MORTARINO ENRICO (US)
JOHNSON SARA (US)
| WO2019067745A1 | 2019-04-04 |
| CLAIMS 1. An article comprising a fabric and a coating, wherein the coating comprises a reducing agent and silk fibroin fragments having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, from between about 5 kDa and about 10 kDa, from between about 6 kDa and about 17 kDa, from between about 10 kDa and about 15 kDa, from between about 14 kDa and about 30 kDa, from between about 15 kDa and about 20 kDa, from between about 17 kDa and about 39 kDa, from between about 20 kDa and about 25 kDa, from between about 25 kDa and about 30 kDa, from between about 30 kDa and about 35 kDa, from between about 35 kDa and about 40 kDa, from between about 39 kDa and about 54 kDa, from between about 39 kDa and about 80 kDa, from between about 40 kDa and about 45 kDa, from between about 45 kDa and about 50 kDa, from between about 50 kDa and about 55 kDa, from between about 55 kDa and about 60 kDa, from between about 60 kDa and about 100 kDa, or from between about 80 kDa and about 144 kDa, and a poly dispersity selected from between 1 and about 5. 2. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between 1 and about 1.5. 3. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 1.5 and about 3.0. 4. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 1.5 and about 2. 5. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 2 and about 2.5. 6. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 2.5 and about 3. 7. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 3 and about 3.5 or from between about 3.5 and about 4.0. 8. The article of claim 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 4.0 and about 4.5 or from between about 4.5 and about 5.0. 9. The article of any one of claims 1 to 8, wherein the silk fibroin fragments comprise one or more of low molecular weight silk fibroin fragments and medium molecular weight silk fibroin fragments. 10. The article of any one of claims 1 to 9, further comprising about 0.01% (w/w) to about 10% (w/w) sericin relative to the silk fibroin fragments. 11. The article of any one of claims 1 to 9, further comprising about 0.01% (w/w) to about 10% (w/w) sericin relative to total weight of the article. 12. The article of any one of claims 1 to 11, wherein the silk fibroin fragments 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 article. 13. The article of any one of claims 1 to 12, wherein the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool. 14. The article of any one of claims 1 to 12, wherein the fabric comprises one or more of natural wool, chlorine-descaled wool, or non-chlorine descaled wool. 15. The article of any one of claims 1 to 14, wherein the reducing agent is selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin. 16. The article of any one of claims 1 to 14, wherein the reducing agent is L-cysteine. 17. The article of any one of claims 1 to 16, wherein the reducing agent is physically adsorbed to a surface of the fabric. 18. The article of any one of claims 1 to 16, wherein the reducing agent is chemically linked to a surface of the fabric. 19. The article of any one of claims 1 to 18, wherein the coating further comprises a crosslinker. 20. The article of claim 19, wherein the crosslinker is glycerol diglycidyl ether (GDE). 21. A method of making a silk fibroin coated fabric, comprising: applying to the fabric a solution comprising a reducing agent; applying to the fabric a silk fibroin solution; and drying the fabric. 22. A method of improving size retention on laundering in a fabric, comprising: applying to the fabric a solution comprising a reducing agent; applying to the fabric a silk fibroin solution; and drying the fabric. 23. The method of claim 21 or 22, wherein upon laundering, the fabric substantially retains its initial size prior to laundering. 24. The method of claim 21 or 22, wherein upon laundering, the fabric retains a substantially higher fraction of its initial size prior to laundering compared to a similar fabric not similarly treated with the reducing agent and the silk fibroin solution. 25. The method of any one of claims 21 to 24, wherein the silk fibroin solution comprises low molecular weight silk fibroin fragments. 26. The method of any one of claims 21 to 24, wherein the silk fibroin solution comprises low molecular weight silk fibroin fragments, medium molecular weight silk fibroin fragments, or a combination thereof. 27. The method of any one of claims 21 to 26, wherein the concentration of the silk fibroin solution is between about 0.1% w/v and about 0.5% w/v, between about 0.5% w/v and about 1% w/v, between about 1% w/v and about 1.5% w/v, between about 1.5% w/v and about 2% w/v, between about 2% w/v and about 3% w/v, between about 3% w/v and about 4% w/v, between about 4% w/v and about 5% w/v, between about 5% w/v and about 6% w/v, between about 6% w/v and about 7% w/v, between about 7% w/v and about 8% w/v, between about 8% w/v and about 9% w/v, or between about 9% w/v and about 10% w/v. 28. The method of any one of claims 21 to 26, wherein the concentration of the silk fibroin solution is about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9% w/v, or about 1% w/v. 29. The method of any one of claims 21 to 26, wherein the concentration of the silk fibroin solution is about 1% w/v, about 2% w/v, 3% w/v, about 4% w/v, about 5% w/v, or about 6%. 30. The method of any one of claims 21 to 29, wherein the drying comprises heating. 31. The method of claim 30, wherein the heating does not substantially modify silk fibroin coating performance. 161 32. The method of any one of claims 21 to 31, wherein the pH of the silk fibroin solution is acidic. 33. The method of any one of claims 21 to 31, wherein the pH of the silk fibroin solution is about 4, about 5, or about 6. 34. The method of any one of claims 21 to 31, wherein the pH of the silk fibroin solution is between about 3 and less than 7. 35. The method of any one of claims 21 to 31, wherein the pH of the silk fibroin solution is between about 3.5 and about 5.5. 36. The method of any one of claims 21 to 35, wherein the fabric is dried after applying the solution comprising a reducing agent and before applying the silk fibroin solution. 37. The method of any one of claims 21 to 36, wherein the concentration of the reducing agent in the solution ranges from about 1 g/L to about 12 g/L. 38. The method of any one of claims 21 to 37, wherein the silk fibroin solution further comprises a reducing agent. 39. The method of any one of claims 21 to 37, wherein the silk fibroin solution and the solution comprising a reducing agent are applied at substantially the same time. 40. The method of any one of claims 21 to 37, wherein the silk fibroin solution and the solution comprising a reducing agent are substantially one solution. 41. The method of any one of claims 21 to 40, wherein the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool. 42. The method of any one of claims 21 to 40, wherein the fabric comprises one or more of natural wool, chlorine-descaled wool, or non-chlorine descaled wool. 43. The method of any one of claims 21 to 42, wherein the reducing agent is independently selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2- mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin. 44. The method of any one of claims 21 to 42, wherein the reducing agent is independently at each occurrence L-cysteine. 162 45. The method of any one of claims 21 to 44, further comprising applying to the fabric a crosslinker. 46. The method of claim 45, wherein the crosslinker is glycerol diglycidyl ether (GDE). 47. The method of any one of claims 21 to 46, wherein the silk fibroin solution comprises silk fibroin fragments having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, from between about 5 kDa and about 10 kDa, from between about 6 kDa and about 17 kDa, from between about 10 kDa and about 15 kDa, from between about 14 kDa and about 30 kDa, from between about 15 kDa and about 20 kDa, from between about 17 kDa and about 39 kDa, from between about 20 kDa and about 25 kDa, from between about 25 kDa and about 30 kDa, from between about 30 kDa and about 35 kDa, from between about 35 kDa and about 40 kDa, from between about 39 kDa and about 54 kDa, from between about 39 kDa and about 80 kDa, from between about 40 kDa and about 45 kDa, from between about 45 kDa and about 50 kDa, from between about 50 kDa and about 55 kDa, from between about 55 kDa and about 60 kDa, from between about 60 kDa and about 100 kDa, or from between about 80 kDa and about 144 kDa, and a poly dispersity selected from between 1 and about 5. 48. The method of any one of claims 21 to 47, wherein the silk fibroin solution further comprises about 0.01% (w/w) to about 10% (w/w) sericin relative to the silk fibroin fragments. 49. The method of any one of claims 21 to 48, wherein the silk fibroin solution comprises silk fibroin fragments which 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 silk fibroin solution. 50. An article comprising a fabric prepared by the method of any one of claims 21 to 49. 163 |
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is an international application claiming the benefit of U.S. Provisional Application No. 63/085,618, filed on September 30, 2020, incorporated herein by reference in its entirety.
FIELD
[0002] The present disclosure is in the field of fabrics coated with silk fibroin-based proteins after pretreatment with a reducing agent, such as L-cysteine.
BACKGROUND
[0003] Silk is a natural polymer produced by a variety of insects and spiders, and comprises a filament core protein, silk fibroin, and a glue-like coating consisting of a non-filamentous protein, sericin. Silk fibers are light weight, breathable, and hypoallergenic. Silk is comfortable when worn next to the skin and insulates very well; keeping the wearer warm in cold temperatures and is cooler than many other fabrics in warm temperatures.
SUMMARY
[0004] In an embodiment, the disclosure provides an article comprising a fabric and a coating, wherein the coating comprises a reducing agent and silk fibroin fragments having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, from between about 5 kDa and about 10 kDa, from between about 6 kDa and about 17 kDa, from between about 10 kDa and about 15 kDa, from between about 14 kDa and about 30 kDa, from between about 15 kDa and about 20 kDa, from between about 17 kDa and about 39 kDa, from between about 20 kDa and about 25 kDa, from between about 25 kDa and about 30 kDa, from between about 30 kDa and about 35 kDa, from between about 35 kDa and about 40 kDa, from between about 39 kDa and about 54 kDa, from between about 39 kDa and about 80 kDa, from between about 40 kDa and about 45 kDa, from between about 45 kDa and about 50 kDa, from between about 50 kDa and about 55 kDa, from between about 55 kDa and about 60 kDa, from between about 60 kDa and about 100 kDa, or from between about 80 kDa and about 144 kDa, and a poly dispersity ranging from 1 to about 5.
[0005] In some embodiments, the silk fibroin fragments have a poly dispersity from about 1.5 to about 3.0.
[0006] In some embodiments, the silk fibroin fragments comprise one or more of low molecular weight silk fibroin fragments and medium molecular weight silk fibroin fragments.
[0007] In some embodiments, the article further comprises about 0.01% (w/w) to about 10% (w/w) sericin relative to the silk fibroin fragments.
[0008] In some embodiments, the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool.
[0009] In some embodiments, the fabric comprises one or more of natural wool, chlorinedescaled wool, or non-chlorine descaled wool.
[0010] In some embodiments, the reducing agent is selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thiogly colic acid, thiosalicylic acid, and pseudothiohydantoin,.
[0011] In some embodiments, the reducing agent is L-cysteine.
[0012] In some embodiments, the coating further comprises a crosslinker.
[0013] In some embodiments, the crosslinker is glycerol diglycidyl ether (GDE).
[0014] In an embodiment, the disclosure provides a method of making a silk fibroin coated fabric, including applying to the fabric a solution comprising a reducing agent, applying to the fabric a silk fibroin solution, and drying the fabric.
[0015] In an embodiment, the disclosure provides a method of improving size retention on laundering in a fabric, including applying to the fabric a solution comprising a reducing agent, applying to the fabric a silk fibroin solution, and drying the fabric;
[0016] In some embodiments, upon laundering, the fabric substantially retains its initial size prior to laundering.
[0017] In some embodiments, upon laundering, the fabric retains a substantially higher fraction of its initial size prior to laundering compared to a similar fabric not similarly treated with the reducing agent and the silk fibroin solution [0018] In some embodiments, the silk fibroin solution comprises low molecular weight silk fibroin fragments.
[0019] In some embodiments, the silk fibroin solution comprises medium molecular weight silk fibroin fragments.
[0020] In some embodiments, the concentration of the silk fibroin solution is between about 0.1% w/v and about 1% w/v.
[0021] In some embodiments, the concentration of the silk fibroin solution is about 0.5% w/v. [0022] In some embodiments, the drying comprises heating.
[0023] In some embodiments, the heating does not substantially modify silk fibroin coating performance.
[0024] In some embodiments, the pH of the silk fibroin solution is acidic. [0025] In some embodiments, the pH of the silk fibroin solution is about 4. [0026] In some embodiments, the fabric is dried after applying the solution comprising a reducing agent and before applying the silk fibroin solution.
[0027] In some embodiments, the concentration of the reducing agent in the solution ranges from about 1 g/L to about 12 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 1 g/L to about 15 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 1 g/L to about 25 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 1 g/L to about 5 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 5 g/L to about 10 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 10 g/L to about 50 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 10 g/L to about 15 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from about 1 g/L to about 100 g/L.
[0028] In some embodiments, the silk fibroin solution further comprises a reducing agent.
[0029] In some embodiments, the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool.
[0030] In some embodiments, the fabric comprises one or more of natural wool, chlorinedescaled wool, or non-chlorine descaled wool. [0031] In some embodiments, the reducing agent is independently selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin,.
[0032] In some embodiments, the reducing agent is independently at each occurrence L-cysteine. [0033] In an embodiment, the disclosure provides an article prepared by a method of the disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Fig. l is a flow chart showing various embodiments for producing silk fibroin protein fragments (SPFs) of the present disclosure.
[0035] Fig. 2 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.
[0036] Fig. 3 is a graph showing the dimensional stability of Chlorine-Descaled wool. L- cysteine pretreat followed by 0.5% silk D coating showed a significant reduction in dimensional shrinkage. L-cysteine pretreat followed by 0.5% silk C coating showed a slight reduction in dimensional shrinkage, while L-cysteine pretreat followed by water showed no impact.
[0037] Fig. 4 is a graph showing the dimensional stability of Non-Chlorine-Descaled wool. L- cysteine (12 g/L) pretreat followed by 0.5% silk D coating showed a significant reduction in dimensional shrinkage. L-cysteine (1 g/L) pretreat followed by 0.5% silk D coating showed a slight reduction in dimensional shrinkage, while 0.5% silk D only without pretreat showed no impact.
[0038] Fig. 5 is a graph showing the dimensional stability of Natural wool. L-cysteine pretreat followed by 0.5% silk D coating showed a reduction in dimensional shrinkage, while 0.5% silk D without L-cysteine pretreat showed no impact.
DETAILED DESCRIPTION
[0039] The disclosure provides fabrics treated with silk fibroin and a reducing agent, which exhibit improved anti-shrinkage properties. In one embodiment, silk fibroin can be used with a natural reducing agent L-cysteine on wool to improve the dimensional stability after laundering, therefore improve the anti-shrinkage performance.
[0040] Wool fiber exhibits soft handle, warm sensation and excellent elasticity, and therefore is widely used as a high quality textile material. Felting, or shrinkage, is a unique undesirable property of natural wool due to the presence of the scales on the cuticle cells. The natural wool textile products require Dry Clean Only to avoid the shrinkage issue.
[0041] There are different levels of anti-shrinkage treatment. The traditional high-level antishrinkage treatment is carried out by a chlorine descaling followed by a deposition of polyamide resin from solution (chlorine-Hercosett process). The resulting products are machine-washable and tumble-dryable. However, textile effluents containing a large amount of resin left from treatment would be released into wastewater, generating environmental concerns. Next-to-skin wool products treated by synthetic polyamide resin increase the risk of skin irritation. Another type of anti-shrinkage treated wool is hand-washable with air drying, for which treatment only involves a descaling process using reduction or oxidation to remove the scale. Without masking the cuticle surface by a polymer such as the Hercosett polyamide, the anti-shrinkage performance is not as satisfying. Therefore, it is meaningful to identify a safe and effective polymer to achieve a better anti-shrinkage performance.
[0042] Silk is a natural polymer produced by a variety of insects and spiders. Silk produced by Bombyx mori (silkworm) comprises a filament core protein, silk fibroin, and a glue-like coating consisting of a nonfilamentous protein, sericin. Silk fibroin is a FDA approved, edible, non-toxic, and relative inexpensive silkworm cocoon derived proteins. The structure and content of amino acids in the silk fibroin protein are very similar to the tissue of the human body.
[0043] Methods of making silk fibroin or silk fibroin-based protein 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, all of which are incorporated herein in their entireties.
[0044] The recent advancements in silk material technology include the emergence of the top- down and bottom-up engineering of silk cocoon, specifically regenerate cocoon into an aqueous silk solution and to use genetic engineering to produce recombinant silk with molecularly defined composition (Tran et al., A review of the emerging role of silk for the treatment of the eye, Pharm. Res., 2018, vol. 35, pp. 1-16). In recent years, silk fibroin proteins are reported to have found applications in ocular tissue reconstruction, corneal tissue engineering and in ocular surface repair due to their biocompatibility, tunable properties, and transparency. Silk films have been found to support corneal cell growth and to develop stratified epithelial cell sheets equivalent to amniotic membrane substrates (Lawrence et al., Silk film biomaterials for cornea tissue engineering, Biomaterials, 2009; 30(7): 1299-308; Harkin et al., Silk fibroin in ocular tissue reconstruction, Biomaterials, 2011; Chirtla et al., Bombyx mori silk fibroin membranes as potential substrata for epithelial constructs used in the management of ocular surface disorders. Tissue Engineering Pan A, 2008; 14(7): 1203- 11.). Silk fibroin protein and hydrolyzed peptide fragments have been shown to inhibit transcription and upstream activation of NF-KB protein subunits and proinflammatory molecules that are classically under the control of NF-KB (Hayden et al., Cell Research, 2011. 21(2): 223-244; Chon et al., International Journal of Molecular Medicine, 2012. 30(5): 1203-1210; Kim et al., J. Neurosurg., 2011. 114(2): 485-90; J. Microbiol. Biotechnol., 2012. 22(4): 494-500).
Definitions
[0045] 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 disclosure belongs. All patents and publications referred to herein are incorporated by reference in their entireties.
[0046] All percentages, parts and ratios are based upon the total weight of the eye care compositions of the present disclosure, 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.
[0047] As used herein, the term “a”, “an”, or “the” generally is construed to cover both the singular and the plural forms.
[0048] The term “about” as used herein, generally refers to a particular numeric value that include variation and 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 zero variation, and a range of ±20%, ±10%, or ±5% of a given numeric value.
[0049] 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.
[0050] 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 Mhis 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: Water-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.
[0051] 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.
[0052] 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
[0053] 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.
[0054] 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.
[0055] As used herein, the term “fast-dissolving solid forms” refers to fast-dissolving solid forms including freeze dried forms (cakes, wafers, thin films), and compressed tablets. [0056] As used herein, the terms “peptide” or “protein” refers to a chain of amino acids that are held together by peptide bonds (also called amide bonds). The basic distinguishing factors for proteins and peptides are size and structure. Peptides are smaller than proteins. Traditionally, peptides are defined as molecules that consist of between 2 and 50 amino acids, whereas proteins are made up of 50 or more amino acids. In addition, peptides tend to be less well defined in structure than proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures.
[0057] As used herein, the term “fibroin” or “silk protein” is a type of structural protein produced by certain spider and insect species that produce silk (See definition provided in WIPO Pearl -WIPO’s Multilingual Terminology Portal database, https://wipopearl.wipo.int/en/linguistic). Fibroin may include silkworm fibroin, insect or spider silk protein (e.g., spidroin), recombinant spider protein, silk proteins present in other spider silk types, e.g., tubuliform silk protein (TuSP), flagelliform silk protein, minor ampullate silk proteins, aciniform silk protein, pyriform silk protein, aggregate silk glue), silkworm fibroin produced by genetically modified silkworm, or recombinant silkworm fibroin.
[0058] As used herein, the term “silk fibroin” refers to silkworm fibroin, silk fibroin produced by genetically modified silkworm, or recombinant silkworm fibroin (See (1) Narayan Ed., Encyclopedia of Biomedical Engineering, Vol. 2, Elsevier, 2019; (2) Kobayashi et al. Eds, Encyclopedia of Polymeric Nanomaterials, Springer, 2014, https://link.springer.com/referenceworkentry/10.1007%2F978-3 -642-36199-9_323-l). In an embodiment, silk fibroin is obtained from Bombyx mori.
[0059] The term “solid solution” as used herein, refers to the active agent molecularly dissolved in the solid excipient matrix such as hydrophobic polymers, wherein the active agent is miscible with the polymer matrix excipient.
[0060] The term “solid dispersion” as used herein, refers to the active agent dispersed as crystalline or amorphous particles, wherein the active agent is dispersed in an amorphous polymer and is distributed at random between the polymer matrix excipient.
[0061] 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 also refer to an even distribution of a component or an additive, for example, silk fibroin-based protein fragments, dermatologically acceptable carrier, etc., throughout the silk eye care composition.
[0062] As used herein, the term “surface tension” refers to the tendency of fluid surfaces to shrink into the minimum surface area possible. At liquid-air interfaces, surface tension results from the greater attraction of liquid molecules to each other (due to cohesion) than to the molecules in the air (due to adhesion). The net effect is an inward force at its surface that causes the liquid to behave as if its surface were covered with a stretched elastic membrane. Because of the relatively high attraction of water molecules to each other through a web of hydrogen bonds, water has a higher surface tension (72.8 mN/m at 20 °C) than most other liquids.
[0063] SPF Definitions and Properties
[0064] As used herein, “silk protein fragments” (SPF) include, without limitation, one or more of: “silk fibroin fragments” as defined herein; “recombinant silk fragments” as defined herein; “spider silk fragments” as defined herein; “silk fibroin-like protein fragments” as defined herein; “chemically modified silk fragments” as defined herein; and/or “sericin or sericin fragments” as defined herein. 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.
[0065] SPF Molecular Weight and Poly dispersity
[0066] 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 composition 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 having 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 115 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 weight 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 between 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 235 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 embodiment, 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.
[0067] 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:
[0068] 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 kDa to about 38 kDa, about 14 kDa to about 30 kDa, or about 6 kDa to about 17 kDa. 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 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa, about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, or about 38 kDa.
[0069] 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, a target medium molecular weight for certain SPF may be weight average molecular weight of about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, or about 55 kDa.
[0070] 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.
[0071] In 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.
[0072] 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 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 2.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 2.0 to about 2.5. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 2.5 to about 3.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 3.0 to about 3.5. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 3.5 to about 4.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 4.0 to about 4.5. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity selected from between about 4.5 to about 5.0.
[0073] In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of 1. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity of about 1.1. In an embodiment, SPF in a composition of the present 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 dispersity of 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 poly dispersity of 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 dispersity of about 2.0. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity 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 poly dispersity 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 dispersity of 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 dispersity of 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 disclosure 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 dispersity of 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 dispersity of 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 poly dispersity of about 4.4. In an embodiment, SPF in a composition of the present disclosure have a poly dispersity 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.
[0074] 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. [0075] Silk Fibroin Fragments
[0076] 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 primary proteins: silk fibroin (approximately 75%) and sericin (approximately 25%). Silk fibroin 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 having a weight average molecular weight of about 350,000 Da (H chain), and a light chain 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. [0077] 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.
[0078] As used herein, the term “fibroin” includes silk worm 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-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. 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.
[0079] The raw silk cocoons from the silkworm Bombyx mori was cut into pieces. The pieces silk cocoons were processed in an aqueous solution of Na2CCh 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 Na2CCh 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 about 100 °C for about 60 minutes to achieve complete dissolution of the native silk protein. The resulting silk fibroin solution was filtered and dialyzed 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 water.
[0080] 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 semiautomated equipment, for example a tangential flow filtration system.
[0081] 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.
[0082] 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.
[0083] 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.
[0084] 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 polydispersity (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 is 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.
[0085] FIG. l 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. 2, 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 Na2CCh (sodium carbonate) is added to the boiling water until the Na2CCh is completely dissolved. The raw silk is added to the boiling water/Na2CO3 (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 Na2CCh volume equals about 0.848 x raw silk weight. In an embodiment, the water volume equals 0.1 x raw silk weight and the Na2CCh volume is maintained at 2.12 g/L.
[0086] Subsequently, the water dissolved Na2CCh solution is drained and excess water/Na2CO3 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, typically 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 resulting 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). 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 would result in liquid silk protein, without any alteration of the protein structure, free of sericin.
[0087] 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.
[0088] The temperature at which the silk fibroin extract is added to the LiBr solution (or vice versa) has an effect on the time required to completely dissolve the fibroin and on the resulting molecular weight and poly dispersity of the final SPF mixture solution. 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 varying 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.
[0089] 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. [0090] 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., silk worm 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 disclosed 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).
[0091] 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 ranging 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 water-based 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.
[0092] An assay for LiBr and Na2CCh 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.
[0093] The analytical method developed for the quantitation of Na2CCh and LiBr in silk protein formulations was 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 can be applied for the quantitative determination of sodium carbonate and lithium bromide in silk protein formulations.
[0094] FIG. 2 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 polydispersity. It should be understood that not all of the steps illustrated are necessarily required to fabricate all silk solutions of the present disclosure.
[0095] In an embodiment, silk protein fragment solutions useful for a wide variety of 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 Na2CCh water solution for about 60 minutes, wherein a volume of the water equals about 0.4 x raw silk weight and the amount of Na2CCh 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 dry silk 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 polydispersity; filtering the solution to remove any remaining debris from the silkworm; diluting the solution with water to result in a 1.0 wt. % silk solution; and removing 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.
[0096] Without 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.
[0097] 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 washing 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.
[0098] 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.
[0099] In 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.
[0100] 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; 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 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.
[0101] 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 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 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 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 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 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 %.
[0102] 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 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 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 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 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%.
[0103] 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 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, 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 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 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 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. 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 wt. % and a vitamin content of at least 20 wt. %.
[0104] 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 poly dispersity 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). Poly dispersity was calculated using Cirrus GPC Online GPC/SEC Software Version 3.3 (Agilent).
[0105] Differences in the processing parameters can result in regenerated silk fibroins that vary in molecular weight, and peptide chain size distribution (poly dispersity, PD). This, in turn, influences the regenerated silk fibroin performance, including mechanical strength, water solubility etc.
[0106] 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 varying the extraction time. Tables A-G summarize the results. Below is a summary:
- A sericin extraction time of 30 minutes resulted in larger molecular weight than a sericin extraction time of 60 minutes
- Molecular weight decreases with time in the oven
- 140 °C LiBr and oven resulted in the low end of the confidence interval to be below a molecular weight of 9500 Da
- 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).
[0107] Experiments were carried out to determine the effect of varying the extraction temperature. Table G summarizes the results. Below is a summary:
- Sericin extraction at 90 °C resulted in higher MW than sericin extraction at 100 °C extraction
- Both 90 °C and 100 °C show decreasing MW over time in the oven.
[0108] Experiments were carried out to determine the effect of varying the Lithium Bromide (LiBr) temperature when added to silk. Tables H-I summarize the results. Below is a summary:
— No impact on molecular weight or confidence interval (all CI -10500-6500 Da)
- 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
[0109] Experiments were carried out to determine the effect of v oven/dis solution temperature. Tables J-N summarize the results. Below is a summary:
- 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.
- 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
- The 140 °C oven resulted in a low end in the confidence interval at -6000 Da.
[0110] 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 Na2CCh (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 Na2CCh 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.
[OHl] 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 molecular weight selected from between about 6 kDa to about 16 kDa, about 17 kDa to about 39 kDa, and about 39 kDa to about 80 kDa and a 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.
[0112] 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 diafiltered 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.
[0113] Six (6) silk solutions were utilized in standard silk structures with the following results: [0114] 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).
[0115] Solution #2 is a silk concentration of 6.4 wt. % (made with a 30 min boil extraction, 60 °C LiBr dissolution for 4 hrs).
[0116] Solution #3 is a silk concentration of 6.17 wt. % (made with a 30 min boil extraction 100 °C LiBr dissolution for 1 hour).
[0117] 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.
[0118] Solution #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. 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.
[0119] 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.
[0120] The preparation of silk fibroin solutions with higher molecular weights is given in Table O
Table O. Preparation and properties of silk fibroin solutions.
[0121] Silk aqueous coating composition for application to fabrics are given in Tables P and Q below.
[0122] Three (3) silk solutions were utilized in film making with the following results:
[0123] 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).
[0124] Solution #2 is a silk concentration of 6.4 % (made with a 30 min boil extraction, 60 °C
LiBr dissolution for 4 hrs).
[0125] Solution #3 is a silk concentration of 6.17 % (made with a 30 min boil extraction, 100 °C LiBr dissolution for 1 hour).
[0126] Films were 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 mL 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 was filled with water. 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 water in spite of the water annealing treatment.
[0127] Silk solutions of various molecular weights and/or combinations of molecular weights can be optimized for gel applications. The following provides an example of this process but it not intended to be limiting in application or formulation. Three (3) silk solutions were utilized in gel making with the following results:
[0128] 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).
[0129] Solution #2 is a silk concentration of 6.4 % (made with a 30 min boil extraction, 60 °C LiBr dissolution for 4 hrs).
[0130] Solution #3 is a silk concentration of 6.17 % (made with a 30 min boil extraction, 100 °C LiBr dissolution for 1 hour).
[0131] “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.
[0132] Solutions #2 and #3 were gelled in accordance with the published horseradish peroxidase (HRP) protocol. Behavior seemed typical of published solutions.
[0133] Materials and Methods: the following equipment and material are used in determination of Silk Molecular weight: Agilent 1100 with chemstation software ver. 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). [0134] Procedural Steps:
[0135] A) Preparation of 1 L Mobile Phase (0.1 M Sodium Chloride solution in 0.0125 M Sodium phosphate buffer)
[0136] 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 filter. 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.
[0137] B) Preparation of Dextran Molecular Weight Standard solutions
[0138] 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 weights. Dissolve the dextran molecular weight standards in 5 mL of mobile phase to make a 1 mg/mL standard solution.
[0139] C) Preparation of Sample Solutions
[0140] 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. Depending 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.
[0141] D) HPLC analysis of the samples
[0142] 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:
[0143] Data analysis and calculations - Calculation of Average Molecular Weight using Cirrus Software
[0144] 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 (M w ), number average molecular weight (Mn), peak average molecular weight (M p ), and poly dispersity for each injection of the sample.
[0145] Spider Silk Fragments
[0146] 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 copolymer-like arrangement, in which two basic sequences, crystalline [poly(A) or poly(GA)] and less crystalline (GGX or GPGXX (SEQ ID NO: 6)) polypeptides alternate. Dragline silk is 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 (SEQ ID NO: 6)) polypeptides alternate in core domain. Specific secondary structures have been assigned to poly(A)/(GA), GGX and GPGXX (SEQ ID NO: 6) motifs including P-sheet, a-helix and P- 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 fibre formation in a spinning duct.
[0147] The main difference between MaSpl and MaSp2 is the presence of proline (P) residues accounting for 15% of the total amino acid content in 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 fibres; 81% MaSpl and 19% MaSp2. Different spiders have different ratios of MaSpl and MaSp2. For example, a dragline silk fibre 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 fibre.
[0148] At least seven different types 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 outstanding mechanical properties including strength, toughness and elasticity. On an equal weight basis, spider silk has a higher toughness than steel and Kevlar. 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.
[0149] 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. nanowires, nanofibers, surface coatings).
[0150] 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 diademalus. 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.
[0151] Examples 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: 52 to 54, which is also described in U.S. Patent No. 9,051,453, which is incorporated by reference herein in its entirety, or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 52 to 54, which is also described in U.S. Patent No. 9,051,453, which is incorporated by reference herein in its entirety. 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 coli 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.
[0152] In the formula (1), the REP1 indicates polyalanine. 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.
[0153] In the major dragline silk, the REP1 corresponds to a crystal region in a fiber where a crystal P 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 flexibility. 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.
[0154] Recombinant Silk Fragments
[0155] 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 Ar aneoids. 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 o Araneoids. In some embodiments, the recombinant silk protein fragment disclosed herein include block copolymer having synthetic repetitive units derived from spider silk polypeptides of Araneidae or Araneoids and non-repetitive units derived from natural repetitive units of spider silk polypeptides of Araneidae or Araneoids.
[0156] Recent advances in genetic engineering have provided a route to produce various types of recombinant silk proteins. Recombinant DNA technology has been used to provide a more practical source of silk proteins. As used herein “recombinant silk protein” refers to synthetic proteins produced heterologously in prokaryotic or eukaryotic expression systems using genetic engineering methods.
[0157] 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 gram-negative, rod-shaped bacterium E. coli is a well-established host for industrial scale production of proteins. Therefore, the majority of recombinant silks have been produced in E. colt. E. coli which is easy to manipulate, has a short generation time, is relatively low cost and can be scaled up for larger amounts protein production.
[0158] 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.
[0159] 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 necessary for 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.
[0160] The prokaryotic systems include Gram-negative bacteria or Gram-positive bacteria. The prokaryotic 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, Bacillus megaterium, Corynebacterium glutamicum, Anabaena, Caulobacter, 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.
[0161] 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.
[0162] 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.
[0163] In 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. coli. 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). [0164] 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.
[0165] 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. coh, Sacchromyces cerevisiae. Pseudomonas sp., Rhodopseudomonas sp., Bacillus sp., and Strepomyces. See EP 0230702, which is incorporate by reference herein by its entirety.
[0166] 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)
[0167] 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 units spontaneously aggregate into P-sheet structures, similar to natural silk fibroin protein, where in the silk protein-like multiblock polymers having any weight average molecular weight described herein.
[0168] 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.
[0169] In some embodiments, this disclosure provides B. mori silkworm recombinant proteins composed of the (GAGAGS)ie (SEQ ID NO: 55) repetitive fragment. In some embodiments, this disclosure provides recombinant proteins composed of the (GAGAGS)ie (SEQ ID NO: 55) repetitive fragment and the non-repetitive (GAGAGS)ie -F-COOH (SEQ ID NO: 56), (GAGAGS)ie -F-F-COOH (SEQ ID NO: 57), (GAGAGS)ie -F-F-F-COOH (SEQ ID NO: 58), (GAGAGS)ie -F-F-F-F-COOH (SEQ ID NO: 59), (GAGAGS)ie -F-F-F-F-F-F-F-F-COOH (SEQ ID NO: 60), (GAGAGS)ie -F-F-F-F-F-F-F-F-F-F-F-F-COOH (SEQ ID NO: 61) produced by E. coli, where F has the following amino acid sequence SGFGPVANGGSGEASSESDFGSSGFGPVANASSGEASSESDFAG (SEQ ID NO: 5), and where in the silk protein-like multiblock polymers having any weight average molecular weight described herein.
[0170] 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 7, 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 dragline 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 orbweb spinning spider is described in U.S. Pat. No. 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, andPichia 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 geometricus, 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. [0171] 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.
[0172] In 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 oiAraneidae or Araneoids.
[0173] 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: 20)) or GPGQQ (SEQ ID NO: 15)) 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 (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50) and/or ADF-4 (SEQ ID NO: 51). 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 (SEQ ID NO: 48), MaSpII (SEQ ID NO: 49), ADF-3 (SEQ ID NO: 50) and/or ADF-4 (SEQ ID NO: 51)but having one or more amino acid substitution at specific amino acid positions.
[0174] As 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: 6), wherein X is an amino acid selected from A, S, G, Y, P and Q; (ii) GGX, wherein X is an amino acid selected from Y, P, R, S, A, T, N and Q, preferably Y, P and Q; (iii) A x , wherein x is an integer from 5 to 10.
[0175] The consensus peptide sequences GPGXX (SEQ ID NO: 6) 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: 6) motif forms turn spiral structures, which imparts elasticity to the silk polypeptide. Major ampullate and flagelliform silks both have a GPGXX (SEQ ID NO: 6) motif. The iterated GGX motif is associated with 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.
[0176] 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: 7) and GGRPSSSYG (SEQ ID NO: 8) derived from Resilin. Resilin is an elastomeric protein found in most arthropods that provides low stiffness and high strength.
[0177] 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-type non-repetitive (carboxy terminal) unit), preferably within ADF-3 (SEQ ID NO:50), ADF-4 (SEQ ID NO: 51), NR3 (SEQ ID NO: 62), NR4 (SEQ ID NO: 63) of the spider Araneus diademalus. 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 protein eADF4, molecular weight of 47.7 kDa, AMSilk) comprising the 16 repeats of the sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), 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.
[0178] 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: 64, which is also 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: 64, particularly functional equivalents, functional derivatives and salts of this sequence are also included.
[0179] 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.
[0180] 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.
[0181] Recombinant silk is also described in other patents and patent applications, incorporated by reference herein: US 2004590196, US 7,754,851, US 2007654470, US 7,951,908, US 2010785960, US 8,034,897, US 20090263430, US 2008226854, US 20090123967, US 2005712095, US 2007991037, US 20090162896, US 200885266, US 8,372,436, US 2007989907, US 2009267596, US 2010319542, US 2009265344, US 2012684607, US 2004583227, US 8,030,024, US 2006643569, US 7,868,146, US 2007991916, US 8,097,583, US 2006643200, US 8,729,238, US 8,877,903, US 20190062557, US 20160280960, US 20110201783, US 2008991916, US 2011986662, US 2012697729, US 20150328363, US 9,034,816, US 20130172478, US 9,217,017, US 20170202995, US 8,721,991, US 2008227498, US 9,233,067, US 8,288,512, US 2008161364, US 7,148,039, US 1999247806, US 2001861597, US 2004887100, US 9,481,719, US 8,765,688, US 200880705, US 2010809102, US 8,367,803, US 2010664902, 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.
[0182] 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.
[0183] 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: 6), GGX and A x as defined herein.
[0184] 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: 10), GPGSG (SEQ ID NO: 11), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13), GPGGA (SEQ ID NO: 14), GPGQQ (SEQ ID NO: 15), GPGGG (SEQ ID NO: 16), GPGQG (SEQ ID NO: 17), GPGGS (SEQ ID NO: 18), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 19), AAAAAA (SEQ ID NO: 20), AAAAAAA (SEQ ID NO: 21), AAAAAAAA (SEQ ID NO: 22), AAAAAAAAA (SEQ ID NO: 23), AAAAAAAAAA (SEQ ID NO: 24), GGRPSDTYG (SEQ ID NO: 7) and GGRPSSSYG (SEQ ID NO: 8), (i) GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 25), (ii) GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 9), (iii) GPGQQGPGQQGPGQQGPGQQ (SEQ ID NO: 26): (iv) GPGGAGGPYGPGGAGGPYGPGGAGGPY (SEQ ID NO: 27), (v) GGTTIIEDLDITIDGADGPITISEELTI (SEQ ID NO: 28), (vi) PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 29), (vii) SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), (viii) GGAGGAGGAGGSGGAGGS (SEQ ID NO: 31), (ix) GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 32), (x) GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 33), (xi) GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 34), (xii) GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 35), (xiii) GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 36), (xiv) GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO: 37), or 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: 10), GGY, GPGSG (SEQ ID NO: 11) in the peptide chain, or sequential order of AAAAAAAA (SEQ ID NO: 22), GPGGY (SEQ ID NO: 12), GPGGP (SEQ ID NO: 13) in the peptide chain, sequential order of AAAAAAAA (SEQ ID NO: 22), GPGQG (SEQ ID NO: 17), GGR in the peptide chain.
[0185] 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 multiblock peptides comprise a repeating unit of amino acids corresponding to one of the sequences (I), (II), (III) and/or (IV) below.
[0186] [(XGG)w(XGA)(GXG)x(AGA) y (G)zAG]p (SEQ ID NO: 38) Formula (I) in which: X corresponds to tyrosine or to glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer and having any weight average molecular weight described herein, and/or
[0187] [(GPG2YGPGQ2)a(X’)2S(A)b]p (SEQ ID NO: 39) Formula (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
[0188] [(GR)(GA)i(A)m(GGX)n(GA)i(A)m] P (SEQ ID NO: 40) Formula (III) and/or [(GGX”)n(GA)m(A)i] p (SEQ ID NO: 41) Formula (IV) in which: X” corresponds to tyrosine, glutamine or alanine, 1 is an integer from 1 to 6, m is an integer from 0 to 4, n is an integer from 1 to 4, and p is an integer. [0189] 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):
[(Xaa Gly Gly) w (Xaa Gly Ala)(Gly Xaa Gly) x (Ala Gly Ala) y (Gly) z Ala Gly] P Formula (V), wherein Xaa is tyrosine or glutamine, w is an integer equal to 2 or 3, x is an integer from 1 to 3, y is an integer from 5 to 7, z is an integer equal to 1 or 2, and p is an integer.
[0190] 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.
[0191] 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-terminus in these proteins, which makes them more hydrophilic. These COOH-terminal amino acids are absent in spider silk proteins expressed in microbial hosts.
[0192] In some embodiments, the recombinant spider silk protein in this disclosure comprises water soluble recombinant spider silk protein C16 modified with an amino or carboxyl terminal selected from the amino acid sequences consisting of: GCGGGGGG (SEQ ID NO: 42), GKGGGGGG (SEQ ID NO: 43), GCGGSGGGGSGGGG (SEQ ID NO: 44), GKGGGGGGSGGGG (SEQ ID NO: 45), and GCGGGGGGSGGGG (SEQ ID NO: 46). In some embodiments, the recombinant spider silk protein in this disclosure comprises CieNR.4, C32NR.4, C16, C32, NR4C16NR.4, NR4C32NR.4, NR3C16NR.3, or NR3C32NR.3 such that the molecular weight of the protein ranges as described herein.
[0193] 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 A. 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: 47) or SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 30), as described in U.S. Pat. No. 8,367,803, which is incorporated by reference herein in its entirety.
[0194] In some embodiments, this disclosure provides recombinant spider proteins composed of the GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 32) repetitive fragment and having a molecular weight as described herein.
[0195] 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), egg sac silk (Cylindriform gland silk), egg case silk (Tubuliform silk), non-sticky dragline silk (Ampullate gland silk), attaching 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. 9,051,453, 9,617,315, 9,689,089, 8,173,772, and 8,642,734.
[0196] 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 types 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).
[0197] 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. MaSp2 silks are characterized by poly alanine, GGX, and GPX motifs. Minor Ampullate (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.
[0198] 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. 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.
[0199] 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.
[0200] The recombinant block copolymer polypeptides based on spider silk sequences produced by gene expression in a recombinant prokaryotic or eukaryotic system 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.
[0201] 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.
[0202] A solution of such polypeptides (i.e., recombinant silk protein) may then be prepared and used as described herein.
[0203] 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.
[0204] 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 fragments 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 typically 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 or NT-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.
[0205] 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 pyrogens are actively removed at this stage.
[0206] 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” and “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, typically 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 composition 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.
[0207] 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.
[0208] 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.
[0209] 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 the 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. 4.2-6.3 promotes rapid polymerization,
[0210] 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.
[0211] 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.
[0212] Without 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.
[0213] 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.
[0214] 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. [0215] 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.
[0216] According 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,
[0217] 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.
[0218] 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.
[0219] 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. [0220] An amino acid sequence represented by SEQ ID NO: 52, which is also described in 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: 53, which is also described in U.S. Patent No. 9,051,453, is identical to an amino acid sequence represented by SEQ ID NO: 52, which is also described in 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: 54, which is also described in U.S. Patent No. 9,051,453, is identical to an amino acid sequence represented by SEQ ID NO: 52 from which 29 residues have been removed from the C-terminal.
[0221] 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: 52 to 54 or an amino acid sequence having a homology of 90% or more with the amino acid sequence represented by any of SEQ ID NOS: 52 to 54, which are also described in U.S. Patent No. 9,051,453, is a polypeptide having an amino acid sequence represented by SEQ ID NO: 65, which is also described in 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: 65, which is also described in 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: 66, which is also described in 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, 1 st to 13 111 repetitive regions are about doubled and the translation ends at the 1154 th amino acid residue. In the polypeptide having the amino acid sequence represented by SEQ ID NO: 65, which is also described in U.S. Patent No. 9,051,453, the C-terminal sequence is identical to the amino acid sequence represented by SEQ ID NO: 54.
[0222] 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: 52 to 54, which are also described in 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: 52 to 54, which are also described in U.S. Patent No. 9,051,453, may be a protein that has an amino acid sequence represented by SEQ ID NO: 65, which is also described in 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.
[0223] 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 ADF4 having an amino acid sequence represented by SEQ ID NO: 67, which is also described in 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: 67, which is also described in U.S. Patent No.
9,051,453, is an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 66, which is also described in 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 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: 67, which is also described in 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: 68, which is also described in 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: 68, which is also described in of U.S. Patent No. 9,051,453, is an amino acid sequence obtained by adding the amino acid sequence (SEQ ID NO: 66, which is also described in 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: 68, which is also described in 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.
[0224] Examples 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.
[0225] In the formula (2), the REP 3 indicates an amino acid sequence composed of Gly-Pro- Gly-Gly-X (SEQ ID NO: 69), where X indicates an amino acid selected from the group consisting of Ala, Ser, Tyr and Vai.
[0226] 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.
[0227] 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: 70, which is also described in 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: 70, which is also described in U.S. Patent No. 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 1220 th residue to the 1659 th residue 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 NCBI database (NCBI Accession No.: AAC38847, GI: 2833649), specifically, a C-terminal amino acid sequence thereof from the 816 th residue to the 907 th residue from the C-terminal, and thereafter adding the amino acid sequence (SEQ ID NO: 66, which is also described in 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: 70, which is also described in 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 an amorphous region.
[0228] 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.
[0229] 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 like 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.
[0230] 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 strength-elongation and toughness and of being synthesized easily.
[0231] Accordingly, the recombinant silk protein (e.g., the recombinant spider silk-based 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.
[0232] Silk Fibroin-like Protein Fragments
[0233] 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, fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) 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%.
[0234] As described herein, a protein such as native silk protein, fibroin heavy chain, fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) 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, fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) 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, fibroin light chain, or any protein comprising one or more GAGAGS (SEQ ID NO: 2) 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.
[0235] In 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 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, or about 39% alanine. In some embodiments, 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.
[0236] Sericin or Sericin Fragments
[0237] The main body of the raw silk is silk fibroin fiber, and the silk fibroin fiber is coated with an adhesive substance silk sericin. Sericin is a colloidal silk protein that covers the surface of the silk thread and is composed of bulky amino acids rich in chemical reactivity such as serine, threonine, and aspartic acid, in addition to glycine and alanine. In the various processes of producing silk from raw silk, sericin is important in controlling the solubility of silk and producing high quality silk. Moreover, it plays an extremely important role as an adhesion functional protein. When silk fiber is used as a clothing material, most of the silk sericin covering the silk thread is removed and discarded, so sericin is a valuable unused resource.
[0238] In some embodiments, the silk protein fragments described herein include sericin or sericin fragments. Methods of preparing sericin or sericin fragments and their applications in various fields are known and are described herein , and are also described, for example, in U.S. Patents Nos. 7,115,388, 7,157,273, and 9,187,538, all of which are incorporated by reference herein in their entireties.
[0239] In some embodiments, sericin removed from the raw silk cocoons, such as in a degumming step, can be collected and used in the methods described herein. Sericin can also be reconstituted from a powder, and used within the compositions and methods of the disclosure.
[0240] Other Properties of SPF
[0241] 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.
[0242] 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.
[0243] 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 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.
[0244] 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.
[0245] 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.
[0246] 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.
[0247] 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% crystalline 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% crystalline 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% crystalline portion and a 60% amorphous region. In an embodiment, the SPF of a composition of the present disclosure 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.
[0248] 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.
[0249] 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 to 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.
[0250] 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.
[0251] 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 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.
[0252] 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.
[0253] 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 SPF 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. %.
[0254] 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 embodiment, 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. %. [0255] 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 ranges 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. %.
[0256] 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 from 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 0.5 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. %. [0257] 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. %.
[0258] 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 storage conditions, percent SPF, and number of shipments and shipment conditions. 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, 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.
[0259] 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.
[0260] In 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 disclosure 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.
[0261] In an embodiment, a composition of the present disclosure having SPF, has non- detectable levels of Na 2 CO 3 residuals. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 100 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 200 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 300 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 400 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 500 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 600 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 700 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 800 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 900 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is less than 1000 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is non-detectable to 500 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is non-detectable to 450 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is non-detectable to 400 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is non-detectable to 350 ppm. In an embodiment, the amount of the Na 2 CO 3 residuals in a composition of the present disclosure is non-detectable to 300 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is non-detectable to 250 ppm. In an embodiment, the amount of the Na2CCh 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 Na2CCh residuals in a composition of the present disclosure is non-detectable to 100 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is 100 ppm to 200 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is 200 ppm to 300 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is 300 ppm to 400 ppm. In an embodiment, the amount of the Na2CCh residuals in a composition of the present disclosure is 400 ppm to 500 ppm.
[0262] 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. 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 stability 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.
[0263] Table R below shows shelf stability test results for embodiments of SPF compositions of the present disclosure.
[0264] 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.
[0265] In some embodiments, the process of annealing may involve inducing beta-sheet 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 presence 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.
[0266] In some embodiments, annealing is carried out by so-called “water-annealing” or “water vapor annealing” in which water vapor is used as an intermediate plasticizing agent or catalyst to promote the packing of beta- sheets. In some embodiments, the process of water 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. [0267] The important feature of the water annealing process is to drive the formation of crystalline 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 water 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.
[0268] 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 40 minutes, about 5 minutes to about 50 minutes, about 5 minutes to about 60 minutes, about 5 minutes to about 70 minutes, about 5 minutes to about 80 minutes, about 5 minutes to about 90 minutes, about 5 minutes to about 100 minutes, about 5 minutes to about 110 minutes, about 5 minutes to about 120 minutes, about 5 minutes to about 130 minutes, about 10 minutes to about 40 minutes, about 10 minutes to about 50 minutes, about 10 minutes to about 60 minutes, about 10 minutes to about 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 minutes, 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.
[0269] 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.
[0270] 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 powders 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.
[0271] As used herein, the terms “substantially sericin free” or “substantially devoid of sericin” refer to silk fibers in which a majority of the sericin protein has been removed. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.01 wt. % to about 10.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having about 0.01 wt. % to about 9.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.01 wt. % to about 8.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.01 wt. % to about 7.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.01 wt. % to about 6.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.01 wt. % to about 5.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.05 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.1 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 0.5 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 1.0 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 1.5 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 2.0 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having from about 2.5 wt. % to about 4.0 wt. % sericin. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content from about 0.01 wt. % to about 0.1 wt. %. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.1 wt. %. In an embodiment, silk fibroin that is substantially devoid of sericin refers to silk fibroin having a sericin content below about 0.05 wt. %. In an embodiment, when a silk source is added to a boiling (100 °C) aqueous solution of sodium carbonate for a treatment time of between about 30 minutes to about 60 minutes, a degumming loss of about 26.0 wt. % to about 31.0 wt. % is obtained.
[0272] 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.
[0273] 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 SPF 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. %.
[0274] 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 embodiment, 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. %.
[0275] 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 ranges 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. %. [0276] 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 from 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. %.
[0277] 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. %.
[0278] In some embodiments, the silk fibroin-based 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 storage conditions, percent SPF, and number of shipments and shipment conditions. 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, 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.
[0279] 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.
[0280] In an embodiment, a selected property of the SPF coated articles that may be enhanced as compared to non-coated articles may include one or more of dimensional stability to laundering, dimensional stability to dry cleaning, appearance after laundering, appearance after dry cleaning, colorfastness to laundering, colorfastness to dry cleaning, colorfastness to non-chlorine bleach, seam torque/spirality (on knits), colorfastness to crocking, colorfastness to rubbing, colorfastness to water, colorfastness to light, colorfastness to perspiration, colorfastness to chlorinated pool water, colorfastness to sea water, tensile strength, seam slippage, tearing strength, seam breaking strength, abrasion resistance, pilling resistance, stretch recovery, bursting strength, colorfastness to die transfer in storage (labels), colorfastness to ozone, pile retention, bowing and skewing, colorfastness to saliva, snagging resistance, wrinkle resistance (e.g., appearance of apparel, retention of creases in fabrics, smooth appearance of fabrics), water repellency, water resistance, stain repellant (e.g., water repellency, oil repellency, water/alcohol repellency), vertical wicking, water absorption, dry rate, soil release, air permeability, wicking, antimicrobial properties, ultraviolet protection, resistance to torque, malodor resistant, biocompatibility, wetting time, absorption rate, spreading speed, accumulative one-way transport, flame retardant properties, coloring properties, fabric softening properties, a pH adjusting property, an antifelting property, and overall moisture management capability.
[0281] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is dimensional stability to laundering, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
[0282] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is size retention on laundering, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
[0283] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is resistance to shrinkage, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
Compositions and Processes Including Silk Protein Fragment Coatings
[0284] In one aspect, the disclosure provides an article comprising a fabric and a coating, wherein the coating comprises a reducing agent and silk protein fragments of the disclosure. [0285] In an embodiment, the invention may include textiles, such as fibers, yarns, fabrics, or other materials and combinations thereof, that may be coated with an SPF mixture solution (i.e., silk fibroin solution (SFS)) as described herein to produce a coated article. In an embodiment, the coated articles described herein may be treated with additional chemical agents that may enhance the properties of the coated article. In an embodiment, the SFS may include one or more chemical agents that may enhance the properties of the coated article.
[0286] In an embodiment, textiles may be flexible materials (woven or non-woven) that include a network of natural and/or man-made fibers, thread, yarn, or a combination thereof. SFS may be applied at any stage of textile processing from individual fibers, to yarn, to fabric, to thread, or a combination thereof.
[0287] In an embodiment, fibers may be natural fibers that may include a natural fiber cellulose base, wherein the natural fiber cellulose base may include one or more of: (1) a baste such as flax, hemp, kenaf, jute, linen, and/or ramie; (2) a leaf such as flax, hemp, sisal, abaca, banana, henequen, ramie, sunn, and/or coir; and (3) seed hair such as cotton and/or kapok. In an embodiment, fibers may be natural fibers that may include a natural fiber protein base, wherein the natural fiber protein base may include one or more of: (1) hair such as alpaca, camel, cashmere, llama, mohair, and/or vicuna; (2) wool such as sheep; (3) filament such as silk. In an embodiment, fibers may be natural fibers that may include a natural fiber mineral base, including asbestos. In an embodiment, fibers may be man-made fibers that may include a man-made fiber organic natural polymer base, which may include one or more of: (1) a cellulose base such as bamboo, rayon, lyocell, acetate, and/or triacetate; (2) a protein base such as azlon; (3) an alginate; and (4) rubber. In an embodiment, fibers may be man-made fibers that may include a man-made fiber organic synthetic base, which may include one or more of acrylic, anidex, aramid, fluorocarbon, modacrylic, novoloid, nylon, nytril, olefin, PBI, polycarbonate, polyester, rubber, saran, spandex, vinal vinvon. In an embodiment, fibers may be man-made fibers that may include a man-made fiber inorganic base, which may include one or more of a glass material, metallic material, and carbon material.
[0288] In an embodiment, yarn may include natural fibers that may include a natural fiber cellulose base, wherein the natural fiber cellulose base may be from: (1) a baste such as flax, hemp, kenaf, jute, linen, and/or ramie; (2) a leaf such as flax, hemp, sisal, abaca, banana, henequen, ramie, sunn, and/or coir; or (3) seed hair such as cotton and/or kapok. In an embodiment, yarn may include natural fibers that may include a natural fiber protein base, wherein the natural fiber protein base may be from: (1) hair such as alpaca, camel, cashmere, llama, mohair, and/or vicuna; (2) wool such as sheep; or (3) filament such as silk. In an embodiment, yarn may include natural fibers that may include a natural fiber mineral base, including asbestos. In an embodiment, yarn may include man-made fibers that may include a man-made fiber organic natural polymer base, which may include: (1) a cellulose base such as bamboo, rayon, lyocell, acetate, and/or triacetate; (2) a protein base such as azlon; (3) an alginate; or (4) rubber. In an embodiment, yarn may include man-made fibers that may include a man-made fiber organic synthetic base, which may include acrylic, anidex, aramid, fluorocarbon, modacrylic, novoloid, nylon, nytril, olefin, PBI, polycarbonate, polyester, rubber, saran, spandex, vinal and/or vinvon. In an embodiment, yarn may include man-made fibers that may include a man-made fiber inorganic base, which may include a glass material, metallic material, carbon material, and/or specialty material.
[0289] In an embodiment, fabrics may include natural fibers and/or yarn that may include a natural fiber cellulose base, wherein the natural fiber cellulose base may be from: (1) a baste such as flax, hemp, kenaf, jute, linen, and/or ramie; (2) a leaf such as flax, hemp, sisal, abaca, banana, henequen, ramie, sunn, and/or coir; or (3) seed hair such as cotton and/or kapok. In an embodiment, fabric may include natural fibers and/or yarn that may include a natural fiber protein base, wherein the natural fiber protein base may be from: (1) hair such as alpaca, camel, cashmere, llama, mohair, and/or vicuna; (2) wool such as sheep; or (3) filament such as silk. In an embodiment, fabric may include natural fibers and/or yarn that may include a natural fiber mineral base, including asbestos. In an embodiment, fabric may include man-made fibers and/or yarn that may include a man-made fiber organic natural polymer base, which may include: (1) a cellulose base such as bamboo, rayon, lyocell, acetate, and/or triacetate; (2) a protein base such as azlon; (3) an alginate; or (4) rubber. In an embodiment, fabric may include man-made fibers and/or yarn that may include a man-made fiber organic synthetic base, which may include acrylic, anidex, aramid, fluorocarbon, modacrylic, novoloid, nylon, nytril, olefin, PBI, polycarbonate, polyester, rubber, saran, spandex, vinal and/or vinvon. In an embodiment, fabric may include man-made fibers and/or yarn that may include a man-made fiber inorganic base, which may include a glass material, metallic material, carbon material, and/or specialty material. [0290] In some embodiments, the fabric may comprise alpaca fiber, alpaca fleece, alpaca wool, lama fiber, lama fleece, lama wool, cotton, sheep fleece, sheep wool, byssus, chiengora, qiviut, yak, rabbit, lambswool, mohair wool, tibetan wool, lopi, camel hair, pashmina, angora wool, silkworm silk, spider silk, abaca fiber, coir fiber, flax fiber, jute fiber, kapok fiber, kenaf fiber, raffia fiber, bamboo fiber, hemp, modal fiber, pina, ramie, sisal, soy protein fiber, polyester, polyamide, polyaramid, polytetrafluoroethylene, polyethylene, polypropylene, polyurethane, silicone, mixtures of polyurethane and polyethyleneglycol, ultrahigh molecular weight polyethylene, high-performance polyethylene, nylon, LYCRA (polyester-polyurethane copolymer, also known as SPANDEX and elastomer), or a mixture thereof. In some embodiments, the fabric comprises wool. In some embodiments, the fabric comprises an inert synthetic material, such as polyester, polyamide, polyaramid, polytetrafluoroethylene, polyethylene, polypropylene, polyurethane, silicone, mixtures of polyurethane and polyethyleneglycol, ultrahigh molecular weight polyethylene, high-performance polyethylene, nylon, LYCRA (polyester-polyurethane copolymer, also known as SPANDEX and elastomer), rayon, or a mixture thereof.
[0291] In some embodiments, the fabric comprises one or more selected from the group consisting of cotton, silk, alpaca fleece, alpaca wool, lama fleece, lama wool, cotton, cashmere, sheep fleece, sheep wool, and combinations thereof. In some embodiments, the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool. [0292] In some embodiments, an article described herein may include a wool component in an amount, by weight of the article (w/w), of greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%,
26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%,
42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,
58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, where the balance of the article, by weight (w/w), is a non-wool component, as described herein. In some embodiments, an article described herein may include a wool component in an amount, by weight of the article (w/w), of less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%,
34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,
50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,
66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,
82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99%, where the balance of the article, by weight (w/w), is a non-wool component, as described herein. In some embodiments, an article described herein may include a wool component in an amount, by weight of the article (w/w), of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,
41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, where the balance of the article, by weight (w/w), is a non-wool component, as described herein.
[0293] In some embodiments, the fabric is chlorine descaled. In a non-limiting example, fabric can be treated with a cationic cross-linkable polymer such as Hercosett® (supplied by Hercules, USA) after chlorination treatment. In some embodiments, the fabric is non-chlorine descaled. In some embodiments, the fabric is non-chlorine descaled. In a non-limiting example, the fabric is treated with a descaling process using reduction or oxidation to remove the scale. In some embodiments, the fabric is natural. In some embodiments, the fabric comprises one or more of natural wool, chlorine-descaled wool, or non-chlorine descaled wool.
[0294] Any reducing agent useful in the field of fabrics and textiles is contemplated by the disclosure. Non-limiting examples of reducing agents include amino acids such as L-cysteine, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin.
[0295] In some embodiments, the reducing agent is an amino acid. Non-limiting examples of amino acids include naturally-occurring and non-naturally occurring amino acids. Non-limiting examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Vai. In some embodiments, the amino acid a genetically encoded amino acid, a naturally occurring non-genetically encoded amino acid, or a synthetic amino acid. Non-limiting examples of amino acids include 2-aminoadipic acid, 3 -aminoadipic acid, 2,3-diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2’-diaminopimelic acid, 6-aminohexanoic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosine, ornithine, citrulline, N-methylisoleucine, norleucine, tert-leucine, phenylglycine, t-butylglycine, N-methylglycine, sarcosine, N-ethylglycine, cyclohexylglycine, 4- oxo-cyclohexylglycine, N-ethylasparagine, cyclohexylalanine, t-butylalanine, naphthylalanine, pyridylalanine, 3 -chloroalanine, 3 -benzothienylalanine, 4-halophenylalanine, 4- chlorophenylalanine, 2-fluorophenylalanine, 3 -fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine, methionine sulfoxide, homoarginine, norarginine, nor-norarginine, N-acetyllysine, N-aminophenylalanine, N-methylvaline, homocysteine, homoserine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine, norvaline, O-allyl-serine, O-allyl-threonine, alpha-aminohexanoic acid, alpha-aminovaleric acid, and pyroglutamic acid. In some embodiments, the amino acid a an L-amino acid or a D-amino acid. Additional non-limiting examples of amino acids include alpha- and beta-amino acids.
[0296] In some embodiments, the reducing agent is L-cysteine. In some embodiments, the reducing agent has physical and/or chemical properties similar to L-cysteine. [0297] In some embodiments, the reducing agent is covalently linked to the surface of the fabric. In a non-limiting example, the reducing agent is covalently linked to a sulfur group attached to the surface of the fabric. In some embodiments, the silk protein fragment is covalently linked to the reducing agent. In some embodiments, the silk protein is covalently linked to the surface of the fabric. In some embodiments, the reducing agent is ionically bonded to the surface of the fabric. In a non-limiting example, the positively charged amine group (-NH3 + ) of an amino acid, such as L-cysteine, is ionically bonded to the negatively charged surface of the fabric.
[0298] In some embodiments, the coating further comprises a crosslinker. In some embodiments, any SPF described herein, including silk fibroin or silk fibroin-based protein fragments are chemically modified with a precursor linker comprising a crosslinker to form silk conjugates. In some embodiments, the fabric is covalently linked to the crosslinker. In some embodiments, the crosslinker is covalently linked to the reducing agent. In some embodiments, the crosslinker is covalently linked to the fabric and to the reducing agent. In some embodiments, the crosslinker is covalently linked to the fabric and an SPF. In some embodiments, Non-limiting examples of crosslinkers include:
Example of
Class of Crosslinker chemical group Example reactivity
Succinimidyl Amine
Bis[Sulfosuccinimidyl] glutarate
Carbodiimide Carboxyls Acyl chloride Amines/Hydroxyls
2,3-Dibromopropionyl chloride
Carbonyldiimidazole Amine/Hydroxyls
N,N'-Carbonyldiimidazole
NHS-maleimide crosslinking, Thiol
Succinimidyl-4- [N- maleimidomethyl]cyclohexane-l- carboxylate
Imidoester crosslinking Amine dimethyl pimelimidate dicyclohexyl carbodiimide Amines crosslinking
Methacrylate Epoxide Hydroxyl/amines
Glycerol diglycidyl ether (GDE)
Silanes Hydroxyls
TEOS Tetraethyl orthosilicate
Alkyne- Click Chemistry Azide
Azide-click Chemistry Alkyne
Aldehyde Amines
Formaldehyde Amines
Thioester Thiols, amines, hydroxyls
Photo-crosslinker Amines
N-Sulfosuccinimidyl-6-[4'-azido-2'- nitrophenylamino] hexanoate pyridyl thiol, Amines and sulfosuccinimidyl 6-[3’-(2- sulfhydryl pyridyldithio)propionamido] hexanoate hydrazide Aldehydes, carbohydrates alkoxyamine Aldehydes reductive amination Amine aryl azide Alkyne Diazirine, NHS diazirine, Amine (through succinimidyl 4,4’- NHS group) to azipentanoate amine (UV 350 nm) azide-phosphine
Aryl halide, l,5-Difluoro-2,4- Primary amines dinitrobenzene
[0299] Precursor linkers can be selected from any of the following natural crosslinkers: caffeic acid, tannic acid, genipin, proanthocyanidin, and the like. Precursor crosslinking can be selected from any of the following enzymatic crosslinking: transglutaminase transferase crosslinking, hydrolase crosslinking, peptidase crosslinking (e.g., sortase SrtA from Staphylococcus aureus), oxidoreductase crosslinking, tyrosinase crosslinking, laccase crosslinking, peroxidase crosslinking (e.g., horseradish peroxidase), lysyl oxidase crosslinking, peptide ligases (e.g., butelase 1, peptiligase, subtiligase, etc.), and the like.
[0300] In some embodiments, silk fibroin or silk fibroin-based protein fragments are chemically modified with a precursor linker to form silk conjugates with a crosslinker or an activator independently selected from a N-hydroxysuccinimide ester crosslinker, an imidoester crosslinker, a sulfosuccinimidyl aminobenzoate, a methacrylate, a silane, a silicate, an alkyne compound, an azide compound, an aldehyde, a carbodiimide crosslinker, a dicyclohexyl carbodiimide activator, a dicyclohexyl carbodiimide crosslinker, a maleimide crosslinker, a haloacetyl crosslinker, a pyridyl disulfide crosslinker, a hydrazide crosslinker, an alkoxyamine crosslinker, a reductive amination crosslinker, an aryl azide crosslinker, a diazirine crosslinker, an azide-phosphine crosslinker, a transferase crosslinker, a hydrolase crosslinker, a transglutaminase crosslinker, a peptidase crosslinker, an oxidoreductase crosslinker, a tyrosinase crosslinker, a laccase crosslinker, a peroxidase crosslinker, a lysyl oxidase crosslinker, and any combinations thereof. Some chemically modified silk fibroin has been described in J Mater Chem. 2009, June 23, 19(36), 6443-6450, including cyanuric chloride-activated coupling, carbodiimide coupling, arginine masking, chlorosulfonic acid reaction, diazonium coupling, tyrosinase-catalyzed grafting, and poly(methacrylate) grafting.
[0301] In some embodiments, the article further comprising a crosslinking agent. In some embodiments, the crosslinking agent is a polyphenol compound comprising 12 phenolic hydroxyl groups, having a molecular weight of about 500-4000 Da, and exhibiting about 5-7 aromatic rings per 1000 Da. In some embodiments, the crosslinking agent is a polyphenol compound selected from the group consisting of curcumin, desmethoxycurcumin, bis- desmethoxycurcumin, resveratrol, caffeic acid, tannin, gallotannin, procyanidin, hydrolysable tannin, phlorotannin, gallic acid, chlorogenic acid, camosol, capsaicin, 6-shogaol, 6-gingerol, flavonoid, flavanol, neoflavonoid, arbutin, cynarin, apigenin, isocuttelarein, luteolin, nobiletin, tangeretin, tectochrysin, galangin, kaempferol, myricetin, quercetin, rutin, citrin, curcurocitrin, eriodictyol, hesperidin, naringenin, naringin, pinocembrin, quercitrin, biochanin A, chrysin, daidzein, equol, formononetin, genistein, glycetein, ipriflavone, lactuin, pycnogenol, silymarin, lignin, and combinations thereof.
[0302] In some embodiments, the crosslinker is glycerol diglycidyl ether (GDE).
[0303] In an embodiment, a water-soluble silk coating may be used as an adhesive or binder for binding particles to fabrics or for binding fabrics. In an embodiment, an article comprises a fabric bound to another fabric using a silk coating. In an embodiment, an article comprises a fabric with particles bound to the fabric using a silk adhesive. [0304] In an embodiment, the coating is applied to an article including a fabric at the yam level. In an embodiment, the coating is applied at the fabric level. In an embodiment, the coating has a thickness selected from the group consisting of about 5 nm, about 10 nm, about 15 nm, about 20 nm, about 25 nm, about 50 nm, about 100 nm, about 200 nm, about 500 nm, about 1 pm, about 5 pm, about 10 pm, and about 20 pm. In an embodiment, the coating has a thickness range selected from the group consisting of about 5 nm to about 100 nm, about 100 nm to about 200 nm, about 200 nm to about 500 nm, about 1 pm to about 2 pm, about 2 pm to about 5 pm, about 5 pm to about 10 pm, and about 10 pm to about 20 pm.
[0305] In an embodiment, fabric is treated with a polymer, such as polyglycolide (PGA), polyethylene glycols, copolymers of glycolide, glycolide/L-lactide copolymers (PGA/PLLA), glycolide/trimethylene carbonate copolymers (PGA/TMC), polylactides (PLA), stereocopolymers of PLA, poly-L-lactide (PLLA), poly-DL-lactide (PDLLA), L-lactide/DL- lactide copolymers, co-polymers of PLA, lactide/tetram ethylglycolide copolymers, lactide/trimethylene carbonate copolymers, lactide/6- valerolactone copolymers, lactide/s- caprolactone copolymers, polydepsipeptides, PLA/polyethylene oxide copolymers, unsymmetrically 3,6-substituted poly-1, 4-dioxane-2, 5-diones, poly-P-hydroxybutyrate (PHBA), PHBA/p-hydroxyvalerate copolymers (PHBA/HVA), poly-P-hydroxypropionate (PHP A), poly- p-dioxanone (PDS), poly-6-valerolactone, poly-s-caprolactone, methylmethacrylate-N-vinyl pyrrolidine copolymers, polyesteramides, polyesters of oxalic acid, polydihydropyrans, polyalkyl-2-cyanoacrylates, polyurethanes (PU), polyvinylalcohols (PVA), polypeptides, poly-P- malic acid (PMLA), poly-P-alkanoic acids, polyvinylalcohol (PVA), polyethyleneoxide (PEO), chitine polymers, polyethylene, polypropylene, polyasetal, polyamides, polyesters, polysulphone, polyether ether ketone, polyethylene terephthalate, polycarbonate, polyaryl ether ketone, and polyether ketone ketone.
[0306] In an embodiment, textiles may be manufactured via one or more of the following processes weaving processes, knitting processes, and non-woven processes. In an embodiment, weaving processes may include plain weaving, twill weaving, and/or satin weaving. In an embodiment, knitting processes may include weft knitting (e.g., circular, flat bed, and/or full fashioned) and/or warp knitting (e.g., tricot, Raschel, and/or crochet). In an embodiment, nonwoven processes may include stable fiber (e.g., dry laid and/or wet laid) and/or continuous filament (e.g., spun laid and/or melt blown). [0307] In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric used for human apparel, including performance and/or athletic apparel. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, and wherein the fabric exhibits improved moisture management properties and/or resistance to microbial growth. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric used for home upholstery. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is used for automobile upholstery. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is used for aircraft upholstery. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is used for upholstery in transportation vehicles for public, commercial, military, or other use, including buses and trains. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is used for upholstery of a product that requires a high degree of resistance to wear as compared to normal upholstery.
[0308] In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric fabricated as trim on automobile upholstery. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a steering wheel. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a headrest. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as an armrest. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as an automobile floor mat. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as automobile or vehicle carpet. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as automotive trim. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a children’s car seat. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a seat belt or safety harness. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a dashboard. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a seat. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a seat panel. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as an interior panel. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as an airbag cover. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as an airbag. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a sunvisor. In an embodiment, the invention provides an article comprising a fabric coated with silk protein fragments, wherein the fabric is a fabric product fabricated as a wiring harness. In an embodiment, the invention provides an article coated with silk protein fragments, wherein the article is a cushion. In an embodiment, the invention provides an article coated with silk protein fragments, wherein the product is automotive, aircraft, or other vehicular insulation. In some embodiments, the coating comprises an article coated with silk protein fragments, thereof having a weight average molecular weight range of about 1 kDa to about 350 kDa, wherein the silk protein fragments have an average weight average molecular weight range selected from the group consisting of about 5 to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk protein fragments have a poly dispersity of between about 1.5 and about 3.0, or about 1.0 and about 5.0, and optionally wherein the proteins or protein fragments, prior to coating the fabric, do not spontaneously or gradually gelate and do not visibly change in color or turbidity when in a solution for at least 10 days. The coating comprises silk protein fragments having a weight average molecular weight range of about 5 kDa and about 144 kDa, wherein the silk protein fragments have an average weight average molecular weight range selected from the group consisting of about 5 to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk protein fragments have a poly dispersity of between about 1.5 and about 3.0, and optionally wherein the proteins or protein fragments, prior to coating the fabric, do not spontaneously or gradually gelate and do not visibly change in color or turbidity when in a solution for at least 10 days.
[0309] In an embodiment, the invention provides an article comprising fabric coated with silk protein fragments. In an embodiment, the article is a fabric used in the manufacture of tents, sleeping bags, ponchos, and soft-walled coolers. In an embodiment, the fabric is a fabric used in the manufacture of athletic equipment. In an embodiment, the fabric is a fabric used in the manufacture of outdoor gear. In an embodiment, the fabric is a fabric used in the manufacture of hiking gear, such as harnesses and backpacks. In an embodiment, the fabric is a fabric used in the manufacture of climbing gear. In an embodiment, the fabric is canvass. In an embodiment, the fabric is a fabric used in the manufacture of a hat. In an embodiment, the fabric is a fabric used in the manufacture of an umbrella. In an embodiment, the fabric is a fabric used in the manufacture of a tent. In an embodiment, the fabric is a fabric used in the manufacture of a baby sleeper, a baby blanket, or a baby pajama. In an embodiment, the fabric is a fabric used in the manufacture of a glove, such as a driving glove or an athletic glove. In an embodiment, the fabric is a fabric used in the manufacture of athletic pants, such as sweat pants, jogging pants, yoga pants, or pants for use in competitive sports. In an embodiment, the fabric is a fabric used in the manufacture of athletic shirts, such as sweat shirts, jogging shirts, yoga shirts, or shirts for use in competitive sports. In an embodiment, the fabric is a fabric used in the manufacture of beach equipment, such as beach umbrellas, beach chairs, beach blankets, and beach towels. In an embodiment, the fabric is a fabric used in the manufacture of jackets or overcoats. In an embodiment, the fabric is a fabric used in the manufacture of medical garments, such as surgical drapes, surgical gowns, surgical sleeves, laboratory sleeves, laboratory coats, wound dressings, sterilization wraps, surgical face masks, retention bandages, support devices, compression bandages, shoe covers, surgical blankets, and the like. The coating comprises silk based proteins or fragments thereof having a weight average molecular weight range of about 5 kDa to about 144 kDa. [0310] In an embodiment, the invention provides an article comprising a textile coated with silk fibroin-based proteins or fragments thereof. In an embodiment, the textile is a textile used in the manufacture of tents, sleeping bags, ponchos, and soft-walled coolers. In an embodiment, the textile is a textile used in the manufacture of athletic equipment. In an embodiment, the textile is a textile used in the manufacture of outdoor gear. In an embodiment, the textile is a textile used in the manufacture of hiking gear, such as harnesses and backpacks. In an embodiment, the textile is a textile used in the manufacture of climbing gear. In an embodiment, the textile is canvass. In an embodiment, the textile is a textile used in the manufacture of a hat. In an embodiment, the textile is a textile used in the manufacture of an umbrella. In an embodiment, the textile is a textile used in the manufacture of a tent. In an embodiment, the textile is a textile used in the manufacture of a baby sleeper, a baby blanket, or a baby pajama. In an embodiment, the textile is a textile used in the manufacture of a glove, such as a driving glove or an athletic glove. In an embodiment, the textile is a textile used in the manufacture of athletic pants, such as sweat pants, jogging pants, yoga pants, or pants for use in competitive sports. In an embodiment, the textile is a textile used in the manufacture of athletic shirts, such as sweat shirts, jogging shirts, yoga shirts, or shirts for use in competitive sports. In an embodiment, the textile is a textile used in the manufacture of beach equipment, such as beach umbrellas, beach chairs, beach blankets, and beach towels. In an embodiment, the textile is a textile used in the manufacture of jackets or overcoats. In an embodiment, the textile is a textile used in the manufacture of medical garments, such as surgical drapes, surgical gowns, surgical sleeves, laboratory sleeves, laboratory coats, wound dressings, sterilization wraps, surgical face masks, retention bandages, support devices, compression bandages, shoe covers, surgical blankets, and the like. The coating comprises silk based proteins or fragments thereof having a weight average molecular weight range of about 1 kDa to about 350 kDa, wherein the silk based proteins or protein fragments thereof have an average weight average molecular weight range selected from the group consisting of about 5 to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk based proteins or fragments thereof have a poly dispersity of between about 1.0 and about 5.0, and optionally wherein the proteins or protein fragments, prior to coating the fabric, do not spontaneously or gradually gelate and do not visibly change in color or turbidity when in a solution for at least 10 days. [0311] In an embodiment, the invention provides a shoe coated with silk fibroin-based proteins or fragments thereof. In an embodiment, the invention provides a shoe coated with silk fibroin- based proteins or fragments thereof, wherein the shoe exhibits an improved property relative to an uncoated shoe. In an embodiment, the invention provides a shoe coated with silk fibroin- based proteins or fragments thereof, wherein the shoe exhibits an improved property relative to an uncoated shoe, and wherein the improved property is stain resistance. In an embodiment, the invention provides a shoe coated with silk fibroin-based proteins or fragments thereof, wherein the shoe exhibits an improved property relative to an uncoated shoe, and wherein the shoe is made of natural leather or synthetic leather. The coating comprises silk based proteins or fragments thereof having a weight average molecular weight range of about 1 kDa to about 350 kDa, or about 5 kDa to about 144 kDa, wherein the silk based proteins or protein fragments thereof have an average weight average molecular weight range selected from the group consisting of about 5 to about 10 kDa, about 6 kDa to about 17 kDa, about 17 kDa to about 39 kDa, about 39 kDa to about 80 kDa, about 60 to about 100 kDa, and about 80 kDa to about 144 kDa, wherein the silk based proteins or fragments thereof have a poly dispersity of between about 1.0 and about 5.0, or about 1.5 and about 3.0, and optionally wherein the proteins or protein fragments, prior to coating the fabric, do not spontaneously or gradually gelate and do not visibly change in color or turbidity when in a solution for at least 10 days.
[0312] In one aspect, the disclosure provides methods of making a silk coated fabric and/or articles using the silk protein fragments of the disclosure. In some embodiments, the silk coated fabric is a silk fibroin coated fabric. In some embodiments, the silk coated article is a silk fibroin coated article. In some embodiments, the disclosure also includes an article prepared by the methods of the disclosure. In some embodiments, the disclosure also includes an article comprising a coated fabric prepared by the methods of the disclosure. In some embodiments, the disclosure also includes a coated fabric prepared by the methods of the disclosure.
[0313] In some embodiments, the disclosure includes a method of making a silk fibroin coated fabric, comprising applying to the fabric a solution comprising a reducing agent, applying to the fabric a silk fibroin solution, and drying the fabric.
[0314] In some embodiments, the disclosure includes a method of improving size retention on laundering in a fabric, comprising applying to the fabric a solution comprising a reducing agent, applying to the fabric a silk fibroin solution, and drying the fabric. [0315] In one aspect, the disclosure includes a method of improving size retention on laundering in a fabric comprising coating a surface of the fabric with a solution comprising a reducing agent, preparing a silk fibroin solution comprising silk protein fibroin fragments, coating a surface of the fabric with the silk fibroin solution, and drying the surface of the fabric that has been coated with the silk fibroin solution, wherein upon laundering, the coated fabric substantially retains its initial size prior to laundering.
[0316] Any reducing agent is contemplated by the present disclosure. In a non-limiting example, the reducing agent is used to pretreat the surface of the fabric in order to improve the surface affinity between the silk protein fragments and the fabric. In some embodiments, the reducing agent is a natural reducing agent. In some embodiments, the reducing agent is a synthetic reducing agent. In some embodiments, the reducing agent is selected from an amino acid such as L-cysteine, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin. In some embodiments, the reducing agent is L- cysteine.
[0317] In some embodiments, the concentration of the reducing agent in the solution ranges from 0.01 g/L to about 100 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from 0.1 g/L to about 50 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from 0.5 g/L to about 25 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from 1 g/L to about 12 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from 0.5 g/L to about 1.5 g/L. In some embodiments, the concentration of the reducing agent in the solution ranges from 11 g/L to about 13 g/L. In some embodiments, the concentration of the reducing agent is about 1 g/L. In some embodiments, the concentration of the reducing agent is about 12 g/L.
[0318] In some embodiments, the silk fibroin solution comprises low molecular weight silk fibroin-based protein fragments, medium molecular weight silk fibroin-based protein fragments, and/or high molecular weight silk fibroin-based protein fragments. In some embodiments, the silk fibroin solution comprises low molecular weight silk fibroin-based protein fragments. In some embodiments, the silk fibroin solution comprises medium molecular weight silk fibroin- based protein fragments. [0319] In some embodiments, drying the surface of the fabric comprises heating the surface of the fabric without substantially modifying silk fibroin coating performance.
[0320] In some embodiments, the method includes an additional step of drying the surface of the fabric. In some embodiments, the additional drying step is performed after coating a surface of the fabric with the solution comprising a reducing agnet. In some embodiments, the additional drying step is performed before coating the surface with the silk fibroin solution.
[0321] In some embodiments, the silk fibroin solution further comprises a reducing agent. In some embodiments, the reducing agent is the same reducing agent as used in the solution comprising a reducing agent. In some embodiments, the reducing agent is a different reducing agent as used in the solution comprising a reducing agent.
[0322] In one aspect, the disclosure provides a method of making a silk fibroin coated fabric, comprising preparing a silk fibroin solution comprising a reducing agent and silk protein fibroin fragments, coating a surface of the fabric with the silk fibroin solution, and drying the surface of the material that has been coated with the silk fibroin solution to provide the silk fibroin coated fabric
[0323] In some embodiments, upon laundering, the fabric substantially retains its initial size prior to laundering. In some embodiments, upon laundering, the fabric retains a substantially higher fraction of its initial size prior to laundering compared to a similar fabric not similarly treated with the reducing agent and the silk fibroin solution
[0324] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is dimensional stability to laundering, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
[0325] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is size retention on laundering, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
[0326] In any of the foregoing embodiments, at least one property of the article is improved, wherein the property that is improved is resistance to shrinkage, and wherein the property is improved by an amount relative to an uncoated article selected from the group consisting of at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 125%, at least 150%, at least 200%, at least 300%, at least 400%, and at least 500%.
[0327] In an embodiment, the foregoing improved property, or any other improved property described herein, is determined after a period of machine washing (e.g., by home laundering machine washing) cycles selected from the group consisting of 0 cycles, 1 cycle, 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, 11 cycles, 12 cycles, 13 cycles, 14 cycles, 15 cycles, 20 cycles, 25 cycles, 30 cycles, 35 cycles, 40 cycles, 45 cycles, and 50 cycles.
[0328] In an embodiment, the concentration of the silk fibroin solution is less than 30.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 25.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 20.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 19.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 18.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 17.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 16.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 15.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 14.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 13.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 12.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 11.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 9.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 8.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 7.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 6.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 5.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 2.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 1.0% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.9% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.8% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.7% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.6% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.5% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.4% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.3% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.2% w/v. In an embodiment, the concentration of the silk fibroin solution is less than 0.1% w/v.
[0329] In an embodiment, the concentration of the silk fibroin solution is greater than 0.1% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.2% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.3% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.4% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.5% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.6% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.7% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.8% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 0.9% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 1.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 2.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 5.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 6.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 7.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 8.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 9.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 11.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 12.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 13.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 14.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 15.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 16.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 17.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 18.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 19.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 20.0% w/v. In an embodiment, the concentration of the silk fibroin solution is greater than 25.0% w/v.
[0330] In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 30.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 25.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 20.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 15.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 9.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 8.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 7.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 6.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 6.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 5.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 5.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 4.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 3.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 2.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 2.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 2.4% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 5.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 4.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 3.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.5% w/v to about 2.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 3.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 2.5% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 2.4% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 2.0% w/v.
[0331] In an embodiment, the concentration of the silk fibroin solution ranges from about 20.0% w/v to about 30.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 1.0% w/v to about 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 2% w/v to about 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 0.1% w/v to about 6.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 6.0% w/v to about 10.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 6.0% w/v to about 8.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 6.0% w/v to about 9.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 10.0% w/v to about 20.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 11.0% w/v to about 19.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 12.0% w/v to about 18.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 13.0% w/v to about 17.0% w/v. In an embodiment, the concentration of the silk fibroin solution ranges from about 14.0% w/v to about 16.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 1.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 0.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 1.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 2.0 wt.%. In an embodiment, the concentration of the silk fibroin solution is about 2.4% w/v. In an embodiment, the concentration of the silk fibroin solution is 3.0% w/v. In an embodiment, the concentration of the silk fibroin solution is 3.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 4.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 4.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 5.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 5.5% w/v. In an embodiment the concentration of the silk fibroin solution is about 6.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 6.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 7.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 7.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 8.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 8.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 9.0% w/v. In an embodiment, the concentration of the silk fibroin solution is about 9.5% w/v. In an embodiment, the concentration of the silk fibroin solution is about 10.0% w/v.
[0332] In some embodiments, the SFS includes an acidic agent. In some embodiments, an acidic agent is a Bronsted acid. In an embodiment, the acidic agent includes one or more of citric acid and acetic acid. In an embodiment, the acidic agent aids the deposition and coating of SPF mixtures (i.e., SFS coating) on the textile to be coated as compared to the absence of such acidic agent. In an embodiment, the acidic agent improves crystallization of the SPF mixtures at the textile to be coated. [0333] In an embodiment, the acidic agent is added at a concentration by weight (% w/w or % w/v) or by volume (v/v) of greater than about 0.001 % , or greater than about 0.002 %, or greater than about 0.003 %, or greater than about 0.004 %, or greater than about 0.005 %, or greater than about 0.006 %, or greater than about 0.007 %, or greater than about 0.008 %, or greater than about 0.009 %, or greater than about 0.01 %, or greater than about 0.02 %, or greater than about 0.03 %, or greater than about 0.04 %, or greater than about 0.05 %, or greater than about 0.06 %, or greater than about 0.07 %, or greater than about 0.08 %, or greater than about 0.09 %, or greater than about 0.1 %, or greater than about 0.2 %, or greater than about 0.3 %, or greater than about 0.4 %, or greater than about 0.5 %, or greater than about 0.6 %, or greater than about 0.7 %, or greater than about 0.8 %, or greater than about 0.9 %, or greater than about 1.0 % or greater than about 2.0 %, or greater than about 3.0 %, or greater than about 4.0 %, or greater than about 5.0% .
[0334] In an embodiment, the acidic agent is added at a concentration by weight (% w/w or % w/v) or by volume (v/v) of less than about 0.001 %, or less than about 0.002 %, or less than about 0.003 %, or less than about 0.004 % , or less than about 0.005 %, or less than about 0.006 %, or less than about 0.007 %, or less than about 0.008 %, or less than about 0.009 %, or less than about 0.01 %, or less than about 0.02 %, or less than about 0.03 %, or less than about 0.04 %, or less than about 0.05 %, or less than about 0.06 %, or less than about 0.07 %, or less than about 0.08 %, or less than about 0.09 %, or less than about 0.1 %, or less than about 0.2 %, or less than about 0.3 %, or less than about 0.4 %, or less than about 0.5 %, or less than about 0.6%, or less than about 0.7 %, or less than about 0.8 %, or less than about 0.9 %, or less than about 1.0 % or less than about 2.0 %, or less than about 3.0 %, or less than about 4.0 %, or less than about 5.0 %.
[0335] In some embodiments, SFS may have a pH of less than about 9, or less than about 8.5, or less than about 8, or less than about 7.5, or less than about 7, or less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4, or greater than about 3.5, or greater than about 4, or greater than about 4.5, or greater than about 5, or greater than about 5.5, or greater than about 6, or greater than about 6.5, or greater than about 7, or greater than about 7.5, or greater than about 8, or greater than about 8.5.
[0336] In some embodiments, SFS may include an acidic agent, and may have a pH of less than about 9, or less than about 8.5, or less than about 8, or less than about 7.5, or less than about 7, or less than about 6.5, or less than about 6, or less than about 5.5, or less than about 5, or less than about 4.5, or less than about 4, or greater than about 3.5, or greater than about 4, or greater than about 4.5, or greater than about 5, or greater than about 5.5, or greater than about 6, or greater than about 6.5, or greater than about 7, or greater than about 7.5, or greater than about 8, or greater than about 8.5.
[0337] In some embodiments, SFS has a pH ranging from about 3 to 5. In some embodiments, SFS has a pH of about 4.
[0338] In some embodiments, SFS may be applied to fibers and/or yarn having a diameter of less than about 100 nm, or less than about 200 nm, or less than about 300 nm, or less than about 400 nm, or less than about 500 nm, or less than about 600 nm, or less than about 700 nm, or less than about 800 nm, or less than about 900 nm, or less than about 1000 nm, or less than about 2 pm, or less than about 5 pm, or less than about 10 pm, or less than about 20 pm, or less than about 30 pm, or less than about 40 pm, or less than about 50 pm, or less than about 60 pm, or less than about 70 pm, or less than about 80 pm, or less than about 90 pm, or less than about 100 pm, or less than about 200 pm, or less than about 300 pm, or less than about 400 pm, or less than about 500 pm, or less than about 600 pm, or less than about 700 pm, or less than about 800 pm, or less than about 900 pm, or less than about 1000 pm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9 mm, or less than about 10 mm, or less than about 20 mm, or less than about 30 mm, or less than about 40 mm, or less than about 50 mm, or less than about 60 mm, or less than about 70 mm, or less than about 80 mm, or less than about 90 mm, or less than about 100 mm, or less than about 200 mm, or less than about 300 mm, or less than about 400 mm, or less than about 500 mm, or less than about 600 mm, or less than about 700 mm, or less than about 800 mm, or less than about 900 mm, or less than about 1000 mm.
[0339] In some embodiments, SFS may be applied to fibers and/or yarn having a diameter of greater than about 100 nm, or greater than about 200 nm, or greater than about 300 nm, or greater than about 400 nm, or greater than about 500 nm, or greater than about 600 nm, or greater than about 700 nm, or greater than about 800 nm, or greater than about 900 nm, or greater than about 1000 nm, or greater than about 2 pm, or greater than about 5 pm, or greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 gm, or greater than about 90 gm, or greater than about 100 gm, or greater than about 200 gm, or greater than about 300 gm, or greater than about 400 gm, or greater than about 500 gm, or greater than about 600 gm, or greater than about 700 gm, or greater than about 800 gm, or greater than about 900 gm, or greater than about 1000 gm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9 mm, or greater than about 10 mm, or greater than about 20 mm, or greater than about 30 mm, or greater than about 40 mm, or greater than about 50 mm, or greater than about 60 mm, or greater than about 70 mm, or greater than about 80 mm, or greater than about 90 mm, or greater than about 100 mm, or greater than about 200 mm, or greater than about 300 mm, or greater than about 400 mm, or greater than about 500 mm, or greater than about 600 mm, or greater than about 700 mm, or greater than about 800 mm, or greater than about 900 mm, or greater than about 1000 mm.
[0340] In some embodiments, SFS may be applied to fibers and/or yarn having a length of less than about 100 nm, or less than about 200 nm, or less than about 300 nm, or less than about 400 nm, or less than about 500 nm, or less than about 600 nm, or less than about 700 nm, or less than about 800 nm, or less than about 900 nm, or less than about 1000 nm, or less than about 2 pm, or less than about 5 pm, or less than about 10 pm, or less than about 20 pm, or less than about 30 pm, or less than about 40 pm, or less than about 50 pm, or less than about 60 pm, or less than about 70 pm, or less than about 80 pm, or less than about 90 pm, or less than about 100 pm, or less than about 200 pm, or less than about 300 pm, or less than about 400 pm, or less than about 500 pm, or less than about 600 pm, or less than about 700 pm, or less than about 800 pm, or less than about 900 pm, or less than about 1000 pm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9 mm, or less than about 10 mm, or less than about 20 mm, or less than about 30 mm, or less than about 40 mm, or less than about 50 mm, or less than about 60 mm, or less than about 70 mm, or less than about 80 mm, or less than about 90 mm, or less than about 100 mm, or less than about 200 mm, or less than about 300 mm, or less than about 400 mm, or less than about 500 mm, or less than about 600 mm, or less than about 700 mm, or less than about 800 mm, or less than about 900 mm, or less than about 1000 mm. [0341] In some embodiments, SFS may be applied to fibers and/or yarn having a length of greater than about 100 nm, or greater than about 200 nm, or greater than about 300 nm, or greater than about 400 nm, or greater than about 500 nm, or greater than about 600 nm, or greater than about 700 nm, or greater than about 800 nm, or greater than about 900 nm, or greater than about 1000 nm, or greater than about 2 pm, or greater than about 5 pm, or greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 pm, or greater than about 90 pm, or greater than about 100 pm, or greater than about 200 pm, or greater than about 300 pm, or greater than about 400 pm, or greater than about 500 pm, or greater than about 600 pm, or greater than about 700 pm, or greater than about 800 pm, or greater than about 900 pm, or greater than about 1000 pm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9 mm, or greater than about 10 mm, or greater than about 20 mm, or greater than about 30 mm, or greater than about 40 mm, or greater than about 50 mm, or greater than about 60 mm, or greater than about 70 mm, or greater than about 80 mm, or greater than about 90 mm, or greater than about 100 mm, or greater than about 200 mm, or greater than about 300 mm, or greater than about 400 mm, or greater than about 500 mm, or greater than about 600 mm, or greater than about 700 mm, or greater than about 800 mm, or greater than about 900 mm, or greater than about 1000 mm.
[0342] In some embodiments, SFS may be applied to fibers and/or yarn having a weight (g/m 2 ) of less than about 1 g/m 2 , or less than about 2 g/m 2 , or less than about 3 g/m 2 , or less than about 4 g/m 2 , or less than about 5 g/m 2 , or less than about 6 g/m 2 , or less than about 7 g/m 2 , or less than about 8 g/m 2 , or less than about 9 g/m 2 , or less than about 10 g/m 2 , or less than about 20 g/m 2 , or less than about 30 g/m 2 , or less than about 40 g/m 2 , or less than about 50 g/m 2 , or less than about 60 g/m 2 , or less than about 70 g/m 2 , or less than about 80 g/m 2 , or less than about 90 g/m 2 , or less than about 100 g/m 2 , or less than about 200 g/m 2 , or less than about 300 g/m 2 , or less than about 400 g/m 2 , or less than about 500 g/m 2 .
[0343] In some embodiments, SFS may be applied to fibers and/or yarn having a weight (g/m 2 ) of at greater than about 1 g/m 2 , or greater than about 2 g/m 2 , or greater than about 3 g/m 2 , or greater than about 4 g/m 2 , or greater than about 5 g/m 2 , or greater than about 6 g/m 2 , or greater than about 7 g/m 2 , or greater than about 8 g/m 2 , or greater than about 9 g/m 2 , or greater than about 10 g/m 2 , or greater than about 20 g/m 2 , or greater than about 30 g/m 2 , or greater than about 40 g/m 2 , or greater than about 50 g/m 2 , or greater than about 60 g/m 2 , or greater than about 70 g/m 2 , or greater than about 80 g/m 2 , or greater than about 90 g/m 2 , or greater than about 100 g/m 2 , or greater than about 200 g/m 2 , or greater than about 300 g/m 2 , or greater than about 400 g/m 2 , or greater than about 500 g/m 2 .
[0344] In some embodiments, SFS may be applied to fabric having a thickness of less than about 100 nm, or less than about 200 nm, or less than about 300 nm, or less than about 400 nm, or less than about 500 nm, or less than about 600 nm, or less than about 700 nm, or less than about 800 nm, or less than about 900 nm, or less than about 1000 nm, or less than about 2 pm, or less than about 5 pm, or less than about 10 pm, or less than about 20 pm, or less than about 30 pm, or less than about 40 pm, or less than about 50 pm, or less than about 60 pm, or less than about 70 pm, or less than about 80 pm, or less than about 90 pm, or less than about 100 pm, or less than about 200 pm, or less than about 300 pm, or less than about 400 pm, or less than about 500 pm, or less than about 600 pm, or less than about 700 pm, or less than about 800 pm, or less than about 900 pm, or less than about 1000 pm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9 mm, or less than about 10 mm.
[0345] In some embodiments, SFS may be applied to fabric having a thickness of greater than about 100 nm, or greater than about 200 nm, or greater than about 300 nm, or greater than about 400 nm, or greater than about 500 nm, or greater than about 600 nm, or greater than about 700 nm, or greater than about 800 nm, or greater than about 900 nm, or greater than about 1000 nm, or greater than about 2 pm, or greater than about 5 pm, or greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 pm, or greater than about 90 pm, or greater than about 100 pm, or greater than about 200 pm, or greater than about 300 pm, or greater than about 400 pm, or greater than about 500 pm, or greater than about 600 pm, or greater than about 700 pm, or greater than about 800 pm, or greater than about 900 pm, or greater than about 1000 pm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9 mm, or greater than about 10 mm.
[0346] In some embodiments, SFS may be applied to fabric having a width of less than about 100 nm, or less than about 200 nm, or less than about 300 nm, or less than about 400 nm, or less than about 500 nm, or less than about 600 nm, or less than about 700 nm, or less than about 800 nm, or less than about 900 nm, or less than about 1000 nm, or less than about 2 pm, or less than about 5 pm, or less than about 10 pm, or less than about 20 pm, or less than about 30 pm, or less than about 40 pm, or less than about 50 pm, or less than about 60 pm, or less than about 70 pm, or less than about 80 pm, or less than about 90 pm, or less than about 100 pm, or less than about 200 pm, or less than about 300 pm, or less than about 400 pm, or less than about 500 pm, or less than about 600 pm, or less than about 700 pm, or less than about 800 pm, or less than about 900 pm, or less than about 1000 pm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9 mm, or less than about 10 mm, or less than about 20 mm, or less than about 30 mm, or less than about 40 mm, or less than about 50 mm, or less than about 60 mm, or less than about 70 mm, or less than about 80 mm, or less than about 90 mm, or less than about 100 mm, or less than about 200 mm, or less than about 300 mm, or less than about 400 mm, or less than about 500 mm, or less than about 600 mm, or less than about 700 mm, or less than about 800 mm, or less than about 900 mm, or less than about 1000 mm, or less than about 2 m, or less than about 3 m, or less than about 4 m, or less than about 5 m.
[0347] In some embodiments, SFS may be applied to fabric having a width of greater than about 100 nm, or greater than about 200 nm, or greater than about 300 nm, or greater than about 400 nm, or greater than about 500 nm, or greater than about 600 nm, or greater than about 700 nm, or greater than about 800 nm, or greater than about 900 nm, or greater than about 1000 nm, or greater than about 2 pm, or greater than about 5 pm, or greater than about 10 pm, or greater than about 20 pm, or greater than about 30 pm, or greater than about 40 pm, or greater than about 50 pm, or greater than about 60 pm, or greater than about 70 pm, or greater than about 80 pm, or greater than about 90 pm, or greater than about 100 pm, or greater than about 200 pm, or greater than about 300 pm, or greater than about 400 pm, or greater than about 500 pm, or greater than about 600 pm, or greater than about 700 pm, or greater than about 800 pm, or greater than about 900 pm, or greater than about 1000 pm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9 mm, or greater than about 10 mm, or greater than about 20 mm, or greater than about 30 mm, or greater than about 40 mm, or greater than about 50 mm, or greater than about 60 mm, or greater than about 70 mm, or greater than about 80 mm, or greater than about 90 mm, or greater than about 100 mm, or greater than about 200 mm, or greater than about 300 mm, or greater than about 400 mm, or greater than about 500 mm, or greater than about 600 mm, or greater than about 700 mm, or greater than about 800 mm, or greater than about 900 mm, or greater than about 1000 mm, or greater than about 2 m, or greater than about 3 m, or greater than about 4 m, or greater than about 5 m.
[0348] In some embodiments, SFS may be applied to fabric having a length of less than about 100 nm, or less than about 200 nm, or less than about 300 nm, or less than about 400 nm, or less than about 500 nm, or less than about 600 nm, or less than about 700 nm, or less than about 800 nm, or less than about 900 nm, or less than about 1000 nm, or less than about 2 pm, or less than about 5 pm, or less than about 10 pm, or less than about 20 pm, or less than about 30 pm, or less than about 40 pm, or less than about 50 pm, or less than about 60 pm, or less than about 70 pm, or less than about 80 pm, or less than about 90 pm, or less than about 100 pm, or less than about 200 pm, or less than about 300 pm, or less than about 400 pm, or less than about 500 pm, or less than about 600 pm, or less than about 700 pm, or less than about 800 pm, or less than about 900 pm, or less than about 1000 pm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9 mm, or less than about 10 mm, or less than about 20 mm, or less than about 30 mm, or less than about 40 mm, or less than about 50 mm, or less than about 60 mm, or less than about 70 mm, or less than about 80 mm, or less than about 90 mm, or less than about 100 mm, or less than about 200 mm, or less than about 300 mm, or less than about 400 mm, or less than about 500 mm, or less than about 600 mm, or less than about 700 mm, or less than about 800 mm, or less than about 900 mm, or less than about 1000 mm.
[0349] In some embodiments, SFS may be applied to fabric having a length of greater than about 100 nm, or greater than about 200 nm, or greater than about 300 nm, or greater than about 400 nm, or greater than about 500 nm, or greater than about 600 nm, or greater than about 700 nm, or greater than about 800 nm, or greater than about 900 nm, or greater than about 1000 nm, or greater than about 2 pm, or greater than about 5 pm, or greater than about 10 pm, or greater than about 20 pm, or greater than about 30 gm, or greater than about 40 gm, or greater than about 50 pm, or greater than about 60 gm, or greater than about 70 gm, or greater than about 80 gm, or greater than about 90 gm, or greater than about 100 gm, or greater than about 200 gm, or greater than about 300 gm, or greater than about 400 gm, or greater than about 500 gm, or greater than about 600 pm, or greater than about 700 gm, or greater than about 800 gm, or greater than about 900 pm, or greater than about 1000 gm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9 mm, or greater than about 10 mm, or greater than about 20 mm, or greater than about 30 mm, or greater than about 40 mm, or greater than about 50 mm, or greater than about 60 mm, or greater than about 70 mm, or greater than about 80 mm, or greater than about 90 mm, or greater than about 100 mm, or greater than about 200 mm, or greater than about 300 mm, or greater than about 400 mm, or greater than about 500 mm, or greater than about 600 mm, or greater than about 700 mm, or greater than about 800 mm, or greater than about 900 mm, or greater than about 1000 mm.
[0350] In some embodiments, SFS may be applied to fabric having a stretch percentage of less than about 1 %, or less than about 2 %, or less than about 3 %, or less than about 4 %, or less than about 5 %, or less than about 6 %, or less than about 7 %, or less than about 8 %, or less than about 9 %, or less than about 10 %, or less than about 20 %, or less than about 30 %, or less than about 40 %, or less than about 50 %, or less than about 60 %, or less than about 70 % , or less than about 80 %, or less than about 90 %, or less than about 100, or less than about 110 %, or less than about 120 %, or less than about 130 %, or less than about 140 %, or less than about 150 %, or less than about 160 %, or less than about 170 %, or less than about 180 %, or less than about 190 %, or less than about 200 %. Stretch percentage may be determined for a fabric having an unstretched width and stretching the fabric to a stretched width, then subtracting the unstretched width from the stretched width to yield the net stretched width, then dividing the net stretched width and multiplying the quotient by 100 to find the stretch percentage (%) (.
[0351] In some embodiments, SFS may be applied to fabric having a stretch percentage of greater than about 1 %, or greater than about 2 %, or greater than about 3 %, or greater than about 4 %, or greater than about 5 %, or greater than about 6 %, or greater than about 7 %, or greater than about 8 %, or greater than about 9 %, or greater than about 10 %, or greater than about 20 %, or greater than about 30 %, or greater than about 40 %, or greater than about 50 %, or greater than about 60 %, or greater than about 70 % , or greater than about 80 %, or greater than about 90 %, or greater than about 100, or greater than about 110 %, or greater than about 120 %, or greater than about 130 %, or greater than about 140 %, or greater than about 150 %, or greater than about 160 %, or greater than about 170 %, or greater than about 180 %, or greater than about 190 %, or greater than about 200 %
[0352] In some embodiments, SFS may be applied to fabric having a tensile energy (N/cm 2 ) of less than about 1 cN/cm 2 , or less than about 2 cN/cm 2 , or less than about 3 cN/cm 2 , or less than about 4 cN/cm 2 , or less than about 5 cN/cm 2 , or less than about 5 cN/cm 2 , or less than about 6 cN/cm 2 , or less than about 7 cN/cm 2 , or less than about 8 cN/cm 2 , or less than about 9 cN/cm 2 , or less than about 10 cN/cm 2 , or less than about 20 cN/cm 2 , or less than about 30 cN/cm 2 , or less than about 40 cN/cm 2 , or less than about 50 cN/cm 2 , or less than about 60 cN/cm 2 , or less than about 70 cN/cm 2 , or less than about 80 cN/cm 2 , or less than about 90 cN/cm 2 , or less than about 100 cN/cm 2 , or less than about 2 N/cm 2 , or less than about 3 N/cm 2 , or less than about 4 N/cm 2 , or less than about 5 N/cm 2 , or less than about 6 N/cm 2 , or less than about 7 N/cm 2 , or less than about 8 N/cm 2 , or less than about 9 N/cm 2 , or less than about 10 N/cm 2 , or less than about 20 N/cm 2 , or less than about 30 N/cm 2 , or less than about 40 N/cm 2 , or less than about 50 N/cm 2 , or less than about 60 N/cm 2 , or less than about 70 N/cm 2 , or less than about 80 N/cm 2 , or less than about 90 N/cm 2 , or less than about 100 N/cm 2 , or less than about 150 N/cm 2 , or less than about 200 N/cm 2 .
[0353] In some embodiments, SFS may be applied to fabric having a tensile energy (N/cm 2 ) of greater than about 1 cN/cm 2 , or greater than about 2 cN/cm 2 , or greater than about 3 cN/cm 2 , or greater than about 4 cN/cm 2 , or greater than about 5 cN/cm 2 , or greater than about 5 cN/cm 2 , or greater than about 6 cN/cm 2 , or greater than about 7 cN/cm 2 , or greater than about 8 cN/cm 2 , or greater than about 9 cN/cm 2 , or greater than about 10 cN/cm 2 , or greater than about 20 cN/cm 2 , or greater than about 30 cN/cm 2 , or greater than about 40 cN/cm 2 , or greater than about 50 cN/cm 2 , or greater than about 60 cN/cm 2 , or greater than about 70 cN/cm 2 , or greater than about 80 cN/cm 2 , or greater than about 90 cN/cm 2 , or greater than about 100 cN/cm 2 , or greater than about 2 N/cm 2 , or greater than about 3 N/cm 2 , or greater than about 4 N/cm 2 , or greater than about 5 N/cm 2 , or greater than about 6 N/cm 2 , or greater than about 7 N/cm 2 , or greater than about 8 N/cm 2 , or greater than about 9 N/cm 2 , or greater than about 10 N/cm 2 , or greater than about 20 N/cm 2 , or greater than about 30 N/cm 2 , or greater than about 40 N/cm 2 , or greater than about 50 N/cm 2 , or greater than about 60 N/cm 2 , or greater than about 70 N/cm 2 , or greater than about 80 N/cm 2 , or greater than about 90 N/cm 2 , or greater than about 100 N/cm 2 , or greater than about 150 N/cm 2 , or greater than about 200 N/cm 2 .
[0354] In some embodiments, SFS may be applied to fabric having a shear rigidity (N/cm- degree) of less than about 1 cN/cm-degree, or less than about 2 cN/cm-degree, or less than about 3 cN/cm-degree, or less than about 4 cN/cm-degree, or less than about 5 cN/cm-degree, or less than about 5 cN/cm-degree, or less than about 6 cN/cm-degree, or less than about 7 cN/cm- degree, or less than about 8 cN/cm-degree, or less than about 9 cN/cm-degree, or less than about 10 cN/cm-degree, or less than about 20 cN/cm-degree, or less than about 30 cN/cm-degree, or less than about 40 cN/cm-degree, or less than about 50 cN/cm-degree, or less than about 60 cN/cm-degree, or less than about 70 cN/cm-degree, or less than about 80 cN/cm-degree, or less than about 90 cN/cm-degree, or less than about 100 cN/cm-degree, or less than about 2 N/cm- degree, or less than about 3 N/cm-degree, or less than about 4 N/cm-degree, or less than about 5 N/cm-degree, or less than about 6 N/cm-degree, or less than about 7 N/cm-degree, or less than about 8 N/cm-degree, or less than about 9 N/cm-degree, or less than about 10 N/cm-degree, or less than about 20 N/cm-degree, or less than about 30 N/cm-degree, or less than about 40 N/cm- degree, or less than about 50 N/cm-degree, or less than about 60 N/cm-degree, or less than about 70 N/cm-degree, or less than about 80 N/cm-degree, or less than about 90 N/cm-degree, or less than about 100 N/cm-degree, or less than about 150 N/cm-degree, or less than about 200 N/cm- degree.
[0355] In some embodiments, SFS may be applied to fabric having a shear rigidity (N/cm- degree) of greater than about 1 cN/cm-degree, or greater than about 2 cN/cm-degree, or greater than about 3 cN/cm-degree, or greater than about 4 cN/cm-degree, or greater than about 5 cN/cm-degree, or greater than about 5 cN/cm-degree, or greater than about 6 cN/cm-degree, or greater than about 7 cN/cm-degree, or greater than about 8 cN/cm-degree, or greater than about 9 cN/cm-degree, or greater than about 10 cN/cm-degree, or greater than about 20 cN/cm-degree, or greater than about 30 cN/cm-degree, or greater than about 40 cN/cm-degree, or greater than about 50 cN/cm-degree, or greater than about 60 cN/cm-degree, or greater than about 70 cN/cm- degree, or greater than about 80 cN/cm-degree, or greater than about 90 cN/cm-degree, or greater than about 100 cN/cm-degree, or greater than about 2 N/cm-degree, or greater than about 3 N/cm-degree, or greater than about 4 N/cm-degree, or greater than about 5 N/cm-degree, or greater than about 6 N/cm-degree, or greater than about 7 N/cm-degree, or greater than about 8 N/cm-degree, or greater than about 9 N/cm-degree, or greater than about 10 N/cm-degree, or greater than about 20 N/cm-degree, or greater than about 30 N/cm-degree, or greater than about 40 N/cm-degree, or greater than about 50 N/cm-degree, or greater than about 60 N/cm-degree, or greater than about 70 N/cm-degree, or greater than about 80 N/cm-degree, or greater than about 90 N/cm-degree, or greater than about 100 N/cm-degree, or greater than about 150 N/cm-degree, or greater than about 200 N/cm-degree.
[0356] In some embodiments, SFS may be applied to fabric having a bending rigidity (N»cm 2 /cm) of less than about 1 cN»cm 2 /cm, or less than about 2 cN»cm 2 /cm, or less than about 3 cN»cm 2 /cm, or less than about 4 cN»cm 2 /cm, or less than about 5 cN»cm 2 /cm, or less than about 5 cN»cm 2 /cm, or less than about 6 cN»cm 2 /cm, or less than about 7 cN»cm 2 /cm, or less than about 8 cN»cm 2 /cm, or less than about 9 cN»cm 2 /cm, or less than about 10 cN»cm 2 /cm, or less than about 20 cN»cm 2 /cm, or less than about 30 cN»cm 2 /cm, or less than about 40 cN»cm 2 /cm, or less than about 50 cN»cm 2 /cm, or less than about 60 cN»cm 2 /cm, or less than about 70 cN»cm 2 /cm, or less than about 80 cN»cm 2 /cm, or less than about 90 cN»cm 2 /cm, or less than about 100 cN»cm 2 /cm, or less than about 2 N»cm 2 /cm, or less than about 3 N»cm 2 /cm, or less than about 4 N»cm 2 /cm, or less than about 5 N»cm 2 /cm, or less than about 6 N»cm 2 /cm, or less than about 7 N»cm 2 /cm, or less than about 8 N»cm 2 /cm, or less than about 9 N»cm 2 /cm, or less than about 10 N»cm 2 /cm, or less than about 20 N»cm 2 /cm, or less than about 30 N»cm 2 /cm, or less than about 40 N»cm 2 /cm, or less than about 50 N»cm 2 /cm, or less than about 60 N»cm 2 /cm, or less than about 70 N»cm 2 /cm, or less than about 80 N»cm 2 /cm, or less than about 90 N»cm 2 /cm, or less than about 100 N»cm 2 /cm, or less than about 150 N»cm 2 /cm, or less than about 200 N*cm 2 /cm.
[0357] In some embodiments, SFS may be applied to fabric having a bending rigidity (N»cm 2 /cm) of greater than about 1 cN»cm 2 /cm, or greater than about 2 cN»cm 2 /cm, or greater than about 3 cN»cm 2 /cm, or greater than about 4 cN»cm 2 /cm, or greater than about 5 cN»cm 2 /cm, or greater than about 5 cN»cm 2 /cm, or greater than about 6 cN»cm 2 /cm, or greater than about 7 cN»cm 2 /cm, or greater than about 8 cN»cm 2 /cm, or greater than about 9 cN»cm 2 /cm, or greater than about 10 cN»cm 2 /cm, or greater than about 20 cN»cm 2 /cm, or greater than about 30 cN»cm 2 /cm, or greater than about 40 cN»cm 2 /cm, or greater than about 50 cN»cm 2 /cm, or greater than about 60 cN»cm 2 /cm, or greater than about 70 cN»cm 2 /cm, or greater than about 80 cN»cm 2 /cm, or greater than about 90 cN»cm 2 /cm, or greater than about 100 cN»cm 2 /cm, or greater than about 2 N»cm 2 /cm, or greater than about 3 N»cm 2 /cm, or greater than about 4 N»cm 2 /cm, or greater than about 5 N»cm 2 /cm, or greater than about 6 N»cm 2 /cm, or greater than about 7 N»cm 2 /cm, or greater than about 8 N»cm 2 /cm, or greater than about 9 N»cm 2 /cm, or greater than about 10 N»cm 2 /cm, or greater than about 20 N»cm 2 /cm, or greater than about 30 N»cm 2 /cm, or greater than about 40 N»cm 2 /cm, or greater than about 50 N»cm 2 /cm, or greater than about 60 N»cm 2 /cm, or greater than about 70 N»cm 2 /cm, or greater than about 80 N»cm 2 /cm, or greater than about 90 N»cm 2 /cm, or greater than about 100 N»cm 2 /cm, or greater than about 150 N*cm 2 /cm, or greater than about 200 N*cm 2 /cm.
[0358] In some embodiments, SFS may be applied to fabric having a compression energy (N»cm/cm 2 ) of less than about 1 cN»cm/cm 2 , or less than about 2 cN»cm/cm 2 , or less than about 3 cN»cm/cm 2 , or less than about 4 cN»cm/cm 2 , or less than about 5 c N»cm/cm 2 , or less than about 5 cN»cm/cm 2 , or less than about 6 cN»cm/cm 2 , or less than about 7 cN»cm/cm 2 , or less than about 8 cN»cm/cm 2 , or less than about 9 cN»cm/cm 2 , or less than about 10 cN»cm/cm 2 , or less than about 20 cN»cm/cm 2 , or less than about 30 cN»cm/cm 2 , or less than about 40 cN»cm/cm 2 , or less than about 50 cN»cm/cm 2 , or less than about 60 cN»cm/cm 2 , or less than about 70 cN»cm/cm 2 , or less than about 80 cN»cm/cm 2 , or less than about 90 cN»cm/cm 2 , or less than about 100 cN»cm/cm 2 , or less than about 2 N»cm/cm 2 , or less than about 3 N»cm/cm 2 , or less than about 4 N»cm/cm 2 , or less than about 5 N»cm/cm 2 , or less than about 6 N»cm/cm 2 , or less than about 7 N»cm/cm 2 , or less than about 8 N»cm/cm 2 , or less than about 9 N»cm/cm 2 , or less than about 10 N»cm/cm 2 , or less than about 20 N»cm/cm 2 , or less than about 30 N»cm/cm 2 , or less than about 40 N»cm/cm 2 , or less than about 50 N»cm/cm 2 , or less than about 60 N»cm/cm 2 , or less than about 70 N»cm/cm 2 , or less than about 80 N»cm/cm 2 , or less than about 90 N»cm/cm 2 , or less than about 100 N»cm/cm 2 , or less than about 150 N»cm/cm 2 , or less than about 200 N»cm/cm 2 .
[0359] In some embodiments, SFS may be applied to fabric having a compression energy (N»cm/cm 2 ) of greater than about 1 cN»cm/cm 2 , or greater than about 2 cN»cm/cm 2 , or greater than about 3 cN»cm/cm 2 , or greater than about 4 cN»cm/cm 2 , or greater than about 5 cN»cm/cm 2 , or greater than about 5 cN»cm/cm 2 , or greater than about 6 cN»cm/cm 2 , or greater than about 7 cN»cm/cm 2 , or greater than about 8 cN»cm/cm 2 , or greater than about 9 cN»cm/cm 2 , or greater than about 10 cN»cm/cm 2 , or greater than about 20 cN»cm/cm 2 , or greater than about 30 cN»cm/cm 2 , or greater than about 40 cN»cm/cm 2 , or greater than about 50 cN»cm/cm 2 , or greater than about 60 cN»cm/cm 2 , or greater than about 70 cN»cm/cm 2 , or greater than about 80 cN»cm/cm 2 , or greater than about 90 cN»cm/cm 2 , or greater than about 100 cN»cm/cm 2 , or greater than about 2 N»cm/cm 2 , or greater than about 3 N»cm/cm 2 , or greater than about 4 N»cm/cm 2 , or greater than about 5 N»cm/cm 2 , or greater than about 6 N»cm/cm 2 , or greater than about 7 N»cm/cm 2 , or greater than about 8 N»cm/cm 2 , or greater than about 9 N»cm/cm 2 , or greater than about 10 N»cm/cm 2 , or greater than about 20 N»cm/cm 2 , or greater than about 30 N»cm/cm 2 , or greater than about 40 N»cm/cm 2 , or greater than about 50 N»cm/cm 2 , or greater than about 60 N»cm/cm 2 , or greater than about 70 N»cm/cm 2 , or greater than about 80 N»cm/cm 2 , or greater than about 90 N»cm/cm 2 , or greater than about 100 N»cm/cm 2 , or greater than about 150 N*cm/cm 2 , or greater than about 200 N*cm/cm 2 .
[0360] In some embodiments, SFS may be applied to fabric having a coefficient of friction of less than about 0.04, or less than about 0.05, or less than about 0.06, or less than about 0.07, or less than about 0.08, or less than about 0.09, or less than about 0.10, or less than about 0.10, or less than about 0.15, or less than about 0.20, or less than about 0.25, or less than about 0.30, or less than about 0.35, or less than about 0.40, or less than about 0.45, or less than about 0.50, or less than about 0.55, or less than about 0.60, or less than about 0.65, or less than about 0.70, or less than about 0.75, or less than about 0.80, or less than about 0.85, or less than about 0.90, or less than about 0.95, or less than about 1.00, or less than about 1.05.
[0361] In some embodiments, SFS may be applied to fabric having a coefficient of friction of greater than about 0.04, or greater than about 0.05, or greater than about 0.06, or greater than about 0.07, or greater than about 0.08, or greater than about 0.09, or greater than about 0.10, or greater than about 0.10, or greater than about 0.15, or greater than about 0.20, or greater than about 0.25, or greater than about 0.30, or greater than about 0.35, or greater than about 0.40, or greater than about 0.45, or greater than about 0.50, or greater than about 0.55, or greater than about 0.60, or greater than about 0.65, or greater than about 0.70, or greater than about 0.75, or greater than about 0.80, or greater than about 0.85, or greater than about 0.90, or greater than about 0.95, or greater than about 1.00, or greater than about 1.05.
[0362] In some embodiments, chemical finishes may be applied to textiles before or after such textiles are coated with SFS. In an embodiment, chemical finishing may be intended as the application of chemical agents and/or SFS to textiles, including fibers, yarn, and fabric, or to garments that are prepared by such fibers, yarn, and fabric to modify the original textile’s or garment’s properties and achieve properties in the textile or garment that would be otherwise absent. With chemical finishes, textiles treated with such chemical finishes may act as surface treatments and/or the treatments may modify the elemental analysis of treated textile base polymers.
[0363] In an embodiment, a type of chemical finishing may include the application of certain silk-fibroin based solutions to textiles. For example, SFS may be applied to a fabric after it is dyed, but there are also scenarios that may require the application of SFS during processing, during dyeing, or after a garment is assembled from a selected textile or fabric, thread, or yarn. In some embodiments, after its application, SFS may be dried with the use of heat. SFS may then be fixed to the surface of the textile in a processing step called curing.
[0364] In some embodiments, SFS may be supplied in a concentrated form suspended in water. In some embodiments, SFS may have a concentration by weight (% w/w or % w/v) or by volume (v/v) of less than about 50 %, or less than about 45%, or less than about 40%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 5%, or less than about 4%, or less than about 3%, or less than about 2%, or less than about 1%, or less than about 0.1%, or less than about 0.01%, or less than about 0.001%, or less than about 0.0001%, or less than about 0.00001%. In some embodiments, SFS may have a concentration by weight (% w/w or % w/v) or by volume (v/v) of greater than about 50 %, or greater than about 45%, or greater than about 40%, or greater than about 35%, or greater than about 30%, or greater than about 25%, or greater than about 20%, or greater than about 15%, or greater than about 10%, or greater than about 5%, or greater than about 4%, or greater than about 3%, or greater than about 2%, or greater than about 1%, or greater than about 0.1%, or greater than about 0.01%, or greater than about 0.001%, or greater than about 0.0001%, or greater than about 0.00001%.
[0365] In some embodiments, the solution concentration and the wet pick of the material determines the amount of silk fibroin solution (SFS), which may include silk-based proteins or fragments thereof, that may be fixed or otherwise adhered to the textile being coated. The wet pick up may be expressed by the following formula: [0367] The total amount of SFS added to the textile material may be expressed by the following formula: 00
[0369] Regarding methods for applying SFS to textiles more broadly, SFS may be applied to textiles through a pad or roller application on process, a saturation and removal process, and/or a topical application process. Moreover, the methods of silk application (i.e., SFS application or coating) may include bath coating, kiss rolling, spray coating, and/or two-sided rolling. In some embodiments, the coating processes (e.g., bath coating, kiss rolling, spray coating, two-sided rolling, roller application, saturation and removal application, and/or topical application), drying processes, and curing processes may be varied as described herein to modify one or more selected textile (e.g., fabric) properties of the resulting coated textile wherein such properties include, but are not limited to wetting time, absorption rate, spreading speed, accumulative oneway transport, and/or overall moisture management capability. In some embodiments, the aforementioned selected properties may be enhanced by varying one or more of the coating processes, drying processes, and curing processes as described herein.
[0370] In an embodiment, the padder application may be used on dry or wet textile. For example, it may be applied on fabric after the dyeing process. The fabric may be fed into a water bath solution and may reach saturation. The fabric to be coated may then pass through a set of rollers that, based on multiple variables, extract the bath solution in excess to the desired wet pick up %. The variables that affect the wet pick up % are the roller pressure and materials, the fabric composition and construction, and the SFS viscosity. The use of any padder roller known in the art is contemplated.
[0371] In an embodiment, the padder application on wet textile may be used to reduce the cost of drying the fabric post dyeing. The fabric exiting the pad rollers may maintain a higher weight % than the incoming fabric to maintain a SFS deposit on the fabric; and the SFS solution may need to account for any dilution taking place due to water present on the incoming fabric.
[0372] In an embodiment, the saturation and removal application is a low wet pick up method that may, for example, solve some of the issues associated with removing large amounts of water during drying processes. Since fabric may dry in an oven from the outside surface towards the inside, water may move from the inside to the outside resulting in a higher coating concentration on the outside surface. With less water content, migration may be reduced due to a higher viscosity in the solution. However, decreased wet pick up may result in an uneven solution deposit.
[0373] In an embodiment, vacuum extraction may be used as a method for low wet pick up. Saturated fabric may be subject to a vacuum that pulls solution out of the fabric and returns it to an application loop. Air jet ejection may be a method for providing low wet pick up. The saturated fabric may be subjected to high pressure steam that removes solution out of the fabric and returns it to an application loop.
[0374] In an embodiment, a porous bowl method may be used for low wet pick up. Solid pad rollers may be substituted with rubber coated fiber rollers. Saturated fabric may be subjected to the pressure of the roller since the porosity of the rollers may allow for more solution to be squeezed from the fabric.
[0375] In an embodiment, a transfer padding method may be used for low wet pick up. Saturated fabric may be passed through two continuous dry non-woven fabrics and may be pressed at low pressure. The non-woven fabrics may extract excess solution from the fabric being treated.
[0376] In an embodiment, topical application may be used as a low wet pick up method of application that deposits the desired amount of SFS to the fabric without removing any excess material. The methods described above may be used for one-sided coating applications, but there are variations that may allow for two-sided coating.
[0377] In an embodiment, kiss rolling may be used as a topical method of application that transfers the SFS from a roller (i.e., a kiss roller) to one side of the fabric. The solution viscosity, roller surface finish, speed of the roller, speed of the fabric, contact angle of the fabric on the roller and properties of the fabric are parameters that control the amount of solution deposited on the fabric. The use of any kiss roller known in the art is contemplated.
[0378] In an embodiment, a variation to the kiss roller technique may be the Triatex MA system that uses two moisture content sensors to determine the solution pick up at the kiss roller and adjust the kiss roller controllable variable to maintain consistent the solution deposit onto the fabric.
[0379] In an embodiment, a loop transfer application may be used as a topical method of application that transfers the SFS from a saturated loop fabric to the fabric to be coated between low pressure pad rollers. There is a two rollers version that may allow for minimum contact with the fabric and a three rollers version that allows for greater contact with the fabric. [0380] In an embodiment, an engrave roller application may be used as a topical method of application that may transfer a metered amount of SFS onto the fabric. This may be achieved by engraving a pattern on the surface of the roller with precise depth and design that contains a controlled amount of SFS. A blade may be used to remove any solution that is deposited on the surface of the roller in order to maintain a consistent transfer of solution to the fabric to be coated.
[0381] In an embodiment, rotary screen printing may be used as a topical method of application that may deposit SFS onto the fabric by seeping the solution through a roller screen. The solution may be contained in the screen print roller core at a set level while a blade may be used to remove any excess solution from the interior roller wall, providing a clean surface for the next revolution of the screen printer roller.
[0382] In an embodiment, magnetic roller coating may be used as a topical method of application that may deposit SFS from a kiss roller onto the fabric to be coated. The kiss roller is semi-submersed in a bath solution while a magnetic field created in the fabric driving roller determines the amount of pressure applied by the kiss roller, controlling the solution pick up rate. [0383] In an embodiment, spraying may be used as a topical method of application that may transfer SFS onto the fabric by nebulizing the solution. The spray pattern may be controlled by the nozzle pattern, size, and the air flow. Spray application may be used for one side application or also two sided application.
[0384] In an embodiment, foam application may be used a topical method of application that may transfer SFS onto the fabric. Foam may be made by substituting part of the water in the solution with air therefore reducing the amount of water to be applied to the fabric. Foam application may be used for one-sided application or two-sided application where the same foam may be deposited through a squeeze roller or different foam solutions may be provided through transfer rolls or through a slot applicator.
[0385] In an embodiment, the application of SFS may take place after a garment is assembled. In an embodiment, the process may take place in a washing and dyeing machine or in a spray booth. For example, a washing and dyeing machine may be similar in shape to a household front loader washing machine, it allows the process to take place at exhaustion post dyeing or with an independent processing cycle. In an embodiment, a spray booth machine may include a manual or a fully automated process. For example, a garment may be held by a mannequin while an operator or an anthropomorphic robot may spray the solution onto the fabric.
[0386] In an embodiment, SFS may be a water based solution that, after its application to the textile, may require thermal vaporization to infuse the SFS onto the textile. Thermal vaporization may be applied by heat transfer through radiation with equipment such as infrared or radio frequency dryer.
[0387] In an embodiment, thermal vaporization may be applied by convection through heated air circulating in an oven to the required temperature, while the fabric is clamped and is transported by a conveyor. This allows full control on fabric width dimension.
[0388] In an embodiment, thermal vaporization may be applied by conduction through contacting the textile with heated cylinder or calendar cylinder. Since the fabric is not clamp there is minimal control on fabric width.
[0389] In an embodiment, curing of the SFS on the textile may be completed with the same equipment used for the thermal vaporization in a continuous cycle or in a separate cycle.
[0390] In an embodiment, curing time temperature may be dependent the textile polymer content and the binding method of preference for the SFS with the specific polymer. The curing process may not start until the thermal vaporization is completed.
[0391] In some embodiments, sensor may be used to monitor SFS deposition on the textile and the drying and curing steps.
[0392] In some embodiments, for monitoring the deposition of SFS, a contactless sensor, like the one supplied by Pleva model AF120 based on microwave absorption of water, may be used. Measurement of the material moisture may be based on microwave absorption by water. A semiconductor oscillator transmits microwave energy through the web. The non-absorbed part of the energy may be received on the opposite side by a microwave receiver. The amount of absorption is a measurement of the absolute moisture content. The microwave sensor is capable of detecting and measuring water content from a minimum of 0 up to 2000 g FhO/m 2 .
[0393] In some embodiments, for wide fabric processing multiple sensor may be paired side by side, delivering the data analysis to a centralized control system loop capable to add more solution in the area of the fabric that is low.
[0394] In some embodiments, another sensor may be used that is based on microwave technology, such as Aqualot by Mahlo. The sensor may evaluate the shift in the resonant frequency of the two standing waves with respect to each other rather than the attenuation of the microwaves by the quantity of water molecules in the measuring gap.
[0395] In some embodiments, another contactless sensor for SFS may be the IR-3000 by MoistTech based on near infrared sensing technology. The sensor measures the amount of near infrared energy reflected at a given wavelength that is inversely proportional to the quantity of absorbing molecules in the fabric.
[0396] In some embodiments, the residual moister at the end of the curing process may be measured to further confirm the drying and curing process. In addition to the above sensor, a contact sensor such as the Textometer RMS by Mahlo may be used for measuring moister through conductivity.
[0397] In some embodiments, monitoring the end of the drying process phase may be achieved by measuring the fabric temperature with a contactless temperature sensor. When wet product enters the dryer, it first heats up to the cooling limit temperature. In some embodiments, when the water content drops to residual moisture levels, the product temperature may begin to rise again. The closer the product temperature approaches the circulation air temperature in the dryer, the slower the temperature continues to rise. In some embodiments, at a certain temperature threshold (called the fixing temperature) the temperature necessary for processing, fixing, or condensing is reached.
[0398] In some embodiments, to determine the dwell time for a desired product temperature, the surface temperature of the product may be measured without contact at several locations in the dryer using high-temperature resistant infrared pyrometers. Mahlo Permaset VMT is an infrarem Pyrometer that may be assembled in multiple units to monitors temperature through the dryer. Setex is another manufacturer offering fabric temperature sensors for use in dryers and oven like the models WTM VI 1, V21, and V41.
[0399] In some embodiments, SFS may be applied to a textile during exhaust dyeing. In some embodiments, the process may involve loading fabric into a bath, originally known as a batch, and allowing it to come into equilibrium with the solution. Exhaust dyeing may be the ability of the silk fibroin molecules to move from the solution onto the fibers or thread of a textile (substantivity). The substantivity of the silk fibroin may be influenced by temperature or additives, such as salt. [0400] In some embodiments, an exhaust dyeing process may take anywhere from a few minutes to a few hours. When the fabric has been absorbed, or fixed, as much silk fibroin as it can, the bath may be emptied and the fabric may be rinsed to remove any excess solution.
[0401] In some embodiments, an important parameter in exhaust dyeing may be what is known as the specific liquor ratio. This describes the ratio of the mass of the fabric to the volume of the SFS bath and determines the amount of silk fibroin deposited on a textile.
[0402] In some embodiments, SFS can be applied to a textile during jet dyeing processes. A jet dyeing machine may formed by closed tubular system where the fabric is placed. For transporting the fabric through the tube, a jet of dye liquor is supplied through a venturi. The jet may create turbulence. This may help in SFS penetration along with preventing the fabric from touching the walls of the tube. For example, as the fabric is often exposed to comparatively higher concentrations of liquor within the transport tube, a small SFS bath is needed in the bottom of the vessel. This arrangement may be enough for the smooth movement from rear to front of the vessel.
[0403] In some embodiments, SFS may be applied during Paddle dyeing. Paddle dyeing machines may be generally used to many forms of textiles but the method best suits to garments. Heat may be generated through steam injection directly into the coating bath. In an embodiment, a paddle dyeing machine operates through a paddle that circulates both the bath and garments in a perforated central island. It is here that the SFS, water, and steam for heat are added. The overhead paddle machine may be described as a vat with a paddle that has blades of full width. The blades may generally dip a few centimeters into the vat. This action may stir the bath and push garments to be died down, thus keeping them submerged in the dye liquor.
[0404] In some embodiments, the processing methods set forth herein may be used to apply SFS to textiles with one or more of the following parameters including, but not limited to, fabric speed, solution viscosity, solution added to fabric, fabric range width, drying temperature, drying time, curing time, fabric tension, padder pressure, padder roller shore hardness, stenter temperature, and common drying and curing temperatures. In an embodiment, the processing method parameters may also include a condensation temperature, which may vary depending upon the chemical recipe used to apply the SFS to the textiles.
[0405] In an embodiment, the fabric speed for the processes of the invention may be less than about 0.1 m/min, or less than about 0.2 m/min, or less than about 0.3 m/min, or less than about 0.4 m/min, or less than about 0.5 m/min, or less than about 0.6 m/min, or less than about 0.7 m/min, or less than about 0.8 m/min, or less than about 0.9 m/min, or less than about 1 m/min, or less than about 2 m/min, or less than about 3 m/min, or less than about 4 m/min, or less than about 5 m/min, or less than about 6 m/min, or less than about 7 m/min, or less than about 8 m/min, or less than about 9 m/min, or less than about 10 m/min, or less than about 20 m/min, or less than about 30 m/min, or less than about 40 m/min, or less than about 50 m/min, or less than about 60 m/min.
[0406] In an embodiment, the fabric speed for the processes of the invention may be greater than about 0.1 m/min, or greater than about 0.2 m/min, or greater than about 0.3 m/min, or greater than about 0.4 m/min, or greater than about 0.5 m/min, or greater than about 0.6 m/min, or greater than about 0.7 m/min, or greater than about 0.8 m/min, or greater than about 0.9 m/min, or greater than about 1 m/min, or greater than about 2 m/min, or greater than about 3 m/min, or greater than about 4 m/min, or greater than about 5 m/min, or greater than about 6 m/min, or greater than about 7 m/min, or greater than about 8 m/min, or greater than about 9 m/min, or greater than about 10 m/min, or greater than about 20 m/min, or greater than about 30 m/min, or greater than about 40 m/min, or greater than about 50 m/min, or greater than about 60 m/min. [0407] In an embodiment, the solution viscosity for the processes of the invention may be less than about 1000 mPas, or less than about 1500 mPas, or less than about 2000 mPas, or less than about 2500, or less than about 3000 mPas, or less than about 4000 mPas, or less than about 4500 mPas, or less than about 5000 mPas, or less than about 5500 mPas, or less than about 6000 mPas, or less than about 6500 mPas, or less than about 7000 mPas, or less than about 7500 mPas, or less than about 8000 mPas, or less than about 8500 mPas, or less than about 9000 mPas, or less than about 9500 mPas, or less than about 10000 mPas, or less than about 10500 mPas, or less than about 11000 mPas, or less than about 11500 mPas, or less than about 12000 mPas.
[0408] In an embodiment, the solution viscosity for the processes of the invention may be greater than about 1000 mPas, or greater than about 1500 mPas, or greater than about 2000 mPas, or greater than about 2500, or greater than about 3000 mPas, or greater than about 4000 mPas, or greater than about 4500 mPas, or greater than about 5000 mPas, or greater than about 5500 mPas, or greater than about 6000 mPas, or greater than about 6500 mPas, or greater than about 7000 mPas, or greater than about 7500 mPas, or greater than about 8000 mPas, or greater than about 8500 mPas, or greater than about 9000 mPas, or greater than about 9500 mPas, or greater than about 10000 mPas, or greater than about 10500 mPas, or greater than about 11000 mPas, or greater than about 11500 mPas, or greater than about 12000 mPas.
[0409] In an embodiment, the solution may be added to a textile (e.g., fabric) for the processes of the invention in less than about 0.01 g/m 2 , or less than about 0.02 g/m 2 , or less than about 0.03 g/m 2 , or less than about 0.04 g/m 2 , or less than about 0.05 g/m 2 , or less than about 0.06 g/m 2 , or less than about 0.07 g/m 2 , or less than about 0.08 g/m 2 , or less than about 0.09 g/m 2 , or less than about 0.10 g/m 2 , or less than about 0.2 g/m 2 , or less than about 0.3 g/m 2 , or less than about 0.4 g/m 2 , or less than about 0.5 g/m 2 , or less than about 0.6 g/m 2 , or less than about 0.7 g/m 2 , or less than about 0.8 g/m 2 , or less than about 0.9 g/m 2 , or less than about 1 g/m 2 , or less than about 2 g/m 2 , or less than about 3 g/m 2 , or less than about 4 g/m 2 , or less than about 5 g/m 2 , or less than about 6 g/m 2 , or less than about 7 g/m 2 , or less than about 8 g/m 2 , or less than about 9 g/m 2 , or less than about 10 g/m 2 , or less than about 20 g/m 2 , or less than about 30 g/m 2 , or less than about 40 g/m 2 , or less than about 50 g/m 2 , or less than about 60 g/m 2 , or less than about 70 g/m 2 , or less than about 80 g/m 2 , or less than about 90 g/m 2 , or less than about 100 g/m 2 .
[0410] In an embodiment, the solution may be added to a textile (e.g., fabric) for the processes of the invention in greater than about 0.01 g/m 2 , or greater than about 0.02 g/m 2 , or greater than about 0.03 g/m 2 , or greater than about 0.04 g/m 2 , or greater than about 0.05 g/m 2 , or greater than about 0.06 g/m 2 , or greater than about 0.07 g/m 2 , or greater than about 0.08 g/m 2 , or greater than about 0.09 g/m 2 , or greater than about 0.10 g/m 2 , or greater than about 0.2 g/m 2 , or greater than about 0.3 g/m 2 , or greater than about 0.4 g/m 2 , or greater than about 0.5 g/m 2 , or greater than about 0.6 g/m 2 , or greater than about 0.7 g/m 2 , or greater than about 0.8 g/m 2 , or greater than about 0.9 g/m 2 , or greater than about 1 g/m 2 , or greater than about 2 g/m 2 , or greater than about 3 g/m 2 , or greater than about 4 g/m 2 , or greater than about 5 g/m 2 , or greater than about 6 g/m 2 , or greater than about 7 g/m 2 , or greater than about 8 g/m 2 , or greater than about 9 g/m 2 , or greater than about 10 g/m 2 , or greater than about 20 g/m 2 , or greater than about 30 g/m 2 , or greater than about 40 g/m 2 , or greater than about 50 g/m 2 , or greater than about 60 g/m 2 , or greater than about 70 g/m 2 , or greater than about 80 g/m 2 , or greater than about 90 g/m 2 , or greater than about 100 g/m 2 .
[0411] In an embodiment, the fabric range width for the processes of the invention may be less than about 1 mm, or less than about 2 mm, or less than about 3 mm, or less than about 4 mm, or less than about 5 mm, or less than about 6 mm, or less than about 7 mm, or less than about 8 mm, or less than about 9, or less than about 10 mm, or less than about 20 mm, or less than about 30 mm, or less than about 40 mm, or less than about 50 mm, or less than about 60 mm, or less than about 70 mm, or less than about 80 mm, or less than about 90 mm, or less than about 100 mm, or less than about 200, or less than about 300 mm, or less than about 400 mm, or less than about 500 mm, or less than about 600 mm, or less than about 700 mm, or less than about 800 mm, or less than about 900 mm, or less than about 1000 mm, or less than about 2000 mm, or less than about 2000 mm, or less than about 3000 mm, or less than about 4000 mm, or less than about 5000 mm.
[0412] In an embodiment, the fabric range width for the processes of the invention may be greater than about 1 mm, or greater than about 2 mm, or greater than about 3 mm, or greater than about 4 mm, or greater than about 5 mm, or greater than about 6 mm, or greater than about 7 mm, or greater than about 8 mm, or greater than about 9, or greater than about 10 mm, or greater than about 20 mm, or greater than about 30 mm, or greater than about 40 mm, or greater than about 50 mm, or greater than about 60 mm, or greater than about 70 mm, or greater than about 80 mm, or greater than about 90 mm, or greater than about 100 mm, or greater than about 200, or greater than about 300 mm, or greater than about 400 mm, or greater than about 500 mm, or greater than about 600 mm, or greater than about 700 mm, or greater than about 800 mm, or greater than about 900 mm, or greater than about 1000 mm, or greater than about 2000 mm, or greater than about 2000 mm, or greater than about 3000 mm, or greater than about 4000 mm, or greater than about 5000 mm.
[0413] In an embodiment, the drying and/or curing temperature for the processes of the invention may be less than about 70 °C, or less than about 75 °C, or less than about 80 °C, or less than about 85 °C, or less than about 90 °C, or less than about 95 °C, or less than about 100 °C, or less than about 110 °C, or less than about 120 °C, or less than about 130 °C, or less than about 140 °C, or less than about 150 °C, or less than about 160 °C, or less than about 170 °C, or less than about 180 °C, or less than about 190 °C, or less than about 200 °C, or less than about 210 °C, or less than about 220 °C, or less than about 230 °C.
[0414] In an embodiment, the drying and/or curing temperature for the processes of the invention may be greater than about 70 °C, or greater than about 75 °C, or greater than about 80 °C, or greater than about 85 °C, or greater than about 90 °C, or greater than about 95 °C, or greater than about 100 °C, or greater than about 110 °C, or greater than about 120 °C, or greater than about 130 °C, or greater than about 140 °C, or greater than about 150 °C, or greater than about 160 °C, or greater than about 170 °C, or greater than about 180 °C, or greater than about 190 °C, or greater than about 200 °C, or greater than about 210 °C, or greater than about 220 °C, or greater than about 230 °C.
[0415] In an embodiment, the drying time for the processes of the invention may be less than about 10 seconds, or less than about 20 seconds, or less than about 30 seconds, or less than about 40 seconds, or less than about 50 seconds, or less than about 60 seconds, or less than about 2 minutes, or less than about, 3 minutes, or less than about 4 minutes, or less than about 5 minutes, or less than about 6 minutes, or less than about 7 minutes, or less than about 8 minutes, or less than about 9 minutes, or less than about 10 minutes, or less than about 20 minutes, or less than about 30 minutes, or less than about 40 minutes, or less than about 50 minutes, or less than about 60 minutes.
[0416] In an embodiment, the drying time for the processes of the invention may be greater than about 10 seconds, or greater than about 20 seconds, or greater than about 30 seconds, or greater than about 40 seconds, or greater than about 50 seconds, or greater than about 60 seconds, or greater than about 2 minutes, or greater than about, 3 minutes, or greater than about 4 minutes, or greater than about 5 minutes, or greater than about 6 minutes, or greater than about 7 minutes, or greater than about 8 minutes, or greater than about 9 minutes, or greater than about 10 minutes, or greater than about 20 minutes, or greater than about 30 minutes, or greater than about 40 minutes, or greater than about 50 minutes, or greater than about 60 minutes.
[0417] In an embodiment, the curing time for the processes of the invention may be less than about 1 second, or less than about 2 seconds, or less than about 3 seconds, or less than about 4 seconds, or less than about 5 seconds, or less than about 6 seconds, or less than about 7 seconds, or less than about 8 seconds, or less than about 9 seconds, or less than about 10 seconds, or less than about 20 seconds, or less than about 30 seconds, or less than about 40 seconds, or less than about 50 seconds, or less than about 60 seconds, or less than about 2 minutes, or less than about 3 minutes, or less than about 4 minutes, or less than about 5 minutes, or less than about 6 minutes, or less than about 7 minutes, or less than about 8 minutes, or less than about 9 minutes, or less than about 10 minutes, or less than about 20 minutes, or less than about 30 minutes, or less than about 40 minutes, or less than about 50 minutes, or less than about 60 minutes. [0418] In an embodiment, the curing time for the processes of the invention may be greater than about 1 second, or greater than about 2 seconds, or greater than about 3 seconds, or greater than about 4 seconds, or greater than about 5 seconds, or greater than about 6 seconds, or greater than about 7 seconds, or greater than about 8 seconds, or greater than about 9 seconds, or greater than about 10 seconds, or greater than about 20 seconds, or greater than about 30 seconds, or greater than about 40 seconds, or greater than about 50 seconds, or greater than about 60 seconds, or greater than about 2 minutes, or greater than about 3 minutes, or greater than about 4 minutes, or greater than about 5 minutes, or greater than about 6 minutes, or greater than about 7 minutes, or greater than about 8 minutes, or greater than about 9 minutes, or greater than about 10 minutes, or greater than about 20 minutes, or greater than about 30 minutes, or greater than about 40 minutes, or greater than about 50 minutes, or greater than about 60 minutes.
[0419] In an embodiment, the fabric tension for the processes of the invention may be less than about 1 N, or less than about 2 N, or less than about 3 N, or less than about 4 N, or less than about 5 N, or less than about 6 N, or less than about 7 N, or less than about 8 N, or less than about 9 N, or less than about 10 N, or less than about 20 N, or less than about 30 N, or less than about 40 N, or less than about 50 N, or less than about 60 N, or less than about 70 N, or less than about 80 N, or less than about 90 N, or less than about 100 N, or less than about 150 N, or less than about 200 N, or less than about 250 N, or less than about 300 N.
[0420] In an embodiment, the fabric tension for the processes of the invention may be greater than about 1 N, or greater than about 2 N, or greater than about 3 N, or greater than about 4 N, or greater than about 5 N, or greater than about 6 N, or greater than about 7 N, or greater than about 8 N, or greater than about 9 N, or greater than about 10 N, or greater than about 20 N, or greater than about 30 N, or greater than about 40 N, or greater than about 50 N, or greater than about 60 N, or greater than about 70 N, or greater than about 80 N, or greater than about 90 N, or greater than about 100 N, or greater than about 150 N, or greater than about 200 N, or greater than about 250 N, or greater than about 300 N.
[0421] In an embodiment, the padder pressure for the processes of the invention may be less than about 1 N/mm, or less than about 2 N/mm, or less than about 3 N/mm, or less than about 4 N/mm, or less than about 4 N/mm, or less than about 5 N/mm, or less than about 6 N/mm, or less than about 7 N/mm, or less than about 8 N/mm, or less than about 9 N/mm, or less than about 10 N/mm, or less than about 20 N/mm, or less than about 30 N/mm, or less than about 40 N/mm, or less than about 50 N/mm, or less than about 60 N/mm, or less than about 70 N/mm, or less than about 80 N/mm, or less than about 90 N/mm.
[0422] In an embodiment, the padder pressure for the processes of the invention may be greater than about 1 N/mm, or greater than about 2 N/mm, or greater than about 3 N/mm, or greater than about 4 N/mm, or greater than about 4 N/mm, or greater than about 5 N/mm, or greater than about 6 N/mm, or greater than about 7 N/mm, or greater than about 8 N/mm, or greater than about 9 N/mm, or greater than about 10 N/mm, or greater than about 20 N/mm, or greater than about 30 N/mm, or greater than about 40 N/mm, or greater than about 50 N/mm, or greater than about 60 N/mm, or greater than about 70 N/mm, or greater than about 80 N/mm, or greater than about 90 N/mm.
[0423] In an embodiment, the padder roller shore hardness for the processes of the invention may be less than about 70 shore A, or less than about 75 shore A, or less than about 80 shore A, or less than about 85 shore A, or less than about 90 shore A, or less than about 95 shore A, or less than about 100 shore A.
[0424] In an embodiment, the padder roller shore hardness for the processes of the invention may be greater than about 70 shore A, or greater than about 75 shore A, or greater than about 80 shore A, or greater than about 85 shore A, or greater than about 90 shore A, or greater than about 95 shore A, or greater than about 100 shore A.
[0425] In an embodiment, the stenter temperature for the processes of the invention may be less than about 70 °C, or less than about 75 °C, or less than about 80 °C, or less than about 85 °C, or less than about 90 °C, or less than about 95 °C, or less than about 100 °C, or less than about 110 °C, or less than about 120 °C, or less than about 130 °C, or less than about 140 °C, or less than about 150 °C, or less than about 160 °C, or less than about 170 °C, or less than about 180 °C, or less than about 190 °C, or less than about 200 °C, or less than about 210 °C, or less than about 220 °C, or less than about 230 °C.
[0426] In an embodiment, the stenter temperature for the processes of the invention may be greater than about 70 °C, or greater than about 75 °C, or greater than about 80 °C, or greater than about 85 °C, or greater than about 90 °C, or greater than about 95 °C, or greater than about 100 °C, or greater than about 110 °C, or greater than about 120 °C, or greater than about 130 °C, or greater than about 140 °C, or greater than about 150 °C, or greater than about 160 °C, or greater than about 170 °C, or greater than about 180 °C, or greater than about 190 °C, or greater than about 200 °C, or greater than about 210 °C, or greater than about 220 °C, or greater than about 230 °C.
[0427] In an embodiment, the common drying temperatures for the processes of the invention may be less than about 110 °C, or less than about 115 °C, or less than about 120 °C, or less than about 125 °C, or less than about 130 °C, or less than about 135 °C, or less than about 140 °C, or less than about 145 °C, or less than about 150 °C.
[0428] In an embodiment, the common drying temperatures for the processes of the invention may be greater than about 110 °C, or greater than about 115 °C, or greater than about 120 °C, or greater than about 125 °C, or greater than about 130 °C, or greater than about 135 °C, or greater than about 140 °C, or greater than about 145 °C, or greater than about 150 °C.
[0429] In some embodiments, a silk fibroin coated material (e.g., fabric) may be heat resistant to a selected temperature where the selected temperature is chosen for drying, curing, and/or heat setting a dye that may be applied to the material (e.g., LYCRA). As used herein, a “heat resistant” may refer to a property of the silk fibroin coating deposited on the material where the silk fibroin coating and/or silk fibroin protein does not exhibit a substantial modification (i.e., “substantially modifying”) in silk fibroin coating performance as compared to a control material having a comparable silk fibroin coating that was not subjected to the selected temperature for drying, curing, wash cycling, and/or heat setting purposes. In some embodiments, the selected temperature is the glass transition temperature (Tg) for the material upon which the silk fibroin coating is applied. In some embodiments, the selected temperature is greater than about 65 °, or greater than about 70 °C, or greater than about 80 °C, or greater than about 90 °C, or greater than about 100 °C, or greater than about 110 °C, or greater than about 120 °C, or greater than about 130 °C, or greater than about 140 °C, or greater than about 150 °C, or greater than about 160 °C, or greater than about 170 °C, or greater than about 180 °C, or greater than about 190 °C, or greater than about 200 °C, or greater than about 210 °C, or greater than about 220 °C. In some embodiments, the selected temperature is less than about 65 °C, or less than about 70 °C, or less than about 80 °C, or less than about 90 °C, or less than about 100 °C, or less than about 110 °C, or less than about 120 °C, or less than about 130 °C, or less than about 140 °C, or less than about 150 °C, or less than about 160 °C, or less than about 170 °C, or less than about 180 °C, or less than about 190 °C, or less than about 200 °C, or less than about 210 °C, or less than about 220
O, [0430] In an embodiment, “substantially modifying” silk fibroin coating performance may be a decrease in a selected property of silk fibroin coating, such as wetting time, absorption rate, spreading speed, accumulative one-way transport, or overall moisture management capability as compared to a control silk fibroin coating that was not subjected to the selected temperature for drying, curing, wash cycling, and/or heat setting purposes, where such decrease is less than about a 1% decrease, or less than about a 2 % decrease, or less than about a 3 % decrease, or less than about a 4 % decrease, or less than about a 5 % decrease, or less than about a 6 % decrease, or less than about a 7 % decrease, or less than about an 8 % decrease, or less than about a 9 % decrease, or less than about a 10 % decrease, or less than about a 15 % decrease, or less than about a 20 % decrease, or less than about a 25 % decrease, or less than about a 30 % decrease, or less than about a 35 % decrease, or less than about a 40 % decrease, or less than about a 45 % decrease, or less than about a 50 % decrease, or less than about a 60% decrease, or less than about a 70 % decrease, or less than about a 80 % decrease, or less than about a 90 % decrease, or less than about 100 % decrease in wetting time, absorption rate, spreading speed, accumulative one-way transport, or overall moisture management capability as compared to a control silk fibroin coating that was not subjected to the selected temperature for drying, curing, wash cycling, and/or heat setting purposes. In some embodiments, “wash cycling” may refer to at least one wash cycle, or at least two wash cycles, or at least three wash cycles, or at least four wash cycles, or at least five wash cycles.
[0431] In an embodiment, “substantially modifying” silk fibroin coating performance may be an increase in a selected property of silk fibroin coating, such as wetting time, absorption rate, spreading speed, accumulative one-way transport, or overall moisture management capability as compared to a control silk fibroin coating that was not subjected to the selected temperature for drying, curing, wash cycling, and/or heat setting purposes, where such increase is less than about a 1% increase, or less than about a 2 % increase, or less than about a 3 % increase, or less than about a 4 % increase, or less than about a 5 % increase, or less than about a 6 % increase, or less than about a 7 % increase, or less than about an 8 % increase, or less than about a 9 % increase, or less than about a 10 % increase, or less than about a 15 % increase, or less than about a 20 % increase, or less than about a 25 % increase, or less than about a 30 % increase, or less than about a 35 % increase, or less than about a 40 % increase, or less than about a 45 % increase, or less than about a 50 % increase, or less than about a 60% increase, or less than about a 70 % increase, or less than about a 80 % increase, or less than about a 90 % increase, or less than about 100 % increase in wetting time, absorption rate, spreading speed, accumulative one-way transport, or overall moisture management capability as compared to a control silk fibroin coating that was not subjected to the selected temperature for drying, curing, wash cycling, and/or heat setting purposes. In some embodiments, “wash cycling” may refer to at least one wash cycle, or at least two wash cycles, or at least three wash cycles, or at least four wash cycles, or at least five wash cycles.
[0432] In some embodiments, the SFS coated article may be subjected to heat setting in order to set one or more dyes that may be applied to the SFS coated article in order to permanently set the one or more dyes on the SFS coated article. In some embodiments, the SFS coated article may be heat setting resistant, wherein the SFS coating on the SFS coated article may resist a heat setting temperature of greater than about 100 °C, or greater than about 110 °C, or greater than about 120 °C, or greater than about 130 °C, or greater than about 140 °C, or greater than about 150 °C, or greater than about 160 °C, or greater than about 170 °C, or greater than about 180 °C, or greater than about 190 °C, or greater than about 200 °C, or greater than about 210 °C, or greater than about 220 °C. In some embodiments, the selected temperature is less than about 100 °C, or less than about 110 °C, or less than about 120 °C, or less than about 130 °C, or less than about 140 °C, or less than about 150 °C, or less than about 160 °C, or less than about 170 °C, or less than about 180 °C, or less than about 190 °C, or less than about 200 °C, or less than about 210 °C, or less than about 220 °C.
[0433] In an embodiment, a material coated by the silk fibroin coating as described herein may partially dissolved or otherwise partially incorporated within a portion of the material after the silk fibroin coated material is subjected to heating and/or curing as described herein. Without being limited to any one theory of the invention, where the silk fibroin coated material is heated to greater than about the glass transition temperature (Tg) for the material that is coated, the silk fibroin coating may become partially dissolved or otherwise partially incorporated within a portion of the material.
[0434] In some embodiments, a material coated by the silk fibroin coating as described herein may be sterile or may be sterilized to provide a sterilized silk fibroin coated material. Alternatively, or in addition thereto, the methods described herein may include a sterile SFS prepared from sterile silk fibroin. [0435] In some embodiments, the fabric constructions that are compatible with the processes of the invention include woven fabrics, knitted fabrics, and non-woven fabrics.
[0436] In some embodiments, the coating pattern provided by the processes of the invention include one side coating, two side coating, and/or throughout coating.
[0437] In some embodiments, the equipment manufacturers that are capable of producing equipment configured to continuously coat SFS on textiles include, but are not limited to, Aigle, Amba Projex, Bombi, Bruckner, Cavitec, Crosta, Dienes Apparatebau, Eastsign, Europlasma, Fermor, Fontanet, Gaston Systems, Hansa Mixer, Harish, Has Group, Icomatex, Idealtech, Interspare, Isotex, Klieverik, KTP, M P, Mageba, Mahr Feinpruef, Matex, Mathis, Menzel LP, Meyer, Monforts, Morrison Textile, Mtex, Muller Frick, Muratex Textile, Reliant Machinery, Rollmac, Salvade, Sandvik Tps, Santex, Chmitt-Machinen, Schott & Meissner, Sellers, Sicam, Siltex, Starlinger, Swatik Group India, Techfull, TMT Manenti, Unitech Textile Machinery, Weko, Willy, Wumag Texroll, Yamuna, Zappa, and Zimmer Austria.
[0438] In some embodiments, the equipment manufactures that are capable of producing equipment configured to dry SFS coated on textiles include, but are not limited to, Aiea, Alkan Makina, Anglada, Atac Makina, Bianco, Bruckner, Campen, CHTC, CTMTC, Dilmenler, Elteksmak, Erbatech, Fontanet, Harish, Icomatex, Ilsung, Inspiron, Interspare, Master, Mathis, Monfongs, Monforts, Salvade, Schmitt-Maschinen, Sellers, Sicam, Siltex, Swastik Group India, Tacome, Tubetex, Turbang, Unitech Textile Machinery, and Yamuna.
[0439] The following clauses describe certain embodiments.
[0440] Clause 1. An article comprising a fabric and a coating, wherein the coating comprises a reducing agent and silk fibroin fragments having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, from between about 5 kDa and about 10 kDa, from between about 6 kDa and about 17 kDa, from between about 10 kDa and about 15 kDa, from between about 14 kDa and about 30 kDa, from between about 15 kDa and about 20 kDa, from between about 17 kDa and about 39 kDa, from between about 20 kDa and about 25 kDa, from between about 25 kDa and about 30 kDa, from between about 30 kDa and about 35 kDa, from between about 35 kDa and about 40 kDa, from between about 39 kDa and about 54 kDa, from between about 39 kDa and about 80 kDa, from between about 40 kDa and about 45 kDa, from between about 45 kDa and about 50 kDa, from between about 50 kDa and about 55 kDa, from between about 55 kDa and about 60 kDa, from between about 60 kDa and about 100 kDa, or from between about 80 kDa and about 144 kDa, and a poly dispersity selected from between 1 and about 5.
[0441] Clause 2. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between 1 and about 1.5.
[0442] Clause 3. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 1.5 and about 3.0.
[0443] Clause 4. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 1.5 and about 2.
[0444] Clause 5. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 2 and about 2.5.
[0445] Clause 6. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 2.5 and about 3.
[0446] Clause 7. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 3 and about 3.5 or from between about 3.5 and about 4.0.
[0447] Clause 8. The article of clause 1, wherein the silk fibroin fragments have a poly dispersity selected from between about 4.0 and about 4.5 or from between about 4.5 and about 5.0.
[0448] Clause 9. The article of any one of clauses 1 to 8, wherein the silk fibroin fragments comprise one or more of low molecular weight silk fibroin fragments and medium molecular weight silk fibroin fragments.
[0449] Clause 10. The article of any one of clauses 1 to 9, further comprising about 0.01% (w/w) to about 10% (w/w) sericin relative to the silk fibroin fragments.
[0450] Clause 11. The article of any one of clauses 1 to 9, further comprising about 0.01% (w/w) to about 10% (w/w) sericin relative to total weight of the article.
[0451] Clause 12. The article of any one of clauses 1 to 11, wherein the silk fibroin fragments 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 article.
[0452] Clause 13. The article of any one of clauses 1 to 12, wherein the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool.
[0453] Clause 14. The article of any one of clauses 1 to 12, wherein the fabric comprises one or more of natural wool, chlorine-descaled wool, or non-chlorine descaled wool. [0454] Clause 15. The article of any one of clauses 1 to 14, wherein the reducing agent is selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2-mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin.
[0455] Clause 16. The article of any one of clauses 1 to 14, wherein the reducing agent is L- cysteine.
[0456] Clause 17. The article of any one of clauses 1 to 16, wherein the reducing agent is physically adsorbed to a surface of the fabric.
[0457] Clause 18. The article of any one of clauses 1 to 16, wherein the reducing agent is chemically linked to a surface of the fabric.
[0458] Clause 19. The article of any one of clauses 1 to 18, wherein the coating further comprises a crosslinker.
[0459] Clause 20. The article of clause 19, wherein the crosslinker is glycerol diglycidyl ether (GDE).
[0460] Clause 21. A method of making a silk fibroin coated fabric, comprising: applying to the fabric a solution comprising a reducing agent; applying to the fabric a silk fibroin solution; and drying the fabric.
[0461] Clause 22. A method of improving size retention on laundering in a fabric, comprising: applying to the fabric a solution comprising a reducing agent; applying to the fabric a silk fibroin solution; and drying the fabric.
[0462] Clause 23. The method of clause 21 or 22, wherein upon laundering, the fabric substantially retains its initial size prior to laundering.
[0463] Clause 24. The method of clause 21 or 22, wherein upon laundering, the fabric retains a substantially higher fraction of its initial size prior to laundering compared to a similar fabric not similarly treated with the reducing agent and the silk fibroin solution.
[0464] Clause 25. The method of any one of clauses 21 to 24, wherein the silk fibroin solution comprises low molecular weight silk fibroin fragments.
[0465] Clause 26. The method of any one of clauses 21 to 24, wherein the silk fibroin solution comprises low molecular weight silk fibroin fragments, medium molecular weight silk fibroin fragments, or a combination thereof. [0466] Clause 27. The method of any one of clauses 21 to 26, wherein the concentration of the silk fibroin solution is between about 0.1% w/v and about 0.5% w/v, between about 0.5% w/v and about 1% w/v, between about 1% w/v and about 1.5% w/v, between about 1.5% w/v and about 2% w/v, between about 2% w/v and about 3% w/v, between about 3% w/v and about 4% w/v, between about 4% w/v and about 5% w/v, between about 5% w/v and about 6% w/v, between about 6% w/v and about 7% w/v, between about 7% w/v and about 8% w/v, between about 8% w/v and about 9% w/v, or between about 9% w/v and about 10% w/v.
[0467] Clause 28. The method of any one of clauses 21 to 26, wherein the concentration of the silk fibroin solution is about 0.1% w/v, about 0.2% w/v, about 0.3% w/v, about 0.4% w/v, about 0.5% w/v, about 0.6% w/v, about 0.7% w/v, about 0.8% w/v, about 0.9% w/v, or about 1% w/v. [0468] Clause 29. The method of any one of clauses 21 to 26, wherein the concentration of the silk fibroin solution is about 1% w/v, about 2% w/v, 3% w/v, about 4% w/v, about 5% w/v, or about 6%.
[0469] Clause 30. The method of any one of clauses 21 to 29, wherein the drying comprises heating.
[0470] Clause 31. The method of clause 30, wherein the heating does not substantially modify silk fibroin coating performance.
[0471] Clause 32. The method of any one of clauses 21 to 31, wherein the pH of the silk fibroin solution is acidic.
[0472] Clause 33. The method of any one of clauses 21 to 31, wherein the pH of the silk fibroin solution is about 4, about 5, or about 6.
[0473] Clause 34. The method of any one of clauses 21 to 31, wherein the pH of the silk fibroin solution is between about 3 and less than 7.
[0474] Clause 35. The method of any one of clauses 21 to 31, wherein the pH of the silk fibroin solution is between about 3.5 and about 5.5.
[0475] Clause 36. The method of any one of clauses 21 to 35, wherein the fabric is dried after applying the solution comprising a reducing agent and before applying the silk fibroin solution. [0476] Clause 37. The method of any one of clauses 21 to 36, wherein the concentration of the reducing agent in the solution ranges from about 1 g/L to about 12 g/L.
[0477] Clause 38. The method of any one of clauses 21 to 37, wherein the silk fibroin solution further comprises a reducing agent. [0478] Clause 39. The method of any one of clauses 21 to 37, wherein the silk fibroin solution and the solution comprising a reducing agent are applied at substantially the same time.
[0479] Clause 40. The method of any one of clauses 21 to 37, wherein the silk fibroin solution and the solution comprising a reducing agent are substantially one solution.
[0480] Clause 41. The method of any one of clauses 21 to 40, wherein the fabric comprises one or more of natural wool, synthetic wool, alpaca fleece, alpaca wool, lama fleece, lama wool, cashmere, sheep fleece, sheep wool, mohair wool, camel hair, or angora wool.
[0481] Clause 42. The method of any one of clauses 21 to 40, wherein the fabric comprises one or more of natural wool, chlorine-descaled wool, or non-chlorine descaled wool.
[0482] Clause 43. The method of any one of clauses 21 to 42, wherein the reducing agent is independently selected from an amino acid, sodium sulfite, sodium bisulfite, ascorbic acid, 2- mercaptoethanol, sodium thioglycolate, dithiothreitol, sodium sulphide, sodium hydrosulfide, thioglycolic acid, thiosalicylic acid, and pseudothiohydantoin.
[0483] Clause 44. The method of any one of clauses 21 to 42, wherein the reducing agent is independently at each occurrence L-cysteine.
[0484] Clause 45. The method of any one of clauses 21 to 44, further comprising applying to the fabric a crosslinker.
[0485] Clause 46. The method of clause 45, wherein the crosslinker is glycerol diglycidyl ether (GDE).
[0486] Clause 47. The method of any one of clauses 21 to 46, wherein the silk fibroin solution comprises silk fibroin fragments having an average weight average molecular weight selected from between about 1 kDa and about 5 kDa, from between about 5 kDa and about 10 kDa, from between about 6 kDa and about 17 kDa, from between about 10 kDa and about 15 kDa, from between about 14 kDa and about 30 kDa, from between about 15 kDa and about 20 kDa, from between about 17 kDa and about 39 kDa, from between about 20 kDa and about 25 kDa, from between about 25 kDa and about 30 kDa, from between about 30 kDa and about 35 kDa, from between about 35 kDa and about 40 kDa, from between about 39 kDa and about 54 kDa, from between about 39 kDa and about 80 kDa, from between about 40 kDa and about 45 kDa, from between about 45 kDa and about 50 kDa, from between about 50 kDa and about 55 kDa, from between about 55 kDa and about 60 kDa, from between about 60 kDa and about 100 kDa, or from between about 80 kDa and about 144 kDa, and a poly dispersity selected from between 1 and about 5.
[0487] Clause 48. The method of any one of clauses 21 to 47, wherein the silk fibroin solution further comprises about 0.01% (w/w) to about 10% (w/w) sericin relative to the silk fibroin fragments.
[0488] Clause 49. The method of any one of clauses 21 to 48, wherein the silk fibroin solution comprises silk fibroin fragments which 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 silk fibroin solution.
[0489] Clause 50. An article comprising a fabric prepared by the method of any one of clauses 21 to 49.
EXAMPLES
[0490] The embodiments encompassed herein are now described with reference to the following examples. These examples are provided for the purpose of illustration only and the disclosure encompassed herein should in no way be construed as being limited to these examples, but rather should be construed to encompass any and all variations which become evident as a result of the teachings provided herein.
[0491] The compositions of this disclosure may be made by various methods known in the art. Such methods include those of the following examples, as well as the methods specifically exemplified below. Modifications of such methods that involve techniques commonly practiced in the art of fabric products may also be used.
[0492] Example 1: Silk Fibroin Used on Wool to Improve Anti-shrinkage Performance
[0493] As described in this Example, silk fibroin can be used with a natural reducing agent L- cysteine on wool to improve the dimensional stability after laundering, therefore improve the anti-shrinkage performance.
[0494] Pretreat of wool sample: Different amounts of L-cysteine (1 g/L - 12 g/L) was dissolved in tap water at room temperature. Wool panel was pretreated in the L-cysteine solution by soaking for 30 min with a liquor to goods ratio of 25 : 1. Excess solution was removed by hydro extraction until the wet-pick-up achieved 90-110%. The wool sample was cured in an oven at 120 °C for 10 min. See also Du et al., “Recycling keratin polypeptides for anti-felting treatment of wool based on L-cysteine pretreatment,” Journal of Cleaner Production 183, 810-817 (2018), which is incorporated by reference herein in its entirety.
[0495] Silk fibroin treatment of wool sample: Silk solution was prepared by diluting the Mid or Low molecular weight silk to the desired concentration (0.5%), and pH was adjusted by citric acid to 4. The pre-treated wool panel was soaked in silk solution at the liquor to goods ratio of 25: 1 for 30 min. After soaking, the wool panel was transferred to the hydro extractor to achieve 90-110% wet-pick-up, while the silk solution remained in the bath. The wool panel was dried in an oven at 120 °C for 10 min. The dried wool panel was immersed in the same bath of the silk solution for another 30 min. Excess solution was hydro extracted until the wet-pick-up of the wool sample reached 90-100%. The wool panel was dried in a tumble dryer at 70 °C (+/- 5 °C fluctuation) for 20 min. The wool panel was steam-pressed (10 second steam with 6 bar pressure, 10 second vacuum) on a steaming table to remove wrinkles and creases generated during the tumble-drying process.
[0496] Wool panel anti-shrinkage performance test: Wool anti-shrinkage performance was tested following EBN In-House TM31. Dimension of wool panel was measured before wash (Original Measurement), after one-time relaxation wash (Relaxation Measurement), and after certain times of felting wash (Felting Measurement) according to the required care claim:
Hand Wash - Flat Dry Care Claim: 1 x 7A
Machine Wash - Flat Dry Care Claim: 2 x 5A
Machine Wash - Tumble Dry Care Claim: 5 x 5A
Where: 7A and 5 A are wash cycles defined by TM31
[0497] Shrinkage was calculated as follows: Shrinkage % = (FM-RM)/RM x 100
Where: FM=Felting Measurement
RM=Relaxation Measurement
[0498] Performance Results: Fig. 3 shows experimental data demonstrating the dimensional stability of Chlorine-Descaled wool. L-cysteine pretreat followed by 0.5% silk D coating showed a significant reduction in dimensional shrinkage. L-cysteine pretreat followed by 0.5% silk C coating showed a slight reduction in dimensional shrinkage, while L-cysteine pretreat followed by water showed no impact. Fig. 4 shows experimental data demonstrating the dimensional stability of Non-Chlorine-Descaled wool. L-cysteine (12g/L) pretreat followed by 0.5% silk D coating showed a significant reduction in dimensional shrinkage. L-cysteine ( 1 g/L) pretreat followed by 0.5% silk D coating showed a slight reduction in dimensional shrinkage, while 0.5% silk D only without pretreat showed no impact. Fig. 5 shows experimental data demonstrating the dimensional stability of Natural wool. L-cysteine pretreat followed by 0.5% silk D coating showed a reduction in dimensional shrinkage, while 0.5% silk D without L- cysteine pretreat showed no impact.
[0499] The process is repeated and data generated for additional types of wool material, including chlorine-descaled wool, non-chlorine descaled wool, and natural wool. For chlorinedescaled wool, silk only without L-cysteine pretreat data is included. For non-chlorine descaled wool, L-cysteine pretreat followed by water control data is included. For natural wool, L- cysteine pretreat followed by water control data is included. In some embodiments, L-cysteine is mixed with silk and applied to wool together in one bath. In some embodiments, L-cysteine pretreat is followed by silk treatment without drying in between.
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