mid, and High Molecular Weight Silk.

[0093] FIG. 49A and FIG. 49B are analytical SEC-MALS of Low Skid silk / modified silk compositions and the constituent AS compositions as separated by Q-SEC (Q- eluent). FIG 49A: Average molecular weight in kDa of Low Skid silk (LS) and AS77- AS81 are shown. FIG. 49B: Poly dispersity (PDI) measurements are shown. The numerical data is presented in Table 24.

[0094] FIG. 50A and FIG. 50B are analytical SEC-MALS of Low Skid silk / modified silk compositions and the constituent AS compositions as separated by SEC. FIG. 50A: Average molecular weight in kDa of Low Skid silk (LS) and AS82-AS89 are shown. FIG. 50B: Polydispersity' (PDI) measurements are shown. The numerical data is presented in Table 25.

[0095] FIG. 51 A and FIG. 5 IB are analytical SEC-MALS of Low Skid silk / modified silk compositions and the constituent AS compositions as separated by Q-HIC-SEC (Q-HIC-Eluent). FIG. 51 A: Average molecular weight in kDa of Low Skid silk (LS) and AS90-AS94 are shown. FIG. 5 IB. Poly dispersity (PDI) measurements are shown. The numerical data is presented in Table 26.

[0096] FIG. 52A and FIG. 52B are analytical SEC-MALS of Low Skid silk / modified silk compositions and the constituent AS compositions as separated by Q-HIC-SEC (Q-HIC-Flowthrough). FIG. 52A: Average molecular weight in kDa of Low Skid silk

[0097] 19

[0098] DBl / 165497080.5(LS) and AS95-AS100 are shown. FIG. 52B: Poly dispersity (PDI) measurements are shown. The numerical data is presented in Table 26.

[0099] FIG. 53A and FIG. 53B are analytical SEC-MALS of Mid Skid silk / modified silk compositions and the constituent AS compositions as separated by Q— SEC (Q- flow through). FIG. 53A: Average molecular weight in kDa of Mid Skid silk (MS) and AS101-AS105 are shown. FIG. 53B:Polydispersity (PDI) measurements are shown. The numerical data is presented in Table 27.

[0100] FIG. 54A and FIG. 54B are analytical SEC-MALS of Mid Skid silk / modified silk compositions and the constituent AS compositions as separated by SEC. FIG. 54A: Average molecular weight in kDa of Mid Skid silk (MS) and AS106-AS111 are shown. FIG. 54B: Polydispersity (PDI) measurements are shown. The numerical data is presented in Table 28.

[0101] FIG. 55A-55E: 27P penetrates the intact skin barrier and binds to dermal fibroblasts in both 3D skin equivalents and 2D in vitro cultures. Fig. 101A) Schematic of experiment design to assess 27P penetration in 3D reconstituted Mattek skin equivalent model. Fig. 101B) Quantification of mean fluorescence intensity of 27P- AF594 binding to different compartments in skin with 1 day (24h) and 4 day (24h+72h) topical treatments, without and with addition of 50 pg / mL VC to culture media, respectively. 4-day treatments included 2 topical applications - first for 24h in the absence of VC and second for 72h in the presence of VC in the media. Data are averaged from 1-2 regions of stitched images from 2-4 slices of full depth tissue from 2-3 replicate samples of 2-3 experiments. Fig. 101C) Representative images of 27P- 594 binding in epidermis, papillary dermis and reticular dermis regions from untreated and 27P treated 10 pm section of 3D skin samples. Zoomed images show specific binding of 27P-AF594 to fibroblast cells in papillary dermis. Important to highlight is that the signal from cell-associated 27P AF594 is much higher than the background intensity in the different compartments and the background intensity w as subtracted from the cell-associated signals. Fig. 101D) Representative images of AF594-labeled 27P binding to 2D dermal fibroblast cells (white arrows in zoom 20

[0102] DBl / 165497080.5images) defined by WGA cell mask (grey). Fig. 101E) 27P-AF594 dose-dependently binds to dermal fibroblast cells (n=3 donors). Specific binding of 27P to fibroblasts was quantified to be dose-dependent (Figures 108A-108C). All data was plotted in GraphPad Prism 10.3.0 with standard error of mean as error bars and considered significant if p < 0.05 in unpaired t-test between treated and respective untreated samples of different tissue regions. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001; ns: no significance. See also Figures 108A-108C.

[0103] FIGS. 56A-56F: 27P supports the sustained synthesis and secretion of collagen by dermal fibroblasts. Fig. 56A) Significant induction of pro-Cl secretion by topical application of 27P on Mattek 3D skin equivalent models during the first 24h (without VC; N=5) and subsequent 72h (with 50 pg / mL VC in media; N=2). Fig. 56B) Dose-dependent (0.25 - 2.0 mg / mL) induction of secreted pro-Cl levels in dermal fibroblasts as assessed by ELISA shows 3.4-fold increase over 24h 27P treatment, compared to 2.0-fold induction by 10 ng / mL TGFp. Combination of 27P treatment (0.25 - 2.0 mg / mL) and VC (50 pg / mL) shows further enhanced pro-Cl secretion over 24h. Fig. 56C) Representative immunofluorescence images of intracellular Coll Al protein (red) accumulated over 24h treatment with 27P and benchmarking ingredients; TGFP treatment was considered as positive control. Significant depletion of intracellular Coll Al was observed with VC that was rescued with 27P (0.5 mg / mL) and VC combination. Fig. 56D) Quantification of %CollAlHigh cells shows significant increases in de novo CollAl with 24h treatment of low doses (0.25 - 0.5 mg / mL) of 27P, while significant depletion was observed with 1 pM RA and 50 pg / mL VC treatments. Fig. 56E) Accumulated intracellular CollAl with 27P (0.25 - 2.0 mg / mL) and VC (50 pg / mL) combination over 24h and 72h shows 27P dose-dependent rescues depletion resulted by VC alone over first 24h treatment. Fig. 56F) mRNA expression of CollAl after 6h and 24h 27P treatments show no significant changes, while TGFP significantly induces CollAl mRNA. Data from at least 3 technical repeats for 3-4 donors were plotted using GraphPad Prism 10.2.2 and unpaired t-test was used to determine statistical significance between treated and untreated samples and considered significant if p <

[0104] 21

[0105] DBl / 165497080.50.05. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001; ns: no significance. See also Figures 61A-61F.

[0106] FIGS. 57A- 57E: 27P enhances collagen matrix deposition by dermal fibroblasts. Fig. 57A) Representative images of sirius red stained total collagen (red) deposited by dermal fibroblasts over 3 days (72h) of 27P and VC (50 pg / mL) treatment. Fig. 57B) 27P and VC combination induced significant increase in % total collagen deposition over 3 days (72h) and 5 (120h) days, comparable to TGFP treatment. Fig. 57C) Representative immunofluorescence images of total extracellular Coll Al deposition during 5 days. Quantification of area of Fig. 57D) total extracellular CollAl, classified as Fig. 67E) unorganized deposits vs. matrix- associated organized filaments shows dose-response increase in organized collagen filaments with 27P and decrease in unorganized deposits. Data from at least 3 technical repeats for 3-4 donors were plotted using GraphPad Prism 10.2.2 and unpaired t-lest was used to determine statistical significance between treated and untreated samples and considered significant if p < 0.05. * p<0.05; ***p<0.005, ****p<0.0001, **** p<0.0001; ns: no significance. See also Figure 62.

[0107] FIGS. 58A-58D: 27P supported collagen production without impacting pro- fibrotic markers. Fig. 58A) Representative immunofluorescence images of fibrosis marker, aSMA protein (magenta) in dermal fibroblasts treated with 27P or 10 ng / mL TGFP for 24h. Fig. 58B) Quantification of %ciSMAHigh cells showed no significant induction of SMA protein expression with 27P treatment over up to 120h. TGFP strongly induced aSMA expression by 12.0-fold by 120h. mRNA levels of fibrosis- associated markers, Fig. 58C) aSMA and Fig. 58D) TGFP show no significant effects with 27P treatment and strong induction with TGFP treatment for 24h and 6h, respectively. Data from at least 3 technical repeats for 3-4 donors were plotted using GraphPad Prism 10.2.2 and unpaired t-test was used to determine statistical significance between treated and untreated samples and considered significant if p < 0.05. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001; ns: no significance

[0108] 22

[0109] DBl / 165497080.5FIGS. 59A-59D: 27P does not induce inflammatory cytokines in dermal fibroblasts. mRNA expression of pro-inflammatory markers, Fig. 59A) IL6 and Fig.

[0110] 105C) IL8 after 6h of 27P treatment showed no significant induction and significant increases at 24h. Secreted Fig. 59B) IL6 protein and Fig. 59D) IL8 protein measured by ELISA remained unchanged with 6h and 24h 27P treatments while both IL6 and IL8 secretion were strongly induced by RA treatments. TGFP also strongly induced IL6 mRNA and protein levels. Relative gene expression was calculated against GAPDH for each gene tested using double delta Ct analysis and data from technical repeats for each sample from 3-4 donors was averaged and plotted using GraphPad Prism 10. Tests for statistical significance were performed using unpaired t-test and considered significant if p < 0.05 when compared with untreated samples. *p<0.05; **p<0.01; ***p<0.005; ****p<0.0001; ns: no significance.

[0111] FIGS. 60A-60C: Specific binding of 27P to keratinocytes and fibroblast cells in vitro. (Related to Figures 55A-55E) Fig. 60A) Far-western blot showing interaction of biotinylated 27P with purified keratin- 1. Purified Keratin 1 (KRT1), Keratin 10 (KRT10), and Bovine Serum Albumin (BSA) as a negative control were loaded onto SDS-PAGE and transferred to a PVDF membrane. The membranes were labeled with biotinylated 27P or anti-His-tag conjugated to HRP (KRT1 and KRT10 are His- tagged) (1 pg protein in each lane). Fig. 60B) Coomassie blue-stained SDS-PAGE gel of KRTL KRT10 and BSA (4 pg protein in each lane). Estimated molecular weights of the target proteins: KRT1 - 67 kDa; KRT10 - 62.8 kDa; BSA - 66.4 kDa. Fig. 60C) Quantification of intensity of labeled-27P binding to fibroblasts and background area, representing non-specific binding.

[0112] FIGS. 61A-61F: 27P maintains prolonged pro-Cl secretion without inducing proliferation, and has similar effects on Coll Al and Col3A synthesis. (Related to Figures 56A-56F) Fig. 61 A) Combination of 27P treatment (0.25 - 2.0 mg / mL) and VC (50 pg / mL) shows pronounced increase in pro-Cl secretion measured at 72h and 120h. Fig. 6 IB) Quantification of nuclear counts shows 27P does not induce proliferation of dermal fibroblasts over 24h. Fig. 61C) Representative

[0113] 23

[0114] DBl / 165497080.5immunofluorescence images and Fig. 61) quantification of intracellular Col3A shows significant increase in intracellular Col3A protein synthesis with 24h of 27P. Fig. 61E) Accumulated intracellular Col3A with 27P (0.25 - 2.0 mg / mL) and VC (50 pg / mL) combination over 24h shows 27P dose-dependent rescue of depletion resulted by VC alone. Fig. 61 F) Col3A mRNA expression after 6h and 24h 27P treatments show no significant changes, directly indicating lack of transcriptional regulation of collagens by 27P. Pooled data from 3-4 donors were plotted using GraphPad Prism 10.2.2 and unpaired t-test was used to determine statistical significance between treated and untreated samples and considered significant if p < 0.05. *p<0.05;

[0115] **p<0.01; ***p<0.005; ****p<0.0001; ns: no significance.

[0116] FIG. 62: 27P does not alter MMP1 mRNA expression. (Related to Figure 57) MMP1 mRNA expression after 6h and 24h 27P treatments show no significant changes, while TGFP significantly induces CollAl mRNA and reduces MMP1 mRNA levels. Pooled data from 3 donors were plotted using GraphPad Prism 10.2.2 and unpaired t-test was used to determine statistical significance between treated and untreated samples and considered significant if p < 0.05. *p<0.05; **p<0.01;

[0117] ***p<0.005; ****p<0.0001; ns: no significance.

[0118] FIGS. 63A and 63B illustrates Activated Silk™ KDF-P supports increased natural keratinocyte differentiation.

[0119] FIG. 64 illustrates the results of the skin penetration study on a 3D skin equivalent model (MatTek).

[0120] FIG. 65 illustrates mean fluorescence intensity' in KDF-P vs. a control.

[0121] FIG. 66 illustrates that differentiation is signaled by the ‘keratin switch’ effect, showing the fold change in K5 cells and the fold increase in KI cells.

[0122] FIG. 67 illustrates that increased differentiation signals tight junction proteins. FIG. 68 illustrates how- increased differentiation drives additional protective proteins.

[0123] FIG. 69 illustrates the regeneration of damaged skin.

[0124] 24

[0125] DBl / 165497080.5FIG. 70 illustrates a significant and rapid improvement in appearance of damaged skin.

[0126] FIGS. 71A-71D are images illustrating visible improvement in damaged skin site after two days. Regarding Fig. 71B -71D, RGB Color Analysis: Measure of the ratio of red light vs. green light reflected from skin The presence of skin redness visually quantified on a scale of 1 (black) to 28 (white).

[0127] FIG. 72 shows low Skid silk attenuates secretion of pro-inflammaroty cytokines in THP1 derived macrophages. Secretion of pro and anti-inflammatory cytokines from PMA differentiated macrophages. Supernatant from PMA induced macrophages treated with LPS from E.coli 0111 :B7 was analyzed with DuoSet ELISA assays and the amount of released cytokine was quantified. Results were analyzed with unpaired t-test (n=3).

[0128] FIG. 73 shows mid skid silk attenuates secretion of pro-inflammatory cytokines IL-6 and TNF-a. A. Human PBMC macrophages were treated with LPS (0.125ng / mL) and Mid Skid silk at the shown concentrations. IL-6 and TNF-a was quantified in the supernatants after 24h. B. Kinetics of IL-6 secretion from human PBMC derived macrophages. Macrophages were treated with LPS, Mid Skid silk. Samples were collected from the supernatant at the indicative time points and IL-6 amount was determined. Data are the mean of 3 technical replicates.

[0129] FIG. 74 is a graph illustrating Perception Question Responses, Day 1 vs. Day 0 using a balm described herein vs. hydrocortisone.

[0130] FIG. 75 is a graph illustrating Perception Question Responses, Day 2 vs. Day 0 using a balm described herein vs. hydrocortisone.

[0131] Figures 76A- 76B shows emulsification capability of hydrogel Pl-alpha and Pl-beta. (A): the viscosity and texture of emulsions made with 1% Pl-alpha at 1%, 20% and 50% oil levels; (B): the viscosity and texture of emulsions made with 1% Pl -beta at 1%, 20% and 50% oil levels.

[0132] 25

[0133] DBl / 165497080.5Figure 77A - Figure 77C show the sequence listing for fibroin heavy chain.

[0134] [SEQ ID NO: 49],

[0135] Figure 78 shows the sequence listing for fibroin light chain. [SEQ ID NO: 50], Figure 79 shows the sequence listing for fibrohexamerin. [SEQ ID NO: 51], While the above-identified drawings set forth presently disclosed embodiments, other embodiments are also contemplated, as noted in the discussion. This disclosure presents illustrative embodiments by way of representation and not limitation. Numerous other modifications and embodiments can be devised by those skilled in the art which fall within the scope and spirit of the principles of the presently disclosed embodiments.

[0136] DETAILED DESCRIPTION

[0137] Methods of making silk fibroin or silk fibroin fragments are known and are described for example in U.S. Patents Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177. Methods of using silk fibroin or silk fibroin fragments in coating applications, including coating applications of animal hair, are known and are described for example in U.S. Patent Application Publications Nos. 20160222579, and 20160281294. Compositions and methods of using silk fibroin or silk fibroin fragments in cosmetic applications are known and are described for example in U.S. Patent Application Publications Nos. 20180280274 and 20180008522, International Patent Application Publication No. WO 2019005848, and International Patent Application Publication No. WO 2024059862. All of the publications cited herein are incorporated by reference herein in their entireties.

[0138] Definitions

[0139] As used in the preceding sections and throughout the rest of this specification, unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art to which this invention

[0140] 26

[0141] DBl / 165497080.5belongs. All patents and publications referred to herein are incorporated by reference in their entireties.

[0142] All percentages, parts and ratios are based upon the total weight of the collagen boosting compositions of the present invention, unless otherwise specified. All such weights as they pertain 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 denoted as “wt. %” or % w / w herein.

[0143] As used herein, the term '‘a”, “an”, or “the” generally is construed to cover both the singular and the plural forms.

[0144] As used herein, the term '‘about” generally refers to a particular numeric value that is within an acceptable error range as determined by one of ordinary skill in the art, which will depend in part on how the numeric 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 a given numeric value.

[0145] As used herein, the term '‘dermatologically acceptable carrier” means a carrier suitable for use in contact with mammalian keratinous tissue without causing any adverse effects such as undue toxicity, incompatibility, instability, allergic response, for example. A dermatologically acceptable carrier may include, without limitations, water, liquid or solid emollients, humectants, solvents, and the like.

[0146] 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, as determined by calculating values for the different regions of the molecule, as described by Griffin's method HLB = 20 * Mh / M, where Mh is the molecular mass of the hydrophilic portion of the surfactant, and M is the molecular mass of the entire surfactant molecule, giving a result on a scale of 0 to 20. A HLB value of 0 corresponds to a completely lipophilic molecule, and a value of 20 corresponds to a completely hydrophilic molecule. The HLB value can be used to predict the surfactant properties of a molecule: HLB < 10: Lipid-soluble (water-insoluble), HLB >10:

[0147] 27

[0148] DBl / 165497080.5Water-soluble (lipid-insoluble), HLB = 1-3: anti-foaming agent, 3-6: W / O (water-in- oil) emulsifier, 7-9: wetting and spreading agent, 8-16: O / W (oil-in-water) emulsifier, 13-16: detergent, 16-18: solubilizer or hydrotrope.

[0149] As used herein, “average weight average molecular weight’' refers to an average of two or more values of weight average molecular weight of silk fibroin or fragments thereof of the same compositions, the two or more values determined by two or more separate experimental readings.

[0150] As used herein, the term polymer “poly dispersity (PD)” is generally used as a measure of the broadness of a molecular weight distribution of a polymer, and is defined by the formula poly dispersity PD = ^ .

[0151] As used herein, the term “substantially homogeneous” may refer to silk fibroin- based protein fragments that are distributed in a normal distribution about an identified molecular weight. As used herein, the term “substantially homogeneous” may refer to an even distribution of a component or an additive, for example, silk fibroin fragments, dermatologically acceptable carrier, etc., throughout a composition of the present disclosure.

[0152] 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 acids units are defined when molecular size becomes an important parameter.

[0153] As used herein, “Fibroin Glycoside (Pl -alpha Hydrogel)” refers to a silk fibroin / xanthan gum formulation as described in WO 2025199286. In some embodiments, “Fibroin Glycoside (Pl -alpha Hydrogel)” comprises a lyophilized mixture of mid-MW silk fibroin polypeptides and a polysaccharide, e.g., xanthan gum.”

[0154] 28

[0155] DBl / 165497080.5SPF Definitions and Properties

[0156] As used herein, “silk protein fragments” (SPF) include one or more of: “silk fibroin fragments” as defined herein; “recombinant silk fragments” as defined herein; “spider silk fragments” as defined herein; “silk fibroin-like protein fragments” as defined herein; and / or “chemically modified silk fragments” as defined herein. SPF may have any molecular weight values or ranges described herein, and any poly dispersity values or ranges described herein. As used herein, in some embodiments the term “silk protein fragment” also refers to a silk protein that comprises or consists of at least two identical repetitive units which each independently selected from naturally-occurring silk polypeptides or of variations thereof, amino acid sequences of naturally-occurring silk polypeptides, or of combinations of both.

[0157] SPF Molecular Weight and Polydispersity

[0158] In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 1 to about 5 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 5 to about 10 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 10 to about 15 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 15 to about 20 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 14 to about 30 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 20 to about 25 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 25 to about 30 kDa. In an embodiment, a

[0159] 29

[0160] DBl / 165497080.5composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 30 to about 35 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 35 to about 40 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 39 to about 54 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 40 to about 45 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 45 to about 50 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 50 to about 55 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 55 to about 60 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 60 to about 65 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 65 to about 70 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 70 to about 75 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 75 to about 80 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 80 to about 85 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 85 to about 90 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 90 to about 95 kDa. In an embodiment, a composition of the present disclosure includes SPF 30

[0161] DBl / 165497080.5having an average weight average molecular weight selected from between about 95 to about 100 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 100 to about 105 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 105 to about 110 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 110 to about 115 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 1 15 to about 120 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 120 to about 125 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 125 to about 130 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 130 to about 135 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 135 to about 140 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 140 to about 145 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 145 to about 150 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 150 to about 155 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 155 to about 160 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 160 to about 165 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular 31

[0162] DBl / 165497080.5weight selected from between about 165 to about 170 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 170 to about 175 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 175 to about 180 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 180 to about 185 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 185 to about 190 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 190 to about 195 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 195 to about 200 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 200 to about 205 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 205 to about 210 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from betw een about 210 to about 215 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 215 to about 220 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 220 to about 225 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 225 to about 230 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 230 to about 235 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 32

[0163] DBl / 165497080.5235 to about 240 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 240 to about 245 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 245 to about 250 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 250 to about 255 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 255 to about 260 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 260 to about 265 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 265 to about 270 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 270 to about 275 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 275 to about 280 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 280 to about 285 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 285 to about 290 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 290 to about 295 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 295 to about 300 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 300 to about 305 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 305 to about 310 kDa. In an

[0164] 33

[0165] DBl / 165497080.5embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 310 to about 315 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 315 to about 320 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 320 to about 325 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 325 to about 330 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 330 to about 335 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 335 to about 340 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 340 to about 345 kDa. In an embodiment, a composition of the present disclosure includes SPF having an average weight average molecular weight selected from between about 345 to about 350 kDa.

[0166] In some embodiments, compositions of the present disclosure include SPF compositions selected from compositions #1001 to #2450, having weight average molecular weights selected from about 1 kDa to about 145 kDa, and a poly dispersity selected from between 1 and about 5 (including, without limitation, a poly dispersity of 1), between 1 and about 1.5 (including, without limitation, a poly dispersity 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:

[0167] 34

[0168] DBl / 165497080.5

[0169]

[0170] 35

[0171] DBl / 165497080.5

[0172]

[0173] 36

[0174] DBl / 165497080.5

[0175]

[0176] 37

[0177] DBl / 165497080.5

[0178]

[0179] As used herein, “low molecular weight,” “low MW,” or “low-MW” SPF may include SPF having a weight average molecular weight, or average weight average molecular weight selected from between about 5 kDato about 38 kDa, about 14 kDa to about 30 kDa, or about 6 kDa to about 17 kDa. In some embodiments, low MW may be referred to as “27P,” and can have a MW selected from between 12 to 20 kDa, and PDI of < 3.4. In some embodiments, molecular weight refers to Mw. In some embodiments, a target low molecular weight for certain SPF may be weight average molecular weight of about 5 kDa, about 6 kDa, about 7 kDa, about 8 kDa, about 9 kDa, about 10 kDa, about 11 kDa, about 12 kDa, about 13 kDa. about 14 kDa, about 38

[0180] DB1 / 165497080.515 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa. about 36 kDa, about 37 kDa, or about 38 kDa.

[0181] As used herein, “medium molecular weight,’" “medium MW,” or “mid-MW” SPF may include SPF having a weight average molecular weight, or average weight average molecular weight selected from between about 31 kDa to about 55 kDa, or about 39 kDa to about 54 kDa. In some embodiments, medium MW may be referred to as “KDF,” and can have a MW selected from between about 21 to about 41 kDa, and PDI of < 3.4 In some embodiments, molecular weight refers to Mw. In some embodiments, a target medium molecular weight for certain SPF may be weight average molecular weight of about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa. about 29 kDa, about 30 kDa, about 31 kDa. about 32 kDa, about 33 kDa. about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, 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.

[0182] As used herein, “high molecular weight,” “high MW,” or “high-MW” SPF may include SPF having a weight average molecular weight, or average weight average molecular weight selected from between about 55 kDa to about 150 kDa. In some embodiments, a target high molecular weight for certain SPF may be about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa. about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, about 75 kDa, about 76 kDa, about 77 kDa, about 78 kDa, about 79 kDa, or about 80 kDa.

[0183] 39

[0184] DBl / 165497080.5In some embodiments, the molecular weights described herein (e.g., low molecular weight silk, medium molecular weight silk, high molecular weight silk) may be converted to the approximate number of amino acids contained within the respective SPF, as would be understood by a person having ordinary skill in the art. For example, the average weight of an amino acid may be about 110 daltons (i.e., 110 g / mol). Therefore, in some embodiments, dividing the molecular weight of a linear protein by 110 daltons may be used to approximate the number of amino acid residues contained therein.

[0185] In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between 1 to about 5.0, including, without limitation, a poly dispersity of 1. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 1.5 to about 3.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between 1 to about 1.5, including, without limitation, a polydispersity of 1. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 1.5 to about 2.0. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 2.0 to about 2.5. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 2.5 to about 3.0. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 3.0 to about 3.5. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 3.5 to about 4.0. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 4.0 to about 4.5. In an embodiment, SPF in a composition of the present disclosure have a polydispersity selected from between about 4.5 to about 5.0.

[0186] In an embodiment, SPF in a composition of the present disclosure have a polydispersity of 1. In an embodiment, SPF in a composition of the present disclosure have a polydispersity of about 1.1. In an embodiment. SPF in a composition of the

[0187] 40

[0188] DBl / 165497080.5present disclosure have a poly dispersity of about 1.2. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.3. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 1.4. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.5. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.6. In an embodiment, SPF in a composition of the present disclosure have a polydispersity7of about 1.7. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.8. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.9. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 2.0. In an embodiment, SPF in a composition of the present disclosure have a polydispersity of about 2.1. In an embodiment. SPF in a composition of the present disclosure have a poly dispersity of about 2.2. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 2.3. In an embodiment, SPF in a composition of the present disclosure have a polydispersity of about 2.4. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 2.5. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 2.6. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 2.7. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 2.8. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 2.9. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 3.1. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.2. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.3. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.4. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.5. In an embodiment, SPF in a composition of the present 41

[0189] DBl / 165497080.5disclosure have a poly dispersity of about 3.6. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.7. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 3.8. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 3.9. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity7of about 4.1. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.2. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.3. In an embodiment, SPF in a composition of the present disclosure have a polydispersity7of about 4.4. In an embodiment, SPF in a composition of the present disclosure have a polydispersity of about 4.5. In an embodiment. SPF in a composition of the present disclosure have a poly dispersity of about 4.6. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.7. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.8. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 4.9. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 5.0.

[0190] In some embodiments, in compositions described herein having combinations of low, medium, and / or high molecular weight SPF, such low, medium, and / or high molecular weight SPF may have the same or different polydispersities.

[0191] Silk Fibroin Fragments

[0192] Methods of making silk fibroin or silk fibroin protein fragments and their applications in various fields are known and are described for example in U.S. Patents Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177, 10,287,728 and 10,301,768, all of which are incorporated herein in their entireties. Raw silk from silkworm Bombyx mori is composed of two primary7proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk 42

[0193] DBl / 165497080.5fibroin is a fibrous protein with a semi-crystalline structure that provides stiffness and strength. As used herein, the term “silk fibroin” means the fibers of the cocoon of Bombyx mori having a weight average molecular weight of about 370,000 Da. The crude silkworm fiber consists of a double thread of fibroin. The adhesive substance holding these double fibers together is sericin. The silk fibroin is composed of a heavy chain [SEQ ID NO: 49] having a weight average molecular weight of about 350,000 Da (H chain), and a light chain [SEQ ID NO: 50] having a weight average molecular weight about 25,000 Da (L chain). Silk fibroin is an amphiphilic polymer with large hydrophobic domains occupying the major component of the polymer, which has a 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-chain contain a repetitive hexapeptide sequence of Gly-Ala-Gly-Ala-Gly-Ser and repeats of Gly-Ala / Ser / Tyr dipeptides, which can form stable anti-parallel-sheet crystallites. The amino acid sequence of the L-chain is non- repetitive, so the L-chain is more hydrophilic and relatively elastic. The hydrophilic (Tyr, Ser) and hydrophobic (Gly, Ala) chain segments in silk fibroin molecules are arranged alternatively such that allows self-assembling of silk fibroin molecules.

[0194] Provided herein are methods for producing pure and highly scalable silk fibroin-protein fragment mixture solutions that may be used across multiple industries for a variety of applications. Without wishing to be bound by any particular theory, it is believed that these methods are equally applicable to fragmentation of any SPF described herein, including without limitation recombinant silk proteins, and fragmentation of silk-like or fibroin-like proteins.

[0195] As used herein, the term “fibroin” includes silkw orm fibroin and insect or spider silk protein. In an embodiment, 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-cry stalline structure that provides stiffness and strength. As used herein, the term “silk fibroin” means the fibers of the cocoon of Bombyx mori having

[0196] 43

[0197] DBl / 165497080.5a weight average molecular weight of about 370,000 Da. Conversion of these insoluble silk fibroin fibrils into water-soluble silk fibroin protein fragments requires the addition of a concentrated neutral salt (e g., 8-10 M lithium bromide), which interferes with inter- and intramolecular ionic and hydrogen bonding that would otherwise render the fibroin protein insoluble in water. Methods of making silk fibroin protein fragments, and / or compositions thereof, are known and are described for example in U.S. Patents Nos. 9,187,538, 9,511,012, 9,517,191, 9,522,107, 9,522,108, 9,545,369, and 10,166,177.

[0198] The raw silk cocoons from the silkworm Bombyx mori was cut into pieces. The pieces silk cocoons were processed in an aqueous solution of Na2COs at about 100 °C for about 60 minutes to remove sericin (degumming). The volume of the water used equals about 0.4 x raw silk weight and the amount of Na2CO?, is about 0.848 x the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed with deionized water three times at about 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. The excess water from the degummed silk cocoon pieces was removed. After the DI w ater washing step, the wet degummed silk cocoon pieces were dried at room temperature. The degummed silk cocoon pieces w ere mixed with a LiBr solution, and the mixture was heated to about 100 °C. The warmed mixture was placed in a dry oven and w as heated at about 100 °C for about 60 minutes to achieve complete dissolution of the native silk protein. The resulting silk fibroin solution was filtered and dialy zed using Tangential Flow Filtration (TFF) and a 10 kDa membrane against deionized water for 72 hours. The resulting silk fibroin aqueous solution has a concentration of about 8.5 wt. %. Then, 8.5 % silk solution was diluted with water to result in 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 to w ater.

[0199] Dialyzing the silk through a series of water changes is a manual and time intensive process, which could be accelerated by changing certain parameters, for example diluting the silk solution prior to dialysis. The dialysis process could be

[0200] 44

[0201] DBl / 165497080.5scaled for manufacturing by using semi-automated equipment, for example a tangential flow filtration system.

[0202] In some embodiments, the silk solutions are prepared under various preparation condition parameters such as: 90 °C 30 min, 90 °C 60 min, 100 °C 30 min, and 100 °C 60 min. Briefly, 9.3 M LiBr was prepared and allowed to sit at room temperature for at least 30 minutes. 5 mL of LiBr solution was added to 1.25 g of silk and placed in the 60 °C oven. Samples from each set were removed at 4, 6, 8, 12, 24, 168 and 192 hours.

[0203] In some embodiments, the silk solutions are prepared under various preparation condition parameters such as: 90 °C 30 min, 90 °C 60 min, 100 °C 30 min, and 100 °C 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60 °C, 80 °C, 100 °C or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the 60 °C oven. Samples from each set were removed at 1, 4 and 6 hours.

[0204] In some embodiments, the silk solutions are prepared under various preparation condition parameters such as: Four different silk extraction combinations were used: 90 °C 30 min, 90 °C 60 min, 100 °C 30 min. and 100 °C 60 min. Briefly, 9.3 M LiBr solution was heated to one of four temperatures: 60 °C. 80 °C, 100 °C or boiling. 5 mL of hot LiBr solution was added to 1.25 g of silk and placed in the oven at the same temperature of the LiBr. Samples from each set were removed at 1, 4 and 6 hours. 1 mL of each sample was added to 7.5 mL of 9.3 M LiBr and refrigerated for viscosity testing.

[0205] In some embodiments, SPF are obtained by dissolving raw unscoured, partially scoured, or scoured silkworm fibers with a neutral lithium bromide salt. The raw silkworm silks are processed under selected temperature and other conditions in order to remove any sericin and achieve the desired weight average molecular weight (Mw) and poly dispersity (PD) of the fragment mixture. Selection of process parameters may be altered to achieve distinct final silk protein fragment characteristics depending upon the intended use. The resulting final fragment solution 45

[0206] DBl / 165497080.5is silk fibroin protein fragments and water with parts per million (ppm) to non- detectable levels of process contaminants, levels acceptable in the pharmaceutical, medical and consumer eye care markets. The concentration, size and poly dispersity of SPF may further be altered depending upon the desired use and performance requirements.

[0207] Fig. 5 is a flow chart showing various embodiments for producing pure silk fibroin protein fragments (SPFs) of the present disclosure. It should be understood that not all of the steps illustrated are necessarily required to fabricate all 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. If starting from raw silk cocoons from Bombyx mori, the cocoons can be cut into small pieces, for example pieces of approximately equal size, step Bl. The raw silk is then extracted and rinsed to remove any sericin, step Cl a. This results in substantially sericin free raw silk. In an embodiment, water is heated to a temperature between 84 °C and 100 °C (ideally boiling) and then Na2COs (sodium carbonate) is added to the boiling water until the NaiCO is completely dissolved. The raw silk is added to the boiling water / Na2COs (100 °C) and submerged for approximately 15 - 90 minutes, where boiling for a longer time results in smaller silk protein fragments. In an embodiment, the water volume equals about 0.4 x raw silk weight and the Na2COs volume equals about 0.848 x raw silk weight. In an embodiment, the water volume equals 0.1 x raw silk weight and the Na2COs volume is maintained at 2.12 g / L.

[0208] Subsequently, the water dissolved Na2COs solution is drained and excess water / Na2COs is removed from the silk fibroin fibers (e.g., ring out the fibroin extract by hand, spin cycle using a machine, etc.). The resulting silk fibroin extract is rinsed with warm to hot water to remove any remaining adsorbed sericin or contaminate, ty pically at a temperature range of about 40 °C to about 80 °C, changing the volume of water at least once (repeated for as many times as required). The resulting silk fibroin extract is a substantially sericin-depleted silk fibroin. In an embodiment, the

[0209] 46

[0210] DBl / 165497080.5resulting silk fibroin extract is rinsed with water at a temperature of about 60 °C. In an embodiment, the volume of rinse water for each cycle equals 0.1 L to 0.2 L x raw silk weight. It may be advantageous to agitate, turn or circulate the rinse water to maximize the rinse effect. After rinsing, excess water is removed from the extracted silk fibroin fibers (e.g., ring out fibroin extract by hand or using a machine).

[0211] Alternatively, methods known to one skilled in the art such as pressure, temperature, or other reagents or combinations thereof may be used for the purpose of sericin extraction. Alternatively, the silk gland (100% sericin free silk protein) can be removed directly from a worm. This w ould result in liquid silk protein, without any alteration of the protein structure, free of sericin.

[0212] The extracted fibroin fibers are then allowed to dry completely. Once dry, the extracted silk fibroin is dissolved using a solvent added to the silk fibroin at a temperature between ambient and boiling, step Clb. In an embodiment, the solvent is a solution of Lithium bromide (LiBr) (boiling for LiBr is 140 °C). Alternatively, the extracted fibroin fibers are not dried but wet and placed in the solvent; solvent concentration can then be varied to achieve similar concentrations as to when adding dried silk to the solvent. The final concentration of LiBr 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 dissolving solvent. Other solvents may be used including, but not limited to, phosphate phosphoric acid, calcium nitrate, calcium chloride solution or other concentrated aqueous solutions of inorganic salts. To ensure complete dissolution, the silk fibers should be fully immersed within the already heated solvent solution and then maintained at a temperature ranging from about 60 °C to about 140 °C for 1-168 hrs. In an embodiment, the silk fibers should be fully immersed within the solvent solution and then placed into a dry oven at a temperature of about 100 °C for about 1 hour.

[0213] The temperature at w hich 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 and on the resulting molecular weight and poly dispersity of the final SPF mixture

[0214] 47

[0215] DBl / 165497080.5solution. In an embodiment, silk solvent solution concentration is less than or equal to 20% w / v. In addition, agitation during introduction or dissolution may be used to facilitate dissolution at vary ing temperatures and concentrations. The temperature of the LiBr solution will provide control over the silk protein fragment mixture molecular weight and poly dispersity created. In an embodiment, a higher temperature will more quickly dissolve the silk offering enhanced process scalability and mass production of silk solution. In an embodiment, using a LiBr solution heated to a temperature from 80 °C to 140 °C reduces the time required in an oven in order to achieve full dissolution. Varying time and temperature at or above 60 °C of the dissolution solvent will alter and control the MW and poly dispersity of the SPF mixture solutions formed from the original molecular weight of the native silk fibroin protein.

[0216] Alternatively, whole cocoons may be placed directly into a solvent, such as LiBr, bypassing extraction, step B2. This requires subsequent filtration of silk worm particles from the silk and solvent solution and sericin removal using methods know in the art for separating hydrophobic and hydrophilic proteins such as a 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 common examples and without limitation for standard protein separation methods, step C2. Non-heat treated cocoons with the silkworm removed, may alternatively be placed into a solvent such as LiBr, bypassing extraction. The methods described above may be used for sericin separation, with the advantage that non-heat treated cocoons will contain significantly less worm debris.

[0217] Dialysis may be used to remove the dissolution solvent from the resulting dissolved fibroin protein fragment solution by dialyzing the solution against a volume of water, step EL Pre-filtration prior to dialysis is helpful to remove any debris (i.e. , silkworm remnants) from the silk and LiBr solution, step D. In one example, a 3 pm or 5 pm filter is used with a flow-rate of 200-300 mL / min to filter a 0.1% to 1.0% silk-LiBr solution prior to dialysis and potential concentration if desired. A method

[0218] 48

[0219] DBl / 165497080.5disclosed herein, as described above, is to use time and / or temperature to decrease the concentration from 9.3 M LiBr to a range from 0.1 M to 9.3 M to facilitate filtration and downstream dialysis, particularly when considering creating a scalable process method. Alternatively, without the use of additional time or temperate, a 9.3 M LiBr- silk protein fragment solution may be diluted with water to facilitate debris filtration and dialysis. The result of dissolution at the desired time and temperate filtration is a translucent particle-free room temperature shelf-stable silk protein fragment-LiBr solution of a known MW and polydispersity. It is advantageous to change the dialysis water regularly until the solvent has been removed (e.g., change water after 1 hour. 4 hours, and then every 12 hours for a total of 6 water changes). The total number of water volume changes may be varied based on the resulting concentration of solvent used for silk protein dissolution and fragmentation. After dialysis, the final silk solution maybe further filtered to remove any remaining debris (i. e. , silk worm remnants).

[0220] Alternatively, Tangential Flow Filtration (TFF). which is a rapid and efficient method for the separation and purification of biomolecules, may be used to remove the solvent from the resulting dissolved fibroin solution, step E2. TFF offers a highly pure aqueous silk protein fragment solution and enables scalability of the process in order to produce large volumes of the solution in a controlled and repeatable manner. The silk and LiBr solution may be diluted prior to TFF (20 % down to 0.1 % silk in either water or LiBr). Pre-filtration as described above prior to TFF processing may maintain filter efficiency and potentially avoids the creation of silk gel boundary layers on the filter's surface as the result of the presence of debris particles. Pre- filtration prior to TFF is also helpful to remove any remaining debris (i.e., silk worm remnants) from the silk and LiBr solution that may cause spontaneous or long-term gelation of the resulting water only solution, step D. TFF, recirculating or single pass, may be used for the creation of water-silk protein fragment solutions ranging from 0.1 % silk to 30.0 % silk (more preferably, 0.1 % - 6.0 % silk). Different cutoff size TFF membranes may be required based upon the desired concentration, molecular weight and poly dispersity of the silk protein fragment mixture in solution. Membranes 49

[0221] DBl / 165497080.5ranging from 1-100 kDa may be necessary’ for varying molecular weight silk solutions created for example by varying the length of extraction boil time or the time and temperate in dissolution solvent (e.g., LiBr). In an embodiment, a TFF 5 or 10 kDa membrane is used to purify the silk protein fragment mixture solution and to create the final desired silk-to-water ratio. As well, TFF single pass, TFF, and other methods known in the art, such as a falling film evaporator, may be used to concentrate the solution following removal of the dissolution solvent (e.g., LiBr) (with resulting desired concentration ranging from 0.1% to 30 % silk). This can be used as an alternative to standard HFIP concentration methods known in the art to create a waterbased solution. A larger pore membrane could also be utilized to filter out small silk protein fragments and to create a solution of higher molecular weight silk with and / or without tighter poly dispersity values.

[0222] An assay for LiBr and Na2COs detection can be performed using an HPLC system equipped with evaporative light scattering detector (ELSD). The calculation was performed by linear regression of the resulting peak areas for the analyte plotted against concentration. More than one sample of a number of formulations of the present disclosure was used for sample preparation and analysis. Generally, four samples of different formulations were weighed directly in a 10 mL volumetric flask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH 3.0) and kept at 2-8 °C for 2 hours with occasional shaking to extract 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 HPLC vials and injected into the HPLC-ELSD system for the estimation of sodium carbonate and lithium bromide.

[0223] The analytical method developed for the quantitation of Na2COs and LiBr in silk protein formulations w as found to be linear in the range 10 - 165 pg / mL, with RSD for injection precision as 2% and 1% for area and 0.38% and 0.19% for retention time for sodium carbonate and lithium bromide respectively . The analytical method

[0224] 50

[0225] DBl / 165497080.5can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein formulations.

[0226] Fig. 6 is a flow chart showing various parameters that can be modified during the process of producing a silk protein fragment solution of the present disclosure during the extraction and the dissolution steps. Select method parameters may be altered to achieve distinct final solution characteristics depending upon the intended use, e.g., molecular weight and poly dispersity. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure.

[0227] In an embodiment, silk protein fragment solutions useful for a wide variety7of applications are prepared according to the following steps: forming pieces of silk cocoons from the Bombyx mori silkworm; extracting the pieces at about 100 °C in a Na2CO3 water solution for about 60 minutes, wherein a volume of the water equals about 0.4 x raw silk weight and the amount of Na2CO3 is about 0.848 x the weight of the pieces to form a silk fibroin extract; triple rinsing the silk fibroin extract at about 60 °C for about 20 minutes per rinse in a volume of rinse water, wherein the rinse water for each cycle equals about 0.2 L x the weight of the pieces; removing excess water from the silk fibroin extract; drying the silk fibroin extract; dissolving the dry7silk fibroin extract in a LiBr solution, wherein the LiBr solution is first heated to about 100 °C to create a silk and LiBr solution and maintained; placing the silk and LiBr solution in a dry oven at about 100 °C for about 60 minutes to achieve complete dissolution and further fragmentation of the native silk protein structure into mixture with desired molecular weight and poly dispersity; 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 solvent from the solution using Tangential Flow Filtration (TFF). In an embodiment, a 10 kDa membrane is utilized to purify the silk solution and create the final desired silk-to-water ratio. TFF can then be used to further concentrate the silk solution to a concentration of 2.0 wt. % silk in water.

[0228] 51

[0229] DBl / 165497080.5Without wishing to be bound by any particular theory, varying extraction (i.e., time and temperature), LiBr (i.e., temperature of LiBr solution when added to silk fibroin extract or vice versa) and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, homogeneities, and colors. Also, without wishing to be bound by any particular theory, increasing the temperature for extraction, lengthening the extraction time, using a higher temperature LiBr solution at emersion and over time when dissolving the silk and increasing the time at temperature (e.g., in an oven as shown here, or an alternative heat source) all resulted in less viscous and more homogeneous solvent and silk solutions.

[0230] The extraction step could be completed in a larger vessel, for example an industrial washing machine where temperatures at or in between 60 °C to 100 °C can be maintained. The rinsing step could also be completed in the industrial w ashing machine, eliminating the manual rinse cycles. Dissolution of the silk in LiBr solution could occur in a vessel other than a convection oven, for example a stirred tank reactor. Dialyzing the silk through a series of water changes is a manual and time intensive process, which could be accelerated by changing certain parameters, for example diluting the silk solution prior to dialysis. The dialysis process could be scaled for manufacturing by using semi-automated equipment, for example a tangential flow filtration system.

[0231] Varying extraction (i.e., time and temperature), LiBr (i.e., temperature of LiBr solution when added to silk fibroin extract or vice versa) and dissolution (i.e., time and temperature) parameters results in solvent and silk solutions with different viscosities, homogeneities, and colors. Increasing the temperature for extraction, lengthening the extraction time, using a higher temperature LiBr solution at emersion and over time when dissolving the silk and increasing the time at temperature (e.g.. in an oven as shown here, or an alternative heat source) all resulted in less viscous and more homogeneous solvent and silk solutions. While almost all parameters resulted in a viable silk solution, methods that allow' complete dissolution to be achieved in fewer than 4 to 6 hours are preferred for process scalability.

[0232] 52

[0233] DBl / 165497080.5In an embodiment, solutions of silk fibroin protein fragments having a weight average selected from between about 6 kDa to about 17 kDa are prepared according to 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 to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising non- detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 60 °C to about 140 °C; maintaining the solution of silk fibroin-lithium bromide in an oven having a temperature of about 140 °C for a period of at most 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 between about 6 kDa to about 17 kDa, and a poly dispersity of between 1 and about 5, or between about 1.5 and 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 comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The aqueous solution of silk fibroin protein fragments may be lyophilized. In some embodiments, the silk fibroin protein fragment solution may be further processed into various forms including gel, powder, and nanofiber.

[0234] In an embodiment, solutions of silk fibroin protein fragments having a weight average molecular weight selected from between about 17 kDa to about 39 kDa are 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 between about 30 minutes to about 60 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin;

[0235] 53

[0236] DBl / 165497080.5draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80 °C to about 140 °C; maintaining the solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60 °C to about 100 °C for a period of at most 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk fibroin protein fragments, wherein the aqueous solution of silk fibroin protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, wherein the aqueous solution of silk protein fragments comprises sodium carbonate residuals of between about 10 ppm and about 100 ppm, wherein the aqueous solution of silk fibroin protein fragments comprises fragments having a weight average molecular weight selected from between about 17 kDa to about 39 kDa, and a poly dispersity of between 1 and about 5, or between about 1.5 and 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 comprise lithium bromide residuals of less than 300 ppm as measured using a high- performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay.

[0237] 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 to about 17 kDa includes the steps of degumming a silk source by adding the silk source to a boiling (100 °C) aqueous solution of sodium carbonate for a treatment time of betw een about 30 minutes to about 60 minutes; removing sericin from the solution to produce a silk fibroin extract comprising non- detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 60 °C to about 140 °C; maintaining the solution of silk fibroin- 54

[0238] DBl / 165497080.5lithium bromide 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 between about 6 kDato about 17 kDa, and a poly dispersity of between 1 and about 5, or between about 1.5 and 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 comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay . The aqueous solution of pure silk fibroin protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high- performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous solution of pure silk fibroin protein fragments. The method may further comprise 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 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 comprise 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 comprise adding hyaluronic acid or its salt form 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 comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50,0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin protein fragments. A gel may be fabricated from the aqueous solution of pure silk 55

[0239] DBl / 165497080.5fibroin protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2 % and a vitamin content of at least 20 %.

[0240] 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 to about 39 kDa includes the steps of: adding a silk source to a boiling (100 °C) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non- detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80 °C to about 140 °C; maintaining the solution of silk fibroinlithium bromide in a dry oven having a temperature in the range between 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 pure silk fibroin protein fragments, wherein the aqueous solution of pure silk fibroin protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, wherein the aqueous solution of silk protein fragments comprises sodium carbonate residuals of between about 10 ppm and about 100 ppm, wherein the aqueous solution of pure silk fibroin protein fragments comprises fragments having an average weight average molecular weight selected from between about 17 kDa to about 39 kDa, and a polydispersity of between 1 and about 5, or between about 1.5 and 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 comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of pure silk fibroin protein fragments may comprise sodium carbonate residuals of less than 100 ppm as measured using a high-performance liquid chromatography sodium carbonate assay. The method may further comprise adding a therapeutic agent to the aqueous 56

[0241] DBl / 165497080.5solution of pure silk fibroin protein fragments. The method may further comprise 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 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 comprise 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 comprise adding hyaluronic acid or its salt form 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 comprise adding at least one of zinc oxide or titanium dioxide. A film may be fabricated from the aqueous solution of pure silk fibroin protein fragments produced by this method. The film may comprise from about 1 ,0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 wt. % to about 20.0 wt. %. The film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2% and a vitamin content of at least 20%.

[0242] In an embodiment, solutions of silk fibroin protein fragments having a weight average molecular weight selected from between about 39 kDa to about 80 kDa are 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 so as to result in degumming; removing sericin from the solution to produce a silk fibroin extract comprising non-detectable levels of sericin; draining the solution from the silk fibroin extract; dissolving the silk fibroin extract in a solution of lithium bromide having a starting temperature upon placement of the silk fibroin extract in the lithium bromide solution that ranges from about 80 °C to about 140 °C; maintaining the 57

[0243] DBl / 165497080.5solution of silk fibroin-lithium bromide in a dry oven having a temperature in the range between about 60 °C to about 100 °C for a period of at most 1 hour; removing the lithium bromide from the silk fibroin extract; and producing an aqueous solution of silk fibroin protein fragments, wherein the aqueous solution of silk fibroin protein fragments comprises lithium bromide residuals of between about 10 ppm and about 300 ppm, sodium carbonate residuals of between about 10 ppm and about 100 ppm, fragments having a weight average molecular weight selected from between about 39 kDa to about 80 kDa, and a poly dispersity of between 1 and about 5, or between about 1.5 and 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 comprise lithium bromide residuals of less than 300 ppm as measured using a high-performance liquid chromatography lithium bromide assay. The aqueous solution of silk fibroin protein fragments may comprise sodium carbonate residuals of less than 100 ppm 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., 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 ly ophilized. The method may further comprise 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 comprise adding hyaluronic acid or its salt form 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 fabricated from the aqueous solution of pure silk fibroin protein fragments produced by this method. The film may comprise from about 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivative thereof. The film may have a water content ranging from about 2.0 w t. % to about 20.0 wt. %. The 58

[0244] DBl / 165497080.5film may comprise from about 30.0 wt. % to about 99.5 wt. % of pure silk fibroin protein fragments. A gel may be fabricated from the aqueous solution of pure silk fibroin protein fragments produced by this method. The gel may comprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivative thereof. The gel may have a silk content of at least 2 wt. % and a vitamin content of at least 20 wt. %.

[0245] Molecular weight of the silk protein fragments may be controlled based upon the specific parameters utilized during the extraction step, including extraction time and temperature; specific parameters utilized during the dissolution step, including the LiBr temperature at the time of submersion of the silk in to the lithium bromide and time that the solution is maintained at specific temperatures; and specific parameters utilized during the filtration step. By controlling process parameters using the disclosed methods, it is possible to create silk fibroin protein fragment solutions with poly dispersity equal to or lower than 2.5 at a variety of different molecular weight selected from between 5 kDa to 200 kDa, or between 10 kDa and 80 kDa. By altering process parameters to achieve silk solutions with different molecular weights, a range of fragment mixture end products, with desired poly dispersity' of equal to or less than 2.5 may be targeted based upon the desired performance requirements. For example, a higher molecular weight silk film containing an ophthalmic drug may have a controlled slow release rate compared to a lower molecular weight film making it ideal for a delivery vehicle in eye care products. Additionally, the silk fibroin protein fragment solutions with a polydispersity of greater than 2.5 can be achieved. Further, two solutions with different average molecular weights and poly dispersity can be mixed to create combination solutions. Alternatively, a liquid silk gland (100% sericin free silk protein) that has been removed directly from a worm could be used in combination with any of the silk fibroin protein fragment solutions of the present disclosure. 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).

[0246] 59

[0247] DBl / 165497080.5Differences in the processing parameters can result in regenerated silk fibroins that vary in molecular weight, and peptide chain size distribution (poly dispersity7, PD). This, in turn, influences the regenerated silk fibroin performance, including mechanical strength, water solubility7etc.

[0248] Parameters were varied during the processing of raw silk cocoons into the silk solution. Varying these parameters affected the MW of the resulting silk solution. Parameters manipulated included (i) time and temperature of extraction, (ii) temperature of LiBr, (iii) temperature of dissolution oven, and (iv) dissolution time. Experiments were carried out to determine the effect of vary ing the extraction time.

[0249] Tables 1-7 summarize the results. Below is a summary:

[0250] - A sericin extraction time of 30 minutes resulted in larger molecular weight than a sericin extraction time of 60 minutes

[0251] - Molecular weight decreases with time in the oven

[0252] - 140 °C LiBr and oven resulted in the low end of the confidence inter al to be below a molecular weight of 9500 Da

[0253] - 30 min extraction at the 1 hour and 4 hour time points have undigested silk - 30 min extraction at the 1 hour time point resulted in a significantly high molecular weight with the low end of the confidence interval being 35,000 Da - The range of molecular weight reached for the high end of the confidence interval was 18000 to 216000 Da (important for offering solutions with specified upper limit).

[0254]

[0255] 60

[0256] DBl / 165497080.5

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] DBl / 165497080.5

[0263]

[0264]

[0265] Experiments were carried out to determine the effect of varying the extraction temperature. Table 7 summarizes the results. Below is a summary:

[0266] - Sericin extraction at 90 °C resulted in higher MW than sericin extraction at 100 °C extraction

[0267] - Both 90 °C and 100 °C show decreasing MW over time in the oven.

[0268]

[0269] 62

[0270] DBl / 165497080.5Experiments were carried out to determine the effect of varying the Lithium Bromide (LiBr) temperature when added to silk. Tables 8-9 summarize the results. Below is a summary :

[0271] — No impact on molecular weight or confidence interval (all CI -10500-6500 Da)

[0272] - Studies illustrated that the temperature of LiBr-silk dissolution, as LiBr is added and begins dissolving, rapidly drops below the original LiBr temperature due to the majority of the mass being silk at room temperature

[0273]

[0274] 63

[0275] DBl / 165497080.5

[0276]

[0277] Experiments were carried out to determine the effect of v oven / dissolution temperature. Tables 10-14 summarize the results. Below is a summary’:

[0278] - Oven temperature has less of an effect on 60 min extracted silk than 30 min extracted silk. Without wishing to be bound by theory’, it is believed that the 30 min silk is less degraded during extraction and therefore the oven temperature has more of an effect on the larger MW, less degraded portion of the silk.

[0279] - For 60 °C vs. 140 °C oven the 30 min extracted silk showed a very’ significant effect of lower MW at higher oven temp, while 60 min extracted silk had an effect but much less

[0280] - The 140 °C oven resulted in a low end in the confidence interval at -6000 Da.

[0281]

[0282] 64

[0283] DBl / 165497080.5

[0284]

[0285]

[0286]

[0287]

[0288] 65

[0289] DBl / 165497080.5

[0290]

[0291] The raw silk cocoons from the silkworm Bombyx mori was cut into pieces. The pieces of raw silk cocoons were boiled in an aqueous solution of Na2COs (about 100 °C) for a period of time between about 30 minutes to about 60 minutes to remove sericin (degumming). The volume of the water used equals about 0.4 x raw silk weight and the amount of Na2COs is about 0.848 x the weight of the raw silk cocoon pieces. The resulting degummed silk cocoon pieces were rinsed with deionized water three times at about 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. The excess water from the degummed silk cocoon pieces was removed. After the DI water washing step, the wet degummed silk cocoon pieces were dried at room temperature. The degummed silk cocoon pieces were mixed with a LiBr solution, and the mixture was heated to about 100 °C. The warmed mixture was placed in a dry oven and was heated at a temperature ranging from about 60 °C to about 140 °C for about 60 minutes to achieve complete dissolution of the native silk protein. The resulting solution was allowed to cool to room temperature and then was dialyzed to remove LiBr salts using a 3.500 Da MWCO membrane. Multiple exchanges were performed in Di water until Br ions were less than 1 ppm as determined in the hydrolyzed fibroin solution read on an Oakton Bromide (Br ) double-junction ion-selective electrode.

[0292] The resulting silk fibroin aqueous solution has a concentration of about 8.0 % w / v containing pure silk fibroin protein fragments having an average weight average 66

[0293] DBl / 165497080.5molecular weight selected from between about 6 kDato about 16 kDa, about 17 kDa to about 39 kDa, and about 39 kDa to about 80 kDa and a poly dispersity of between about 1.5 and about 3.0. The 8.0 % w / v was diluted with DI water to provide a 1.0 % w / v, 2.0 % w / v, 3.0 % w / v, 4.0 % w / v, 5.0 % w / v by the coating solution.

[0294] A variety of % 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. Solution is diafdtered in the TFF to remove lithium bromide. Once below a specified level of residual LiBr, solution undergoes ultrafiltration to increase the concentration through removal of water. See examples below.

[0295] Six (6) silk solutions were utilized in standard silk structures with the following results:

[0296] Solution #1 is a silk concentration of 5.9 wt. %, average MW of 19.8 kDa and 2.2 PDI (made with a 60 min boil extraction, 100 °C LiBr dissolution for 1 hour).

[0297] Solution #2 is a silk concentration of 6.4 wt. % (made with a 30 min boil extraction, 60 °C LiBr dissolution for 4 hrs).

[0298] Solution #3 is a silk concentration of 6.17 wt. % (made with a 30 min boil extraction 100 °C LiBr dissolution for 1 hour).

[0299] Solution #4 is a silk concentration of 7.30 wt. %: A 7.30 % silk solution was produced beginning with 30 minute extraction batches of 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100 °C 9.3 M LiBr in a 100 °C oven for 1 hour. 100 g of silk fibers were dissolved per batch to create 20% silk in LiBr. Dissolved silk in LiBr was then diluted to 1 % silk and filtered through a 5 pm filter to remove large debris. 15,500 mL of 1 %, filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 1300 mL. 1262 mL of 7.30 % silk was then collected. Water was added to the feed to help remove the remaining solution and 547 mL of 3.91 % silk was then collected.

[0300] 67

[0301] DBl / 165497080.5Solution #5 is a silk concentration of 6.44 wt. %: A 6.44 wt. % silk solution was produced beginning with 60 minute extraction batches of a mix of 25, 33, 50, 75 and 100 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100 °C 9.3 M LiBr in a 100 °C oven for 1 hour. 35, 42, 50 and 71 g per batch of silk fibers were dissolved to create 20 % silk in LiBr and combined. Dissolved silk in LiBr was then diluted to 1 % silk and filtered through a 5 pm filter to remove large debris.

[0302] 17,000 mL of 1 %, filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 3000 mL. 1490 mL of 6.44 % silk was then collected. Water was added to the feed to help remove the remaining solution and 1454 mL of 4.88 % silk was then collected.

[0303] Solution #6 is a silk concentration of 2.70 wt. %: A 2.70 % silk solution was produced beginning with 60-minute extraction batches of 25 g silk cocoons per batch. Extracted silk fibers were then dissolved using 100 °C 9.3 M LiBr in a 100 °C oven for 1 hour. 35.48 g of silk fibers were dissolved per batch to create 20 % silk in LiBr. Dissolved silk in LiBr was then diluted to 1% silk and filtered through a 5 pm filter to remove large debris. 1000 mL of 1%, filtered silk solution was used as the starting volume / diafiltration volume for TFF. Once LiBr was removed, the solution was ultrafiltered to a volume around 300 mL. 312 mL of 2.7 % silk was then collected.

[0304] The preparation of silk fibroin solutions with higher molecular weights is given in Table 15.

[0305] Table 15. Preparation and properties of silk fibroin solutions.

[0306]

[0307] DBl / 165497080.5

[0308]

[0309] Silk aqueous coating composition for application to fabrics are given in Tables 16 and 17 below.

[0310]

[0311]

[0312] DBl / 165497080.5

[0313]

[0314] Three (3) silk solutions were utilized in film making with the following results:

[0315] Solution #1 is a silk concentration of 5.9 %, average MW of 19.8 kDa and 2.2 PD (made with a 60 min boil extraction, 100 °C LiBr dissolution for 1 hr).

[0316] Solution #2 is a silk concentration of 6.4 % (made with a 30 min boil extraction, 60 °C LiBr dissolution for 4 hrs).

[0317] Solution #3 is a silk concentration of 6.17 % (made with a 30 min boil extraction, 100 °C LiBr dissolution for 1 hour).

[0318] Films w ere made in accordance with Rockwood et al. (Nature Protocols; Vol. 6; No. 10; published on-line Sep. 22, 2011; doi: 10.1038 / nprot.2011.379). 4 rnL of 1% or 2% (wt / vol) aqueous silk solution was added into 100 mm Petri dish (Volume of silk can be varied for thicker or thinner films and is not critical) and allowed to dry overnight uncovered. The bottom of a vacuum desiccator w as filled with w ater. Dry films were placed in the desiccator and vacuum applied, allowing the films to water anneal for 4 hours prior to removal from the dish. Films cast from solution #1 did not result in a structurally continuous film; the film was cracked in several pieces. These pieces of film dissolved in w ater in spite of the w ater annealing treatment.

[0319] 70

[0320] DB1 / 165497080.5Silk 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 not intended to be limiting in application or formulation. Three (3) silk solutions were utilized in gel making with the following results:

[0321] Solution #1 is a silk concentration of 5.9 %, average MW of 19.8 kDa and 2.2 PD (made with a 60 min boil extraction, 100 °C LiBr dissolution for 1 hr).

[0322] Solution #2 is a silk concentration of 6.4 % (made with a 30 min boil extraction, 60 °C LiBr dissolution for 4 hrs).

[0323] Solution #3 is a silk concentration of 6.17 % (made with a 30 min boil extraction, 100 °C LiBr dissolution for 1 hour).

[0324] “Egel” is an electrogelation process as described in Rockwood of al. Briefly, 10 ml of aqueous silk solution is added to a 50 ml conical tube and a pair of platinum wire electrodes immersed into the silk solution. A 20 volt potential was applied to the platinum electrodes for 5 minutes, the power supply turned off and the gel collected. Solution #1 did not form an EGEL over the 5 minutes of applied electric current.

[0325] Solutions #2 and #3 were gelled in accordance with the published horseradish peroxidase (HRP) protocol. Behavior seemed typical of published solutions.

[0326] Materials and Methods: the following equipment and material are used in determination of Silk Molecular weight: Agilent 1100 with chemstation software ver.

[0327] 10.01; Refractive Index Detector (RID); analytical balance; volumetric flasks (1000 mL, 10 mL and 5 mL); HPLC grade water; ACS grade sodium chloride; ACS grade sodium phosphate dibasic heptahydrate; phosphoric acid; dextran MW Standards - Nominal Molecular Weights of 5 kDa, 11.6 kDa, 23.8 kDa, 48.6 kDa, and 148 kDa; 50 mL PET or polypropylene disposable centrifuge tubes; graduated pipettes; amber glass HPLC vials with Teflon caps; Phenomenex PolySep GFC P-4000 column (size: 7.8 mm x 300 mm).

[0328] Procedural Steps:

[0329] 71

[0330] DBl / 165497080.5A) Preparation of 1 L Mobile Phase (0.1 M Sodium Chloride solution in 0.0125 M Sodium phosphate buffer)

[0331] Take a 250 mL clean and dry beaker, place it on the balance and tare the weight. Add about 3.3509 g of sodium phosphate dibasic heptahydrate to the beaker. Note down the exact weight of sodium phosphate dibasic weighed. Dissolve the weighed sodium phosphate by adding 100 mL of HPLC water into the beaker. Take care not to spill any of the content of the beaker. Transfer the solution carefully into a clean and dry' 1000 mL volumetric flask. Rinse the beaker and transfer the rinse into the volumetric flask. Repeat the rinse 4-5 times. In a separate clean and dry 250 mL beaker weigh exactly about 5.8440 g of sodium chloride. 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 with phosphoric acid. Make up the volume in volumetric flask with HPLC water to 1000 mL and shake it vigorously to homogeneously mix the solution. Filter the solution through 0.45 pm polyamide membrane fdter. Transfer the solution to a clean and dry solvent bottle and label the bottle. The volume of the solution can be varied to the requirement by correspondingly varying the amount of sodium phosphate dibasic heptahydrate and sodium chloride.

[0332] B) Preparation of Dextran Molecular Weight Standard solutions At least five different molecular weight standards are used for each batch of samples that are run so that the expected value of the sample to be tested is bracketed by the value of the standard used. Label six 20 mL scintillation glass vials respective to the molecular weight standards. Weigh accurately about 5 mg of each of dextran molecular weight standards and record the w eights. Dissolve the dextran molecular weight standards in 5 mL of mobile phase to make a 1 mg / mL standard solution.

[0333] C) Preparation of Sample solutions

[0334] When preparing sample solutions, if there are limitations on how' much sample is available, the preparations may be scaled as long as the ratios are maintained.

[0335] 72

[0336] DBl / 165497080.5Depending on sample type and silk protein content in sample weigh enough sample in a 50 mL disposable centrifuge tube on an analytical balance to make a 1 mg / mL sample solution for analysis. Dissolve the sample in equivalent volume of mobile phase make a 1 mg / mL solution. Tightly cap the tubes and mix the samples (in solution). Leave the sample solution for 30 minutes at room temperature. Gently mix the sample solution again for 1 minute and centrifuge at 4000 RPM for 10 minutes.

[0337] D) HPLC analysis of the samples

[0338] Transfer 1.0 mL of all the standards and sample solutions into individual HPLC vials. Inject the molecular weight standards (one injection each) and each sample in duplicate. Analyze all the standards and sample solutions using the following HPLC conditions:

[0339]

[0340] E) Data analysis and calculations - Calculation of Average Molecular Weight using Cirrus Software

[0341] Upload the chromatography data files of the standards and the analytical samples into Cirrus SEC data collection and molecular weight analysis software. Calculate the weight average molecular weight (Mw), number average molecular weight (Mn), peak average molecular weight (MP), and poly dispersity for each injection of the sample.

[0342] DBl / 165497080.5Spider Silk Fragments

[0343] Spider silks are natural polymers that consist of three domains: a repetitive middle core domain that dominates the protein chain, and non-repetitive N-terminal and C-terminal domains. The large core domain is organized in a block copolymerlike arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides alternate. Dragline silk is the protein complex composed of major ampullate dragline silk protein 1 (MaSpl) and major ampullate dragline silk protein 2 (MaSp2), Both silks are approximately 3500 amino acid long. MaSpl can be found in the fibre core and the periphery, whereas MaSp2 forms clusters in certain core areas. The large central domains of MaSpl and MaSp2 are organized in block copolymer-like arrangements, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX) polypeptides alternate in core domain. Specific secondary structures have been assigned to poly(A) / (GA), GGX and GPGXX motifs including P-sheet, a-helix and - spiral respectively. The primary sequence, composition and secondary' structural elements of the repetitive core domain are responsible for mechanical properties of spider silks; whereas, non-repetitive N- and C-terminal domains are essential for the storage of liquid silk dope in a lumen and fiber formation in a spinning duct.

[0344] The main difference between MaSpl and MaSp2 is the presence of proline (P) residues accounting for 15% of the total amino acid content m MaSp2. whereas MaSpl is proline-free. By calculating the number of proline residues in N. clavipes dragline silk, it is possible to estimate the presence of the two proteins in fibers; 81% MaSpl and 19% MaSp2. Different spiders have different ratios of MaSpl andMaSp2. For example, a dragline silk fiber from the orb weaver Argiope aurantia contains 41% MaSpl and 59% MaSp2. Such changes in the ratios of major ampullate silks can dictate the performance of the silk fiber.

[0345] At least seven different ty pes of silk proteins are known for one orb-weaver species of spider. Silks differ in primary sequence, physical properties and functions. For example, dragline silks used to build frames, radii and lifelines are known for 74

[0346] DBl / 165497080.5outstanding mechanical properties including strength, toughness and elasticity. On an equal weight basis, spider silk has a higher toughness than steel and Kevlar.

[0347] Flageliform silk found in capture spirals has extensibility of up to 500%. Minor ampullate silk, which is found in auxiliary spirals of the orb-web and in prey wrapping, possesses high toughness and strength almost similar to major ampullate silks, but does not supercontract in water.

[0348] Spider silks are known for their high tensile strength and toughness. The recombinant silk proteins also confer advantageous properties to cosmetic or dermatological compositions, in particular to be able to improve the hydrating or softening action, good film forming property’ and low surface density. Diverse and unique biomechanical properties together with biocompatibility and a slow rate of degradation make spider silks excellent candidates as biomaterials for tissue engineering, guided tissue repair and drug delivery’, for cosmetic products (e.g., nail and hair strengthener, skin care products), and industrial materials (e.g. nanoyvires, nanofibers, surface coatings).

[0349] In an embodiment, a silk protein may include a polypeptide derived from natural spider silk proteins. The polypeptide is not limited particularly as long as it is derived from natural spider silk proteins, and examples of the polypeptide include natural spider silk proteins and recombinant spider silk proteins such as variants, analogs, derivatives or the like of the natural spider silk proteins. In terms of excellent tenacity’, the polypeptide may be derived from major dragline silk proteins produced in major ampullate glands of spiders. Examples of the major dragline silk proteins include major ampullate spidroin MaSpl and MaSp2 from Nephila clavipes, and ADF3 and ADF4 from Araneus diadematus, etc. Examples of the polypeptide derived from major dragline silk proteins include variants, analogs, derivatives or the like of the major dragline silk proteins. Further, the polypeptide may be derived from flagelliform silk proteins produced in flagelliform glands of spiders. Examples of the flagelliform silk proteins include flagelliform silk proteins derived from Nephila clavipes, etc.

[0350] 75

[0351] DBl / 165497080.5Examples of the polypeptide derived from major dragline silk proteins include a polypeptide containing two or more units of an amino acid sequence represented by the formula 1: REP1-REP2 (1), preferably a polypeptide containing five or more units thereof and more preferably a polypeptide containing ten or more units thereof. Alternatively, the polypeptide derived from major dragline silk proteins may be a polypeptide that contains units of the amino acid sequence represented by the formula 1 : REP1-REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9.051,453 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No. 9,051,453. In the polypeptide derived from major dragline silk proteins, units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) may be the same or may be different from each other. In the case of producing a recombinant protein using a microbe such as Escherichia coh as a host, the molecular weight of the polypeptide derived from major dragline silk proteins is 500 kDa or less, or 300 kDa or less, or 200 kDa or less, in terms of productivity.

[0352] In the formula (1), the REP1 indicates poly alanine. In the REP1, the number of alanine residues arranged in succession is preferably 2 or more, more preferably 3 or more, further preferably 4 or more, and particularly preferably 5 or more. Further, in the REP1, the number of alanine residues arranged in succession is preferably 20 or less, more preferably 16 or less, further preferably 12 or less, and particularly preferably 10 or less. In the formula (1), the REP2 is an amino acid sequence composed 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 with respect to the total number of amino acid residues contained therein.

[0353] In the major dragline silk, the REP1 corresponds to a crystal region in a fiber where a cry stal sheet is formed, and the REP2 corresponds to an amorphous region in a fiber where most of the parts lack regular configurations and that has more

[0354] 76

[0355] DBl / 165497080.5flexibility. Further, the [REP1-REP2] corresponds to a repetitious region (repetitive sequence) composed of the crystal region and the amorphous region, which is a characteristic sequence of dragline silk proteins.

[0356] Recombinant Silk Fragments

[0357] In some embodiments, the recombinant silk protein refers to recombinant spider silk polypeptides, recombinant insect silk polypeptides, or recombinant mussel silk polypeptides. In some embodiments, the recombinant silk protein fragment disclosed herein include recombinant spider silk polypeptides of Araneidae or Araneoids, or recombinant insect silk polypeptides of Bombyx mori. In some embodiments, the recombinant silk protein fragment disclosed herein include recombinant spider silk polypeptides of Araneidae or Araneoids. In some embodiments, the recombinant silk protein fragment disclosed herein include block copolymer having repetitive units derived from natural spider silk polypeptides of Araneidae or Araneoids . In some embodiments, the recombinant silk protein fragment disclosed herein include block copolymer having synthetic repetitive units derived from spider silk polypeptides i Araneidae ox Araneoids and non-repetitive units derived from natural repetitive units of spider silk polypeptides of Araneidae or Araneoids.

[0358] Recent advances in genetic engineering have provided a route to produce various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein “recombinant silk protein” refers to synthetic proteins produced heterologously in prokaryotic or eukaryotic expression systems using genetic engineering methods.

[0359] Various methods for synthesizing recombinant silk peptides are known and have been described by Ausubel et al., Current Protocols in Molecular Biology § 8 (John Wiley & Sons 1987, (1990)), incorporated herein by reference. A gramnegative. rod-shaped bacterium E. coll is a well-established host for industrial scale production of proteins. Therefore, the majority of recombinant silks have been

[0360] 77

[0361] DBl / 165497080.5produced in E. coll. E. coll which is easy to manipulate, has a short generation time, is relatively low cost and can be scaled up for larger amounts protein production.

[0362] The recombinant silk proteins can be produced by transformed prokaryotic or eukaryotic systems containing the cDNA coding for a silk protein, for a fragment of this protein or for an analog of such a protein. The recombinant DNA approach enables the production of recombinant silks with programmed sequences, secondary structures, architectures and precise molecular weight. There are four main steps in the process: (i) design and assembly of synthetic silk-like genes into genetic ‘cassettes’, (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 the selected clones.

[0363] The term “recombinant vectors”, as used herein, includes any vectors known to the skilled person including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). Said vectors include expression as well as cloning vectors. Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences necessary7for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, or plant) or in in vitro expression systems. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.

[0364] The prokaryotic systems include Gram-negative bacteria or Gram-positive bacteria. The prokary otic expression vectors can include an origin of replication which can be recognized by the host organism, a homologous or heterologous promoter which is functional in the said host, the DNA sequence coding for the spider silk protein, for a fragment of this protein or for an analogous protein. Nonlimiting examples of prokaryotic expression organisms are Escherichia coli, Bacillus subtilis,

[0365] 78

[0366] DBl / 165497080.5Bacillus megaterium, Corynebacterium glutamicum. Anabaena, Caulobacter.

[0367] Gluconobacter, Rhodobacter, Pseudomonas, Para coccus, Bacillus (e.g. Bacillus subtilis) Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium) , Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Propionibacterium, Staphylococcus or Streptomyces cells.

[0368] The eukaryotic systems include yeasts and insect, mammalian or plant cells. In this case, the expression vectors can include a yeast plasmid origin of replication or an autonomous replication sequence, a promoter, a DNA sequence coding for a spider silk protein, for a fragment or for an analogous protein, a polyadenylation sequence, a transcription termination site and, lastly, a selection gene. Nonlimiting examples of eukaryotic expression organisms include yeasts, such as Saccharomyces cerevisiae. Pichia pastoris, basidiosporogenous, ascosporogenous, filamentous fungi, such as Aspergillus niger, Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum, Candida, Hansenula, Kluyveromyces, Saccharomyces (e.g. Saccharomyces cerevisiae). Schizosaccharomyces, Pichia (e.g. Pichia pastoris) or Yarrowia cells etc., mammalian cells, such as HeLa cells, COS cells, CHO cells etc., insect cells, such as Sf9 cells, MEL cells, etc., “insect host cells’’ such as Spodoptera frugiperda or Trichoplusia ni cells. SF9 cells, SF-21 cells or High-Five cells, wherein SF-9 and SF-21 are ovarian cells from Spodoptera frugiperda, and High-Five cells are egg cells from Trichoplusia ni., “plant host cells”, such as tobacco, potato or pea cells.

[0369] A variety of heterologous host systems have been explored to produce different types of recombinant silks. Recombinant partial spidroins as well as engineered silks have been cloned and expressed in bacteria (Escherichia coli), yeast Pichia pastoris), insects (silkworm larvae), plants (tobacco, soybean, potato, Arabidopsis), mammalian cell lines (BHT / hamster) and transgenic animals (mice, goats). Most of the silk proteins are produced with an N- or C-terminal His-tags to make purification simple and produce enough amounts of the protein.

[0370] 79

[0371] DBl / 165497080.5In some embodiments, the host suitable for expressing the recombinant spider silk protein using heterogeneous system may include transgenic animals and plants. In some embodiments, the host suitable for expressing the recombinant spider silk protein using heterogeneous system comprises bacteria, yeasts, mammalian cell lines. In some embodiments, the host suitable for expressing the recombinant spider silk protein using heterogeneous system comprises E. coll. In some embodiments, the host suitable for expressing the recombinant spider silk protein using heterogeneous system comprises transgenic B. mori silkworm generated using genome editing technologies (e.g., CRISPR).

[0372] The recombinant silk protein in this disclosure comprises synthetic proteins which are based on repeat units of natural silk proteins. Besides the synthetic repetitive silk protein sequences, these can additionally comprise one or more natural nonrepetitive silk protein sequences.

[0373] In some embodiments, “recombinant silk protein” refers to recombinant silkworm silk protein or fragments thereof. The recombinant production of silk fibroin and silk sericin has been reported. A variety of hosts are used for the production including E. coli, Sacchromyces cerevisiae. Pseudomonas sp. , Rhodopseudomonas sp. , Bacillus sp., and Strepomyces . See EP 0230702, which is incorporate by reference herein by its entirety7.

[0374] Provided herein also include design and biological-synthesis of silk fibroin protein-like multiblock polymer comprising GAGAGX (SEQ ID NO:1) hexapeptide (X is A, Y, V or S) derived from the repetitive domain of B. mori silk heavy chain (H chain) [SEQ ID NO: 49],

[0375] In some embodiments, this disclosure provides silk protein-like multiblock polymers derived from the repetitive domain of B. mori silk heavy chain (H chain) comprising the GAGAGS (SEQ ID NO:2) hexapeptide repeating units. The GAGAGS (SEQ ID NO:2) hexapeptide is the core unit of H-chain and plays an important role in the formation of crystalline domains. The silk protein-like multiblock polymers containing the GAGAGS (SEQ ID NO:2) hexapeptide repeating 80

[0376] DBl / 165497080.5units spontaneously aggregate into 0-sheet structures, similar to natural silk fibroin protein, where in the silk protein-like multiblock polymers having any weight average molecular weight described herein.

[0377] In some embodiments, this disclosure provides silk-peptide like multiblock copolymers composed of the GAGAGS (SEQ ID NO:2) hexapeptide repetitive fragment derived from H chain of B. mori silk heavy chain and mammalian elastin VPGVG (SEQ ID NO:3) motif produced by E. coli. In some embodiments, this disclosure provides fusion silk fibroin proteins composed of the GAGAGS (SEQ ID NO:2) hexapeptide repetitive fragment derived from H chain of B. mori silk heavy chain and GVGVP (SEQ ID NO:4) produced by E. coli, where in the silk protein-like multiblock polymers having any weight average molecular weight described herein.

[0378] In some embodiments, this disclosure provides B. mori silkworm recombinant proteins composed of the (GAGAGS)ie (SEQ ID NO:5) repetitive fragment. In some embodiments, this disclosure provides recombinant proteins composed of the (GAGAGS)is (SEQ ID NO:5) repetitive fragment and the non-repetitive (GAGAGS)16 -F-COOH, (GAGAGS)16 -F-F-COOH. (GAGAGS)16 -F-F-F-COOH, (GAGAGS)16 -F-F-F-F-COOH, (GAGAGS)16 -F-F-F-F-F-F-F-F-COOH, (GAGAGS)ie -F-F-F-F-F-F-F-F-F-F-F-F-COOH produced by E. coli, where F has the following amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG (SEQ ID NO: 6), and where in the silk protein-like multiblock polymers having any weight average molecular weight described herein.

[0379] In some embodiments, '‘recombinant silk protein” refers to recombinant spider silk protein or fragments thereof. The productions of recombinant spider silk proteins based on a partial cDNA clone have been reported. The recombinant spider silk proteins produced as such comprise a portion of the repetitive sequence derived from a dragline spider silk protein, Spidroin 1, from the spider Nephila clavipes. see Xu et al. (Proc. Natl. Acad. Sci. U.S.A., 87:7120-7124 (1990). cDNA clone encoding a portion of the repeating sequence of a second fibroin protein, Spidroin 2, from

[0380] 81

[0381] DBl / 165497080.5dragline silk of Nephila clavipes and the recombinant synthesis thereof is described in J. Biol. Chem., 1992, volume 267, pp. 19320-19324. The recombinant synthesis of spider silk proteins including protein fragments and variants of Nephila clavipes from transformed E. coli is described in U.S. Pat. Nos. 5,728.810 and 5,989,894. cDNA clones encoding minor ampullate spider silk proteins and the expression thereof is described in U.S. Pat. Nos. 5,733,771 and 5,756,677. cDNA clone encoding the flagelliform silk protein from an orb-web spinning spider is described in U.S. Pat. No.

[0382] 5,994,099. U.S. Pat. No. 6.268,169 describes the recombinant synthesis of spider silk like proteins derived from the repeating peptide sequence found in the natural spider dragline of Nephila clavipes by E. coli. Bacillus subtilis. and Pichia pastoris recombinant expression systems. WO 03 / 020916 describes the cDNA clone encoding and recombinant production of spider spider silk proteins having repeative sequences derived from the major ampullate glands of Nephila madagascariensis . Nephila senegalensis, Tetragnatha kauaiensis, Tetragnatha versicolor. Argiope aurantia, Argiope trifasciata, Gasteracantha mammosa, and Latrodectus geomelricus. the flagelliform glands of Argiope trifasciata, the ampullate glands of Dolomedes tenebrosus. two sets of silk glands from Plectreurys tristis, and the silk glands of the mygalomorph Euagrus chisoseus. Each of the above reference is incorporated herein by reference in its entirety.

[0383] In some embodiments, the recombinant spider silk protein is a hybrid protein of a spider silk protein and an insect silk protein, a spider silk protein and collagen, a spider silk protein and resilin, or a spider silk protein and keratin. The spider silk repetitive unit comprises or consists of an amino acid sequence of a region that comprises or consists of at least one peptide motif that repetitively occurs within a naturally occurring major ampullate gland polypeptide, such as a dragline spider silk polypeptide, a minor ampullate gland polypeptide, a flagelliform polypeptide, an aggregate spider silk polypeptide, an aciniform spider silk polypeptide or a pyriform spider silk polypeptide.

[0384] 82

[0385] DBl / 165497080.5In some embodiments, the recombinant spider silk protein in this disclosure comprises synthetic spider silk proteins derived from repetitive units of natural spider silk proteins, consensus sequence, and optionally one or more natural non-repetitive spider silk protein sequences. The repeated units of natural spider silk polypeptide may include dragline spider silk polypeptides or flagelliform spider silk polypeptides of Araneidae or Arcmeoids.

[0386] As used herein, the spider silk "‘repetitive unit” comprises or consists of at least one peptide motif that repetitively occurs within a naturally occurring major ampullate gland polypeptide, such as a dragline spider silk polypeptide, a minor ampullate gland polypeptide, a flagelliform polypeptide, an aggregate spider silk polypeptide, an aciniform spider silk polypeptide or a pyriform spider silk polypeptide. A “repetitive unit” refers to a region which corresponds in amino acid sequence to a region that comprises or consists of at least one peptide motif (e.g. AAAAAA) (SEQ ID NO:21) or GPGQQ (SEQ ID NO: 16)) that repetitively occurs within a naturally occurring silk polypeptide (e.g. MaSpI. ADF-3, ADF-4, or Flag) (i.e. identical amino acid sequence) or to an amino acid sequence substantially similar thereto (i.e. variational amino acid sequence). A “repetitive unit” having an amino acid sequence which is “substantially similar” to a corresponding amino acid sequence within a naturally occurring silk polypeptide (i.e. wild-type repetitive unit) is also similar with respect to its properties, e.g. a silk protein comprising the “substantially similar repetitive unit” is still insoluble and retains its insolubility. A “repetitive unit” having an amino acid sequence which is “identical” to the amino acid sequence of a naturally occurring silk polypeptide, for example, can be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII, ADF-3 and / or ADF-4. A “repetitive unit” having an amino acid sequence which is “substantially similar” to the amino acid sequence of a naturally occurring silk polypeptide, for example, can be a portion of a silk polypeptide corresponding to one or more peptide motifs of MaSpI, MaSpII. ADF-3 and / or ADF-4. but having one or more amino acid substitution at specific amino acid positions.

[0387] 83

[0388] DBl / 165497080.5As used herein, the term ’‘consensus peptide sequence’’ refers to an amino acid sequence which contains amino acids which frequently occur in a certain position (e.g., “G”) and wherein, other amino acids which are not further determined are replaced by the place holder “X”. In some embodiments, the consensus sequence is at least one of (i) GPGXX (SEQ ID NO:7), wherein X is an amino acid selected from A, S, G, Y, P and Q; (ii) GGX, wherein X is an amino acid selected from Y, P, R, S, A, T, N and Q, preferably Y, P and Q; (iii) Ax, wherein x is an integer from 5 to 10.

[0389] The consensus peptide sequences GPGXX (SEQ ID NO:7) and GGX, i.e., glycine rich motifs, provide flexibility to the silk polypeptide and thus, to the thread formed from the silk protein containing said motifs. In detail, the iterated GPGXX (SEQ ID NO: 7) motif forms turn spiral structures, which imparts elasticity to the silk polypeptide. Major ampullate and flagelliform silks both have a GPGXX (SEQ ID NO: 7) motif. The iterated GGX motif is associated w ith a helical structure having three amino acids per turn and is found in most spider silks. The GGX motif may provide additional elastic properties to the silk. The iterated polyalanine Ax (peptide) motif forms a crystalline P-sheet structure that provides strength to the silk polypeptide, as described for example in WO 03 / 057727.

[0390] In some embodiments, the recombinant spider silk protein in this disclosure comprises two identical repetitive units each comprising at least one, 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. Resilin is an elastomeric protein found in most arthropods that provides low stiffness and high strength.

[0391] As used herein, “non-repetitive units” refers to an amino acid sequence which is “substantially similar” to a corresponding non-repetitive (carboxy terminal) amino acid sequence within a naturally occurring dragline polypeptide (i.e. wild-ty pe non- repetitive (carboxy terminal) unit), preferably within ADF-3, ADF-4, NR3, NR4 of the spider Araneus diadematus. which is also described in U.S. Pat. No. 9,217,017, which is incorporated by reference herein in its entirety, C16 peptide (spider silk 84

[0392] DBl / 165497080.5protein eADF4, molecular weight of 47.7 kDa, AMSilk) comprising the 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 10), an amino acid sequence adapted from the natural sequence of ADF4 from A. diadematus. Non-repetitive ADF-4 and variants thereof display efficient assembly behavior.

[0393] Among the synthetic spider silk proteins, the recombinant silk protein in this disclosure comprises in some embodiments the C16-protein having the polypeptide sequence SEQ ID NO: 1 as described in U.S. Patent No. 8,288,512, which is incorporated by reference herein in its entirety. Besides the polypeptide sequence shown in SEQ ID NO: 1 as described in U.S. Patent No. 8,288,512. particularly functional equivalents, functional derivatives and salts of this sequence are also included.

[0394] As used herein, “functional equivalents” refers to mutant which, in at least one sequence position of the abovementioned amino acid sequences, have an amino acid other than that specifically mentioned.

[0395] In some embodiments, the recombinant spider silk protein in this disclosure comprises, in an effective amount, at least one natural or recombinant silk protein including spider silk protein, corresponding to Spidroin major 1 described by Xu et al., PNAS, USA, 87, 7120, (1990), Spidroin major 2 described by Hinman and Lewis, J. Biol. Chem., 267, 19320, (1922), recombinant spider silk protein as 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, 7,060,260, or alternatively the minor Spidroins described in patent application WO 95 / 25165. Each of the above-cited references is incorporated herein by reference in its entirety. Additional recombinant spider silk proteins suitable for the recombinant RSPF of this disclosure include ADF3 and ADF4 from the “Major Ampullate” gland of Araneus diadematus.

[0396] Recombinant silk is also described in other patents and patent applications, incorporated by reference herein: US 2004590196, US 7,754,851, US 2007654470,

[0397] 85

[0398] DBl / 165497080.5US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US 200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542, US 2009265344, US 2012684607. US 2004583227. US 8,030,024, US 2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US 20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US 2012697729, US 20150328363, US 9,034,816, US 20130172478, US 9,217.017, US 20170202995. US 8,721,991, US 2008227498, US 9,233,067, US 8.288,512, US 2008161364, US 7,148.039, US 1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902, US 7,569,660, US 1999138833, US 2000591632, US 20120065126, US 20100278882, US 2008161352, US 20100015070, US 2009513709, US 20090194317, US 2004559286, US 200589551. US 2008187824, US 20050266242, US 20050227322, and US 20044418.

[0399] Recombinant silk is also described in other patents and patent applications, incorporated by reference herein: US 20190062557, US 20150284565, US 20130225476, US 20130172478, US 20130136779, US 20130109762, US 20120252294, US 20110230911, US 20110201783, US 20100298877, US 10,478.520, US 10,253,213, US 10,072.152, US 9,233,067. US 9,217,017, US 9,034,816, US 8,877,903, US 8,729,238, US 8,721,991, US 8,097,583, US 8,034,897, US 8,030,024, US 7,951,908, US 7,868,146, and US 7,754,851.

[0400] In some embodiments, the recombinant spider silk protein in this disclosure comprises or consists of 2 to 80 repetitive units, each independently selected from GPGXX (SEQ ID NO: 7), GGX and Axas defined herein.

[0401] In some embodiments, the recombinant spider silk protein in this disclosure comprises or consists of repetitive units each independently selected from 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),

[0402] 86

[0403] DBl / 165497080.5GPGGS (SEQ ID NO: 19), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 20), AAAAAA (SEQ ID 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) GPGGAGGPYGPGGAGGPYGPGGAGGPY (SEQ ID NO:31), (v) GGTTIIEDLDITIDGADGPITISEELTI (SEQ ID NO: 32), (vi) PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ IDNO:33), (vii) SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 34), (viii) GGAGGAGGAGGSGGAGGS (SEQ ID NO:35), (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (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) GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO 52), or variants thereof as described in U.S. Pat. No. 8,877,903, for example, a synthetic spider peptide having sequential order of GPGAS (SEQ ID NO: 11), GGY, GPGSG (SEQ ID NO: 12) in the peptide chain, or sequential order of AAAAAAAA (SEQ ID NO:23), GPGGY (SEQ ID NO: 13), GPGGP (SEQ ID NO: 14) in the peptide chain, sequential order of AAAAAAAA (SEQ ID NO:23), GPGQG (SEQ ID NO: 18), GGR in the peptide chain.

[0404] In some embodiments, this disclosure provides silk protein-like multiblock peptides that imitate the repeating units of amino acids derived from natural spider silk proteins such as Spidroin major 1 domain, Spidroin major 2 domain or Spidroin minor 1 domain and the profile of variation between the repeating units without modifying their three-dimensional conformation, wherein these silk protein-like

[0405] 87

[0406] DBl / 165497080.5multiblock peptides comprise a repeating unit of amino acids corresponding to one of the sequences (I), (II), (III) and / or (IV) below.

[0407] [(XGG)w(XGA)(GXG)x(AGA)y(G)zAG]PFormula (I) in which: X corresponds to ty rosine or to glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer and having any weight average molecular weight described herein, and / or [(GPG2YGPGQ2)a(X’)2S(A)b]PFormula (II) in which: X’ corresponds to the amino acid sequence GPS or GPG, a is equal to 2 or 3, b is an integer from 7 to 10, and p is an integer and having any weight average molecular weight described herein, and / or

[0408] [(GR)(GA)i(A)m(GGX)n(GA)i(A)m]PFormula (III) and / or [(GGX”)n(GA)m(A)i]PFormula (IV) in which: X” corresponds to ty rosine, glutamine or alanine, 1 is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer.

[0409] In some embodiments, the recombinant spider silk protein or an analog of a spider silk protein comprising an amino acid repeating unit of sequence (V):

[0410] [(Xaa Gly Gly)w(Xaa Gly Ala)(Gly Xaa Gly)x(Ala Gly Ala)y(Gly)zAla Gly]PFormula (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.

[0411] In some embodiments, the recombinant spider silk protein in this disclosure is selected from the group consisting of ADF-3 or variants thereof, ADF-4 or variants thereof, MaSpI or variants thereof, MaSpII or variants thereof as described in U.S. Pat. No. 9,217,017.

[0412] In some embodiments, this disclosure provides water soluble recombinant spider silk proteins produced in mammalian cells. The solubility' of the spider silk proteins produced in mammalian cells was attributed to the presence of the COOH-

[0413] 88

[0414] DBl / 165497080.5terminus in these proteins, which makes them more hydrophilic. These COOH- terminal amino acids are absent in spider silk proteins expressed in microbial hosts.

[0415] In some embodiments, the recombinant spider silk protein in this disclosure comprises water soluble recombinant spider silk protein C 16 modified with an amino or carboxyl terminal selected from the amino acid sequences consisting of:

[0416] GCGGGGGG (SEQ ID NO:41). GKGGGGGG (SEQ ID NO 42), GCGGSGGGGSGGGG (SEQ ID NO 43), GKGGGGGGSGGGG (SEQ ID NO 44), and GCGGGGGGSGGGG (SEQ ID NO:45). In some embodiments, the recombinant spider silk protein in this disclosure comprises CieNR4, C32NR4, C16, C32, NR4C16NR4. NR4C?2NR4, NR3CieNR3, or NR3C32NR3 such that the molecular weight of the protein ranges as described herein.

[0417] In some embodiments, the recombinant spider silk protein in this disclosure comprises recombinant spider silk protein having a synthetic repetitive peptide segments and an amino acid sequence adapted from the natural sequence of ADF4 from 4. diadematus as described in U.S. Pat. No. 8,877,903. In some embodiments, the RSPF in this disclosure comprises the recombinant spider silk proteins having repeating peptide units derived from natural spider silk proteins such as Spidroin major 1 domain, Spidroin major 2 domain or Spidroin minor 1 domain, wherein the repeating peptide sequence is GSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO:46) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO:47), as described in U.S. Pat. No. 8,367,803, which is incorporated by reference herein in its entirety.

[0418] In some embodiments, this disclosure provides recombinant spider proteins composed of the GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO:48) repetitive fragment and having a molecular weight as described herein.

[0419] As used herein, the term “recombinant silk” refers to recombinant spider and / or silkworm silk protein or fragments thereof. In an embodiment, the spider silk protein is selected from the group consisting of swathing silk (Achniform gland silk),

[0420] 89

[0421] DBl / 165497080.5egg sac silk (Cylindriform gland silk), egg case silk (Tubuliform silk), non-sticky dragline silk (Ampullate gland silk), ataching thread silk (Pyriform gland silk), sticky silk core fibers (Flagelliform gland silk), and sticky silk outer fibers (Aggregate gland silk). For example, recombinant spider silk protein, as described herein, includes the proteins described in U.S. Patent Application No. 2016 / 0222174 and U.S. Patent Nos.

[0422] 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734.

[0423] Some organisms make multiple silk fibers with unique sequences, structural elements, and mechanical properties. For example, orb weaving spiders have six unique types of glands that produce different silk polypeptide sequences that are polymerized into fibers tailored to fit an environmental or lifecycle niche. The fibers are named for the gland they originate from and the polypeptides are labeled with the gland abbreviation (e.g. “Ma”) and “Sp” for spidroin (short for spider fibroin). In orb weavers, these ty pes include Major Ampullate (MaSp, also called dragline), Minor Ampullate (MiSp), Flagelliform (Flag), Aciniform (AcSp), Tubuliform (TuSp), and Pyriform (PySp). This combination of polypeptide sequences across fiber types, domains, and variation amongst different genus and species of organisms leads to a vast array of potential properties that can be harnessed by commercial production of the recombinant fibers. To date, the vast majority of the work with recombinant silks has focused on the Major Ampullate Spidroins (MaSp).

[0424] Aciniform (AcSp) silks tend to have high toughness, a result of moderately high strength coupled with moderately high extensibility. AcSp silks are characterized by large block (“ensemble repeat”) sizes that often incorporate motifs of poly serine and GPX. Tubuliform (TuSp or Cylindrical) silks tend to have large diameters, with modest strength and high extensibility. TuSp silks are characterized by their poly serine and poly threonine content, and short tracts of poly alanine. Major Ampullate (MaSp) silks tend to have high strength and modest extensibility. MaSp silks can be one of two subtypes: MaSpl and MaSp2. MaSpl silks are generally less extensible than MaSp2 silks, and are characterized by poly alanine, GX, and GGX motifs.

[0425] MaSp2 silks are characterized by poly alanine, GGX, and GPX motifs. Minor

[0426] 90

[0427] DBl / 165497080.5Ampullate (MiSp) silks tend to have modest strength and modest extensibility. MiSp silks are characterized by GGX, GA, and poly A motifs, and often contain spacer elements of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high extensibility and modest strength. Flag silks are usually characterized by GPG, GGX, and short spacer motifs.

[0428] Silk polypeptides are characteristically composed of a repeat domain (REP) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In an embodiment, both the C-terminal and N-terminal domains are between 75-350 amino acids in length. The repeat domain exhibits a hierarchical architecture. The repeat domain comprises a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (making up a quasi-repeat domain), throughout the silk repeat domain. The length and composition of blocks varies among different silk types and across different species. Table 1 of U.S.

[0429] Published Application No. 2016 / 0222174, the entirety of which is incorporated herein, lists examples of block sequences from selected species and silk types, with further examples presented in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pg 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences. Science, 291:5513. pg. 2603-2605 (2001). In some cases, blocks may be arranged in a regular pattern, forming larger macro-repeats that appear multiple times (usually 2-8) in the repeat domain of the silk sequence. Repeated blocks inside a repeat domain or macro-repeat, and repeated macro-repeats within the repeat domain, may be separated by spacing elements.

[0430] The construction of certain spider silk block copolymer polypeptides from the blocks and / or macro-repeat domains, according to certain embodiments of the disclosure, is illustrated in U.S. Published Patent Application No. 2016 / 0222174.

[0431] The recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in a recombinant prokaryotic or eukaryotic 91

[0432] DBl / 165497080.5system can be purified according to methods known in the art. In a preferred embodiment, a commercially available expression / secretion system can be used, whereby the recombinant polypeptide is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium. If expression / secretion vectors are not used, an alternative approach involves purifying the recombinant block copolymer polypeptide from cell lysates (remains of cells following disruption of cellular integrity) derived from prokaryotic or eukaryotic cells in which a polypeptide was expressed. Methods for generation of such cell lysates are known to those of skill in the art. In some embodiments, recombinant block copolymer polypeptides are isolated from cell culture supernatant.

[0433] Recombinant block copolymer polypeptide may be purified by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant polypeptide or nickel columns for isolation of recombinant polypeptides tagged with 6-8 histidine residues at their N-terminus or C- terminus Alternative tags may comprise the FLAG epitope or the hemagglutinin epitope. Such methods are commonly used by skilled practitioners.

[0434] A solution of such polypeptides (i.e., recombinant silk protein) may then be prepared and used as described herein.

[0435] In another embodiment, recombinant silk protein may be prepared according to the methods described in U.S. Patent No. 8,642,734, the entirety of which is incorporated herein, and used as described herein.

[0436] In an embodiment, a recombinant spider silk protein is provided. The spider silk protein typically consists of from 170 to 760 amino acid residues, such as from 170 to 600 amino acid residues, preferably from 280 to 600 amino acid residues, such as from 300 to 400 amino acid residues, more preferably from 340 to 380 amino acid residues. The small size is advantageous because longer spider silk proteins tend to form amorphous aggregates, which require use of harsh solvents for solubilization and polymerization. The recombinant spider silk protein may contain more than 760 residues, in particular in cases where the spider silk protein contains more than two 92

[0437] DBl / 165497080.5fragments derived from the N-terminal part of a spider silk protein, The spider silk protein comprises an N-terminal fragment consisting of at least one fragment (NT) derived from the corresponding part of a spider silk protein, and a repetitive fragment (REP) derived from the corresponding internal fragment of a spider silk protein. Optionally, the spider silk protein comprises a C-terminal fragment (CT) derived from the corresponding fragment of a spider silk protein. The spider silk protein comprises ty pically a single fragment (NT) derived from the N-terminal part of a spider silk protein, but in preferred embodiments, the N-terminal fragment include at least two, such as two fragments (NT) derived from the N-terminal part of a spider silk protein. Thus, the spidroin can schematically be represented by the formula NTm-REP, and alternatively NTm-REP-CT, where m is an integer that is 1 or higher, such as 2 or higher, preferably in the ranges of 1-2, 1-4, 1-6, 2-4 or 2-6. Preferred spidroins can schematically be represented by the formulas NT2-REP orNT-REP, and alternatively NT2-REP-CT or NT-REP-CT. The protein fragments are covalently coupled, typically via a peptide bond. In one embodiment, the spider silk protein consists of the NT fragment(s) coupled to the REP fragment, which REP fragment is optionally coupled to the CT fragment.

[0438] In one embodiment, the first step of the method of producing polymers of an isolated spider silk protein involves expression of a polynucleic acid molecule which encodes the spider silk protein in a suitable host, such as Escherichia coli. The thus obtained protein is isolated using standard procedures. Optionally, lipopolysaccharides and other py rogens are actively removed at this stage.

[0439] In the second step of the method of producing polymers of an isolated spider silk protein, a solution of the spider silk protein in a liquid medium is provided. By the terms “soluble’7and “in solution” is meant that the protein is not visibly aggregated and does not precipitate from the solvent at 60,000xg. The liquid medium can be any suitable medium, such as an aqueous medium, preferably a physiological medium, ty pically a buffered aqueous medium, such as a 10-50 mM Tris-HCl buffer or phosphate buffer. The liquid medium has a pH of 6.4 or higher and / or an ion

[0440] 93

[0441] DBl / 165497080.5composition that prevents polymerization of the spider silk protein. That is, the liquid medium has either a pH of 6.4 or higher or an ion composition that prevents polymerization of the spider silk protein, or both.

[0442] Ion compositions that prevent polymerization of the spider silk protein can readily be prepared by the skilled person utilizing the methods disclosed herein. A preferred ion composition that prevents polymerization of the spider silk protein has an ionic strength of more than 300 mM. Specific examples of ion compositions that prevent polymerization of the spider silk protein include above 300 mM NaCl, 100 mM phosphate and combinations of these ions having desired preventive effect on the polymerization of the spider silk protein, e.g. a combination of 10 mM phosphate and 300 mM NaCl.

[0443] The presence of an NT fragment improves the stability of the solution and prevents polymer formation under these conditions. This can be advantageous when immediate polymerization may be undesirable, e.g. during protein purification, in preparation of large batches, or when other conditions need to be optimized. It is preferred that the pH of the liquid medium is adjusted to 6.7 or higher, such as 7.0 or higher, or even 8.0 or higher, such as up to 10.5, to achieve high solubility of the spider silk protein. It can also be advantageous that the pH of the liquid medium is adjusted to the range of 6.4-6.8, which provides sufficient solubility of the spider silk protein but facilitates subsequent pH adjustment to 6.3 or lower.

[0444] In the third step, the properties of the liquid medium are adjusted to a pH of 6.3 or lower and ion composition that allows polymerization. That is. if the liquid medium wherein the spider silk protein is dissolved has a pH of 6.4 or higher, the pH is decreased to 6.3 or lower. The skilled person is well aware of various ways of achieving this, typically involving addition of a strong or weak acid. If the liquid medium wherein the spider silk protein is dissolved has an ion composition that prevents polymerization, the ion composition is changed so as to allow polymerization. The skilled person is well aware of various ways of achieving this, e.g. dilution, dialysis or gel filtration. If required, this step involves both decreasing 94

[0445] DBl / 165497080.5the pH of the liquid medium to 6.3 or lower and changing the ion composition so as to allow polymerization. It is preferred that the pH of the liquid medium is adjusted to 6.2 or lower, such as 6.0 or lower. In particular, it may be advantageous from a practical point of view to limit the pH drop from 6.4 or 6.4-6.8 in the preceding step to 6.3 or 6.0-6.3, e.g. 6.2 in this step. In a preferred embodiment, the pH of the liquid medium of this step is 3 or higher, such as 4.2 or higher. The resulting pH range, e.g.

[0446] 4.2-6.3 promotes rapid polymerization,

[0447] In the fourth step, the spider silk protein is allowed to polymerize in the liquid medium having pH of 6.3 or lower and an ion composition that allows polymerization of the spider silk protein. Although the presence of the NT fragment improves solubility of the spider silk protein at a pH of 6.4 or higher and / or an ion composition that prevents polymerization of the spider silk protein, it accelerates polymer formation at a pH of 6.3 or lower when the ion composition allows polymerization of the spider silk protein. The resulting polymers are preferably solid and macroscopic, and they are formed in the liquid medium having a pH of 6.3 or lower and an ion composition that allows polymerization of the spider silk protein. In a preferred embodiment, the pH of the liquid medium of this step is 3 or higher, such as 4.2 or higher. The resulting pH range, e.g. 4.2-6.3 promotes rapid polymerization. Resulting polymer may be provided at the molecular weights described herein and prepared as a solution form that may be used as necessary for article coatings.

[0448] Ion compositions that allow polymerization of the spider silk protein can readily be prepared by the skilled person utilizing the methods disclosed herein. A preferred ion composition that allows polymerization of the spider silk protein has an ionic strength of less than 300 mM. Specific examples of ion compositions that allow polymerization of the spider silk protein include 150 mM NaCl, 10 mM phosphate, 20 mM phosphate and combinations of these ions lacking preventive effect on the polymerization of the spider silk protein, e.g. a combination of 10 mM phosphate or 20 mM phosphate and 150 mM NaCl. It is preferred that the ionic strength of this liquid medium is adjusted to the range of 1-250 mM.

[0449] 95

[0450] DBl / 165497080.5Without desiring to be limited to any specific theory, it is envisaged that the NT fragments have oppositely charged poles, and that environmental changes in pH affects the charge balance on the surface of the protein followed by polymerization, whereas salt inhibits the same event.

[0451] At neutral pH, the energetic cost of burying the excess negative charge of the acidic pole may be expected to prevent polymerization. However, as the dimer approaches its isoelectric point at lower pH, attractive electrostatic forces will eventually become dominant, explaining the observed salt and pH-dependent polymerization behavior of NT and NT-containing minispidroins. It is proposed that, in some embodiments, pH-induced NT polymerization, and increased efficiency of fiber assembly of NT-minispidroins, are due to surface electrostatic potential changes, and that clustering of acidic residues at one pole of NT shifts its charge balance such that the polymerization transition occurs at pH values of 6.3 or lower.

[0452] In a fifth step, the resulting, preferably solid spider silk protein polymers are isolated from said liquid medium. Optionally, this step involves actively removing lipopolysaccharides and other pyrogens from the spidroin polymers.

[0453] Without desiring to be limited to any specific theory, it has been observed that formation of spidroin polymers progresses via formation of water-soluble spidroin dimers. The present disclosure thus also provides a method of producing dimers of an isolated spider silk protein, wherein the first two method steps are as described above. The spider silk proteins are present as dimers in a liquid medium at a pH of 6.4 or higher and / or an ion composition that prevents polymerization of said spider silk protein. The third step involves isolating the dimers obtained in the second step, and optionally removal of lipopolysaccharides and other pyrogens. In a preferred embodiment, the spider silk protein polymer of the disclosure consists of polymerized protein dimers. The present disclosure thus provides a novel use of a spider silk protein, preferably those disclosed herein, for producing dimers of the spider silk protein.

[0454] 96

[0455] DBl / 165497080.5According to another aspect, the disclosure provides a polymer of a spider silk protein as disclosed herein. In an embodiment, the polymer of this protein is obtainable by any one of the methods therefor according to the disclosure. Thus, the disclosure provides various uses of recombinant spider silk protein, preferably those disclosed herein, for producing polymers of the spider silk protein as recombinant silk based coatings. According to one embodiment, the present disclosure provides a novel use of a dimer of a spider silk protein, preferably those disclosed herein, for producing polymers of the isolated spider silk protein as recombinant silk based coatings. In these uses, it is preferred that the polymers are produced in a liquid medium having a pH of 6.3 or lower and an ion composition that allows polymerization of said spider silk protein. In an embodiment, the pH of the liquid medium is 3 or higher, such as 4.2 or higher. The resulting pH range, e.g. 4.2-6.3 promotes rapid polymerization, Using the method(s) of the present disclosure, it is possible to control the polymerization process, and this allows for optimization of parameters for obtaining silk polymers with desirable properties and shapes.

[0456] In an embodiment, the recombinant silk proteins described herein, include those described in U.S. patent No. 8,642,734, the entirety of which is incorporated by reference.

[0457] In another embodiment, the recombinant silk proteins described herein may be prepared according to the methods described in U.S. Patent No. 9,051,453, the entirety of which is incorporated herein by reference.

[0458] An amino acid sequence represented by SEQ ID NO: lof U.S. Patent No. 9,051,453, is identical to an amino acid sequence that is composed of 50 amino acid residues of an amino acid sequence of ADF3 at the C-terminal (NCBI Accession No.: AAC47010, GI: 1263287). An amino acid sequence represented by SEQ ID NO: 2 of U.S. Patent No. 9,051,453, is identical to an amino acid sequence represented by SEQ ID NO: 1 of U.S. Patent No. 9,051,453, from which 20 residues have been removed from the C-terminal. An amino acid sequence represented by SEQ ID NO: 3 of U.S. Patent No. 9,051,453, is identical to an amino acid sequence represented by SEQ ID 97

[0459] DBl / 165497080.5NO: 1 of U.S. Patent No. 9,051,453 from which 29 residues have been removed from the C -terminal.

[0460] An example of the polypeptide that contains units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No.

[0461] 9,051,453 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No.

[0462] 9,051,453, is a polypeptide having an amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, which is incorporated by reference herein in its entirety. The polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, is obtained by the following mutation: in an amino acid sequence of ADF3 (NCBI Accession No.: AAC47010, GI: 1263287) to the N-terminal of which has been added an amino acid sequence (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) composed of a start codon, His 10 tags and an HRV3C Protease (Human rhinovirus 3C Protease) recognition site, 1stto 13threpetitive regions are about doubled and the translation ends at the 1154thamino acid residue. In the polypeptide having the amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 3 of U.S. Patent No. 9,051.453.

[0463] Further, the polypeptide that contains units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) and that has, at a C-terminal, an amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No.

[0464] 9,051,453, or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 1 to 3 of U.S. Patent No.

[0465] 9,051,453, may be a protein that has an amino acid sequence represented by SEQ ID NO: 8 of U.S. Patent No. 9,051,453, in which one or a plurality of amino acids have been substituted, deleted, inserted and / or added and that has a repetitious region composed of a crystal region and an amorphous region.

[0466] 98

[0467] DBl / 165497080.5Further, an example of the polypeptide containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) is a recombinant protein derived from ADF4 having an amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9.051,453, which is incorporated by reference herein in its entirety. The amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No.

[0468] 9,051,453, is an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) composed of a start codon, Elis 10 tags and an HRV3C Protease (Human rhinovirus 3C Protease) recognition site, to the N- terminal of a partial amino acid sequence of ADF4 obtained from the NCBI database (NCBI Accession No.: AAC47011, GI: 1263289). Further, the polypeptide containing two or more units of the amino acid sequence represented by the formula 1: REP1- REP2 (1) may be a polypeptide that has an amino acid sequence represented by SEQ ID NO: 15 of U.S. Patent No. 9,051.453, in which one or a plurality of amino acids have been substituted, deleted, inserted and / or added and that has a repetitious region composed of a crystal region and an amorphous region. Further, an example of the polypeptide containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) is a recombinant protein derived from MaSp2 that has an amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453, which is incorporated by reference here in its entirety. The amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 5 of U.S. Patent No. 9,051,453.) composed of a start codon, His 10 tags and an HRV3C Protease (Human rhinovirus 3C Protease) recognition site, to the N-terminal of a partial sequence of MaSp2 obtained from the NCBI web database (NCBI Accession No.: AAT75313, GI: 50363147). Furthermore, the polypeptide containing two or more units of the amino acid sequence represented by the formula 1: REP1-REP2 (1) may be a polypeptide that has an amino acid sequence represented by SEQ ID NO: 17 of U.S. Patent No. 9,051,453, in which one or a plurality of amino acids have been substituted, deleted, inserted and / or added and that has a repetitious region composed of a crystal region and an amorphous region.

[0469] 99

[0470] DBl / 165497080.5Examples of the polypeptide derived from flagelliform silk proteins include a polypeptide containing 10 or more units of an amino acid sequence represented by the formula 2: REP3 (2), preferably a polypeptide containing 20 or more units thereof, and more preferably a polypeptide containing 30 or more units thereof. In the case of producing a recombinant protein using a microbe such as Escherichia coli as a host, the molecular weight of the polypeptide derived from flagelliform silk proteins is preferably 500 kDa or less, more preferably 300 kDa or less, and further preferably 200 kDa or less, in terms of productivity.

[0471] In the formula (2), the REP 3 indicates an amino acid sequence composed of Gly-Pro-Gly-Gly-X, where X indicates an amino acid selected from the group consisting of Ala, Ser, Tyr and Vai.

[0472] A major characteristic of the spider silk is that the flagelliform silk does not have a crystal region, but has a repetitious region composed of an amorphous region. Since the major dragline silk and the like have a repetitious region composed of a crystal region and an amorphous region, they are expected to have both high stress and stretchability. Meanwhile, as to the flagelliform silk, although the stress is inferior to that of the major dragline silk, the stretchability is high. The reason for this is considered to be that most of the flagelliform silk is composed of amorphous regions.

[0473] An example of the polypeptide containing 10 or more units of the amino acid sequence represented by the formula 2: REP3 (2) is a recombinant protein derived from flagelliform silk proteins having an amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No. 9.051,453, which is incorporated by reference herein in its entirety. The amino acid sequence represented by SEQ ID NO: 19 of U.S. Patent No.

[0474] 9,051,453, is an amino acid sequence obtained by combining a partial sequence of flagelliform silk protein of Nephila clavipes obtained from the NCBI database (NCBI Accession No.: AAF36090, GI: 7106224), specifically, an amino acid sequence thereof from the 1220thresidue to the 1659thresidue from the N-terminal that corresponds to repetitive sections and motifs (referred to as a PR1 sequence), with a partial sequence of flagelliform silk protein of Nephila clavipes obtained from the 100

[0475] DBl / 165497080.5NCBI database (NCBI Accession No.: AAC38847, GI: 2833649), specifically, a C- terminal amino acid sequence thereof from the 816thresidue to the 907thresidue from the C-terminal, and thereafter adding the amino acid sequence (SEQ ID NO: 5 of U.S. Patent No. 9,051,453) composed of a start codon, His 10 tags and an HRV3C Protease recognition site, to the N-terminal of the combined sequence. Further, the polypeptide containing 10 or more units of the amino acid sequence represented by the formula 2: REP3 (2) may be a polypeptide that has an amino acid sequence represented by SEQ ID NO: 19 ofU.S. PatentNo. 9,051,453, in which one or a plurality of amino acids have been substituted, deleted, inserted and / or added and that has a repetitious region composed of an amorphous region.

[0476] The polypeptide can be produced using a host that has been transformed by an expression vector containing a gene encoding a polypeptide. A method for producing a gene is not limited particularly, and it may be produced by amplifying a gene encoding a natural spider silk protein from a cell derived from spiders by a polymerase chain reaction (PCR), etc., and cloning it. or may be synthesized chemically. Also, a method for chemically synthesizing a gene is not limited particularly, and it can be synthesized as follows, for example: based on information of amino acid sequences of natural spider silk proteins obtained from the NCBI web database, etc., oligonucleotides that have been synthesized automatically with AKTA oligopilot plus 10 / 100 (GE Healthcare Japan Corporation) are linked by PCR, etc. At this time, in order to facilitate the purification and observation of protein, it is possible to synthesize a gene that encodes a protein having an amino acid sequence of the above-described amino acid sequence to the N-terminal of which has been added an amino acid sequence composed of a start codon and His 10 tags.

[0477] Examples of the expression vector include a plasmid, a phage, a virus, and the like that can express protein based on a DNA sequence. The plasmid-type expression vector is not limited particularly as long as it allows a target gene to be expressed in a host cell and it can amplify itself. For example, in the case of using Escherichia coli Rosetta (DE3) as a host, a pET22b(+) plasmid vector, a pCold plasmid vector, and the

[0478] 101

[0479] DBl / 165497080.5like can be used. Among these, in terms of productivity of protein, it is preferable to use the pET22b(+) plasmid vector. Examples of the host include animal cells, plant cells, microbes, etc.

[0480] The polypeptide used in the present disclosure is preferably a polypeptide derived from ADF3, which is one of two principal dragline silk proteins of Araneus diadematus. This polypeptide has advantages of basically having high strengthelongation and toughness and of being synthesized easily.

[0481] Accordingly, the recombinant silk protein (e.g., the recombinant spider silkbased protein) used in accordance with the embodiments, articles, and / or methods described herein, may include one or more recombinant silk proteins described above or recited in U.S. Patent Nos. 8,173,772. 8,278,416, 8,618,255, 8,642,734, 8,691.581, 8,729,235, 9.115,204, 9,157.070, 9,309,299. 9,644,012. 9,708,376, 9.051,453, 9,617,315, 9,968,682, 9,689,089, 9,732,125, 9,856,308, 9,926,348, 10,065,997, 10,316,069, and 10,329,332; and U.S. Patent Publication Nos. 2009 / 0226969, 2011 / 0281273, 2012 / 0041177, 2013 / 0065278, 2013 / 0115698, 2013 / 0316376, 2014 / 0058066. 2014 / 0079674. 2014 / 0245923, 2015 / 0087046, 2015 / 0119554, 2015 / 0141618, 2015 / 0291673, 2015 / 0291674, 2015 / 0239587, 2015 / 0344542, 2015 / 0361144, 2015 / 0374833, 2015 / 0376247, 2016 / 0024464, 2017 / 0066804, 2017 / 0066805, 2015 / 0293076, 2016 / 0222174, 2017 / 0283474, 2017 / 0088675, 2019 / 0135880, 2015 / 0329587. 2019 / 0040109, 2019 / 0135881, 2019 / 0177363, 2019 / 0225646. 2019 / 0233481. 2019 / 0031842, 2018 / 0355120, 2019 / 0186050, 2019 / 0002644, 2020 / 0031887, 2018 / 0273590, 20191 / 094403, 2019 / 0031843, 2018 / 0251501, 2017 / 0066805, 2018 / 0127553, 2019 / 0329526, 2020 / 0031886, 2018 / 0080147, 2019 / 0352349. 2020 / 0043085, 2019 / 0144819, 2019 / 0228449, 2019 / 0340666. 2020 / 0000091. 2019 / 0194710, 2019 / 0151505, 2018 / 0265555, 2019 / 0352330, 2019 / 0248847, and 2019 / 0378191, the entirety of which are incorporated herein by reference.

[0482] 102

[0483] DBl / 165497080.5Silk Fibroin-like Protein Fragments

[0484] The recombinant silk protein in this disclosure comprises synthetic proteins which are based on repeat units of natural silk proteins. Besides the synthetic repetitive silk protein sequences, these can additionally comprise one or more natural nonrepetitive silk protein sequences. As used herein, ‘’silk fibroin-like protein fragments” refer to protein fragments having a molecular weight and poly dispersity as defined herein, and a certain degree of homology to a protein selected from native silk protein, fibroin heavy chain [SEQ ID NO: 49J, fibroin light chain [SEQ ID NO: 50], or any protein comprising one or more GAGAGS hexa amino acid repeating units. In some embodiments, a 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%.

[0485] As described herein, a protein such as native silk protein, fibroin heavy chain [SEQ ID NO: 49], fibroin light chain [SEQ ID NO: 50], or any protein comprising one or more GAGAGS hexa amino acid repeating units includes 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, a protein such as native silk protein, fibroin heavy chain [SEQ ID NO: 49], fibroin light chain [SEQ ID NO: 50], or any protein comprising one or more GAGAGS hexa amino acid repeating units includes 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 native silk protein, fibroin heavy chain [SEQ ID NO: 49], fibroin light chain [SEQ ID NO: 50], or any protein comprising one or more GAGAGS hexa amino acid repeating units includes between 9% and about 12% serine, or about 9% serine, or about 10% serine, or about 11% serine, or about 12% serine.

[0486] 103

[0487] DBl / 165497080.5In some embodiments, a silk fibroin-like protein described herein includes about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%. about 21%, about 22%, about 23 %. about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%. about 49%, about 50%, about 51%, about 52%, about 53%. about 54%. or about 55% glycine. In some embodiments, a silk fibroin-like protein described herein includes about 13%, about 14%, about 15%, about 1 %, 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, a silk fibroin-like protein described herein includes 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, a silk fibroin-like protein described herein may include independently any amino acid known to be included in natural fibroin. In some embodiments, a silk fibroin-like protein described herein may exclude independently any amino acid known to be included in natural fibroin. In some embodiments, on average 2 out of 6 amino acids, 3 out of 6 amino acids, or 4 out of 6 amino acids in a silk fibroin-like protein described herein is glycine. In some embodiments, on average 1 out of 6 amino acids, 2 out of 6 amino acids, or 3 out of 6 amino acids in a silk fibroin-like protein described herein is alanine. In some embodiments, on average none out of 6 amino acids, 1 out of 6 amino acids, or 2 out of 6 amino acids in a silk fibroin-like protein described herein is serine.

[0488] 104

[0489] DBl / 165497080.5Other Properties of SPF

[0490] Compositions of the present disclosure are “biocompatible’' or otherwise exhibit “biocompatibility'’ meaning that the compositions are compatible with living tissue or a living system by not being toxic, injurious, or physiologically reactive and not causing immunological rejection or an inflammatory response. Such biocompatibility can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time 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.

[0491] In some embodiments, compositions described herein, which may be biocompatible compositions (e.g., biocompatible coatings that include silk), may be evaluated and comply with International Standard ISO 10993-1, titled the “Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process.” In some embodiments, compositions described herein, which may be biocompatible compositions, may be evaluated under ISO 106993-1 for one or more of cytotoxicity, sensitization, hemocompatibility, pyrogenicity, implantation, genotoxicity, carcinogenicity, reproductive and developmental toxicity, and degradation.

[0492] Compositions of the present disclosure are “hypoallergenic” meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of 105

[0493] DBl / 165497080.5time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time 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.

[0494] In an embodiment, the stability' of a composition of the present disclosure is about 1 day. In an embodiment, the stability of a composition of the present disclosure is about 2 days. In an embodiment, the stability of a composition of the present disclosure is about 3 days. In an embodiment, the stability of a composition of the present disclosure is about 4 days. In an embodiment, the stability' of a composition of the present disclosure is about 5 days. In an embodiment, the stability' of a composition of the present disclosure is about 6 days. In an embodiment, the stability of a composition of the present disclosure is about 7 days. In an embodiment, the stability of a composition of the present disclosure is about 8 days. In an embodiment, the stability' of a composition of the present disclosure is about 9 days. In an embodiment, the stability' of a composition of the present disclosure is about 10 days.

[0495] In an embodiment, the stability' of a composition of the present disclosure is about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 22 days, about 23 days, about 24 days, about 25 days, about 26 days, about 27 days, about 28 days, about 29 days, or about 30 days.

[0496] In an embodiment, the stability' of a composition of the present disclosure is 10 days to 6 months. In an embodiment, the stability' of a composition of the present disclosure is 6 months to 12 months. In an embodiment, the stability of a composition of the present disclosure is 12 months to 18 months. In an embodiment, the stability of a composition of the present disclosure is 18 months to 24 months. In an embodiment, the stability' of a composition of the present disclosure is 24 months to 106

[0497] DBl / 165497080.530 months. In an embodiment, the stability of a composition of the present disclosure is 30 months to 36 months. In an embodiment, the stability of a composition of the present disclosure is 36 months to 48 months. In an embodiment, the stability of a composition of the present disclosure is 48 months to 60 months.

[0498] In an embodiment, a SPF composition of the present disclosure is not soluble in an aqueous solution due to the crystallinity of the protein. In an embodiment, a SPF composition of the present disclosure is soluble in an aqueous solution. In an embodiment, the SPF of a composition of the present disclosure include a crystalline portion of about two-thirds and an amorphous region of about one-third. In an embodiment, the SPF of a composition of the present disclosure include a crystalline portion of about one-half and an amorphous region of about one-half. In an embodiment, the SPF of a composition of the present disclosure include a 99% cry stalline portion and a 1% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 95% crystalline portion and a 5% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 90% crystalline portion and a 10% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 85% crystalline portion and a 15% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 80% crystalline portion and a 20% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 75% crystalline portion and a 25% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 70% crystalline portion and a 30% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 65% crystalline portion and a 35% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 60% cry stalline portion and a 40% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 50% crystalline portion and a 50% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 40% cry stalline portion and a 60% amorphous region. In an embodiment, the SPF of a composition of the present 107

[0499] DBl / 165497080.5disclosure include a 35% crystalline portion and a 65% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 30% crystalline portion and a 70% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 25% crystalline portion and a 75% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 20% crystalline portion and a 80% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 15% crystalline portion and a 85% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 10% crystalline portion and a 90% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 5% crystalline portion and a 90% amorphous region. In an embodiment, the SPF of a composition of the present disclosure include a 1% crystalline portion and a 99% amorphous region.

[0500] As used herein, the term “substantially free of inorganic residuals” means that the composition exhibits residuals of 0.1 % (w / w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.05% (w / w) or less. In an embodiment, substantially free of inorganic residuals refers to a composition that exhibits residuals of 0.01 % (w / w) or less. In an embodiment, the amount of inorganic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of inorganic residuals is ND to about 500 ppm. In an embodiment, the amount of inorganic residuals is ND to about 400 ppm. In an embodiment, the amount of inorganic residuals is ND to about 300 ppm. In an embodiment, the amount of inorganic residuals is ND to about 200 ppm. In an embodiment, the amount of inorganic residuals is ND to about 100 ppm. In an embodiment, the amount of inorganic residuals is between 10 ppm and 1000 ppm.

[0501] As used herein, the term “substantially free of organic residuals” means that the composition exhibits residuals of 0.1 % (w / w) or less, in an embodiment, substantially free of organic residuals refers to a composition that exhibits residuals of 0.05% (w / w) or less. In an embodiment, substantially free of organic residuals refers

[0502] 108

[0503] DBl / 165497080.5to a composition that exhibits residuals of 0.01% (w / w) or less. In an embodiment, the amount of organic residuals is between 0 ppm (“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount of organic residuals is ND to about 500 ppm. In an embodiment, the amount of organic residuals is ND to about 400 ppm. In an embodiment, the amount of organic residuals is ND to about 300 ppm. In an embodiment, the amount of organic residuals is ND to about 200 ppm. In an embodiment, the amount of organic residuals is ND to about 100 ppm. In an embodiment, the amount of organic residuals is between 10 ppm and 1000 ppm.

[0504] Compositions of the present disclosure exhibit “biocompatibility” meaning that the compositions are compatible with living tissue or a living system by not being toxic, injurious, or physiologically reactive and not causing immunological rejection. Such biocompatibility can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days, in an embodiment, the extended period of time is about 14 days, in an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected from the group consisting of about I 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.

[0505] Compositions of the present disclosure are “hypoallergenic” meaning that they are relatively unlikely to cause an allergic reaction. Such hypoallergenicity can be evidenced by participants topically applying compositions of the present disclosure on their skin for an extended period of time. In an embodiment, the extended period of time is about 3 days. In an embodiment, the extended period of time is about 7 days. In an embodiment, the extended period of time is about 14 days. In an embodiment, the extended period of time is about 21 days. In an embodiment, the extended period of time is about 30 days. In an embodiment, the extended period of time is selected

[0506] 109

[0507] DBl / 165497080.5from 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.

[0508] Following are non-limiting examples of suitable ranges for various parameters in and for preparation of the silk solutions of the present disclosure. The silk solutions of the present disclosure may include one or more, but not necessarily all, of these parameters and may be prepared using various combinations of ranges of such parameters.

[0509] In an embodiment, the percent SPF in the solution is less than 30.0 wt. %. In an embodiment, the percent SPF in the solution is less than 25.0 wt. %. In an embodiment, the percent SPF in the solution is less than 20.0 wt. %. In an embodiment, the percent SPF in the solution is less than 19.0 wt. %. In an embodiment, the percent SPF in the solution is less than 18.0 wt. %. In an embodiment, the percent SPF in the solution is less than 17.0 wt. %. In an embodiment, the percent SPF in the solution is less than 16.0 wt. %. In an embodiment, the percent SPF in the solution is less than 15.0 wt. %. In an embodiment, the percent SPF in the solution is less than 14.0 wt. %. In an embodiment, the percent SPF in the solution is less than 13.0 wt. %. In an embodiment, the percent SPF in the solution is less than 12.0 wt. %. In an embodiment, the percent SPF in the solution is less than 11.0 wt. %. In an embodiment, the percent SPF in the solution is less than 10.0 wt. %. In an embodiment, the percent SPF in the solution is less than 9.0 wt. %. In an embodiment, the percent SPF in the solution is less than 8.0 wt. %. In an embodiment, the percent SPF in the solution is less than 7.0 wt. %. In an embodiment, the percent SPF in the solution is less than 6.0 wt. %. In an embodiment, the percent SPF in the solution is less than 5.0 wt. %. In an embodiment, the percent SPF in the solution is less than 4.0 wt. %. In an embodiment, the percent SPF in the solution is less than 3.0 wt. %. In an embodiment, the percent SPF in the solution is less than 2.0 wt. %. In an embodiment, the percent SPF in the solution is less than 1.0 wt. %. In an embodiment, the percent

[0510] 110

[0511] DBl / 165497080.5SPF in the solution is less than 0.9 wt. %. In an embodiment, the percent SPF in the solution is less than 0.8 wt. %. In an embodiment, the percent SPF in the solution is less than 0.7 wt. %. In an embodiment, the percent SPF in the solution is less than 0.6 wt. %. In an embodiment, the percent SPF in the solution is less than 0.5 wt. %. In an embodiment, the percent SPF in the solution is less than 0.4 wt. %. In an embodiment, the percent SPF in the solution is less than 0.3 wt. %. In an embodiment, the percent SPF in the solution is less than 0.2 wt. %. In an embodiment, the percent SPF in the solution is less than 0.1 wt. %.

[0512] In an embodiment, the percent SPF in the solution is greater than 0.1 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.2 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.3 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.4 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.5 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.6 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.7 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.8 wt. %. In an embodiment, the percent SPF in the solution is greater than 0.9 wt. %. In an embodiment, the percent SPF in the solution is greater than 1.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 2.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 3.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 4.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 5.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 6.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 7.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 8.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 9.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 10.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 11.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 12.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 13.0 wt. %. In an

[0513] 111

[0514] DBl / 165497080.5embodiment, the percent SPF in the solution is greater than 14.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 15.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 16.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 17.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 18.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 19.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 20.0 wt. %. In an embodiment, the percent SPF in the solution is greater than 25.0 wt. %.

[0515] In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 30.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 25.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 20.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 15.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 10.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 9.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 8.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 7.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 6.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 6.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 5.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 5.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 4.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 4.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 3.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 3.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 2.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 2.0 wt. %. In an embodiment, the percent SPF in the solution 112

[0516] DBl / 165497080.5ranges from about 0.1 wt. % to about 2.4 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 5.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 4.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 4.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 3.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 3.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.5 wt. % to about 2.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 4.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 3.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 3.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 2.5 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 2.4 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 2.0 wt. %.

[0517] In an embodiment, the percent SPF in the solution ranges from about 20.0 wt. % to about 30.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 10.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 1.0 wt. % to about 10.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 2 wt. % to about 10.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 0.1 wt. % to about 6.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 6.0 wt. % to about 10.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 6.0 wt. % to about 8.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 6.0 wt. % to about 9.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 10.0 wt. % to about 20.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 11.0 wt. % to about 19.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 12.0 wt. % to about 18.0 wt. %. In an embodiment, the percent SPF in the solution ranges from about 13.0 wt. % to about 17.0 wt. %. In an embodiment, the percent SPF in the solution ranges 113

[0518] DBl / 165497080.5from about 14.0 wt. % to about 16.0 wt. %. In an embodiment, the percent SPF in the solution is about 1.0 wt. %. In an embodiment, the percent SPF in the solution is about 1.5 wt. %. In an embodiment, the percent SPF in the solution is about 2.0 wt.%. In an embodiment, the percent SPF in the solution is about 2.4 wt. %. In an embodiment, the percent SPF in the solution is 3.0 wt. %. In an embodiment, the percent SPF in the solution is 3.5 wt. %. In an embodiment, the percent SPF in the solution is about 4.0 wt. %. In an embodiment, the percent SPF in the solution is about 4.5 wt. %. In an embodiment, the percent SPF in the solution is about 5.0 wt. %. In an embodiment, the percent SPF in the solution is about 5.5 wt. %. In an embodiment the percent SPF in the solution is about 6.0 wt. %. In an embodiment, the percent SPF in the solution is about 6.5 wt. %. In an embodiment, the percent SPF in the solution is about 7.0 wt. %. In an embodiment, the percent SPF in the solution is about 7.5 wt. %. In an embodiment, the percent SPF in the solution is about 8.0 wt. %. In an embodiment, the percent SPF in the solution is about 8.5 wt. %. In an embodiment, the percent SPF in the solution is about 9.0 wt. %. In an embodiment, the percent SPF in the solution is about 9.5 wt. %. In an embodiment, the percent SPF in the solution is about 10.0 wt. %.

[0519] In an embodiment, the percent sericin in the solution is non-detectable to 25.0 wt. %. In an embodiment, the percent sericin in the solution is non-detectable to 5.0 wt. %. In an embodiment, the percent sericin in the solution is 1.0 wt. %. In an embodiment, the percent sericin in the solution is 2.0 wt. %. In an embodiment, the percent sericin in the solution is 3.0 wt. %. In an embodiment, the percent sericin in the solution is 4.0 wt. %. In an embodiment, the percent sericin in the solution is 5.0 wt. %. In an embodiment, the percent sericin in the solution is 10.0 wt. %. In an embodiment, the percent sericin in the solution is 25.0 wt. %.

[0520] In some embodiments, the silk fibroin protein fragments of the present disclosure are shelf stable (they will not slowly or spontaneously gel when stored in an aqueous solution and there is no aggregation of fragments and therefore no increase in molecular weight over time), from 10 days to 3 years depending on

[0521] 114

[0522] DBl / 165497080.5storage conditions, percent SPF, and number of shipments and shipment conditions. Additionally, pH may be altered to extend shelflife and / or support shipping conditions by preventing premature folding and aggregation of the silk. In an embodiment, the stability- of the LiBr-silk fragment solution is 0 to 1 year. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 0 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 2 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 1 to 5 years. In an embodiment, the stability- of the LiBr-silk fragment solution is 2 to 3 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 2 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 4 years. In an embodiment, the stability of the LiBr-silk fragment solution is 3 to 5 years. In an embodiment, the stability of the LiBr-silk fragment solution is 4 to 5 years.

[0523] In an embodiment, the stability of a composition of the present disclosure is 10 days to 6 months. In an embodiment, the stability of a composition of the present disclosure is 6 months to 12 months. In an embodiment, the stability of a composition of the present disclosure is 12 months to 18 months. In an embodiment, the stability of a composition of the present disclosure is 18 months to 24 months. In an embodiment, the stability of a composition of the present disclosure is 24 months to 30 months. In an embodiment, the stability of a composition of the present disclosure is 30 months to 36 months. In an embodiment, the stability of a composition of the present disclosure is 36 months to 48 months. In an embodiment, the stability of a composition of the present disclosure is 48 months to 60 months.

[0524] 115

[0525] DBl / 165497080.5In an embodiment, a composition of the present disclosure having SPF has non-detectable levels of LiBr residuals. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is between 10 ppm and 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 25 ppm. In an embodiment, the amount of the Li Br residuals in a composition of the present disclosure is less than 50 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 75 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non- detectable to 500 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the LiBr residue in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present 116

[0526] DBl / 165497080.5disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the LiBr residuals in a composition of the present disclosure is 400 ppm to 500 ppm.

[0527] In an embodiment, a composition of the present disclosure having SPF, has non-detectable levels of Na2CCh residuals. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the Na2CO? residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the Na2COs 117

[0528] DBl / 165497080.5residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the NazCOs residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is non-detectable to 200 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is non-detectable to 150 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the Na2COs residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the Na2CO3 residuals in a composition of the present disclosure is 400 ppm to 500 ppm.

[0529] A unique feature of the SPF compositions of the present disclosure are shelf stability (they will not slowly or spontaneously gel when stored in an aqueous solution and there is no aggregation of fragments and therefore no increase in molecular weight over time), from 10 days to 3 years depending on storage conditions, percent silk, and number of shipments and shipment conditions.

[0530] Additionally, pH may be altered to extend shelf-life and / or support shipping conditions by preventing premature folding and aggregation of the silk. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 2 weeks at room temperature (RT). In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 4 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf 118

[0531] DBl / 165497080.5stability for up to 6 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 8 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 10 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability for up to 12 weeks at RT. In an embodiment, a SPF solution composition of the present disclosure has a shelf stability ranging from about 4 weeks to about 52 weeks at RT.

[0532] Table 18 below shows shelf stability test results for embodiments of SPF compositions of the present disclosure.

[0533] >

[0534] >

[0535]

[0536] >

[0537] In some embodiments, the water solubility of the silk film derived from silk fibroin protein fragments as described herein can be modified by solvent annealing (water annealing or methanol annealing), chemical crosslinking, enzyme crosslinking and heat treatment.

[0538] In some embodiments, the process of annealing may involve inducing betasheet formation in the silk fibroin protein fragment solutions used as a coating material. Techniques of annealing (e.g., increase crystallinity) or otherwise promoting “molecular packing’" of silk fibroin-protein based fragments have been described. In some embodiments, the amorphous silk film is annealed to introduce beta-sheet in the presence of a solvent selected from the group of water or organic solvent. In some embodiments, the amorphous silk film is annealed to introduce beta-sheet in the

[0539] 119

[0540] DBl / 165497080.5presence of water (water annealing process). In some embodiments, the amorphous silk fibroin protein fragment film is annealed to introduce beta-sheet in the presence of methanol. In some embodiments, annealing (e.g., the beta sheet formation) is induced by addition of an organic solvent. Suitable organic solvents include, but are not limited to methanol, ethanol, acetone, isopropanol, or combination thereof.

[0541] In some embodiments, annealing is carried out by so-called "water-annealing" or “w ater vapor annealing’’ in which w ater vapor is used as an intermediate plasticizing agent or catalyst to promote the packing of beta-sheets. In some embodiments, the process of w ater annealing may be performed under vacuum. Suitable such methods have been described in Jin H-J 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.

[0542] The important feature of the w ater annealing process is to drive the formation of cry stalline beta-sheet in the silk fibroin protein fragment peptide chain to allow' the silk fibroin self-assembling into a continuous film. In some embodiments, the crystallinity of the silk fibroin protein fragment film is controlled by controlling the temperature of w ater vapor and duration of the annealing. In some embodiments, the annealing is performed 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, annealing is performed at a temperature selected from the group 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.

[0543] In some embodiments, the annealing process lasts a period of time selected from the group of about 1 minute to about 40 minutes, about 1 minute to about 50 minutes, about 1 minute to about 60 minutes, about 1 minute to about 70 minutes, about 1 minute to about 80 minutes, about 1 minute to about 90 minutes, about 1 minute to about 100 minutes, about 1 minute to about 110 minutes, about 1 minute to about 120 minutes, about 1 minute to about 130 minutes, about 5 minutes to about 120

[0544] DBl / 165497080.540 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 70 minutes, about 10 minutes to about 80 minutes, about 10 minutes to about 90 minutes, about 10 minutes to about 100 minutes, about 10 minutes to about 110 minutes, about 10 minutes to about 120 minutes, about 10 minutes to about 130 minutes, about 15 minutes to about 40 minutes, about 15 minutes to about 50 minutes, about 15 minutes to about 60 minutes, about 15 minutes to about 70 minutes, about 15 minutes to about 80 minutes, about 15 minutes to about 90 minutes, about 15 minutes to about 100 minutes, about 15 minutes to about 110 minutes, about 15 minutes to about 120 minutes, about 15 minutes to about 130 minutes, about 20 minutes to about 40 minutes, about 20 minutes to about 50 minutes, about 20 minutes to about 60 minutes, about 20 minutes to about 70 minutes, about 20 minutes to about 80 minutes, about 20 minutes to about 90 minutes, about 20 minutes to about 100 minutes, about 20 minutes to about 110 minutes, about 20 minutes to about 120 minutes, about 20 minutes to about 130 minutes, about 25 minutes to about 40 minutes, about 25 minutes to about 50 minutes, about 25 minutes to about 60 minutes, about 25 minutes to about 70 minutes, about 25 minutes to about 80 minutes, about 25 minutes to about 90 minutes, about 25 minutes to about 100 minutes, about 25 minutes to about 110 minutes, about 25 minutes to about 120 minutes, about 25 minutes to about 130 minutes, about 30 minutes to about 40 minutes, about 30 minutes to about 50 minutes, about 30 minutes to about 60 minutes, about 30 minutes to about 70 minutes, about 30 minutes to about 80 minutes, about 30 minutes to about 90 minutes, about 30 minutes to about 100 minutes, about 30 minutes to about 110 minutes, about 30 minutes to about 120 minutes, about 30 minutes to about 130 minutes, about 35 minutes to about 40 minutes, about 35 minutes to about 50

[0545] 121

[0546] DBl / 165497080.5minutes, about 35 minutes to about 60 minutes, about 35 minutes to about 70 minutes, about 35 minutes to about 80 minutes, about 35 minutes to about 90 minutes, about 35 minutes to about 100 minutes, about 35 minutes to about 110 minutes, about 35 minutes to about 120 minutes, about 35 minutes to about 130 minutes, about 40 minutes to about 50 minutes, about 40 minutes to about 60 minutes, about 40 minutes to about 70 minutes, about 40 minutes to about 80 minutes, about 40 minutes to about 90 minutes, about 40 minutes to about 100 minutes, about 40 minutes to about 110 minutes, about 40 minutes to about 120 minutes, about 40 minutes to about 130 minutes, about 45 minutes to about 50 minutes, about 45 minutes to about 60 minutes, about 45 minutes to about 70 minutes, about 45 minutes to about 80 minutes, about 45 minutes to about 90 minutes, about 45 minutes to about 100 minutes, 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 a period of time ranging from about 1 minute to about 60 minutes. In some embodiments, the annealing process lasts a period of time ranging from about 45 minutes to about 60 minutes. The longer water annealing post-processing corresponded an increased crystallinity of silk fibroin protein fragments.

[0547] In some embodiments, the annealed silk fibroin protein fragment film is immersing the wet silk fibroin protein fragment film in 100 % methanol for 60 minutes at room temperature. The methanol annealing changed the composition of silk fibroin protein fragment film from predominantly amorphous random coil to crystalline antiparallel beta-sheet structure.

[0548] In some embodiments, the SPF as described herein can be used to prepare SPF microparticles by precipitation with methanol. Alternative flash drying, fluid-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 powders comprise low molecular weight silk fibroin protein fragments. In some embodiments, the SPF

[0549] 122

[0550] DBl / 165497080.5powders comprise mid-molecular weight silk fibroin protein fragments. In some embodiments, the SPF powders comprise a mixture of low molecular weight silk fibroin protein fragments and mid-molecular weight silk fibroin protein fragment. Silk Protein Fragments in Collagen Boosting Compositions and Methods Thereof The disclosure provides a method of treatment or prevention of a disorder, disease, or condition alleviated by stimulating or modulating collagen expression in a subject in need thereof, comprising administering to the subject a composition comprising silk fibroin fragments, or without limitation any other silk protein fragments described herein, having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, between about 5 kDa and about 10 kDa, between about 6 kDa and about 17 kDa, between about 10 kDa and about 15 kDa, between about 15 kDa and about 20 kDa, between about 14 kDa and about 30 kDa, between about 17 kDa and about 39 kDa, between about 20 kDa and about 25 kDa, between about 25 kDa and about 30 kDa, between about 30 kDa and about 35 kDa, between about 35 kDa and about 40 kDa, between about 39 kDa and about 54 kDa, between about 39 kDa and about 80 kDa, between about 40 kDa and about 45 kDa, between about 45 kDa and about 50 kDa, between about 60 kDa and about 100 kDa, and between about 80 kDa and about 144 kDa, and a polydispersity between 1 and about 5. Any other molecular weight, molecular weight range, and poly dispersity’ of silk fibroin fragments, or without limitation any other silk protein fragments, described herein, can be used in the methods and compositions of the disclosure.

[0551] 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, or without limitation any other silk protein fragments described herein, have a poly dispersity between 1 and about 1.5. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, have a poly dispersity between about 1.5 and about 2.0. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, have a

[0552] 123

[0553] DBl / 165497080.5poly dispersity between about 1.5 and about 3.0. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, have a poly dispersity between about 2.0 and about 2.5. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, have a poly dispersity between about 2.5 and about 3.0. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, are present in the composition at about 0.001 wt. % to about 10.0 wt. % relative to the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the total weight of the composition. In some embodiments, the composition further comprises about 0.001% (w / w) to about 10% (w / w) sericin relative to the silk fibroin fragments, or without limitation any other silk protein fragments described herein. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, do not spontaneously or gradually gelate and do not visibly change in color or turbidity when in an aqueous solution for at least 10 days prior to formulation into the composition. In some embodiments, the silk fibroin fragments, or without limitation any other silk protein fragments described herein, are present in the composition at about 0.01 wt. % to about 10.0 wt. % relative to the total weight of the composition. In some embodiments, the silk fibroin fragments, or without limitation 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 without limitation 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 without limitation 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 w ithout limitation 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

[0554] 124

[0555] DBl / 165497080.5embodiments, the silk fibroin fragments, or without limitation 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 fragments, or without limitation any other silk protein fragments described herein, are present in the composition at about 5.0 wt. % to about 6.0 wt. % relative to the total weight of the composition.

[0556] In some embodiments, the composition is formulated as an injectable composition or as a topical composition. In some embodiments, the composition is formulated for improving look and feel of skin, including without limitation by boosting collagen (e.g., without limitation, stimulating or modulating collagen expression). In some embodiments, the composition is formulated for boosting collagen on skin. In some embodiments, the composition is formulated for boosting collagen intradermally. In some embodiments, the composition is formulated for boosting collagen on 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 nourishing agent 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 125

[0557] DBl / 165497080.5some embodiments, the composition is formulated as a drug for boosting collagen. In some embodiments, the composition is formulated as a therapeutic for boosting collagen. In some embodiments, the composition is formulated as a silk-coated fabric for boosting collagen. In some embodiments, the composition is formulated as a silk- coated non-woven material for boosting collagen.

[0558] 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, an emulsion, a powder, a solution, a dispersion, or an elixir. In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, a jelly, a cream, a lotion, a foam, a slurry, an ointment, an oil, a paste, a suppository , a spray, a semisolid composition, a solid composition, a stick, or a mousse. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of sesame oil, com 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, HA described herein has a molecular weight of 100,000 daltons or greater, 150,000 daltons or greater, 1 million daltons or greater, or 2 million daltons or greater. In some embodiments, 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 salt such as, for example,

[0559] 126

[0560] DBl / 165497080.5sodium hyaluronate. In some embodiments, the HA is crosslinked. Crosslinked HA can be formulated into a variety of shapes, such as membranes, gels, semi-gels, sponges, or microspheres. In some embodiments, the crosslinked HA is in fluid gel form, i.e., it takes the shape of its container. The viscosity of an HA gel or semi-gel can be altered by the addition of unconjugated HA and / or hyaluronate. Viscosity can also be tuned by varying the degree of SPF-SPF, SPF-HA, and / or HA-HA crosslinking as described herein. In some embodiment, about 4% to about 12% of the HA may be crosslinked as HA-HA or HA-SPF.

[0561] In some embodiments, the pharmaceutically acceptable carrier comprises one or more of aliphatic oil, a fatty alcohol, a fatty acid, a glyceride, an acylglycerol, and a phospholipid. In some embodiments, the pharmaceutically acceptable carrier comprises one or more of a monoglyceride, a diglyceride, or a triglyceride. 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 oil, a fatty acid ester, or a cationic quaternary ammonium salt. In some embodiments, a portion of the pharmaceutically acceptable carrier is modified with a cross-linking agent, a cross-linking precursor, or an activating agent selected from a polyepoxy linker, a diepoxy linker, a polyepoxy -PEG, a diepoxy-PEG, a polyglycidyl-PEG, a diglycidyl-PEG, a poly acrylate PEG, a diacrylate PEG, l,4-bis(2,3- epoxypropoxyjbutane, 1,4-bisgly ci dyloxy butane, divinyl sulfone (DVS), 1,4- butanediol diglycidyl ether (BDDE). UV light, glutaraldehyde, l,2-bis(2,3- epoxypropoxyjethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiirmde (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMD A), l-(2,3- epoxypropyl)-2,3-epoxy cyclohexane, a carbodiimide, and any combinations thereof. In some embodiments, the poly epoxy 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,

[0562] 127

[0563] DBl / 165497080.5polytetramethylene 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.

[0564] 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 includes lidocaine. In some embodiments, the concentration of lidocaine in the composition is between about 0.01% and about 1%, including any increment of 0.01%. In some embodiments, the concentration of lidocaine in the composition is about 0.3%.

[0565] In certain embodiments, the compositions described herein can include one or more anesthetic agents in an amount effective to ameliorate or mitigate pain or discomfort at a composition injection site. The local anesthetic can be selected from the group of ambucaine, amolanone, amylocalne, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butamben, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperodon, dicyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine, hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, mepr lcaine. metabutoxy caine, methyl chloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine, parethoxy caine, phenacaine, phenol, piperocaine, piridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine, ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine, zolamine, and salts thereof.

[0566] 128

[0567] DBl / 165497080.5In some embodiments, the compositions described herein may include lidocaine or other anesthetic recited herein at a concentration, 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%.

[0568] In some embodiments, the pharmaceutically acceptable carrier comprises or is formulated as a gel. The gel can be either an injectable gel, for example but without limitation, a tissue filler, or a gel for topical administration. Suitable gels are described for example in WO2019005848, 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 by one or more linker moieties comprising one or more of polyethylene glycol (PEG), polypropylene glycol (PPG), and a secondary alcohol, wherein the linker moieties are 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, are modified or crosslinked. In some embodiments, a portion of the silk fibroin or silk fibroin fragments, or any other SPF described herein, are free. In some embodiments, a portion of the silk fibroin or silk fibroin fragments, or any other SPF described herein, are crosslinked to HA. In some embodiments, a portion of the silk fibroin or silk fibroin fragments, or any other SPF described herein, are 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 129

[0569] DBl / 165497080.5sericin. 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, modification or crosslinking is obtained using as crosslinker a diepoxy -PEG, a polyglycidyl-PEG, a diglycidyl-PEG, a diepoxy-PPG, a polyglycidyl-PPG, a diglycidyl-PPG, or any combinations thereof. In some embodiments, modification or cross-linking is obtained using 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, modification or cross-linking is obtained using 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 includes water. In some embodiments, the gel is monophasic. 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 30 G or 27 G needles. In some embodiments, the gel has a storage modulus (G ’) of from about 5 Pa to about 500 Pa. In some embodiments. G ’ is measured by means of an oscillatory stress of about 1 Hz, about 5 Hz, or about 10 Hz. In some embodiments, the gel has a complex viscosity from about 1 Pa s to about 10 Pa s. In some embodiments, the complex viscosity is measured by means of 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), carboxymethyl cellulose (CMC), starch, alginate, chondroitin- 4-sulfate, chondroitin-6-sulfate, xanthan gum, chitosan, pectin, agar, carrageenan, and guar gum.

[0570] 130

[0571] DBl / 165497080.5In 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 injection, intradermal injection, transdermal injection, or subdermal injection. In some embodiments, the composition is administered by intramuscular injection, intravenous injection, intraperitoneal injection, intraosseous injection, intracardiac injection, intraarticular injection, or intracavemous 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, is biocompatible, biodegradable, bioabsorbable, bioresorbable, or a combination thereof. In some embodiments, the composition provided herein include a fluid component, for example a single fluid or a solution including substantially one or more fluids. In some embodiments, the composition includes water or an aqueous solution. In some embodiments, the composition is injectable, implantable, or deliverable under the skin by any means known in the art such as, for example, following surgical resection of the tissue. In some embodiments, the compositions are dermal fillers. In some embodiments, the compositions are sterile.

[0572] In an embodiment, the percent water content, by weight, 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 131

[0573] DBl / 165497080.557%, 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%.

[0574] In some embodiments, the composition can be administered in and about soft tissue to add volume, add support, or otherwise treat a soft tissue deficiency, in addition to boosting collagen expression. The compositions described herein can be administered at multiple levels beneath the dermis. As used herein, the term ‘’soft tissue” may refer to those tissues that connect, support, or surround other structures and organs of the body. For example, soft tissues described herein may include, without limitation, skin, dermal tissues, subdermal tissues, cutaneous tissues, subcutaneous tissues, intradural tissue, muscles, tendons, ligaments, fibrous tissues, fat, blood vessels and arteries, nerves, and synovial (intradermal) tissues. In some embodiments, the disclosure provides methods of treating a soft tissue condition of an individual, including administering one or more compositions disclosed herein to a site of the soft tissue condition of the individual, wherein the administration of the composition improves the soft tissue condition, thereby treating the soft tissue condition. In some embodiments, a 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 abdominal condition, a buttock condition, an upper leg condition, a lower leg condition including calf condition, a foot condition including plantar fat pad condition, an eye condition, a genital condition, or a condition effecting another body part, region or area.

[0575] In some embodiments, the disclosure provides for compositions and methods of treatment involving a dermal region, including without limitation, the region of

[0576] 132

[0577] DBl / 165497080.5skin comprising the epidermal-dermal junction and the dermis including the superficial dermis (papillary region) and the deep dermis (reticular region). The skin is composed of three primary layers: the epidermis, which provides waterproofing and serves as a barrier to infection; the dermis, which serves as a location for the appendages of skin; and the hypodermis (subcutaneous adipose layer). The epidermis contains no blood vessels, and is nourished by diffusion from the dermis. The main ty pe of cells which make up the epidermis are keratinocytes, melanocytes, Langerhans cells, and Merkels cells.

[0578] In an embodiment, the compositions described herein may be provided in methods of treating one or more conditions in a patient in need thereof. In some embodiments, a therapeutically effective amount of a composition may be delivered into a tissue of a patient in need thereof to treat a condition or other tissue deficiency.

[0579] As used herein, the term ’‘treating,”, '‘treat”, or “treatment” refers to reducing or eliminating in a patient a cosmetic or clinical symptom of a condition, such as a soft tissue condition, or delaying or preventing in an individual the onset of a cosmetic or clinical symptom of a condition.

[0580] In some embodiments, the condition treated by the compositions described herein may include a soft tissue condition. Soft tissue conditions include, without limitation, augmentations, reconstructions, diseases, disorders, defects, or imperfections of a body part, region or area. In one aspect, a soft tissue condition treated by the disclosed compositions include, without limitation, a facial augmentation, a facial reconstruction, a facial disease, a facial disorder, a facial defect, or a facial imperfection. In some embodiments, a soft tissue condition treated by the compositions described herein include, without limitation, skin dehydration, a lack of skin elasticity , skin roughness, a lack of skin tautness, a skin stretch line or mark, skin paleness, a dermal divot, a sunken check, a sunken temple, a thin lip, a urethra defect, a skin defect, a breast defect, a retro-orbital defect, a facial fold, or a wrinkle. In some embodiments, a soft tissue condition treated by the compositions described herein include, without limitation, breast imperfection, defect, disease 133

[0581] DBl / 165497080.5and / or disorder, such as, e.g., a breast augmentation, a breast reconstruction, mastopexy, micromastia, thoracic hypoplasia, Poland’s syndrome, defects due to implant complications like capsular contraction and / or rupture; a facial imperfection, defect, disease or disorder, such as, e.g., a facial augmentation, a facial reconstruction, Parry-Romberg syndrome, lupus erythematosus profundus, dermal divots, sunken cheeks, sunken temples, thin lips, nasal imperfections or defects, retro-orbital imperfections or defects, a facial fold, line and / or wrinkle like a glabellar line, a nasolabial line, a perioral line, and / or a marionette line, and / or other contour deformities or imperfections of the face; a neck imperfection, defect, disease or disorder; a skin imperfection, defect, disease and / or disorder; other soft tissue imperfections, defects, diseases and / or disorders, such as, e.g., an augmentation or a reconstruction of the upper arm, lower arm, hand, shoulder, back, torso including abdomen, buttocks, upper leg, lower leg including calves, foot including plantar fat pad, eye, genitals, or other body part, region or area, or a disease or disorder affecting these body parts, regions or areas; urinary incontinence, fecal incontinence, other forms of incontinence; and gastroesophageal reflux disease (GERD).

[0582] In some embodiments, the compositions described herein may be delivered to soft tissues including, without limitation skin, dermal tissues, subdermal tissues, cutaneous tissues, subcutaneous tissues, intradural tissue, muscles, tendons, ligaments, fibrous tissues, fat, blood vessels and arteries, nerves, and synovial (intradermal) tissues.

[0583] In some embodiments, the compositions described herein can be placed directly in a wound to aid in healing by providing an artificial biodegradable matrix along with cell attachment, migration, and proliferation signals. In some embodiments, the compositions described herein can be coated on a biodegradable mesh or other implanted material, or it can itself be formed into sheets or other structures, or can be maintained in a hydrated form.

[0584] In some embodiments, the amount of a composition used with any of the methods as disclosed herein will be determined based on the alteration and / or 134

[0585] DBl / 165497080.5improvement desired, the reduction and / or elimination of a condition symptom desired, the clinical and / or cosmetic effect desired by the individual and / or physician, and the body part or region being treated. The effectiveness of composition administration may be manifested by one or more of the following clinical and / or cosmetic measures: altered and / or improved soft tissue shape, altered and / or improved soft tissue size, altered and / or improved soft tissue contour, altered and / or improved tissue function, tissue ingrow th support and / or new collagen deposition, sustained engraftment of the composition, improved patient satisfaction and / or quality of life, and decreased use of implantable foreign material. For example, for breast augmentation procedures, effectiveness of the compositions and methods may be manifested by one or more of the following clinical and / or cosmetic measures: increased breast size, altered breast shape, altered breast contour, sustained engraftment, reduction in the risk of capsular contraction, decreased rate of liponecrotic cyst formation, improved patient satisfaction and / or quality of life, and decreased use of breast implant.

[0586] In some embodiments, administering the composition decreases expression of one or more metalloproteinases (MMP) in the subject. In some embodiments, stimulating or modulating collagen expression comprises increasing collagen expression.

[0587] In some embodiments, collagen expression is increased over a base level 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%,

[0588] 135

[0589] DBl / 165497080.5about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%. about 87%, about 88%, about 89%, about 90%, about 91%. about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%.

[0590] In some embodiments, collagen expression is increased over a base level by 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%, about 123%, about 124%, about 125%, about 126%, about 127%, about 128%, about 129%, about 130%, about 131%, about 132%, about 133%, about 134%, about 135%, about 136%, about 137%, about 138%, about 139%, about 140%, about 141%, about 142%, about 143%, about 144%. about 145%, about 146%, about 147%, about 148%, about 149%. about 150%. about 151%, about 152%, about 153%, about 154%, about 155%, about 156%, about 157%, about 158%, about 159%, about 160%, about 161%, about 162%, about 163%, about 164%, about 165%, about 166%, about 167%, about 168%, about 169%, about 170%, about 171%, about 172%, about 173%, about 174%, about 175%. 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%.

[0591] In some embodiments, collagen expression is increased over a base level by about 225%, about 250%, about 275%, about 300%, about 325%. about 350%. about 375%, about 400%, about 425%, about 450%, about 475%, about 500%, about 525%, about 550%, about 575%, about 600%, about 625%, about 650%, about 675%, about 700%, about 725%, about 750%, about 775%, about 800%, about 825%, about 850%, about 875%, about 900%, about 925%, about 950%, about 975%. or about 1000%.

[0592] 136

[0593] DBl / 165497080.5In some embodiments, administering the composition results in one or more of preventing or reversing wrinkles in the subject, preventing or reversing age spots in the subject, preventing or reversing dry skin in the subject, increasing uneven skin tone in the subject, or improving look and feel of skin. Improving look and feel of skin includes without limitation improving look and feel of damaged skin, but also improving look and feel of skin which is not otherwise visibly damaged. In some embodiments, administering the composition results in one or more of preventing or reversing skin sagging in the subject, preventing or reversing skin aging in the subject, preventing or reversing reduced skin tensile strength in the subject, preventing or reversing photodamaged skin in the subject, or preventing or reversing striae distensae (stretch marks) in the subject. In some embodiments, the disorder, disease, or condition comprises wrinkles, age spots, dry skin, uneven skin tone, skin sagging, skin aging, reduced skin tensile strength, photodamaged skin, or striae distensae (stretch marks). In some embodiments, the disorder, disease, or condition comprises a skin condition. In some embodiments, the skin condition can be skin dehydration, lack of skin elasticity, skin roughness, lack of skin tautness, a skin stretch line, a skin stretch mark, skin paleness, a dermal divot, a sunken cheek, sunken temple, a thin lip, a retro-orbital defect, a facial fold, or a wrinkle.

[0594] In some embodiments, the methods of treatment disclosed comprise an augmentation, a reconstruction, treating a disease, treating a disorder, correcting a defect or imperfection of a body part, region or area. In some embodiments, the methods of treatment disclosed comprise a facial augmentation, a facial reconstruction, treating a facial disease, treating a facial disorder, treating a facial defect, or treating a facial imperfection.

[0595] In some embodiments, the methods of treatment provided include one or more of administering a composition of the disclosure before, after, or during a laser treatment, administering a composition of the disclosure before, after, or during a skin peel, administering a composition of the disclosure before, after, or during a radiation treatment. In some embodiments, the methods of treatment provided include one or

[0596] 137

[0597] DBl / 165497080.5more of administering a composition of the disclosure to treat a bum, including without limitation any type of bum (e.g., thermic bum, sunburn, fire bum, hot liquid bum, radiation bum, chemical bum, and the like). In some embodiments, the methods of treatment provided include one or more of administering a composition of the disclosure to treat a bum, including without limitation a first-, second-, or third-degree bum. In some embodiments, the methods of treatment provided include one or more of administering a composition of the disclosure for treating a skin condition due to aging.

[0598] In some embodiments, the disorder, disease, or condition comprises thyroid hormone-induced myocardial hypertrophy. In some embodiments, the disorder, disease, or condition comprises a tendon rupture, damage, or tear. In some embodiments, the tendon is selected from Teres minor tendons, Infraspinatus tendons, Supraspinatus tendons, Subscapularis tendons, Deltoid tendons, Biceps tendons, Triceps tendons, Brachioradialis tendons, Supinator tendons, Flexor carpi radialis tendons, Flexor carpi ulnaris tendons, Extensor carpi radialis tendons, Extensor carpi radialis brevis tendons, Iliopsoas tendons, Obturator intemus tendons. Adductor longus, brevis or magnus tendons, Gluteus maximus or gluteus medius tendons, Quadriceps tendons, patellar tendon, Hamstring tendons, Sartorius tendons, Gastrocnemius tendons, Achilles tendon, Soleus tendons, Tibialis anterior tendons. Peroneus longus tendons. Flexor digitorum longus tendons, Interosseus tendons, Flexor digitorum profundus tendons, Abductor digiti minimi tendons, Opponens pollicis tendons, Flexor pollicis longus tendons, Extensor or abductor pollicis tendons, Flexor hallucis longus tendons, Flexor digitorum brevis tendons, Lumbrical tendons, Abductor hallucis tendons. Flexor digitorum longus tendons, Abductor digiti minimi tendons, Ocular tendons, Levator palpebrae tendons. Masseter tendons, Temporalis tendons, Trapezius tendons, Sternocleidomastoid tendons, Semispinalis capitis or splenius capitis tendons, Mylohyoid or thy rohyoid tendons, Sternohyoid tendons, Rectus abdominis tendons. External oblique tendons, Transversus abdominis tendons, Latissimus dorsi tendons, and Erector spinae tendons. In some embodiments, the disorder, disease, or condition comprises Werner’s syndrome. In some embodiments,

[0599] 138

[0600] DBl / 165497080.5the disorder, disease, or condition comprises diminished 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 diminished 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 aneurysms. In some embodiments, the disorder, disease, or condition comprises a wound. In some embodiments, the disorder, disease, or condition comprises a skin or connective tissue disease. In some embodiments, the disorder, disease, or condition comprises a cartilage disease. In some embodiments, the disorder, disease, or condition is selected from relapsing polychondritis, Tietze’s Syndrome, cellulitis, Ehler's Danlos syndrome, keloids (including acne keloids), mucopolysaddaridosis I, necrobiotic disorders (including granuloma annulare, necrobiosis lipoidica), osteogenesis imperfect, cutis laxa, dermatomyositis, Dupytren’s contracture, homocystinuria, lupus erythematosis (including cutaneous, discoid, panniculitis, systemic and nephritis), marfan syndrome, mixed connective tissue disease, mucinosis (including follicular), mucopolysaccaridoses (1, 11, UU, IV, IV, and VII), myxedema, scleredemo adultorum and synovial cysts, connective tissue neoplasms, Noonan syndrome, osteopoikilosis, panniculitis, including erythema induratum, nodular nonsuppurative and peritoneal, penile induration, pseudoxanthoma elasticum, rheumatic diseases, including arthritis (rheumatoid, juvenile rheumatoid, Caplan’s syndrome, Felty’s syndrome, rheumatoid nodule, ankylosing spondylitis, and still’s disease), hyperostosis, polymyalgia rheumatics, circumscribed scleroderma, and systemic scleroderma (CREST syndrome). In some embodiments, the disorder, disease, or condition is selected from angiolymphoid hyperplasia with eosinophilia; cicatix (including hypertophic); cutaneous fistula, cuis laxa; dermatitis, including acrodermatitis, atopic dermatitis, contact dermatitis (allergic contact, photoallergic, toxicodendron), irritant dermatitis (phototoxic, diaper rash), occupational dermatitis; exfoliative dermatitis, herpetiformis dermatitis, seborrheic dermatitis, drug eruptions 139

[0601] DBl / 165497080.5(such as toxic epidermal necrolysis, erythema nodosum, serum sickness) eczema, including dyshidrotic, intertrigo, neurodermatitis, and radiodermatitis; dermatomyositis; erythema, including chronicum migrans, induratum, infectiosum, multiforme (Stevens-Johnson syndrome), and nodosum (Sweet’s syndrome); exanthema, including subitum; facial dermatosis, including acneiform eruptions (keloid, rosacea, vulgaris and Favre-Racouchot syndrome); foot dermatosis, including tinea pedis; hand dermatoses; keratoacanthoma; keratosis, including callosities, cholesteatoma (including middle ear), ichthyosis (including congenital ichtyosiform erythroderms, epidermolytic hyperkeratosis, lamellar ichthyosis, ichthyosis vulgaris. X-linked ichthyosis, and Sjogren-Larsson syndrome), keratoderma blennorrhagicum, palmoplantar keratoderms, follicularis keratosis, seborrheic keratosis, parakeratosis and porokeratosis; leg dermatosis, mastocytosis (urticaria pigmentosa), necrobiotic disorders (granuloma annulare and necrobiosis lipoidica), photosensitivity disorders (photoallergic or photoxic dermatitis, hydroa vacciniforme, sundum, and xeroderma pigmentosum); pigmentation disorders, including argyria, hyperpigmentation, melanosis, aconthosis nigricans, lentigo, Peutz-Jeghers syndrome, hypopigmentation, albinism, pibaldism, vitiligo, incontinentia pigmenti, urticaria pigmentosa, xeroderma pigmentosum, prurigo; pruritis (including ani and vulvae); pyoderma, including ecthyma and pyoderma gangrenosum; sclap dermatoses; sclerodema adultorum; sclerma neonatorum; skin appenage diseases, including hair diseases (alopecia, folliculitis, hirsutism, hypertichosis, Kinky hair syndrome), nail diseases (nail-patella syndrome, ingrown or malformed nails, onychomycosis, paronychia), sebaceous gland diseases (rhinophyma, neoplasms), sweat gland diseases (hidradenitis, hyperhidrosis, hypohidrosis, miliara, Fox-Fordyce disease, neoplasms); genetic skin diseases, including alfinism, cutis laxa, benign familial pemphigis, porphyria, acrodermatitis, ectodermal dysplasia, Ellis-Van Creveld syndrome, focal dermal hypoplasia, Ehlers-Danlos syndrome, epidermolysis bullosa, ichtysosis; infectious skin diseases, including dermatomycoses, blastomycosis, candidiasis, chromoblastomycosis, maduromycosis, paracoccidioidomycosis, sporotrichosis, tinea; bacterial skin diseases, such as cervicofacial actinomycosis, bacilliary angiomatosis,

[0602] 140

[0603] DBl / 165497080.5ecthyma, erysipelas, erythema chronicum migrans, erythrasma, granuloma inguinale, hidradenitis suppurativa, maduromycosis, paronychia, pinta, rhinoscleroma, staphylococcal skin infections (furuncolosis, carbuncle, 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, moolusum contagiosum, and warts.

[0604] In some embodiments, the amount of a composition used with any of the methods disclosed herein will typically be 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 composition that will elicit 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 those skilled in the art, using the guidance provided herein. For example, an effective amount can be extrapolated from any and all in vitro and in vivo assays as described herein. One skilled in the art will recognize that the condition of the individual can be monitored throughout the course of therapy and that the effective amount of a composition disclosed herein that is administered can be adjusted accordingly.

[0605] In some embodiments, the amount of a composition administered is, without limitation, 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 0.8 g, or at least 0.9 g, or at least 1 g, or at least 2 g. or at least 3 g, or at least 4 g, or at least 5 g, or at least 6 g, or at least 7 g. or at

[0606] 141

[0607] DBl / 165497080.5least 8 g, or at least 9 g, or at least 10 g, or at least 11 g, or at least 12 g, or at least 13 g, or at least 14 g, or at least 15 g, or at least 20 g, or at least 25 g, or at least 30 g, or at least 35 g, or at least 40 g, or at least 45 g, or at least 50 g, or at least 55 g, or at least 60 g, or at least 65 g, or at least 70 g, or at least 75 g, or at least 80 g, or at least 85 g, or at least 90 g, or at least 95 g, or at least 100 g.

[0608] In some embodiments, the amount of a composition administered is, without limitation, at most 0.001 g, or at most 0.002 g, or at most 0.003 g, or at most 0.004 g, or at most 0.005 g, or at most 0.006 g, or at most 0.007 g, or at most 0.008 g, or at most 0.009 g, or at most 0.01 g, or at most 0.02 g, or at most 0.03 g, or at most 0.04 g, or at most 0.05 g, or at most 0.06 g, or at most 0.07 g, or at most 0.08 g, or at most 0.09 g, or at most 0.1 g, or at most 0.2 g, or at most 0.3 g, or at most 0.4 g, or at most 0.5 g, or at most 0.6 g, or at most 0.7 g, or at most 0.8 g, or at most 0.9 g, or at most 1 g, or at most 2 g, or at most 3 g, or at most 4 g, or at most 5 g, or at most 6 g, or at most 7 g, or at most 8 g, or at most 9 g, or at most 10 g, or at most 11 g, or at most 12 g, or at most 13 g, or at most 14 g. or at most 15 g, or at most 20 g, or at most 25 g. or at most 30 g, or at most 35 g, or at most 40 g, or at most 45 g, or at most 50 g, or at most 55 g, or at most 60 g, or at most 65 g, or at most 70 g, or at most 75 g, or at most 80 g, or at most 85 g, or at most 90 g, or at most 95 g, or at most 100 g.

[0609] In some embodiments, the amount of a composition administered is, without limitation, 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 0.6 g, or about 0.7 g, or about 0.8 g, or about 0.9 g. or about 1 g. or about 2 g, or about 3 g, or about 4 g, or about 5 g, or about 6 g, or about 7 g, or about 8 g, or about 9 g, or about 10 g, or about 11 g, or about 12 g, or about 13 g, or about 14 g, or about 15 g, or about 20 g, or about 25 g, or a...

Claims

CLAIMS1. A method of treatment or prevention of a disorder, disease, or condition alleviated by: stimulating or modulating collagen expression in a subject in need thereof; and / or stimulating or modulating claudin-1 expression in a subject in need thereof; and / or stimulating or modulating one more anti-inflammatory genes in a subject in need thereof, the method comprising administering to the subject a composition comprising fibroin heavy chain protein fragments, fibroin light chain protein fragments, and fibrohexamerin protein fragments, wherein:one or more fibroin heavy chain protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 49 selected from Q58E, M64X, N68D, N70D, N77D, M80X, N93D, M103X, Q125E, N132D, Q139E, Q275E, N4191D, Q5216E, and N5262D;one or more fibroin light chain protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 50 selected from N23D, Q24E, N28D, M69X, N105D, N108D, N118D, N136D, N138D, Q149E, N186D, N200D, Q202E, N204D, N240D, N248D, and Q255E; and one or more fibrohexamerin protein fragments comprise at least one amino acid modification at an amino acid position corresponding to an amino acid residue in SEQ ID NO: 51 selected from Q62E, N93D, M120X, N149D, N172D, N174D, and N202D, wherein X is methionine oxide.

2. The method of claim 1, wherein the composition is formulated as an injectable composition or as a topical composition further comprising a pharmaceutically acceptable carrier, wherein one or more of the following apply:403DBl / 165497080.5the pharmaceutically acceptable carrier comprises an aqueous phase;the pharmaceutically acceptable carrier comprises an oil-in-water emulsion or a water-in- oil emulsion;the pharmaceutically acceptable carrier comprises or is formulated as one or more of a cosmeceutical, a suspension, an emulsion, a powder, a solution, a dispersion, or an elixir; the pharmaceutically acceptable carrier comprises or is formulated as one or more of a gel, a jelly, a cream, a lotion, a foam, a slurry, an ointment, an oil, a paste, a suppository, a spray, a semisolid composition, a solid composition, a stick, or a mousse;the pharmaceutically acceptable carrier comprises one or more of aliphatic oil, a fatty alcohol, a fatty acid, a glyceride, an acylglycerol, and a phospholipid;the pharmaceutically acceptable carrier comprises one or more of a monoglyceride, a diglyceride, or a triglyceride; and / orthe pharmaceutically acceptable carrier comprises one or more of a hydrocarbon oil, a fatty acid, a fatty oil, a fatty acid ester, or a cationic quaternary ammonium salt.

3. The method of claim 1 or 2, wherein the composition is formulated for one or more of the following:for administration to an epithelial surface selected from a superficial epidermal area, a stratum corneum, an eye surface, or an intestinal surface;for reducing trans-epidermal water loss;as a barrier formulation;as a wound-closure formulation;for preventing or reversing wrinkles in the subject, preventing or reversing age spots in the subject, preventing or reversing dry skin in the subject, or preventing or reversing uneven skin tone in the subject;404DBl / 165497080.5for preventing or reversing skin sagging in the subject, preventing or reversing skin aging in the subject, preventing or reversing reduced skin tensile strength in the subject, preventing or reversing photodamaged skin in the subject, or preventing or reversing striae distensae (stretch marks) in the subject.

4. The method of any one of claims 1 to 3, wherein the disorder, disease, or condition comprises wrinkles, age spots, dry skin, uneven skin tone, skin sagging, skin aging, reduced skin tensile strength, photodamaged skin, o striae distensae (stretch marks).

5. The method of any one of claims 1 to 3, wherein the disease, disorder or condition is selected from Atopic Dermatitis (Eczema), Psoriasis (e.g., plaque psoriasis), Contact Dermatitis, rosacea, acne (e.g., cystic acne), Ichthyosis (e.g., ichthyosis vulgaris), inflammation, Netherton Syndrome (a genetic disorder that lead to chronic skin inflammation and increased susceptibility to an infection), Xerosis (Dry Skin), Aging Skin, impaired wound healing, Diabetic Dermopathy, Pruritus, Scleroderma, Multiple Sclerosis (MS), Bacterial and fungal Infection, abnormal scarring condition, melanoma, dysbiosis, autoimmune condition like lupus, Systemic Lupus Erythematosus, and rheumatoid arthritis.

6. The method of any one of claims 1 to 3, wherein the disorder, disease, or condition is an inflammatory disease, disorder or condition selected from Rheumatoid Arthritis, Inflammatory Bowel Disease, Systemic Lupus Erythematosus, Multiple Sclerosis (MS), Psoriasis, Asthma, allergic rhinitis, asthma, food allergy, and another autoimmune condition.

7. The method of any one of claims 1 to 3, wherein the disease, disorder or condition is selected from inflammatory bowel disease (IBD), Intestinal Permeability, Crohn’s 405DBl / 165497080.5disease, ulcerative colitis, celiac disease, irritable bowel syndrome (IBS), NASH, cirrhosis, metabolic disorders, and another disease, a disorder or condition involving regulation of a gut epithelial cell.

8. The method of any one of claims 1 to 3, wherein the disease, disorder or condition is a neurodegenerative condition selected from Alzheimer’s Disease (AD), Multiple Sclerosis (MS), Parkinson’s Disease (PD), and Amyotrophic Lateral Sclerosis (ALS).

9. The method of any one of claims 1 to 3, wherein the disease, disorder or condition is a metabolic condition selected from obesity and type 2 Diabetes, and non-alcoholic fatty liver disease (NAFLD).

10. The method of any one of claims 1 to 3, wherein the disease, disorder or condition is a cancer.

11. The method of any one of claims 1 to 3, wherein:the disease, disorder or condition is associated with dysregulation in a gut barrier and / or in a tight junction of the gut;the disease, disorder, or condition is alleviated by decreasing the secretion of TNF-a and IL-6, and / or increasing secretion of IL-10;the disease, disorder, or condition is alleviated by modulating Annexin A2’s role in tumor growth, immune function, neuronal protection, and / or metabolic processes;the disorder, disease, or condition is alleviated by stimulating or modulating a skin barrier-specific protein in a subject in need thereof, wherein the skin barrier-specific protein comprises at least one of desmoplakin (DSP), cystatin A (CSTA), tumor-406DBl / 165497080.5associated calcium signal transducer 2 (TACSTD2), biglycan (BGN), decorin (DCN) and / or epiplakin 1 (EPPK1), wherein at least one of desmoplakin (DSP), cystatin A (CSTA) and / or tumor-associated calcium signal transducer 2 (TACSTD2) is upregulated post-administration, and / or wherein at least one of biglycan (BGN), decorin (DCN) and / or epiplakin 1 (EPPK1) is downregulated post-administration, and / or wherein at least one of the skin barrier-specific protein is a tight junction protein; and / or administering the composition induces secretion of pro-CollAl, Col3A, and / or a-SMA.

12. The method of any one of claims 1 to 11, wherein the protein fragments have a weight average molecular weight selected from between about 1 kDa to about 250 kDa,13. The method of any one of claims 1 to 11, wherein the protein fragments have a weight average molecular weight selected from between about 1 kDa and about 20 kDa, from between about 20 kDa and about 40 kDa, from between about 40 kDa and about 60 kDa, from between about 60 kDa and about 80 kDa, from between about 80 kDa and about 100 kDa, from between about 100 kDa and about 120 kDa, from between about 120 kDa and about 140 kDa, or from between about 140 kDa and about 160 kDa, from between about 160 kDa and about 180 kDa, from between about 180 kDa and about 200 kDa, or from between about 200 kDa and about 250 kDa.

14. The method of any one of claims 1 to 11, wherein the protein fragments have a weight average molecular weight selected from between about 10 kDa and about 20 kDa, from between about 20 kDa and about 40 kDa, from between about 40 kDa and about 60 kDa, from between about 60 kDa and about 80 kDa, from between about 80 kDa and about 100 kDa, from between about 100 kDa and about 120 kDa, from between about 120 kDa and about 140 kDa, from between about 140 kDa and about 160 kDa, or from between about 160 kDa and about 180 kDa.407DBl / 165497080.

515. The method of any one of claims 1 to 11, wherein the protein fragments have a weight average molecular weight selected from between about 21 kDa and about 41 kDa.

16. The method of any one of claims 1 to 11, wherein the protein fragments have a weight average molecular weight selected from between about 12 kDa and about 20 kDa.

17. The method of any one of claims 12 to 16, wherein the protein fragments have apoly dispersity between 1 and 3.4.

18. The method of any one of claims 12 to 16, wherein the protein fragments have apoly dispersity between 1 and about 1.5, between about 1.5 and about 2.0, between about 2.0 and about 2.5, or between about 2.5 and about 3.0.

19. The method of any one of claims 12 to 16, wherein the protein fragments have apoly dispersity between 1 and about 1.7.

20. The method of any one of claims 12 to 16, wherein the protein fragments have apoly dispersity between 1 and about 1.1, between about 1.1 and about 1.2, between about 1.2 and about 1.3, between about 1.3 and about 1.4, or between about 1.4 and about 1.5.

21. The method of any one of claims 1 to 20, wherein each modification is independently ranging in the composition between about 1% to about 99%.408DBl / 165497080.

522. The method of claim 21, wherein a percent modification is defined as (number of protein fragments comprising a modification at a specific position, divided by the total number of protein fragments which include the specific position, whether comprising a modification or not) x 100.

23. The method of any one of claims 1 to 22, wherein a percentage of amino acid modification in the fibroin heavy chain protein fragments at Q58E is from about 1% to about 10%, at M64X is from about 20% to about 90%, at N68D is from about 30% to 100%, at N70D is from about 20% to 100%, at N77D is from about 80% to 100%, at M80X is from about 10% to about 50%, at N93D is from about 1% to about 40%, at M103X is from about 10% to about 40%, at Q125E is from about 1% to about 30%, at N132D is from about 10% to about 90%, at Q139E is from about 1% to about 30%, at Q275E is from about 1% to about 10%, atN4191D is from about 10% to 100%, at Q5216E is from about 1% to about 20%, and at at N5262D is from about 1% to about 50%.

24. The method of any one of claims 1 to 22, wherein a percentage of amino acid modification in the fibroin light chain protein fragments at N23D is from about 70% to 100%, at Q24E is from about 1% to about 10%, at N28D is from about 1% to about 40%, at M69X is from about 20X% to about 80%, at N105D is from about 40% to about 80%, at N 108D is from about 10% to about 50%, at N118D is from about 20% to about 60%, at N136D is from about 10% to about 60%, at N138D is from about 1% to about 10%, at Q149E is from about 1% to about 20%, at N186D is from about 1% to about 40%, at N200D is from about 40% to 100%, at Q202E is from about 1% to about 50%, at N204D is from about 40% to 100%, at N240D is from about 20% to 100%, at N248D is from about 10% to about 80%, and at Q255E is from about 1% to about 10%.409DBl / 165497080.

525. The method of any one of claims 1 to 22, wherein a percentage of amino acid modification in the fibrohexamerin protein fragments at Q62E is from about 1% to about 90%, at N93D is from about 1% to about 15%, at M120X is from about 1% to about 70%, at N149D is from about 20% to about 70%, at N172D is from about 40% to about 80%, at N174D is from about 1% to about 80%, and at N202D is from about 1% to about 30%.

26. The method of any one of claims 1 to 25, wherein:the composition comprises a fibroin fragment subpopulation having an isoelectric point selected from between about 4.4 to about 9.6;the composition comprises a fibroin fragment subpopulation having a self-assembly rate factor selected from between 0 to about 5;the composition comprises a fibroin fragment subpopulation having a self-assembly rate factor selected from between about 0.6 to about 4;the composition comprises a fibroin fragment subpopulation each having a self-assembly factor selected from between about 0.5 to about 0.9;the composition comprises a fibroin fragment subpopulation having a self assembly rate factor selected from between about 5 to about 11;the composition comprises a fibroin fragment subpopulation having an Amax selected from between 0 to about 0.7;the composition comprises a fibroin fragment subpopulation having an Amax selected from between about 0.5 to about 0.8;the composition comprises a fibroin fragment subpopulation having a to.s(min) selected from between 0 to about 1500; and / orthe composition comprises a fibroin fragment subpopulation having a to.s(min) selected from between 0 to about 1000.410DBl / 165497080.

527. The method of any one of claims 1 to 27, wherein the composition comprises a formulation comprising water and one or more of: sodium benzoate (0.50-1.50% w / w), aspen bark (0.50-3.50% w / w), glycerin (0.50-3.50% w / w), fibroin glycoside (Pl-alpha hydrogel, 0.10-1.00% w / w), medium-chain triglyceride oil (MCT, 10-35% w / w), and an acidic agent (0.10-0.50% w / w).

28. The method of claim 27, wherein the acidic agent is citric acid (50% Solution).

29. The method of claim 27 or 28, wherein the protein fragments concentration in the formulation is between about 1% and about 15% w / w.411DBl / 165497080.5

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