Cosmetic and personal care compositions comprising recombinant silk

Abstract

Disclosed herein are compositions comprising recombinant silk polypeptides and their use as a replacement for silicones (both "fluid" and "elastomer") in cosmetic and personal care formulations to affect the properties of skin and hair, which may include benefits to the formulation itself, such as increasing the viscosity of the formulation.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to compositions and uses thereof that contain recombinant silk polypeptides as silicone (both "fluid" and "elastomeric") replacements in cosmetic and personal care formulations to affect the properties of skin and hair, which may include benefits to the formulation itself, such as increased formulation viscosity. For skin, these properties may include long wear, silky soft feel, matte finish, enhanced pigment delivery, spreadability, rapid absorption, wrinkle hiding effect, and UV and pollution protection. For hair, these properties may include long wear, shine, non-greasy, frizz control, added density to hair, styling hold, heat resistance, and UV and pollution protection. [Background technology]

[0002] background Silicone polymers are a broad family of chemicals that share the commonality of alternating silicon and oxygen atoms that make up the polymer's backbone.Generally speaking, "liquid" silicone polymers or "silicone fluids" refer to high molecular weight silicone polymers that may or may not contain functional groups.These polymers may be referred to as "dimethicones."Silicone elastomers refer to a type of silicone in which linear silicone polymers are crosslinked to form a gel-like network.

[0003] Silicone fluids and silicone elastomers are widely used in the beauty and personal care industry. For example, silicone elastomers are known to make skin feel soft and velvety, as well as to give skin a matte finish. Some common uses of silicones include BB creams, anti-aging wrinkle reducers, primers, liquid foundations, mousse foundations, gel eyeshadows, and many more.

[0004] Fluid silicones and silicone elastomers offer benefits for skin, hair, and personal care applications, but silicones can be difficult to wash off and can remain in pores. This is because silicones are hydrophobic and repel water. For this reason, silicone-based products do not wash off easily. Silicones are also not environmentally friendly - they biodegrade very slowly in the environment and therefore can accumulate in living organisms. If silicones are washed down the drain, they can lead to the accumulation of sludge pollution in oceans and waterways and cannot be decomposed for decades or centuries (Horii Y., Kannan K. (2019) Main Uses and Environmental Emissions of Volatile Methylsiloxanes. In: Homem V., Ratola N. (eds) Volatile Methylsiloxanes in the Environment. The Handbook of Environmental Chemistry, vol 89. Springer, Cham. https: / / doi.org / 10.1007 / 698_2019_375 (Non-Patent Document 1)).

[0005] To date, there is an unmet need for alternative silicone elastomer components that meet the same performance levels as silicone elastomers, while being bio-based and biodegradable. There are many component aspects to make "greener" silicone elastomers by replacing cyclosiloxane diluents (usually associated with silicone elastomers) with bio-based alternatives. This is a step in the right direction, but falls far short of solving the current problem that silicone elastomers are still not biodegradable. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Horii Y., Kannan K. (2019) Main Uses and Environmental Emissions of Volatile Methylsiloxanes. In: Homem V., Ratola N. (eds) Volatile Methylsiloxanes in the Environment. The Handbook of Environmental Chemistry, vol 89. Springer, Cham. https: / / doi.org / 10.1007 / 698_2019_375 Summary of the Invention

[0007] [Brief description of the drawings]

[0008] The foregoing and other objects, features, and advantages will be apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, in which like reference characters refer to the same parts throughout the different views, and in which the drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[0009] [Figure 1A] 1 illustrates a top down view of an SPF formulation. [Figure 1B] 1 shows a spider chart plotting data from a blinded study comparison of SPF formulations. [Figure 1C] 1 shows the ingredients used in all SPF formulations (recombinant silk polypeptide and silicone free). [Figure 1D] Illustrates the elastic modulus, viscous modulus, and phase angle versus frequency for an SPF base formulation, an SPF formulation with 1.5% recombinant silk polypeptide, and an SPF formulation with 2% silicone. [Figure 1E]Shown are optical microscope images of the SPF base and the SPF base formulated with 1% b-silk protein (i.e., 18B silk; recombinant silk comprising SEQ ID NO: 2878) or 5% silicone elastomer component. Also shown is a reference image of 1% recombinant silk polypeptide powder suspended in water. [Figure 2A] 1 illustrates a top-down view of a 3-in-1 eye / cheek / lip cream formulation (i.e., color cosmetic) having 1% recombinant silk polypeptide or 10% silicone elastomer. [Figure 2B] 1 illustrates a flow chart for evaluation of color cosmetic products for pigment delivery to the skin and validation. [Figure 2C] Representative images of application of color makeup having i) 1% recombinant silk polypeptide, ii) 5% silicone elastomer, and iii) 10% silicone elastomer and wiping off of the color makeup are shown. [Figure 2D] Shows ingredients used in all color cosmetic formulations (recombinant silk polypeptide and silicone free). [Figure 2E] 1 shows optical microscope images of a color cosmetic base and a base formulated with 1% recombinant silk polypeptide or 5% silicone elastomer ingredient. [Figure 2F] 1 shows charts of elastic modulus, viscous modulus, and phase angle versus frequency for a color cosmetic base, a color cosmetic having 1% recombinant silk polypeptide, and a color cosmetic having 5% silicone elastomer. [Figure 3A] 1 shows optical microscope images of a hair serum formulation with and without 1% recombinant silk polypeptide and 5% silicone elastomer, and a reference image of 1% recombinant silk polypeptide powder suspended in water. [Figure 3B]Shown are SEM images of yak hair at 150x and 800x magnification: i) untreated, ii) treated with serum base, iii) treated with serum base with 1% recombinant silk protein, and iv) treated with serum base with 5% elastomer. [Figure 3C] 1 shows charts of elastic modulus, viscous modulus, and phase angle versus frequency (bottom row) or versus shear strain (top row) for a colored hair serum base, a hair serum base with 1% recombinant silk protein, and a hair serum base with 5% silicone elastomer. [Figure 3D] 1 is a table of ingredients used in the hair serum base formulation (without recombinant silk polypeptides and silicone elastomers). [Figure 4A] 1 shows a comparison of the G' and G" of an industry standard silicone elastomer gel (30% dry solids) and 12% recombinant silk polypeptide on a chart of elastic modulus, viscous modulus, and phase angle vs. frequency (top row) or viscosity vs. shear rate (bottom row). [Figure 4B] 1 shows SEM images of pure recombinant silk polypeptides and silicone elastomer dispersed on yak hair at 150x and 800x magnification. [Diagram 5] 1 shows images of hair swatches after exposure to a curl retention test following application of a leave-in hair serum containing either serum base alone or a serum base having 1% silk polypeptide, 1% keratin component, or 5% silicone elastomer component. [Figure 6] 1 is a table of ingredients used in the rinse-off shampoo formulation (without recombinant silk polypeptide and silicone elastomer) described in Example 6. [Figure 7] 1 is a table of ingredients used in the leave-on skin serum (without recombinant silk polypeptide and silicone elastomer) described in Example 7. [Figure 8] 1 is a table of ingredients used in the leave-on skin primer (without recombinant silk polypeptide and silicone elastomer) described in Example 8. [Figure 9A] 7 is a graph of shear viscosity as a function of shear rate for the compositions of FIG. 6 containing different amounts of recombinant silk polypeptide compared to a placebo. [Figure 9B] FIG. 9B is a chart showing the change in viscosity from placebo for the compositions tested in FIG. 9A. [Figure 10A] FIG. 9B is a chart showing the change in rheology (G′ and G”) of the composition tested in FIG. 9A. [Figure 10B] FIG. 9B is a chart showing the change in rheology (G′ and G”) of the composition tested in FIG. 9A. [Figure 11] 8 is a chart showing the change in viscosity from placebo for the composition of FIG. 7 having different amounts of recombinant silk polypeptide. [Figure 12A] 9 is a graph of shear viscosity as a function of shear rate for the compositions of FIG. 8 containing different amounts of recombinant silk polypeptide compared to a placebo. [Figure 12B] FIG. 12B is a chart showing the change in viscosity from placebo for the compositions tested in FIG. 12A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Detailed Description Details of various embodiments of the invention are set forth in the following description. Other features, objects, and advantages of the invention will become apparent from the description and drawings, as well as the aspects.

[0011] The present invention relates to the use of recombinant silk polypeptides as silicone replacements in personal care and / or cosmetic compositions to provide cosmetics, skin, and hair benefits that would normally be associated with linear silicones and silicone elastomers. The skin, hair, or cosmetic compositions of the present disclosure may have any standard ingredients that are typically included in such compositions, including active ingredients, pigments, and additives. The silicone replacements including recombinant silk polypeptides can be used in combination with any standard hair care, skin care, or cosmetic ingredients to form compositions of the present disclosure in which the silicone (fluid or elastomer) typically included in such compositions is replaced. Any type of skin, hair, or cosmetic composition for use with silicone elastomer replacement components, including but not limited to SPF formulations, color cosmetics, rinse-off hair shampoos, skin serums, skin primers, or hair serums, is contemplated herein. The recombinant silk polypeptides may deliver these benefits at equal or reduced loading levels compared to silicone components. Advantageously, it has been observed that recombinant silk polypeptides as silicone replacements can maintain similar G' and G" rheological curve shapes as those containing silicone. Furthermore, it has been found that in some compositions, recombinant silk polypeptides can significantly increase the viscosity of the formulation compared to an equivalent amount of silicone, thereby allowing for significantly reduced loading levels of recombinant silk polypeptide compared to silicone. Furthermore, while silicone components are not biodegradable, recombinant silk polypeptides are biodegradable and break down in the environment (e.g., when washed down a drain).

[0012] The cosmetic, hair or skin care composition according to the present disclosure comprises a silicone-replaced component comprising recombinant silk polypeptide and one or more active ingredients for cosmetic, skin or hair care.The composition of the present disclosure can be substantially free of silicone.References to "silicone" herein should be understood to include both silicone fluids and silicone elastomers, unless otherwise specified.For example, the composition of the present disclosure can have less than 0.1% silicone.For example, the composition of the present disclosure can have less than 0.1% silicone elastomer.

[0013] Recombinant silk polypeptide is a high molecular weight polypeptide that entropically self-assembles into a cross-linked and semi-crystalline state. The recombinant polypeptide can be included in the composition as a powder. For example, hollow particles of recombinant silk polypeptide can be ground and included in the composition as a ground powder. The silicone-replaced component can include recombinant silk polypeptide that is actually suspended in a solvent. The silicone-replaced component can include recombinant silk polypeptide as a randomly structured gel. At the macro level, this can range from a low-viscosity weak gel (sometimes referred to as a "slurry") suspended in an aqueous solvent to a dry hollow powder particle (<15% moisture content).

[0014] In some embodiments, provided herein are compositions comprising recombinant silk polypeptides and their use as silicone replacements in cosmetic and personal care formulations.

[0015] In some embodiments, the recombinant silk polypeptide is a high molecular weight polypeptide, greater than >100 amino acids in length and less than 90 kDa amino acids in length.

[0016] In some embodiments, recombinant silk polypeptides self-assemble into a semi-crystalline state characterized by beta-sheet cross-linking groups in which the crystalline portions resist solubility in water, other polar and non-polar solvents (hexanol, hexane, benzene), oils, waxes, surfactants (anionic, non-ionic, cationic, amphoteric) at a pH of 3-8, yet disperse readily in these materials to form heterogeneous dispersions.

[0017] In some embodiments, the recombinant silk polypeptide is present as a randomly structured gel. This gel may also include the presence of preservatives and chelating agents. In various aspects, the recombinant silk polypeptide is present as a hollow powder. The hollow powder may be milled so that the powder is incorporated into the composition as a milled powder. The powder may also include or be mixed with preservatives and chelating agents.

[0018] In some embodiments, the recombinant silk polypeptide is present as a hollow powder suspended in an aqueous, polar, non-polar, oil, wax, or surfactant diluent, which mixture may also include the presence of preservatives and chelating agents.

[0019] The recombinant silk polypeptide may be included in the composition in an amount of about 0.01% to about 30% by weight, based on the total weight of the composition. For example, the recombinant silk polypeptide may be included in the composition in an amount of about 0.05% to about 5% by weight, about 0.5% to about 5% by weight, about 0.01% to about 0.5% by weight, about 0.1% to about 0.5% by weight, about 0.05% to about 5% by weight, about 0.5% to about 5% by weight, about 5% to about 20% by weight, about 5% to about 30% by weight, about 25% to about 30% by weight, about 10% to about 25% by weight, or about 1% to about 5% by weight, based on the total weight of the composition.

[0020] In some embodiments, recombinant silk polypeptides form a distinctive and detectable film on skin and hair when applied in leave-on or rinse-off formulations.

[0021] In some embodiments, recombinant silk polypeptides aid in cleaning or exfoliation when applied in a rinse-off formulation.

[0022] In some embodiments, recombinant silk polypeptides can be detected within the formulation (unwashed or washed out) as evidenced by visual inspection with a microscope where the powder can be viewed with a 5-100x objective lens. Additionally, recombinant silk polypeptides can be detected by a high molecular weight peak of 50 kDa to 90 kDa using SEC-HPLC.

[0023] In some embodiments, recombinant silk polypeptides match the performance of silicone elastomers in skin care, hair care, cosmetics, personal care, and antiperspirant / deodorant formulations at the same concentration or less.

[0024] In some embodiments, the recombinant silk polypeptides outperform silicone elastomers in skin care, hair care, cosmetics, personal care, and antiperspirant / deodorant formulations at the same or lower concentrations.

[0025] In some embodiments, the recombinant silk polypeptide replaces the silicone elastomer in a ratio of at least 1:1 to a ratio of up to 1:60, meaning that 1 part silicone elastomer can be replaced with 1 part recombinant silk polypeptide, and up to 60 parts silicone elastomer can be replaced with 1 part recombinant silk polypeptide. For example, a shampoo was produced having silk polypeptide as a silicone replacement, and the silk polypeptide was present in an amount of 0.05% by weight, based on the total weight of the composition, while the same composition required an amount of silicone elastomer of 3% by weight (a 60-fold increase) to achieve the same performance.

[0026] In some embodiments, the performance characteristics of the composition include: a) Silky smooth and silky feel b) Reduced skin greasiness c) Increased hair shine d) Brilliant and effective pigment delivery e) Easy ductility f) Rapid absorption time g) Mattification (i.e., less oily feel) h) Wrinkle-hiding effect i) Hair style retention and heat resistance j) UV and Pollution Protection k) Increase in compound viscosity Contains one or more of the following:

[0027] In some embodiments, the recombinant silk polypeptides are compatible (meaning their structure and performance are maintained) with a wide variety of common cosmetic components, such as polar and non-polar solvents, oils, waxes, fatty acids, humectants, and (sunscreen) active agents.

[0028] Due to the difference in swelling of recombinant silk polypeptide in different solvents, these differences can be used as formulation processing aids.For example, recombinant silk polypeptide can be added to a formulation in a non-swelling state in oil, wax, or non-polar solvent, and then swells when contacted with water.Recombinant silk polypeptide can be completely or partially removed from skin and hair by water, and completely removed by surfactants.

[0029] In some embodiments, recombinant silk polypeptides are superior to silicone elastomers in biodegradation under both anaerobic and aerobic digestion conditions. In the Organization for Economic Cooperation and Development (OECD) 301 degradability test, recombinant silk polypeptides undergo rapid biodegradation of at least 5-15% in the first 3-5 days. During the subsequent 5-90 days of incubation, recombinant silk polypeptides show a steady increase in biodegradation without a plateau for more than 20 days. OECD (1992), Test No. 301: Ready Biodegradability, OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing, Paris, doi.org / 10.1787 / 9789264070349-en, which is incorporated herein by reference in its entirety.

[0030] definition The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0031] The term "stability" as used herein with respect to silk proteins refers to the ability of the product not to gel, discolor, or form haze due to self-aggregation of silk proteins. For example, US Patent Publication No. 2015 / 0079012 (Wray et al.) is directed to the use of humectants, including glycerol, to enhance the storage stability of skin care products containing full-length silk fibroin. US Patent No. 9,187,538 is directed to skin care formulations containing full-length silk fibroin that can be stored for up to 10 days. Both of these publications are incorporated herein by reference in their entirety.

[0032] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlock conformations.

[0033] Unless otherwise indicated, as an example for all sequences described herein under the general format "SEQ ID NO:", "a nucleic acid comprising SEQ ID NO:1" refers to a nucleic acid having, at least in part, either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is determined by the context. For example, if the nucleic acid is used as a probe, the choice between the two is determined by the requirement that the probe is complementary to the desired target.

[0034] An "isolated" RNA, DNA or mixed polymer is one that is substantially separated from other cellular components which naturally accompany the natural polynucleotide in its natural host cell, such as naturally associated ribosomes, polymerases, and genomic sequences.

[0035] An "isolated" organic molecule (e.g., silk protein) is one that is substantially separated from cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it is derived, or from the medium in which the host cell is cultured. The term does not require that the biomolecule be separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.

[0036] The term "recombinant" refers to a biological molecule, such as a gene or protein, that is: (1) removed from its naturally occurring environment, (2) not associated with all or a portion of a polynucleotide with which the gene is found in nature, (3) operably linked to a polynucleotide with which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0037] An endogenous nucleic acid sequence (or its encoded protein product) in the genome of an organism is considered "recombinant" herein when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence such that expression of the endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, regardless of whether the heterologous sequence is itself endogenous (originating from the same host cell or its progeny) or exogenous (originating from a different host cell or its progeny). As an example, a promoter sequence can replace (e.g., by homologous recombination) the native promoter of a gene in the genome of a host cell such that the gene has an altered expression pattern. The gene would now be "recombinant" because it has been separated from at least some of the sequences that naturally flank it.

[0038] A nucleic acid is also considered "recombinant" if it contains any alteration relative to the corresponding nucleic acid in a genome that does not occur naturally. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation that has been artificially introduced, e.g., by human intervention. "Recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, and a nucleic acid construct that exists as an episome.

[0039] The term "peptide" as used herein refers to short polypeptides, e.g., typically less than about 50 amino acids in length, more typically less than about 30 amino acids in length. The term as used herein encompasses analogs and mimetics that mimic structure-function and thus biological function.

[0040] The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. A polypeptide may be monomeric or polymeric. Furthermore, a polypeptide may contain multiple distinct domains, each of which has one or more distinct activities.

[0041] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, in terms of its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where the purity can be determined with respect to the presence of other cellular material (e.g., does not include other proteins from the same species), (3) is expressed by cells from a different species, or (4) does not occur in nature (e.g., it is a fragment of a naturally occurring polypeptide, or it contains amino acid analogs or derivatives not found in nature, or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A polypeptide or protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. When defined in this way, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide so described be physically removed from its natural environment.

[0042] The term "polypeptide fragment" refers to a polypeptide having a deletion, e.g., an amino-terminal and / or carboxy-terminal deletion, compared to a full-length polypeptide. In a preferred embodiment, a polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally occurring sequence. A fragment is typically at least 5, 6, 7, 8, 9 or 10 amino acids in length, preferably at least 12, 14, 16 or 18 amino acids in length, more preferably at least 20 amino acids in length, more preferably at least 25, 30, 35, 40 or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, even more preferably at least 70 amino acids in length.

[0043] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences (thus the term "homologous proteins" is defined to mean that two proteins have similar amino acid sequences). As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted to mean similarity of function.

[0044] When "homologous" is used in reference to proteins or peptides, it is recognized that non-identical residue positions often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (incorporated herein by reference).

[0045] The twenty conventional amino acids and their abbreviations follow conventional usage. nd ed. 1991). Stereoisomers of the twenty conventional amino acids (e.g., D-amino acids), non-naturally occurring amino acids, such as α-,α-disubstituted amino acids, N-alkylamino acids, etc., and other non-conventional amino acids may also be suitable components for the polypeptides of the present invention. Examples of non-conventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminus and the right-hand end corresponds to the carboxy terminus, in accordance with standard usage and convention.

[0046] The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T), 2) aspartic acid (D), glutamic acid (E), 3) asparagine (N), glutamine (Q), 4) arginine (R), lysine (K), 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0047] Polypeptide sequence homology, sometimes referred to as percent sequence identity, is typically measured using sequence analysis software. See, for example, Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG contains programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as between homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1.

[0048] Algorithms useful for comparing a particular polypeptide sequence with a database containing a large number of sequences from different organisms are the computer programs BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0049] The preferred parameters for BLASTp are: expectation: 10 (default); filter: segment (default); cost to open a gap: 11 (default); cost to widen a gap: 1 (default); maximum alignments: 100 (default); word size: 11 (default); number of explanations: 100 (default); penalty matrix: BLOWSUM62.

[0050] The preferred parameters for BLASTp are expectation: 10 (default); filter: segment (default); cost to open a gap: 11 (default); cost to extend a gap: 1 (default); maximum alignment: 100 (default); word size: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences to be compared for homology is generally at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably longer than about 35 residues. When searching a database containing sequences from many different organisms, it is preferable to compare amino acid sequences. Database searches using amino acid sequences can be measured by algorithms other than blastp known in the art. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignments and percent sequence identity of the best overlapping regions between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), incorporated herein by reference. For example, percent sequence identity between amino acid sequences can be determined using FASTA provided in GCG version 6.1, incorporated herein by reference, with its default parameters (word size of 2, and a scoring matrix of PAM250).

[0051] Throughout this specification and the embodiments, the word "comprise" or variations such as "comprises" or "comprising" are understood to mean the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0052] As used herein, the term "glass transition" refers to the transition of a substance or composition from a hard, rigid, or "glassy" state to a more flexible, "rubbery" or "viscous" state.

[0053] As used herein, the term "glass transition temperature" refers to the temperature at which a substance or composition undergoes a glass transition.

[0054] As used herein, the term "melt transition" refers to the transition of a material or composition from a rubbery state to a less ordered liquid phase.

[0055] As used herein, the term "melting temperature" refers to the temperature range over which a material undergoes a melting transition.

[0056] The term "plasticizer" as used herein refers to any molecule that interacts with a polypeptide sequence to prevent the polypeptide sequence from forming tertiary structures and bonds and / or to increase the mobility of the polypeptide sequence.

[0057] The term "powder" as used herein refers to a composition that exists in granular form, which may or may not be complexed or aggregated with a solvent such as water or serum. The term "dry powder" may be used interchangeably with the term "powder", but as used herein, "dry powder" refers simply to the overall appearance of the granulated material and does not intend that the material is completely free of complexed or aggregated solvent, unless otherwise indicated. Dry powders can be produced by spray drying, freeze drying, and / or according to methods known in the art.

[0058] The term "carrier" refers to recombinant proteins used to hydrate surfaces, cleanse surfaces, protect surfaces, detoxify surfaces, exfoliate surfaces, improve surfaces, color, and / or deliver various additives or solvents, including, but not limited to, water, glycerin, alcohol, siloxane, oil, humectants, emollients, occlusive agents, active agents, and / or cosmetic adjuvants, to surfaces such as skin, hair, or nails. Carriers as used herein include the outer shell and the hollow core, e.g., 18B protein.

[0059] As used herein, the term "cosmetics" includes make-up, foundation, skin care, hair care, and nail care products.

[0060] As used herein, the term "make-up" refers to products that leave a dark and brown color on the face, including foundations, i.e., mascara, concealer, eyeliner, eyebrow color, eye shadow, blusher, lip color, powder, solid emulsion compacts, and the like.

[0061] As used herein, the term "foundation" refers to a liquid, cream, mousse, pancake, compact, concealer, etc. product created or reintroduced by cosmetic companies to even out the overall color of the skin.

[0062] The term "skin care product" as used herein refers to anything used to treat or care for the skin, or to moisturize, improve, or cleanse in any way. Products contemplated by the phrase "skin care product" include, but are not limited to, creams, mists, serums, cleansing gels, ampoules, adhesives, patches, bandages, toothpastes, anhydrous occlusive moisturizers, antiperspirants, deodorants, personal cleansing products, powder laundry detergents, fabric softeners, occlusive drug delivery patches, nail polish, powders, tissues, wipes, anhydrous hair conditioners, shaving creams, and the like.

[0063] As used herein, the term "sagging" refers to a condition of loose, flaccid skin resulting from loss, damage, alteration, and / or abnormalities of dermal elastin, muscle, and / or subcutaneous fat.

[0064] The term "treat" or "treatment" as used herein refers to the treatment (e.g., reducing or eliminating symptoms, and / or curing) and / or prevention or inhibition, or alleviation of symptoms, of a condition (e.g., a skin condition).

[0065] Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used to practice the present invention and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0066] Recombinant silk proteins This disclosure describes embodiments of the invention that include fibers synthesized from synthetic proteinaceous copolymers (i.e., recombinant polypeptides). Suitable proteinaceous copolymers are discussed in U.S. Patent Publication No. 2016 / 0222174, published Aug. 45, 2016, U.S. Patent Publication No. 2018 / 0111970, published Apr. 26, 2018, and U.S. Patent Publication No. 2018 / 0057548, published Mar. 1, 2018, each of which is incorporated herein by reference in its entirety.

[0067] In some embodiments, synthetic proteinaceous copolymers are made from silk-like polypeptide sequences. In some embodiments, silk-like polypeptide sequences are 1) block copolymer polypeptide compositions produced by mixing and matching repeat domains derived from silk polypeptide sequences, and / or 2) recombinant expression of block copolymer polypeptides with a size (approximately 40 kDa) large enough to form useful molded body compositions by secretion from industrially scalable microorganisms. Large (approximately 40 kDa to approximately 100 kDa) block copolymer polypeptides engineered from silk repeat domain fragments, including sequences from nearly all published silk polypeptide amino acid sequences, can be expressed in the engineered microorganisms described herein. In some embodiments, silk polypeptide sequences are matched and engineered to produce highly expressed and secreted polypeptides capable of molded body formation.

[0068] In some embodiments, block copolymers are engineered from combinatorial mixtures of silk polypeptide domains spanning the silk polypeptide sequence space. In some embodiments, block copolymers are produced by expression and secretion in scalable organisms (e.g., yeast, fungi, and gram-positive bacteria). In some embodiments, block copolymer polypeptides include zero or more N-terminal domains (NTDs), one or more repeat domains (REPs), and zero or more C-terminal domains (CTDs). In some aspects of the embodiments, the block copolymer polypeptide is a single polypeptide chain of more than 100 amino acids. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a block copolymer polypeptide disclosed in International Publication No. WO / 2015 / 042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated by reference in its entirety.

[0069] Several types of natural spider silk have been identified. The mechanical properties of each type of naturally spun silk are believed to be closely related to the molecular composition of the silk. See, for example, Garb, JE, et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); 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 Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pg.131-85 (2011).

[0070] Tufted (AcSp) silks tend to have high toughness, resulting from a combination of moderately high strength and moderately high extensibility. AcSp silks are often characterized by the size of large blocks ("ensemble repeats") that incorporate polyserine and GPX motifs. Small tubular (TuSp or cylindrical) silks tend to have large diameters with moderate strength and high extensibility. TuSp silks are characterized by their polyserine and polythreonine content, as well as short tracts of polyalanine. Large ampullate (MaSp) silks tend to have high strength and moderate extensibility. MaSp silks can be one of two subtypes: MaSp1 and MaSp2. MaSp1 silks are generally less extensible than MaSp2 silks and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silks are characterized by polyalanine, GGX, and GPX motifs. Ampullate (MiSp) silks tend to have moderate strength and moderate extensibility. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagellar (Flag) silks tend to have very high extensibility and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0071] The properties of each silk type may vary from species to species, and spiders that lead distinct lifestyles (e.g., sedentary termite bugs vs. wandering assassin bugs) or are evolutionarily older may produce silks with properties different from those described above (for a description of spider diversity and classification, see Hormiga, G., and Griswold, C.E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pg. 487-512 (2014); and Blackedge, T.A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, pg. 5229-5234 (2009)). However, synthetic block copolymer polypeptides with sequence similarity and / or amino acid composition similarity to the repeat domains of natural silk proteins can be used to produce consistent bodies on a commercial scale with properties that recapitulate those of corresponding bodies made from natural silk polypeptides.

[0072] In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for related terms, e.g., "spidroin," "fibroin," "MaSp," and the sequences can be pooled with additional sequences obtained by independent sequencing efforts. The sequences are then translated into amino acids, filtered for duplicate entries, and manually divided into domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequences are back-translated into DNA sequences optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, the various silk domains that show successful expression and secretion are then assembled in a combinatorial manner to construct silk molecules capable of forming molded bodies.

[0073] Silk polypeptides are characteristically composed of repetitive domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In embodiments, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repetitive domains exhibit a hierarchical structure. They contain a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (constituting quasi-repetitive domains), throughout the repetitive domain of the silk. The length and composition of the blocks vary between different silk types and across different species. Table 1A lists examples of block sequences from selected species and silk types, and further examples are 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, pg169-184 (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, the blocks are arranged in a regular pattern, forming larger macrorepeats that occur multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Repeated blocks within the repeat domain or macrorepeat, and repeated macrorepeats within the repeat domain, may be separated by spacing elements. In some embodiments, the block sequence comprises a glycine-rich region followed by a polyA region. In some embodiments, a short (about 1-10) amino acid motif occurs multiple times within a block. For purposes of the present invention, blocks from different native silk polypeptides can be selected without reference to circular permutation (i.e., otherwise similar identified blocks between silk polypeptides may not align for circular permutation).Thus, for example, a "block" of SGAGG (SEQ ID NO:2871) is, for purposes of the present invention, the same as GSGAG (SEQ ID NO:2872) and the same as GGSGA (SEQ ID NO:2873), all of which are just circular permutations of each other. The particular permutation selected for a given silk sequence may be determined more by convenience than anything else (usually starting with G). Silk sequences obtained from the NCBI database may be partitioned into blocks and non-repetitive regions.

[0074] (Table 1A) Sample block array JPEG2024540202000001.jpg246160JPEG2024540202000002.jpg237170

[0075] Fiber-forming block copolymer polypeptides from blocks and / or macro-repeat domains according to certain embodiments of the invention are described in International Publication No. WO / 2015 / 042164, which is incorporated by reference. Natural silk sequences obtained from protein databases such as GenBank or by de novo sequencing are dissected by domains (N-terminal domain, repeat domain, and C-terminal domain). The N-terminal and C-terminal domain sequences selected for synthesis and assembly into fibers or molded bodies contain the natural amino acid sequence information and other modifications described herein. The repeat domains are broken down into repeat sequences that typically contain 1-8 representative blocks depending on the type of silk, which incorporates the important amino acid information while reducing the size of the DNA encoding the amino acids into easily synthesizable fragments. In some embodiments, a properly formed block copolymer polypeptide comprises at least one repeat domain that contains at least one repeat sequence, optionally adjacent to the N-terminal and / or C-terminal domain.

[0076] In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is between 150 and 300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repeat sequence comprises multiple macrorepeats. In some embodiments, the blocks or macrorepeats are divided across multiple repeat sequences.

[0077] In some embodiments, the repeat sequence cannot start with glycine and end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some of the repeat sequences may be modified compared to the native sequence. In some embodiments, the repeat sequence may be modified, such as by the addition of a serine to the C-terminus of the polypeptide (to avoid termination at F, Y, W, C, H, N, M, or D). In some embodiments, the repeat sequence may be modified by filling in an incomplete block with a homologous sequence from another block. In some embodiments, the repeat sequence may be modified by rearranging the order of the blocks or macrorepeats.

[0078] In some embodiments, non-repetitive N- and C-terminal domains may be selected for synthesis. In some embodiments, the N-terminal domain may be due to removal of a leading signal sequence, for example, as identified by SignalP (Peterson, TN, et. Al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0079] In some embodiments, the N-terminal domain, repeat sequence, or C-terminal domain sequence is selected from the group consisting of Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope bruennichi, Argiope trifasciata, Argiope trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis, Latrodectus hesperus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipesclavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0080] In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence may be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to another affinity tag, such as 6-8 His residues.

[0081] In some embodiments, the recombinant silk polypeptide is based on a recombinant spider silk protein fragment sequence derived from MaSp2, such as from the species Argiope bruennichii. In some embodiments, the synthesized fibers contain protein molecules comprising 2-20 repeat units, with each repeat unit having a molecular weight greater than about 20 kDa. Within each repeat unit of the copolymer, there are greater than about 60 amino acid residues, often in the range of 60-100 amino acids organized into several "quasi-repeat units." In some embodiments, the repeat units of the polypeptides described herein have at least 95% sequence identity to the MaSp2 dragline silk protein sequence.

[0082] Repeating units of proteinaceous block copolymers that form fibers with good mechanical properties can be synthesized using portions of silk polypeptides. These polypeptide repeating units contain alanine-rich and glycine-rich regions and are 150 amino acids or more in length. Some exemplary sequences that can be used as repeats in the proteinaceous block copolymers of the present disclosure are provided in co-owned PCT Publication No. WO2015 / 042164, which is incorporated by reference in its entirety, and have been shown to be expressed using a Pichia expression system.

[0083] In some embodiments, the silk protein comprises at least two occurrences of a repeating unit, the repeating unit having more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having six or more consecutive amino acids with an alanine content of at least 80%; a glycine-rich region having twelve or more consecutive amino acids with a glycine content of at least 40% and an alanine content of less than 30%, and the fiber comprises at least one property selected from the group consisting of an elastic modulus of greater than 550 cN / tex, an extensibility of at least 10%, and an ultimate tensile strength of at least 15 cN / tex.

[0084] In some embodiments, the recombinant silk protein comprises repeat units, each repeat unit having at least 95% sequence identity to a sequence comprising 2-20 quasi-repeat units, each quasi-repeat unit being selected from the group consisting of {GGY-[GPG-X1] n1 -GPS-(A) n2}, wherein for each quasi-repeat unit, X1 is independently selected from the group consisting of SGGQQ (SEQ ID NO:2874), GAGQQ (SEQ ID NO:2875), GQGOPY (SEQ ID NO:2876), AGQQ (SEQ ID NO:2877), and SQ, n1 is 4 to 8, and n2 is 6 to 10. The repeat unit is composed of multiple quasi-repeat units.

[0085] In some embodiments, three "long" quasi-repeats are followed by three "short" quasi-repeat units. As stated above, short quasi-repeat units are those where n1=4 or 5. Long quasi-repeat units are defined as those where n1=6, 7, or 8. In some embodiments, all of the short quasi-repeats have the same X1 motif at the same position within each quasi-repeat unit of the repeat unit. In some embodiments, no more than three of the six quasi-repeat units share the same X1 motif.

[0086] In additional embodiments, the repeat unit is comprised of quasi-repeat units that do not use the same X1 more than two consecutive occurrences within the repeat unit. In additional embodiments, the repeat unit is comprised of quasi-repeat units, where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 quasi-repeats do not use the same X1 more than two times within a single quasi-repeat unit of the repeat unit.

[0087] In some embodiments, the recombinant silk polypeptide comprises the polypeptide sequence of SEQ ID NO: 2878 (i.e., 18B). In some embodiments, the repeating unit is a polypeptide comprising SEQ ID NO: 2879. These sequences are provided in Table 1B.

[0088] Table 1B. Exemplary polypeptide sequences of recombinant proteins and repeat units JPEG2024540202000003.jpg126170

[0089] In some embodiments, the structure of the fibers formed from the described recombinant silk polypeptides forms a beta-sheet structure, a beta-turn structure, or an alpha-helical structure. In some embodiments, the secondary, tertiary, and quaternary protein structure of the formed fibers is described as having nanocrystalline beta-sheet regions, amorphous beta-turn regions, amorphous alpha-helical regions, randomly spatially distributed nanocrystalline regions embedded in an amorphous matrix, or randomly oriented nanocrystalline regions embedded in an amorphous matrix. Without intending to be bound by theory, it is theorized that the structural properties of proteins in spider silks are related to the mechanical properties of the fibers. The crystalline regions of the fibers are associated with the tensile strength of the fibers, while the amorphous regions are associated with the extensibility of the fibers. Major ampullate (MA) silks tend to be stronger and less extensible than flagelliform silks, and MA silks also have a greater volume fraction of crystalline regions compared to flagelliform silks. Furthermore, theoretical models based on molecular dynamics of crystalline and amorphous regions of spider silk proteins support the assertion that crystalline regions are associated with fiber tensile strength and amorphous regions are associated with fiber extensibility. Theoretical modeling further supports the importance of secondary, tertiary, and quaternary structures to the mechanical properties of recombinant protein fibers (RPFs). For example, both the assembly of nanocrystalline domains in random, parallel, and serial spatial distributions and the strength of interaction forces between entangled chains within the amorphous regions and between the amorphous and nanocrystalline regions influenced the theoretical mechanical properties of the resulting fibers.

[0090] In some embodiments, the molecular weight of the silk protein is between 20 kDa and 2000 kDa, or greater than 20 kDa, or greater than 10 kDa, or greater than 5 kDa, or between 5 and 400 kDa, or between 5 and 300 kDa, or between 5 and 200 kDa, or between 5 and 100 kDa, or between 5 and 50 kDa, or between 5 and 500 kDa, or between 5 and 1000 kDa, or between 5 and 2000 kDa, or between 10 and 400 kDa, or between 10 and 300 kDa, or between 10 and 200 kDa. a, or 10-100 kDa, or 10-50 kDa, or 10-500 kDa, or 10-1000 kDa, or 10-2000 kDa, or 20-400 kDa, or 20-300 kDa, or 20-200 kDa, or 40-300 kDa, or 40-500 kDa, or 20-100 kDa, or 20-50 kDa, or 20-500 kDa, or 20-1000 kDa, or 20-2000 kDa.

[0091] Characterization of impurities and degradation of recombinant spider silk polypeptide powders Different recombinant spider silk polypeptides have different physicochemical properties, such as melting temperature and glass transition temperature, based on the strength and stability of the secondary and tertiary structures formed by the protein. In the form of monomers, silk polypeptides form beta-sheet structures. In the presence of other monomers, silk polypeptides form a three-dimensional crystal lattice of beta-sheet structures. The beta-sheet structures are separated and interspersed with amorphous regions of the polypeptide sequence.

[0092] The beta sheet structure is very stable at high temperatures, with the melting temperature of beta sheets being approximately 257°C as measured by fast scanning calorimetry. See Cebe et al., Beating the Heat-Fast Scanning Melts Silk Beta Sheet Crystals, Nature Scientific Reports 3:1130 (2013). The beta sheet structure is believed to remain intact above the glass transition temperature of silk polypeptides, and therefore it is hypothesized that the structural transition seen at the glass transition temperature of recombinant silk polypeptides is due to increased mobility of the amorphous regions between the beta sheets.

[0093] Plasticizers reduce the glass transition temperature and melting temperature of silk proteins by increasing the mobility of amorphous regions and potentially disrupting beta-sheet formation. Suitable plasticizers for this purpose include, but are not limited to, water and polyalcohols (polyols), such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to, dimethyl isosorbide, adipic acid, amide of dimethylaminopropylamine with caprylic / capric acid, acetamide, and any combination thereof.

[0094] The hydrophilic portion of silk polypeptides can bind ambient water present in the air as humidity, so water is almost always present, and the bound ambient water can plasticize silk polypeptides. In some embodiments, a suitable plasticizer can be glycerol, present either alone or in combination with water or other plasticizers. Other suitable plasticizers are discussed above.

[0095] In addition, when recombinant silk polypeptides are produced by fermentation and recovered therefrom as recombinant silk polypeptide powder, impurities that act as plasticizers or otherwise inhibit the formation of tertiary structure may be present in the recombinant silk polypeptide powder. For example, residual lipids and sugars may act as plasticizers and thus affect the glass transition temperature of the protein by preventing the formation of tertiary structure.

[0096] A variety of established methods can be used to assess the purity and relative composition of recombinant silk polypeptide powders or compositions. Size exclusion chromatography can be used to separate molecules based on relative size and analyze the relative amounts of recombinant silk polypeptide in its full length polymeric and monomeric forms, as well as the amounts of high, low, and medium molecular weight impurities in recombinant silk polypeptide powders. Similarly, rapid high performance liquid chromatography can be used to measure the presence of various compounds in a solution, such as the monomeric form of recombinant silk polypeptide. Ion exchange liquid chromatography can be used to assess the concentration of various trace molecules in a solution, including impurities such as lipids and sugars. Other methods of chromatography and quantification of various molecules, such as mass spectrometry, are well established in the art.

[0097] Depending on the embodiment, the recombinant silk polypeptide may have a purity calculated based on the amount by weight of the recombinant silk polypeptide in monomeric form relative to other components of the recombinant silk polypeptide powder. In various examples, the purity may range from 50% to 90% by weight, depending on the type of recombinant silk polypeptide and the techniques used to recover, separate and work up the recombinant silk polypeptide powder.

[0098] Both size exclusion chromatography and reversed-phase high performance liquid chromatography are useful techniques for measuring full-length recombinant silk polypeptides, and comparing the amount of full-length silk polypeptide in a composition before and after treatment can be useful techniques for determining whether a treatment step has degraded the recombinant silk polypeptide. In various embodiments of the invention, the amount of full-length recombinant silk polypeptide present in a composition before and after treatment can be subject to minimal degradation. The amount of degradation can range from 0.001% to 10% by weight, or 0.01% to 6% by weight, for example, less than 10% by weight, or 8% by weight, or 6% by weight, or less than 5% by weight, less than 3% by weight, or less than 1% by weight.

[0099] Silicone Replacement Ingredients The silicone-replaced component comprises a recombinant silk polypeptide. The silicone-replaced component may consist of a recombinant silk polypeptide. The silicone-replaced component may comprise a recombinant silk polypeptide with a solvent and / or one or more additives, such as a preservative and a chelating agent. The silicone-replaced component may comprise a recombinant silk polypeptide in an amount of about 1% to about 40% by weight based on the total weight of the silicone-replaced component, or any other suitable amount necessary to achieve the final desired loading of the recombinant silk polypeptide in the cosmetic, skin care, or hair care composition.

[0100] Without intending to be bound by theory, in various embodiments of the present invention, derivatization of the silicone-replacement component may be used in applications where it is desirable to prevent the aggregation of monomeric recombinant silk polypeptides into their crystalline polymeric form, or to control the transition of recombinant silk polypeptides to their crystalline polymeric form at a later stage in processing, while in other embodiments, such derivatization is not required.

[0101] In one particular embodiment, the silicone elastomer replacement component can be used to prevent the recombinant silk polypeptide from aggregating before blending the recombinant silk polypeptide with a second polymer. In another particular embodiment, the silicone elastomer replacement component can be used to create a base for a cosmetic or skin care product in which the recombinant silk polypeptide is present in the base in its monomeric form. In this embodiment, having the recombinant silk polypeptide in its monomeric form in the base allows the monomer to be controlled from aggregating into its crystalline polymeric form upon contact with the skin or through various other chemical reactions.

[0102] In various embodiments, the temperature to which the silicone elastomer replacement component with the recombinant silk polypeptide is heated is minimized to minimize or completely prevent degradation of the recombinant silk polypeptide. In certain embodiments, the recombinant silk melt is heated to a temperature of less than 120°C, less than 100°C, less than 80°C, less than 60°C, less than 40°C, or less than 20°C. Often, the melt is at a temperature in the range of 10°C-120°C, 10°C-100°C, 15°C-80°C, 15°C-60°C, 18°C-40°C, or 18°C-22°C during processing. In other embodiments, the silicone elastomer replacement component is not heated. In such embodiments, the presence of heat is not required to form the silicone elastomer replacement component.

[0103] The amount of degradation of the recombinant silk polypeptide may be measured using various techniques. As discussed above, the amount of degradation of the recombinant silk polypeptide may be measured using size exclusion chromatography to measure the amount of full-length recombinant silk polypeptide present. In various embodiments, the recombinant silk polypeptide is degraded in an amount of less than 6.0% by weight after it is formed into a molded body. In another embodiment, the recombinant silk polypeptide is degraded in an amount of less than 4.0% by weight, less than 3.0% by weight, less than 2.0% by weight, or less than 1.0% by weight after molding, resulting in an amount of degradation that may range from 0.001% to 10%, 8%, 6%, 4%, 3%, 2%, or 1% by weight, or from 0.01% to 6%, 4%, 3%, 2%, or 1% by weight. In another embodiment, the recombinant silk protein in the composition is not substantially degraded. In a similar embodiment, the recombinant silk protein in the composition is not substantially degraded over a period of at least 1 day, 1 month, 1 year, or 5 years.

[0104] In some embodiments, the silicone-replaced component is physically stable. In various embodiments, the component remains in its material form, e.g., powder, for an extended period of time and has a long shelf life. In extended use, the silicone-replaced component remains substantially stable. In some embodiments, the silicone-replaced component has substantially the same stability as that of silicone and / or silicone elastomer.

[0105] In some embodiments, the silicone-replaced component has material properties substantially similar to those of the silicone and / or silicone elastomer. In various embodiments, the silicone-replaced component has a rheology substantially similar to that of the silicone and / or silicone elastomer and / or imparts a rheology similar to that of the inclusion of the silicone and / or silicone elastomer to the composition of the present disclosure.

[0106] In most embodiments of the present invention, the silicone-replaced component is in powder form. The silicone-replaced component may include recombinant silk polypeptide in powder. In some embodiments, the silicone-replaced component is spray-dried. In other embodiments, the silicone-replaced component is freeze-dried or vacuum-dried. The terms "spray-dried" and "spray-dried" are used herein for brevity, but those skilled in the art will understand that freeze-drying or lyophilization and vacuum-drying can be substituted for spray-drying if necessary. These silicone-replaced components can be stored dry.

[0107] 18B protein is more stable in dry form than in aqueous slurry. In some embodiments, spray-dried recombinant silk is obtained as follows: A slurry composition containing extracted recombinant silk is kept cool during the drying step. It is pumped into a tall form spray dryer where the moisture content of the resulting powder is tightly controlled. Since protein powders are hygroscopic, the final powder collection and packaging is done to minimize the reintroduction of moisture. The design of the packaging material should minimize exposure to moisture and light.

[0108] In some embodiments, recovery and isolation of recombinant silk polypeptides from cell culture is carried out by: i) extraction and isolation, ii) removal of urea by ultrafiltration, iii) washing by precipitation, iv) removal of salts and concentration of the protein, and v) spray drying.

[0109] In some embodiments, to freeze-dry the composition, the composition is cooled until solidified and placed under vacuum to sublimate most of the volatile components in the composition.The solid residue may form a single mass that requires milling to form a fine powder.Typical freeze-dried powders contain porous irregularly shaped particles and are easily hydrated.Freeze-drying does not require intense heat, so it is used to produce powders that contain volatile components.In some embodiments, the silicone-replaced component is deep freeze-dried at a temperature of less than about -100°C.

[0110] After the formation of the silicone-replaced component, the crystallinity of the silicone-replaced component increases, which can strengthen the composition.In some embodiments, the silicone-replaced component remains the same or decreases.In some embodiments, the crystallinity index of the silicone-replaced component is 2% to 90% when measured by X-ray crystallography.In some other embodiments, the crystallinity index of the silicone-replaced component is at least 3%, at least 4%, at least 5%, at least 6%, or at least 7% when measured by X-ray crystallography.

[0111] In some embodiments of the present invention, the silicone-replaced component is a solid or film. In some embodiments, the silicone-replaced component is a powder. In some embodiments, the solid or film is substantially homogenous, meaning that the material has little or no inclusions or precipitates when examined by optical microscopy. In some embodiments, optical microscopy can be used to measure birefringence, which can be used as a proxy for alignment of the recombinant silk to a three-dimensional lattice. Birefringence is an optical property of a material that has a refractive index that depends on the polarization and propagation of light. Specifically, a high degree of axial order, as measured by birefringence, can be associated with high tensile strength. In some embodiments, recombinant silk solids and films have minimal birefringence. In various embodiments, the solid is a bead. In some other embodiments, the solid functions as an exfoliant. The recombinant silk solid may be in the form of a skin scrub that is gentle on the skin. In some embodiments, the material is in the form of a roll, pellet, sheet, or flake.

[0112] In some embodiments, the recombinant silk protein comprises a hollow core and / or shell. In some embodiments, the recombinant silk protein ranges in diameter from about 1 μm to about 30 μm, about 5 μm to about 20 μm, or about 10 μm to about 50 μm, and the recombinant silk protein in water ranges in diameter from about 20 to about 80 μm, about 30 μm to about 70 μm, or about 40 μm to about 100 μm. Prior to incorporation into the composition of the present disclosure, the recombinant silk protein hollow powder can be ground and incorporated as a ground powder.

[0113] solvent In some embodiments, the silicone-replaced component may include one or more solvents. For example, the recombinant silk polypeptide may be suspended in a solvent. The solvent may be an aqueous solvent, an alcohol, or an oil-based solvent. For example, the solvent may be one or more of water, glycerin, deionized water, olive oil, and pentylene glycol. For example, the recombinant silk polypeptide may be treated with a solvent such that the hollow core contains liquid water or a solvent such as glycerin, either in the form of liquid water itself, or as a liquid aqueous solution, as an emulsion containing liquid water, or as an aqueous dispersion. In some embodiments, the silicone-replaced component comprises about a 25% by weight solution in glycerin.

[0114] In some embodiments, the solvent is water. Without intending to be bound by theory, subjecting recombinant silk polypeptides to a solvent such as water results in an expanded or swollen recombinant silk polypeptide, with the protein acting as a carrier containing the solvent (e.g., water). These compositions can be stored in a dry state and can be partially rehydrated after immersion in water to directly form a liquid or semi-liquid aqueous suspension of swollen particles.

[0115] In some embodiments, the recombinant silk protein can expand a portion of the hollow core. In some other embodiments, the recombinant silk protein can expand a portion of the shell. In such embodiments where the solvent is water, the recombinant silk protein changes into a hydrogel. In other embodiments where the solvent is water, the recombinant silk protein changes into a paste. In various embodiments, heat and / or pressure can be applied to further process the recombinant silk protein composition.

[0116] In some embodiments, the solvent is generally present in a percentage ranging from 55-90% by weight based on the total weight of the recombinant silk polypeptide, including all specified values ​​and subranges therebetween, including 60%, 65%, 70%, 75%, 80%, and 85% by weight. In some embodiments, the recombinant silk protein is insoluble in a variety of solvents, including water, glycerin, alcohols, siloxanes, and oils at a variety of different pH levels.

[0117] In some embodiments, the solvent is of the aqueous type. In such embodiments, the solvent is water. The solvent may have a pH in the range of 6-12. In some embodiments, the solvent has a pH of 6. In some other embodiments, the solvent has a pH in the range of 0-5, 2-7, 4-9, 6-11, 8-13, or 10-14.

[0118] In other embodiments, the solvent comprises a mixture of various volatile organic solvents to obtain a relatively short drying time, hi some embodiments, the solvent is an alcohol.

[0119] Solvents include water, ethyl alcohol, toluene, methylene chloride, isopropanol, n-butyl alcohol, castor oil, organopolysiloxane oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethylsulfoxide, dimethylformamide, and tetrahydrofuran.

[0120] In some embodiments, the organopolysiloxane oil can be volatile, non-volatile, or a mixture of volatile and non-volatile silicones. The term "non-volatile" as used in this context refers to silicones that are liquid under ambient conditions and have a flash point (at one atmospheric pressure) above about 100°C. The term "volatile" as used in this context refers to all other silicone oils. Suitable organopolysiloxanes can be selected from a wide variety of silicones that span a wide range of volatility and viscosity. Suitable silicones are disclosed in U.S. Patent No. 5,069,897, issued December 3, 1991, which is incorporated herein by reference in its entirety. Examples of suitable organopolysiloxanes include, but are not limited to, polyalkylsiloxanes, alkyl-substituted dimethicones, dimethiconols, polyalkylarylsiloxanes, and mixtures thereof. For example, polyalkylsiloxanes, dimethicones, and cyclomethicones can be used.

[0121] In some embodiments, the solvent is vegetable oil and hydrogenated vegetable oil. In some embodiments, the solvent is free fatty acid. Examples of vegetable oil and hydrogenated vegetable oil include safflower oil, castor oil, coconut oil, cottonseed oil, menhaden oil, palm kernel oil, palm oil, peanut oil, soybean oil, rapeseed oil, linseed oil, rice bran oil, pine oil, sesame oil, sunflower seed oil, partially and fully hydrogenated oils from the aforementioned sources, and mixtures thereof. Animal fats and oils, such as cod liver oil, lanolin and its derivatives, such as acetylated lanolin and isopropyl lanolate, can be used. Polypropylene glycol C4-C 20 Alkyl ether, polypropylene glycol C1-C 20 Carboxylic acid esters and di-C8-C 30 Alkyl ethers are also useful and examples include PPG-14 butyl ether, PPG-15 stearyl ether, dioctyl ether, dodecyl octyl ether, and mixtures thereof.

[0122] The compositions of the present invention may be substantially free of semi-solid hydrocarbons such as petrolatum, lanolin and lanolin derivatives, sterols (e.g., ethoxylated soy sterols), high molecular weight polybutenes, and cocoa butter. As used herein, "substantially free" means that the concentration of semi-solid hydrocarbons is less than 10%, or less than 5%, or less than 2%, or 0%.

[0123] Recombinant silk proteins as cosmetic formulations In various embodiments, the recombinant silk protein is incorporated into a silk cosmetic or skin care product (e.g., a solution applied to the skin or hair). Specifically, the recombinant silk protein can be incorporated into a silicone-replaced component used as a base for a cosmetic or skin care product in which the recombinant silk polypeptide is present in the base in its monomeric or low-crystalline form. In some embodiments, the silicone-replaced component can be used as a base for a cosmetic or skin care product in which the recombinant silk polypeptide is present in the base in its semi-crystalline form. In such an embodiment, the recombinant silk polypeptide is not present in the base in its monomeric form.

[0124] In most embodiments, the cosmetic formulation is physically stable. In such embodiments, the recombinant silk protein and any other ingredients remain in the formulation for an extended period of time and have a long shelf life. In extended use, the silicone-replaced components remain substantially stable and the ingredients do not precipitate from the formulation.

[0125] The compositions of the present invention can be used to apply silk proteins to the skin, nails, hair or mucous membranes of a subject by contacting the composition with the skin, nails, hair or mucous membranes of the subject. Preferably, the compositions of the present invention are used on human subjects.

[0126] In most embodiments, the cosmetic formulations are non-toxic or non-allergenic to the host to which the cosmetic is applied. It is also desirable in the art to produce cosmetic compositions for hair and epidermal contact that do not permanently stain tissue and can be removed by normal washing with an aqueous detergent.

[0127] The solids, films, emulsions, hydrogels, and other material forms discussed in various embodiments may contain various humectants, emollients, occlusive agents, active agents, and cosmetic adjuvants, depending on the embodiment and the desired efficacy of the formulation. In some embodiments, recombinant silk protein functions as a carrier. In some embodiments, recombinant silk protein is a carrier that delivers one or more agents to a surface, such as skin, hair, or nails.

[0128] In some embodiments, the cosmetic formulation includes a plasticizer. Suitable concentrations of the plasticizer in the composition range, for example, from 1 to 60%, 10 to 60%, 10 to 50%, 10 to 40%, 15 to 40%, 10 to 30%, or 15 to 30% by weight. In some embodiments, the plasticizer is glycerol. In some embodiments, the plasticizer is triethanolamine, trimethylene glycol, polyethylene glycol, propylene glycol, sorbitol, sucrose, saturated fatty acids, or unsaturated fatty acids.

[0129] When water is used as a plasticizer, suitable concentrations of water in the composition range, by weight, for example, from 5 to 80%, 15 to 70%, 20 to 60%, 25 to 50%, 19 to 43%, or 19 to 27%. When water is used in combination with another plasticizer, water may be present, for example, in the range of 5 to 50%, 15 to 43%, or 19 to 27%.

[0130] In some embodiments, suitable plasticizers may include polyols (e.g., glycerol), water, lactic acid, ascorbic acid, phosphoric acid, ethylene glycol, propylene glycol, triethanolamine, acid acetate, propane-1,3-diol, or any combination thereof. In various embodiments, the amount of plasticizer may vary depending on the purity and relative composition of the recombinant silk protein. For example, powders with higher purity may have fewer impurities, such as low molecular weight compounds that can act as plasticizers, and therefore may require the addition of a higher weight percentage of plasticizer.

[0131] In some embodiments, the composition comprises a humectant or emollient. The term "humectant" as used herein refers to a hygroscopic substance that forms bonds with water molecules. Suitable humectants include, but are not limited to, glycerol, propylene glycol, polyethylene glycol, pentalyene glycol, tremella extract, sorbitol, dicyanamide, sodium lactate, hyaluronic acid, aloe vera extract, alpha hydroxy acid, and pyrrolidone carboxylate (NaPCA).

[0132] The term "emollient" as used herein refers to a compound that fills cracks on the surface of the skin, thereby providing the skin with a soft or supple appearance.Suitable emollients include, but are not limited to, shea butter, cocoa butter, squalene, squalane, octyl octanoate, sesame oil, grape seed oil, natural oils that contain oleic acid (e.g., sweet almond oil, argan oil, olive oil, avocado oil), natural oils that contain gamma linoleic acid (e.g., evening primrose oil, borage oil), natural oils that contain linoleic acid (e.g., safflower oil, sunflower oil), or any combination thereof.

[0133] In some instances, the emollient or moisturizing agent may be an occlusive agent, and the present disclosure contemplates the inclusion of an occlusive agent in the composition in various embodiments. The term "occlusive agent" refers to a compound that forms a barrier on the skin surface to retain moisture. Other suitable occlusive agents may include, but are not limited to, beeswax, canuba wax, ceramides, vegetable waxes, lecithin, and allantoin. Without intending to be bound by theory, the film-forming ability of the silicone-replaced components presented herein creates an occlusive agent that forms a moisture-retaining barrier, since the recombinant silk polypeptide attracts water molecules and also acts as a moisturizing agent.

[0134] Optionally, the cosmetic formulation includes an active agent. The term "active agent" refers to any compound with a known beneficial effect in hair care, skin care, or cosmetic formulations, including pigments in cosmetic formulations. Various active agents may include, but are not limited to, acetic acid (i.e., vitamin C), alpha hydroxyl acid, beta hydroxyl acid, zinc oxide, titanium dioxide, retinol, niacinamide, other recombinant proteins (either as full-length sequences or hydrolyzed into partial sequences or "peptides"), copper peptides, curcuminoids, glycolic acid, hydroquinone, kojic acid, l-ascorbic acid, alpha lipoic acid, azelaic acid, lactic acid, ferulic acid, mandelic acid, dimethylaminoethanol (DMAE), resveratrol, natural extracts containing antioxidants (e.g., green tea extract, pine tree extract), caffeine, alpha arbutin, coenzyme Q-10, and salicylic acid.

[0135] The term "cosmetic adjuvant" refers to a variety of other agents used to create cosmetic products with commercially desirable properties, including, but not limited to, surfactants, emulsifiers, preservatives, and thickeners.

[0136] As described herein, in various embodiments, the recombinant silk protein may form a dispersible semi-solid or gel-like structure. In various embodiments in which the recombinant silk protein is incorporated into a skin care formulation, the recombinant silk protein may form an irreversible three-dimensional structure, such as a gel or film, that transforms into a dispersible liquid on the surface of the skin.

[0137] In various embodiments, recombinant silk protein may be suspended in water ("aqueous suspension protein") to form a silicone-replaced component in the form of a film, gel, or base that may be incorporated (i.e., formulated) into a cosmetic or skin care formulation. Depending on the embodiment, the amount of recombinant silk protein to water in the aqueous suspension protein may vary, and the relative ratio of recombinant silk polypeptide powder to additive in the recombinant silk protein may also vary. In some embodiments, the silicone-replaced component comprises 10-33% by weight of recombinant silk polypeptide powder. In some embodiments, a solvent other than water is used. In some embodiments, the recombinant silk protein is suspended in water to create an aqueous suspension protein that is 1-40% recombinant silk protein and 60-99% water. In certain embodiments, the silicone-replaced component is suspended in water to create an aqueous suspension protein that is 10% by weight of recombinant silk polypeptide powder, 30% by weight of additive, and 60% by weight of water, based on the total weight of the silicone-replaced component. In certain embodiments, the protein is suspended in water to make an aqueous suspended protein that is 6% by weight recombinant silk polypeptide powder, 18% by weight additives, and 76% by weight water, based on the total weight of the silicone-substituted components. In certain embodiments, the protein is suspended in water to make an aqueous suspended protein that is 10% by weight recombinant silk polypeptide powder and 90% by weight water, based on the total weight of the silicone-substituted components.

[0138] Depending on the embodiment, the aqueous suspension protein may be optionally heated and stirred when resuspended in water. In some embodiments, heating and stirring the aqueous suspension protein may cause a phase transition of the recombinant silk polypeptide in the aqueous suspension protein. Specifically, heating and stirring the aqueous suspension protein results in three distinct phases that are evaluated by centrifugation: 1) a gel phase that is separate from the supernatant after centrifugation, 2) a colloid phase that may be filtered from the supernatant after centrifugation, and 3) a solution phase that remains after filtering the colloid phase from the supernatant. Various combinations of heating, stirring and centrifugation can be used, provided that the aqueous suspension protein should not be subjected to heating for long periods of time to prevent degradation of the recombinant silk polypeptide. In certain embodiments, the protein is gently stirred at 90°C for 5 minutes and centrifuged at 16,000 RCF for 30 minutes.

[0139] In various embodiments, any of the various phases of the aqueous suspension protein (i.e., colloid phase, gel phase, and solution) or the aqueous suspension protein may be incorporated into a cosmetic or skin care formulation to provide a source of recombinant silk protein. Depending on the embodiment, the aqueous suspension protein may be subjected to agitation with or without heating prior to incorporation into the skin care formulation. Optionally, the aqueous suspension protein may be separated into the above phases by centrifugation and / or filtration. Depending on the embodiment, the skin care formulation may be an emulsion (e.g., a cream or serum) or a predominantly aqueous solution (e.g., a gel). In certain embodiments, the recombinant silk protein may be incorporated into any of the cosmetic, skin care, or hair care formulations described herein without aqueous resuspension. In these compositions, a homogenizer or similar device may be used to ensure that the recombinant silk protein is uniformly distributed in the composition.

[0140] In some embodiments, the aqueous suspension protein can be subjected to heating and agitation and then cast onto a flat surface and dried into a film. In some embodiments, the aqueous suspension protein can be cast onto a flat surface and dried into a film without being subjected to heating and / or agitation. In such embodiments, the aqueous suspension protein can be cast onto a flat surface and dried into a film without being subjected to additional processing. In some embodiments, the aqueous suspension protein can be incorporated into an emulsion and then cast onto a flat surface and dried into a film. Depending on the embodiment, a variety of different drying conditions can be used. Suitable drying conditions include drying at 60° C. or 80° C. with and without vacuum. In embodiments using vacuum, 15 Hg is a suitable amount of vacuum. Other drying methods are well established in the art.

[0141] In various embodiments, the film containing only the aqueous suspension protein has a low melting temperature. In various embodiments, the film containing only the aqueous suspension protein has a melting temperature lower than body temperature (about 34-36°C) and melts on contact with the skin. Without intending to be bound by theory, the recombinant silk polypeptide forms sufficient intermolecular interactions to create a semi-solid structure (i.e., a film), but this structure is reversible on skin contact and can reform after dispersion on the skin surface. In various embodiments, the film has reduced crystallinity compared to recombinant silk protein or recombinant silk powder as measured by Fourier transform infrared spectroscopy (FTIR). In various embodiments, the film containing the aqueous suspension protein does not melt on contact with the skin. In such embodiments, the film functions as a barrier. In various embodiments, the film is a low density hydrophobic film. The film or barrier can range from about 1 μm to about 50 μm in thickness, about 10 μm to about 30 μm, or about 20 μm to about 40 μm in thickness. Upon contact with the skin, a barrier is formed on the surface of the epidermal layer, providing strong non-specific adhesion to the skin surface, In some embodiments, the thickness of the film varies depending on the concentration of recombinant silk protein and the applied surface area.

[0142] In some embodiments, the barrier is long-lasting and provides protection against one or more environmental stressors including wind, humidity, harsh additives, pollution, abrasion, dirt, and grease, and can withstand wear equivalent to at least 100 rubs, at least 200 rubs, at least 400 rubs, at least 600 rubs, or at least 800 rubs by hand.

[0143] In one particular embodiment, an aqueous suspension of protein or proteins can be incorporated into an emulsion (e.g., homogenized) and then cast onto a flat surface and freeze-dried to create a porous film. Depending on the embodiment, various techniques can be used for freeze-drying, including freezing the film at -80°C for 30 minutes. Other freeze-drying techniques are known to those skilled in the art.

[0144] In various embodiments, the above-mentioned film can be used as a topical skin care agent. The film can be applied directly to the skin and rehydrated to form a dispersible viscous material that is incorporated into the skin. As discussed herein, various emollients, humectants, active agents, and other cosmetic adjuvants can be incorporated into the film. The film can be applied directly to the skin and adsorbed to the skin by contact with the skin or after lightly rubbing the film into the skin. In some embodiments, the film can be applied directly to the skin and adsorbed to the skin without additional friction or contact. In some embodiments, the protein resuspended in an aqueous solution can be applied to the face and then exposed to a coagulant such as propylene glycol via a mist to form a gellable mask.

[0145] Depending on the embodiment, the cast film may be a flat film (i.e., without surface irregularities) or may be cast onto a mold incorporating microstructures, in certain embodiments, a film cast onto a mold incorporating microneedle structures that pierce the surface of the skin and aid in the delivery of the active agent.

[0146] In another embodiment, the aqueous suspension protein can be added to an emulsion that is used as a cosmetic product.The emulsion can be applied to skin or hair, and then can form a film on the surface of the skin when dried.As discussed herein, various emollients, moisturizers, active agents, and other cosmetic adjuvants can be incorporated into the emulsion.

[0147] In some embodiments, the compositions of the present disclosure may be liquid or semi-solid, such as creams, lotions, and gels. The compositions useful in the present invention may be in a wide variety of product forms known in the art. These include, but are not limited to, powders, lotions, creams, gels, patches, serums, ampoules, powders, sticks, sprays, ointments, pastes, mousses, ointments, liquids, emulsions, foams, or aerosols. These product forms may contain several types of additives, including, but not limited to, solutions, aerosols, emulsions, gels, solids, and liposomes, as further discussed herein. The compounds active in the compositions and methods of the present invention may be delivered topically by any means known to those skilled in the art.

[0148] In some other embodiments, the composition may be a basic cosmetic composition, for example, a facial cleanser such as lotion, cream, essence, cleansing foam, and cleansing water; a pack and body oil; a color cosmetic composition such as foundation, lipstick, mascara, and makeup base; a hair product composition such as shampoo, rinse, hair conditioner, and hair gel; a soap, etc. The cosmetic formulation may be prepared by any method known in the art using the silicone-substituted components described herein, optionally together with at least one carrier and / or additive commonly used in the art of preparing cosmetic compositions.

[0149] In some embodiments, the composition comprises at least one cosmetic agent. Examples of cosmetic agents include emollients, humectants, colorants, pigments, fragrances, moisturizers, viscosity adjusters, and any other cosmetic formulations. One or more cosmetic agents can be included in the cosmetic composition. In another embodiment, additional active ingredients known in the art and described herein can also be used, including, but not limited to, skin softeners, skin penetration enhancers, colorants, fragrances, emulsifiers, and thickeners. The cosmetic composition can also further comprise fragrances, pigments, germicides, antioxidants, preservatives, and / or moisturizers, as well as inorganic salts and synthetic polymeric materials, for example, to improve physical properties.

[0150] The composition can also be delivered topically through lotion.Single emulsion skin care preparations, such as oil-in-water and water-in-oil lotions and creams, are well known in the cosmetic field and are useful in the present invention.Multi-phase emulsion compositions, such as water-in-oil-in-water, are also useful in the present invention.In general, such single or multi-phase emulsions contain water, emollients, and emulsifiers as essential components.

[0151] The compositions of the present invention can also be formulated into solid formulations (eg, wax-based sticks, bar soap compositions, powders, beads, exfoliants, or wipes containing liquids or powders).

[0152] The compositions of the present invention can be formulated as gels (e.g., aqueous gels using a suitable gelling agent). Suitable gelling agents for aqueous gels include, but are not limited to, natural gums, acrylic acid and acrylate polymers and copolymers, and cellulose derivatives (e.g., hydroxymethylcellulose and hydroxypropylcellulose). Suitable gelling agents for oils (such as mineral oil) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gels typically contain about 0.1% to 5% by weight of such gelling agents. In some embodiments, such compositions include, but are not limited to, recombinant silk protein, water (Aqua), sodium C14-16 olefin sulfonate, glycerin, cocoa betaine, sodium benzoate, sodium hydroxide, calcium gluconate, sodium hyaluronate, propanediol, xanthan gum, gluconolactone, and tetrasodium glutamate diacetate. In some embodiments, the composition comprises a cleansing detergent, soap, serum, or toner. In certain embodiments, the serum is aqueous based, hi another particular embodiment, the toner is alcohol based.

[0153] Compositions useful in the present invention may be formulated as emulsions. When the composition is an emulsion, in some embodiments, about 1% to about 10% or about 2% to about 5% of the composition comprises an emulsifier. The emulsifier may be nonionic, anionic, or cationic. Suitable emulsifiers are disclosed, for example, in the INCI Handbook, pp. 1673-1686. Lotions and creams may be formulated as emulsions. In some embodiments, the composition is an emulsion and the recombinant silk protein is the emulsifier. In some embodiments, the composition is an emulsion and the recombinant silk protein is the emulsifier, and the composition does not include other emulsifiers.

[0154] Yet another type of composition may be an ointment. An ointment may contain a simple base of animal or vegetable oils or semisolid hydrocarbons. An ointment may contain about 0.1% to about 2% of a thickening agent, as well as about 2% to about 10% of an emollient. Examples of thickening agents include, for example, cellulose derivatives (methylcellulose and hydroxypropylmethylcellulose), synthetic high molecular weight polymers (e.g., carboxyvinyl polymers and polyvinyl alcohol), plant hydrocolloids (e.g., karaya gum and tragacanth gum), clay thickeners (e.g., colloidal magnesium aluminum silicate and bentonite), carboxyvinyl polymers, carboxylic acid polymers, crosslinked polyacrylates, polyacrylamides, xanthan gum, and mixtures thereof.

[0155] In addition to the components described above, the compositions useful in the present invention may contain a wide variety of additional oil-soluble and / or water-soluble materials, at art-established levels, conventionally used in compositions for use on the skin, hair, and nails.

[0156] The compositions of the present invention may be applied directly to the skin or to other delivery devices such as wipes, sponges, brushes, etc. The compositions may be used in products designed to be left on the skin, wiped off the skin, or washed off the skin.

[0157] In some embodiments, the composition improves the appearance of the skin, such as increasing skin firmness / plumpness, increasing elasticity, improving overall skin health, increasing hydration, promoting and / or improving wound healing, improving pollution defense, reducing dermatological aging, reducing skin fragility, preventing and reversing collagen and / or elastin loss, preventing skin atrophy, promoting / accelerating cell turnover, increasing gene expression, improving skin texture, preventing and reducing fine lines and wrinkles, improving skin tone, increasing skin thickness, reducing pore size, minimizing skin discoloration, restoring skin radiance, minimizing signs of fatigue, improving skin barrier function, minimizing skin dryness, preventing, reducing, or treating hyperpigmentation, improving skin mitochondrial function, improving exfoliation, reducing toxicity, mattifying the skin, reducing oxidative stress levels, reducing pollution-induced oxidative stress, reducing UVA or UVB-induced oxidative stress, or any combination thereof.

[0158] The compositions of various embodiments provide protection against pollutants and other irritants, such that many skin conditions, such as acne, redness associated with rosacea (adult acne), and other inflammatory conditions, can be proactively managed by application of cosmetic formulations.

[0159] Coagulant In some embodiments, the composition as described herein and / or recombinant silk polypeptide containing silicone-substituted components are exposed to a coagulant. This can change the properties of the composition / component to facilitate controlled aggregation of silk in silk-based compositions. In some embodiments, the composition / component is immersed in the coagulant. In some embodiments, the composition / component is exposed to a coagulant mist or vapor. In one embodiment, the aqueous protein composition contains, is immersed in, or is mixed with a coagulant. In some embodiments, a silk-based solid or semi-solid, such as a film, is immersed in or exposed to a vapor containing the coagulant. In some embodiments, methanol is used as an effective coagulant.

[0160] In some embodiments, alcohol (e.g., isopropanol, ethanol, or methanol) may be used as the coagulant or solvent. In some embodiments, 60%, 70%, 80%, 90%, or 100% alcohol is used as the coagulant. In some embodiments, a salt may be used as the coagulant. Examples of salts include, but are not limited to, ammonium sulfate, sodium chloride, sodium sulfate, and other protein precipitating salts that are effective at temperatures between 20-60°C.

[0161] In some embodiments, a combination of one or more of water, acids, solvents, and salts can be used as the coagulant, including but not limited to the following chemical classes: Bronsted-Lowry acids, Lewis acids, binary hydride acids, organic acids, metal cation acids, organic solvents, inorganic solvents, alkali metal salts, and alkaline earth metal salts. In some embodiments, the acid comprises dilute hydrochloric acid, dilute sulfuric acid, formic acid, or acetic acid. In some embodiments, the solvent comprises ethanol, methanol, isopropanol, t-butyl alcohol, ethyl acetate, propylene glycol, or ethylene glycol. In some embodiments, the salt comprises LiCl, KCl, BeCl2, MgCl2, CaCl2, NaCl, ZnCl2, FeCl3, ammonium sulfate, sodium sulfate, sodium acetate, or other salts of nitric acid, sulfuric acid, or phosphoric acid. In some embodiments, the coagulant has a pH of 2.5 to 7.5.

[0162] Other Additives In some embodiments, the composition according to the present disclosure and / or its silicone-substituted components may include one or more additives. This can change the properties of the composition when interacting with the skin. In some embodiments, the silk-based composition is immersed in the additive. In some embodiments, the composition / component is exposed to a mist or vapor of the additive. In one embodiment, the aqueous protein composition contains the additive, is immersed in the additive, or is mixed with the additive. In some embodiments, a silk-based solid or semi-solid, such as a film, is immersed or exposed to a vapor containing the additive. In some embodiments, the silk-based gel is exposed to the additive prior to hollow powder formation (e.g., the silk-based gel and the additive are co-spray dried together).

[0163] The additives may be inert by themselves or may have their own dermatological benefits. The additives should also be physically and chemically compatible with the essential components described herein and should not unduly impair the stability, efficacy, or other use benefits associated with the compositions of the present invention. The type of additives utilized in the present invention depends on the type of product form desired for the composition. In some embodiments, the additive is an acid fiber dye.

[0164] Pigments are frequently added to cosmetic formulations to achieve the desired color for application to the skin. Such pigments are known and the concentrations required to achieve the desired coloration can be readily determined. Pigments can be inorganic or organic. Inorganic pigments include iron oxides (red, black, brown), manganese violet, ultramarine (green, blue, pink, red, or purple aluminum sulfate), aquamarine, copper powder, mica, clay, silica, and titanium dioxide. Organic dyes approved for cosmetic use by the U.S. FDA generally have the prefix "D&C" and a color and number suffix (e.g., D&C Green No. 3).

[0165] Certain embodiments of the present invention contain about 0% to about 30%, about 1% to about 20%, about 2% to about 15%, or about 5% to about 15% colorant based on anhydrous pigment weight. These are typically aluminum, barium, calcium salts or lakes. Dyes may be present at concentrations of about 0% to about 3%, pearlescent agents, etc. may be present at concentrations of 0% to about 10%. Such dyes in combination with recombinant silk proteins are stable and have a long shelf life. The shelf life of such compositions may be about 6 months, about 1 year, or about 2 years. In some embodiments, the shelf life of such compositions may be at least 5 years.

[0166] There is no particular limitation regarding the pigment, colorant, or filler powder used in the composition. Each may be an extender pigment, an inorganic white pigment, an inorganic color pigment, a pearling agent, etc. Specific examples include talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silica, titanium dioxide, zinc oxide, red iron oxide, yellow iron oxide, black iron oxide, ultramarine, polyethylene powder, methacrylate powder, polystyrene powder, silk powder, crystalline cellulose, starch, titanium mica, titanium iron oxide mica, bismuth oxychloride, etc.

[0167] Additional pigments / powder fillers include inorganic powders such as gums, chalk, fuller's earth, kaolin, sericite, muscovite, phlogopite, synthetic mica, lepidolite, biotite, lithia mica, vermiculite, aluminum silicate, starch, smectite clays, alkyl and / or trialkylarylammonium smectites, chemically modified magnesium aluminum silicate, organically modified montmorillonite clays, hydrated aluminum silicate, fumed aluminum starch octenyl succinate barium silicate, calcium silicate, magnesium silicate, strontium silicate, metal tungstates, magnesium, silica alumina, zeolites, barium sulfate, calcined calcium sulfate (calcined gypsum), calcium phosphate, fluoroapa Examples of suitable white pigments include, but are not limited to, tite, hydroxyapatite, ceramic powders, metal soaps (zinc stearate, magnesium stearate, zinc myristate, calcium palmitate, and aluminum stearate), colloidal silicon dioxide, and boron nitride; organic powders such as polyamide resin powders (nylon powders), cyclodextrin, polymethylmethacrylate powders, styrene and acrylic acid copolymer powders, benzoguanamine resin powders, poly(tetrafluoroethylene) powders, and carboxyvinyl polymers; cellulose powders such as hydroxyethylcellulose and sodium carboxymethylcellulose; ethylene glycol monostearate; and inorganic white pigments such as magnesium oxide. Other useful powders are disclosed in U.S. Patent No. 5,688,831, issued Nov. 18, 1997 to El-Nokaly et al., which is incorporated herein by reference in its entirety. These pigments and powders can be used independently or in combination.

[0168] In addition to silk proteins, the compositions according to the invention may further comprise a film-forming material, for example, cellulose derivatives, nitrocellulose, acrylic polymers or copolymers, acrylic, styrene, acrylate-styrene and vinyl resins, vinyl copolymers, polyester polymers, arylsulfonamide resins, and alkyd resins.

[0169] In some embodiments, the composition may include an amphoteric surfactant, a phospholipid, or a wax.

[0170] Examples of other additives include cannabidiol, foaming surfactants, depigmenting agents, reflective agents, detangling / wet combing agents, amino acids and their derivatives, antibacterial agents, allergy suppressants, anti-acne agents, anti-aging agents, anti-wrinkle agents, bactericides, analgesics, cough suppressants, antipruritics, local anesthetics, hair loss prevention agents, hair growth promoters, hair growth inhibitors, antihistamines, anti-infective agents, anti-inflammatory agents, antiemetics, anticholinergics, vasoconstrictors, vasodilators, wound healing promoters, peptides, polypeptides and proteins, deodorants and antiperspirants, pharmaceuticals, skin emollients and moisturizers, skin tightening agents, hair care agents, hair conditioners ... These include, but are not limited to, conditioners, hair softeners, hair moisturizers, vitamins, tanning agents, skin lightening agents, anti-fungal agents, depilatories, shaving preparations, topical analgesics, perfumes, anti-irritants, hemorrhoidal agents, insecticides, poison ivy products, poison sumac products, burn medications, diaper rash prevention agents, heat rash agents, makeup preparations, vitamins, herbal extracts, retinoids, flavenoids, sensates, antioxidants, skin conditioners, hair lighteners, chelating agents, cell turnover enhancers, sunscreens, anti-edema agents, collagen enhancers, and mixtures thereof.

[0171] Examples of suitable vitamins non-exclusively include B complex vitamins, including thiamine, nicotinic acid, biotin, pantothenic acid, choline, riboflavin, vitamin B6, vitamin B12, pyridoxine, inositol, camitin; vitamins A, C, D, E, K, and their derivatives, such as vitamin A palmitate and provitamins (e.g., panthenol (provitamin B5) and panthenol triacetate), and mixtures thereof.

[0172] Examples of sunscreens include, but are not limited to, avobenzone, benzophenone, bornelone, butyl paba, cinnamidopropyl trimethyl ammonium chloride, disodium distyryl biphenyl disulfonate, paba, potassium methoxycinnamate, butyl methoxydibenzoylmethane, octyl methoxycinnamate, oxybenzone, octocrylene, octyl salicylate, phenylbenzimidazole sulfonic acid, ethyl hydroxypropyl aminobenzoate, menthyl anthranilate, aminobenzoic acid, cinoxate, diethanolamine methoxycinnamate, glyceryl aminobenzoate, titanium dioxide, zinc oxide, oxybenzone, padimate O, red petrolatum, and mixtures thereof.

[0173] The amount of additive combined with the composition may vary depending, for example, on the ability of the additive to penetrate the skin, hair, or nails, the particular additive selected, the particular benefit desired, the user's sensitivity to the additive, the user's health, age, and condition of the skin, hair, and / or nails, etc. In short, the additive is used in a "safe and effective amount," which is high enough to provide the desired skin, hair, or nail benefit or correct the particular condition being treated, with a reasonable risk-to-benefit ratio within the bounds of sound medical judgment, yet low enough to avoid significant side effects.

[0174] The invention illustratively disclosed herein may suitably be practiced in the absence of any component, ingredient, or step not specifically disclosed herein. In order to further illustrate the nature of the invention and the manner of practicing it, some examples are given below. However, the invention should not be considered as being limited to the details thereof.

[0175] The compositions and methods of the present invention provide equivalent or better performance to the skin in terms of softness, fast absorption, easy spreadability (or "playtime"), lightweight film formation, and non-greasy feel compared to compositions containing silicone elastomers. Additionally, the present invention provides equivalent or better performance in terms of low white cast when the skin has been treated with an SPF composition. The compositions and methods of the present invention provide equivalent or better performance to the hair in terms of long wear, shine, non-greasy feel, frizz control, added density to hair, styling hold, static properties, heat resistance, and UV radiation and pollution protection.

[0176] Equivalence and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. The scope of the present invention is not limited to the above description, but rather is as set forth in the accompanying aspects.

[0177] In aspects, articles such as "a," "an," and "the" can mean one, or more than one, unless the contrary is indicated or otherwise clear from the context. An aspect or description between "or" members that includes one or more members of a group is considered to be satisfied if one, more than one, or all of the group members are present, employed, or otherwise relevant in a given product or process, unless the contrary is indicated or otherwise clear from the context. The invention includes embodiments in which exactly one member of a group is present, employed, or otherwise relevant in a given product or process. The invention includes embodiments in which two or more, or all of the group members are present, employed, or otherwise relevant in a given product or process.

[0178] It should also be noted that the term "comprising" is intended to be open, allowing, but not requiring, the inclusion of additional elements or steps. Thus, when the term "comprising" is used herein, the terms "consisting of" and "consisting essentially of" are also included and disclosed.

[0179] Where ranges are given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise clear from the context and the understanding of one of ordinary skill in the art, it should be understood that values ​​expressed as ranges can assume any specific value or subrange within the ranges described in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0180] All cited sources, e.g., references, publications, databases, database entries, and techniques cited herein, are incorporated by reference into this application, even if not explicitly stated in the citation. In the event of a conflict between the statements of a cited source and this application, the statements of this application shall take precedence.

[0181] The section and table headings are not intended to be limiting. EXAMPLES

[0182] The following are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., amounts, temperatures, etc.), but it is to be understood that some experimental error and deviation should be allowed for.

[0183] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of the art. Such techniques are fully explained in the literature. See, for example, TECreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); ALLehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0184] Example 1: Recombinant Silk Polypeptides as a Replacement for Silicones in SPF Formulations This example shows that recombinant silk polypeptide is comparable to and superior to silicone (i.e., blend of linear silicone and cyclosiloxane) in certain characteristics of SPF skin care products.A mineral SPF formulation originally containing 2% combined linear silicone and cyclosiloxane (1% of each component) was reformulated with 1.5% recombinant silk polypeptide and evaluated for its aesthetic profile.Recombinant silk polypeptide was preferred to silicone formulation in blind comparison of white cast, absorption, filminess, and oiliness, and was comparable to silicone formulation in playtime and softness performance.

[0185] Methodology: The SPF formulation was prepared as per standard techniques in the art. The water phase ingredients were mixed separately and the oil phase ingredients were mixed separately. The two were combined by gentle mixing at an elevated temperature of 80-85°C. The ingredients were continuously mixed until uniform and then cooled to 30-45°C. After the water and oil phases were mixed and cooled to 30-45°C, 18B recombinant silk polypeptide comprising SEQ ID NO: 2878 was added to the formulation. The formulation was continuously mixed while cooling to room temperature. Water was used to make up for the difference in weight percentage of recombinant silk polypeptide and silicone elastomer between the formulations. For this formulation, the powder form of the recombinant silk polypeptide was used.

[0186] A spider chart was prepared in a blind comparative study, where subjects were asked to compare the two formulations on a scale of 1 to 5 for six different attributes (key: 1=unfavorable, 2=poor, 3=satisfactory, 4=good, 5=excellent). Ten different individuals tested the products on their faces over a period of one week. Skin types were different, such as oily, dry, and combination.

[0187] FIG. 1A illustrates a top-down view of an SPF formulation.

[0188] Figure 1B shows a spider chart plotting data from a blinded study comparison of 10 subjects. The 1.5% recombinant silk polypeptide formulation outperformed the 2% silicone formulation in terms of desirable white cast, absorption, filminess, and greasiness. The 1.5% recombinant silk polypeptide formulation performed similarly to the 2% silicone formulation in terms of spreadability and softness.

[0189] FIG. 1C: Table of ingredients used in SPF formulations (excluding recombinant silk polypeptides and silicones).

[0190] FIG. 1D: The SPF formulation containing 1.5% recombinant silk polypeptide outperformed the 2% silicone formulation in terms of desirable white cast, absorption, filminess, and greasiness, but the rheological data shows similarity between the three formulations. Frequency sweeps show similar magnitude and shape of the G' and G" curves. This data supports that the SPF formulations have similar gel-like structure characteristics.

[0191] Figure 1E: Optical microscope images of SPF base and SPF base formulated with 1% recombinant silk polypeptide protein or 5% silicone elastomer components. The SPF base and 5% silicone elastomer samples show typical dispersion morphology with more transparent areas and other areas that are opaque. The 1% recombinant silk polypeptide sample clearly has the visible presence of particles that are hollow, 2-200 μm in diameter, and appear as bright circles. White arrows have been added to the figure to point to exemplary silk powder particles. A reference image of 1% recombinant silk polypeptide powder suspended in water is included.

[0192] Example 2: Recombinant silk polypeptides as a replacement for silicones in color cosmetic products Recombinant silk polypeptides are comparable to and superior to silicone elastomers in certain characteristics of color cosmetic products. Eye / cheek / lip tinted 3-in-1 cream formulations were prepared using either 1% recombinant silk polypeptide or 5% or 10% silicone elastomer and evaluated for their aesthetic profile. The recombinant silk polypeptide formulations outperformed silicone elastomers, providing improved spreadability, as well as uniform pigment delivery and soft dry-down (i.e., not sticky or greasy like the silicone elastomer versions) and were also wipe-resistant.

[0193] Method: Color cosmetic formulations were prepared similar to standard techniques in the art. All waxes, oils, and solvents were either at room temperature or separately at elevated temperatures, e.g., above 37°C but below 90°C. Oils and solvents were mixed with pigments and lightly ground. Waxes were then added to the mixture and cooled. b-Silk protein (i.e., 18B recombinant silk polypeptide containing SEQ ID NO:2878) and / or silicone elastomer components can be added at any stage of the process. Water was used to make up the difference in weight percentage of recombinant silk polypeptide and silicone elastomer between formulations. This formulation used recombinant silk polypeptide in powder form and silicone elastomer component with approximately 30% solids.

[0194] The pigmentation and wiping test included applying 10 mg of product to an area of ​​1 cm x 1 cm and allowing the product to dry for 15 minutes. To test the wiping, a white tissue was placed on the skin and a 200 g bolus was placed on the skin. The tissue was pulled over the skin three times and the wiping characteristics were visually evaluated.

[0195] Rheological measurements were performed using a Kinexus Lab+ rheometer using a 2 degree / 20 mm cone and plate geometry. To measure the G' and G" of the materials, a frequency sweep was performed at 0.1% strain rate. The onset frequency was 100 s -1 and the end frequency is 0.01s -1 It was.

[0196] FIG. 2A: A top-down view of a color cosmetic formulation.

[0197] FIG. 2B: Step-by-step visualization of how color cosmetics were assessed for pigment delivery and firmness on the skin.

[0198] Figure 2C: Representative images of product color application and wiping. Compared to the 5% and 10% elastomeric products, the recombinant silk polypeptide had pigments that spread more evenly on the skin and adhered more firmly to the skin during wiping.

[0199] FIG. 2D: Table of ingredients used in color cosmetic formulations (excluding recombinant silk polypeptides and silicones).

[0200] Figure 2E: Optical microscope images of color cosmetic base and base formulated with 1% b-silk protein or 5% silicone elastomer components. The SPF base and 5% silicone elastomer samples show typical dispersion morphology with more transparent areas and other areas that are opaque. The 1% recombinant silk polypeptide sample clearly has the visible presence of particles that are hollow, 2-200 μm in diameter, and appear as light circles. White arrows have been added to the figure to point to exemplary silk powder particles.

[0201] FIG. 2F: Color cosmetic formulations containing 1% recombinant silk protein are distinct from the 5% and 10% formulations as measured by performance characteristics and microscopy, however, the rheological data shows similarities between the three formulations. Frequency sweeps show similar magnitude and shape of the G' and G" curves. This data indicates that the color cosmetic formulations have similar gel-like structural characteristics.

[0202] Example 3: Recombinant silk polypeptides as a replacement for silicone elastomers in hair serums Leave-on hair serums were prepared with either 1% recombinant silk polypeptide or 5% silicone elastomer, and the formulations performed similarly with respect to styling, including shine, curl retention, and frizz control.

[0203] Method: The hair serum formulation was prepared as per standard techniques in the art. All ingredients were mixed gently until completely homogenous. Water was used to make up for the difference in weight percentage of recombinant silk polypeptide (i.e., 18B silk) and silicone elastomer between the formulations. This formulation used recombinant silk polypeptide in powder form and silicone elastomer component with approximately 30% solids.

[0204] Rheological measurements were performed using a Kinexus Lab+ rheometer using a 2 degree / 20 mm cone and plate geometry. To measure the G' and G" of the materials, amplitude sweeps were performed over a range of 0.01% to 250% shear strain for 1 s. -1 Furthermore, a frequency sweep was performed at a strain rate of 0.1%. The starting frequency was 100 s -1 and the end frequency is 0.01s -1 It was.

[0205] To evaluate the morphology of the formulations on hair, 0.25 g of the formulation was spread evenly onto 0.35 g of yak hair with gentle massage. The hair was then dried with hot air at 200 °C for 5 min until it felt dry to the touch. Scanning electron microscopy (SEM) used a focused electron beam to evaluate the morphology of the materials via secondary electrons. The electron beam was scanned in a raster pattern to collect micrographs at magnifications from 1 mm to 10 nm or 10X to 100,000X. The SEM method used low vacuum (1 to 10 torr) and avoided the need to dehydrate or sputter coat biological samples.

[0206] Optical microscopy images were used to examine the powder morphology of the formulations, and were obtained using a Leica DM750P optical microscope using a 10x objective. The microscope was linked to a complementary PC-based image analysis Leica Application Suite, LAS V4.9, to capture the images.

[0207] Figure 3A: Optical microscope images of hair serum formulations with and without 1% recombinant silk polypeptide and 5% silicone elastomer. The base serum and silicone elastomer samples have a uniform structure as evidenced by the lack of visually distinct features. The 1% recombinant silk polypeptide sample clearly has the visible presence of recombinant silk polypeptide particles that are hollow and range in diameter from 2 to 200 μm.

[0208] FIG. 3B: SEM images of hair serum formulations dispersed on yak hair. Approximately 0.25 g of serum was dispersed on 0.35 g of hair. Compared to the untreated one, all serum samples coat the hair strands. Only the recombinant silk polypeptide serum resulted in a distinct surface morphology marked by areas of smooth film, areas of rough film, and areas of particles.

[0209] Figure 3C: Although the serum formulations containing 1% recombinant silk protein were clearly different when measured by microscopy, the rheological data showed similarities between the three formulations. The amplitude sweeps show similar magnitude and length of the linear viscoelastic region (see G' curve). Additionally, the frequency sweeps show similar magnitude and length of the G' curve. This data indicates that the hair serums have similar gel-like structure properties and perform similarly rheologically.

[0210] FIG. 3D: Table of ingredients used in the hair serum formulation (without recombinant silk polypeptides and silicones).

[0211] Example 4: Rheological and morphological comparison of recombinant silk polypeptides with silicone elastomer components Methods: Rheological measurements were performed with a Kinexus Lab+ rheometer using a 2 degree / 20 mm cone and plate geometry. To measure the G' and G" of the materials, a frequency sweep was performed at 0.1% strain rate. The starting frequency was 100 s -1 and the end frequency is 0.01s -1 Three samples were taken every 10 years. Viscosity measurements were performed at 0.1 s -1 ~1000s -1 The viscosity measurements were performed by shearing the samples at a shear rate of 22-27 °C. Recombinant silk protein solutions (containing 18B silk) were prepared by mixing the powders with DI water in a high shear mixer.

[0212] To evaluate the morphology of the formulations on hair, 0.25 g of the formulation was spread evenly onto 0.35 g of yak hair with gentle massage. The hair was then dried with hot air at 200 °C for 5 min until it felt dry to the touch. Scanning electron microscopy (SEM) used a focused electron beam to evaluate the morphology of the materials via secondary electrons. The electron beam was scanned in a raster pattern to collect micrographs at magnifications from 1 mm to 10 nm or 10X to 100,000X. The SEM method used low vacuum (1 to 10 torr) and avoided the need to dehydrate or sputter coat biological samples.

[0213] FIG. 4A: Comparison of G' and G" of an industry standard silicone elastomer gel (30% dry solids) and 12% recombinant silk polypeptide. Even though silicone elastomer and recombinant silk polypeptide have very different molecular structures, crosslinking chemistries, and macromolecular morphologies, both materials exhibit similar rheological properties. This is represented by the flat G' curve in the rheological frequency sweep. This flat curve indicates that both materials are structured gels. In the viscosity curves, both materials show similar non-Newtonian shear thinning profiles, typical of polymer solutions and suspensions.

[0214] Figure 4B: SEM images of pure recombinant silk polypeptide and silicone elastomer dispersed on yak hair. Approximately 0.25 g of serum was dispersed on 0.35 g of hair. Compared to the untreated one, both the recombinant silk polypeptide and the silicone elastomer deposited a visible film on the hair shaft. The silicone elastomer film is relatively smooth. The recombinant silk polypeptide film is characterized by a rougher surface with visible pits and grains protruding from its surface.

[0215] Viscosity measurements shown in this Example 4 were also obtained for a commercial silicone elastomer in diluent and for 18B recombinant silk polypeptide in water, as follows: 1) Specsil K-13 (Innospec, Englewood, Colorado), 10% w / w in cyclopentasiloxane, 2) Silmer G-162-F5 (Siltech, Toronto, Ontario), 15% w / w in dimethicone, 3) CHT- beausil gel 8055 (CHT, Tubingen, Germany), 5% w / w in dimethicone, 4) Dowsil HMW 2220 non-ionic emulsion (Dow, Midland, Michigan), 60% w / w in C12-13 Pareth-23 and C12-13 Pareth3, and 5) 18B recombinant silk polypeptide (19% recombinant silk polypeptide in water, 22% recombinant silk polypeptide in water, 25% silk polypeptide in water, and 27% silk polypeptide in water). All materials tested showed similar non-Newtonian shear thinning profiles typical of polymer solutions and suspensions.

[0216] A comparison of G' and G" with CHT-beausil Gel 8055, a recombinant silk polypeptide of 18B, 5% w / w in dimethicone and 27% in water, was also performed. Both materials showed similar curves with G' exceeding G" in the magnitude range of 30 Pa up to 16000 Pa over the angular frequency range of 1-100 Hz.

[0217] Example 5: Style retention testing of leave-on hair styling serum containing recombinant silk polypeptides Swatches of untreated hair (medium brown, 3 g, 1 inch wide, 8 inches long, sealed with hot melt) were treated with 1 g of leave-in hair serum. The leave-in hair serum contained serum base only or serum base with either 1% silk polypeptide, 1% keratin component, or 5% silicone elastomer component. The serum base formulations are outlined in FIG. 3D. The hair swatches were tightly rolled up on 3 / 4 inch diameter curlers and attached to a grid board with 1 cm wide markings. The samples were incubated in an oven at 50° C. and 70% humidity for 8 hours. After 8 hours, the samples were removed from the oven, cooled to room temperature, and unrolled.

[0218] Figure 5 shows images of hair swatches after exposure to the curl retention test regime as described above. The recombinant silk polypeptide sample had improved curl retention, outperforming all other samples. Quantification of the length of the hair swatches after exposure to the curl retention test regime is shown in Table 2. The recombinant silk polypeptide showed 35% higher curl retention than the base serum and 28% higher curl retention than the silicone elastomer sample.

[0219] (Table 2) JPEG2024540202000004.jpg99163

[0220] Example 6: Rinse-off Shampoo Formulation Rinse-off shampoo formulations containing a range of silk polypeptides (0.05%, 0.5%, and 1%) were developed. These formulations were compared to a placebo (a formulation in which the silk polypeptide was removed and the difference was made up by adding more water). A silicone elastomer version was also made - the silk polypeptide was omitted and replaced with silicone elastomer at a 3% loading level (the difference in mass was made up by adding less water).

[0221] A shampoo formulation was prepared similar to the standard technique outlined in Example 3. The formulation used recombinant silk polypeptide in powder form and a silicone elastomer component with approximately 60% solids. A list of the ingredients in the formulation (not including silk polypeptide or silicone elastomer) with a range of loading levels is provided in Figure 6.

[0222] Viscosity and rheology were evaluated using the methods of Example 3. Viscosity curves of the formulations showed that all formulations exhibited similar shear thinning behavior. Figures 9A and 9B show the shear thinning behavior from 1 to 1000 s. -1 The results showed that over a range of shear rates, the silk polypeptide samples did not deviate from the placebo by more than 34%. The rheology curves of the formulations showed that in all cases the formulations showed similar shapes, with G' exceeding G". Figures 10A and 10B contain tables of rheology curve data points which showed that over a range of angular frequencies from 0.1 Hz to 10 Hz, the silk polypeptide did not deviate from the placebo by more than 31% for G' and 19% for G". The inclusion of silk polypeptide as described herein did not significantly alter the rheological profile compared to the shampoo placebo.

[0223] Light microscopy was evaluated using the method of Example 3. Silk polypeptides can be visually detected at loading levels of 0.05%.

[0224] For the split head evaluation, each side of the head was wetted and 2 g of shampoo sample was massaged into the hair for approximately 45 seconds. The hair was rinsed for 3 minutes. The hair was combed and then blow-dried for 5-10 minutes. The 0.05% silk polypeptide loading level performed similarly to the silicone elastomer samples in terms of style control. As the silk polypeptide concentration increased, the silk polypeptide samples outperformed the silicone elastomer for style control.

[0225] SEM images of how the various products were deposited on the hair shaft were obtained. Approximately 0.25g of shampoo was dispersed onto 0.35g of hair and massaged for approximately 45 seconds. The hair was then rinsed with DI water for 45 seconds. The hair swatches were allowed to dry at ambient temperature / humidity for 24 hours. The samples were evaluated with SEM using the method of Example 3. No significant differences were detected in how the materials (silk protein vs. silicone elastomer formulation) affected the surface of the hair shaft.

[0226] List of ingredients in the formulation (not including silk polypeptides or silicone elastomers) with a range of loading levels.

[0227] Example 7: Leave-on skin serum Leave-on skin serum formulations containing a range of silk polypeptides (0.1%, 0.25%, 0.5%, and 0.75%) were developed. These formulations were compared to a placebo (a formulation in which the silk polypeptide was removed and the difference was made up by adding more water). A silicone elastomer version was also made - the silk polypeptide was omitted and replaced with silicone elastomer at a loading level of 0.75% (the difference in mass was made up by adding less water).

[0228] A skin serum formulation was prepared similar to the standard techniques outlined in Example 1. The formulation used recombinant silk polypeptide in powder form and a silicone elastomer component with approximately 15% solids. A list of the ingredients in the formulation (not including silk polypeptide or silicone elastomer) with a range of loading levels is provided in Figure 7.

[0229] Viscosity and rheology were evaluated using the methods of Example 3. The viscosity curves of the formulations showed that all formulations had similar shear thinning behavior. Figure 11 shows the viscosity curves from 1 to 100 s. -11 is a table of viscosity curve data points showing that over a range of shear rates, silk polypeptide samples deviated from the placebo by up to 137%, representing a significant and beneficial increase in viscosity. However, the shapes of the G' and G" curves were observed to remain substantially similar between the disclosed compositions and the placebo.

[0230] Light microscopy was evaluated using the method of Example 3. Silk polypeptides can be visually detected at loading levels of 0.1%.

[0231] Example 8: Leave-on skin primer A leave-on skin primer formulation containing 2% silk polypeptide was developed. The formulation was compared to a placebo (a formulation in which the silk polypeptide was removed and the difference was covered by adding more water). A silicone elastomer version was also made - the silk polypeptide was omitted and replaced with silicone elastomer at a loading level of 7.5% (the difference in mass was made up by adding less caprylic / capric triglyceride).

[0232] A skin primer formulation was prepared similar to the standard techniques outlined in Example 1. This formulation used recombinant silk polypeptide in powder form and utilized a silicone elastomer component with approximately 15% solids.

[0233] 12A and 12B, the viscosity curve data points are plotted from 1 to 100 s -1 The results showed that over a range of shear rates, silk polypeptide samples did not deviate from the placebo by more than 35%.

[0234] Other embodiments The words which have been used are words of description rather than of limitation, and it is to be understood that changes may be made within the scope of the appended embodiments without departing from the true scope and spirit of the invention in its broader aspects.

[0235] While the present invention has been described at some length and with some particularity with respect to several described embodiments, it is not intended that it should be limited to any such details or embodiments, or to any particular embodiment, but should be interpreted by reference to the appended aspects in order to provide the broadest possible interpretation of such aspects in view of the prior art, and thus effectively encompass the intended scope of the invention.

[0236] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0237] Aspects 1. A composition comprising a recombinant silk polypeptide and a solvent. 2. The composition of claim 1, wherein the composition is a cosmetic or skin care product. 3. The composition of any one of the above aspects, wherein the composition comprises less than 30%, less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, less than 0.01%, less than 0.005%, or less than 0.001% silicone elastomer. 4. The composition of any one of the above aspects, wherein the composition does not comprise a silicone elastomer. 5. The composition of any one of the above aspects, wherein the composition comprises at least 0.1%, at least 0.5%, at least 1%, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% recombinant silk polypeptide. 6. The composition of any one of the above aspects, wherein the composition is a cosmetic or skin care product. 7. The composition of any one of the above aspects, wherein the length of the recombinant silk polypeptide is greater than 100 amino acids. 8. The composition of any one of the above aspects, wherein the recombinant silk polypeptide is self-assembled into a semi-crystalline state. 9. The composition of embodiment 8, wherein the crystalline portion of the recombinant silk polypeptide is characterized by beta-sheet cross-links. 10. The composition of any one of the above aspects, wherein the recombinant silk has poor solubility in a solvent selected from the group consisting of water, other polar and non-polar solvents (e.g., hexanol, hexane, benzene), oils, waxes, surfactants (e.g., anionic, non-ionic, cationic, amphoteric) at a pH of 3-8. 11. The composition of any one of the above aspects, wherein the recombinant silk forms a heterogeneous dispersion in a solvent selected from the group consisting of water, other polar and non-polar solvents (hexanol, hexane, benzene), oils, waxes, surfactants (anionic, non-ionic, cationic, amphoteric) at a pH of 3-8. 12. The composition of any one of aspects 1-7, wherein the recombinant silk polypeptide forms part of a randomly structured gel. 13. The composition of embodiment 12, wherein the gel comprises a preservative or chelating agent. 14. The composition of any one of aspects 1-7, wherein the recombinant silk polypeptide forms part of a hollow powder. 15. The composition of claim 12, wherein the powder comprises a preservative or chelating agent. 16. The composition of any one of aspects 14 to 15, wherein the hollow powder is suspended in a solvent. 17. The composition according to aspect 16, wherein the solvent is selected from the group consisting of an aqueous solvent, a polar solvent, a non-polar solvent, an oil solvent, a wax solvent, or a surfactant. 18. The composition according to aspect 16 or 17, wherein the solvent comprises a preservative or chelating agent. 19. The composition of any one of the above aspects, comprising 0.01-0.5% recombinant silk polypeptide, 0.05%-5% recombinant silk polypeptide, 0.5%-5% recombinant silk polypeptide, 1%-5% recombinant silk polypeptide, or 5%-20% recombinant silk polypeptide. 20. The composition according to any one of the above aspects, wherein the recombinant silk polypeptide replaces a silicone elastomer traditionally used in the composition. 21. The composition according to aspect 20, wherein the recombinant silk polypeptide replaces the silicone elastomer in a silicone elastomer:silk polypeptide ratio of 1:1 to 10:1. 22. The composition of any one of the above aspects, comprising performance characteristics selected from the group consisting of silky smooth and dry feel, reduced skin shine, enhanced hair shine, vivid and effective pigment delivery, easy spreadability, fast absorption time, mattifying, wrinkle hiding, hair style retention, heat resistance of hair, UV and pollution protection, and increased viscosity. 23. The composition of any one of the above aspects, wherein the recombinant silk polypeptide comprises SEQ ID NO: 2878. 24. The composition of any one of the above aspects, which is an SPF formulation, a color cosmetic, or a hair serum. 25. The composition of any one of aspects 1-24, comprising water. 26. The composition of any one of aspects 1-24, comprising water, glycerin, and sodium benzoate. 27. Zinc Oxide, Aloe Barbadensis Leaf Juice, Water, Butyrospermum Parkii Nut Extract, Camellia Sinesis (Green Tea) Leaf Extract, Caprylic / Capric Triglyceride, Capryloyl Glycerin / Sebacic Acid Copolymer, Caprylyl / Capryl Glucoside, Cetearyl Alcohol, Cetearyl Glucoside, Coco-Caprate / Caprylate, Coco-Glucoside, Coconut Alkanes, Diheptyl Succinate, Dipotassium Glycyrrhizate, Ethylhexylglycerin, Glycerin, Hydrolyzed Jojoba Esters, Isostearic Acid, Lecithin, Phenoxyethanol, Polyglyceryl-3 Polyricinoleate, Polyhydroxystearic Acid, Potassium Sorbate, Pyrus 25. The composition of any one of the preceding aspects, comprising: Citrus aurantium ginseng (Malus) fruit extract, sclerotium gum, sodium benzoate, sodium phytate, squalane, tocopherol, xanthan gum, or any combination thereof. 28. The composition of any one of aspects 1-24, comprising water, brassica glycerides, caprylic / capric triglyceride, cetearyl alcohol, CI 77491, cucumber extract, gluconolactone, glycerin, glyceryl stearate, helianthus annuus seed wax, hydrogenated polycyclopentadiene, iron oxides, jojoba esters, lithospermum erythorhzon root extract, polyacrylate crosspolymer-y, polyglycerin-3, polyglyceryl-6 polyricinoleate, silica, simmondsia chinensis seed oil, sodium benzoate, sodium stearoyl glutamate, tocopherol, or any combination thereof. 29. The composition of any one of aspects 1-24, comprising water, polyacrylate crosspolymer-6, glycerin, citric acid, gluconolactone, sodium benzoate, cetrimonium chloride, or any combination thereof. 30. The composition of any one of the above aspects, wherein the solvent comprises an aqueous solvent, an alcohol, an oil-based solvent, or a silicone. 31. The composition of any one of the above aspects, wherein the solvent is selected from the group consisting of water, glycerin, deionized water, olive oil, and pentylene glycol. 32. A composition according to any one of the above aspects, for cleansing the skin. 33. The composition of any one of the above embodiments, further comprising a dye.

Claims

1. 1. A cosmetic, skin, or hair care composition having a silicone substitute, comprising: one or more cosmetic, skin, or hair care active ingredients; a silicone-substituted component comprising a recombinant silk polypeptide; Including, A composition that is substantially free of silicone.

2. 10. The composition of claim 1, wherein the silicone replacement component is present at a loading level that is less than conventional loading levels of silicone while providing at least the same performance.

3. 3. The composition of claim 2, wherein the performance is one or more of imparting a matte finish, detangling hair, imparting a soft feel to the skin, imparting a silky smooth and silky feel, reducing shine to the skin, enhancing the shine of the hair, delivering vibrant and effective pigment, easy spreadability, fast absorption time, wrinkle hiding effect, hair style retention, hair heat resistance, UV and pollution protection, and increasing viscosity.

4. 10. The composition of claim 1, wherein the recombinant silk polypeptide is in the form of a powder.

5. The composition of claim 4, wherein the silicone replacement composition further comprises a solvent, and the powder is suspended in the solvent.

6. The composition of claim 5 , wherein the solvent is an aqueous solvent, a polar solvent, a non-polar solvent, an oil solvent, a wax solvent, or a surfactant.

7. 7. The composition of claim 5 or 6, wherein the silicone-substituted composition further comprises a preservative or a chelating agent.

8. The composition of any one of claims 1 to 4, wherein the silicone substitute is the recombinant silk polypeptide.

9. The composition of any one of claims 1 to 4, wherein the silicone substitute comprises the recombinant silk polypeptide suspended in a diluent.

10. 4. The composition of claim 1, wherein the silicone elastomer substitute comprises the recombinant silk polypeptide as a randomly structured gel.

11. The composition of claim 10 , wherein the gel comprises a preservative or a chelating agent.

12. 10. The composition of claim 1, wherein 1 part recombinant silk polypeptide can provide a replacement for up to 60 parts silicone.

13. 10. The composition of claim 1, wherein the recombinant silk polypeptide is present in an amount of about 0.01% to about 30% by weight, based on the total weight of the composition.

14. 14. The composition of claim 13, comprising 0.01% to 0.5% by weight of recombinant silk polypeptide, 0.05% to 5% by weight of recombinant silk polypeptide, 0.5% to 5% by weight of recombinant silk polypeptide, 1% to 5% by weight of recombinant silk polypeptide, or 5% to 30% by weight of recombinant silk polypeptide, based on the total weight of the composition.

15. 10. The composition of claim 1, wherein the silicone replacer increases the viscosity of the composition as measured by G' and G" rheological curves compared to a composition having the same amount of silicone, while maintaining G' and G" rheological curves that have substantially the same shape as the G' and G" rheological curves for the composition having the same amount of silicone.

16. 10. The composition of claim 1, wherein the silicone substitute comprises the recombinant silk polypeptide suspended in a solvent.

17. 17. The composition of claim 16, wherein the recombinant silk polypeptide is present in an amount of about 1% to about 40% by weight, based on the total weight of the silicone substitute.

18. 18. The composition according to claim 16 or 17, wherein the solvent is an aqueous solvent, an alcohol, or an oil-based solvent.

19. 18. The composition of claim 16 or 17, wherein the solvent is one or more of water, glycerin, deionized water, olive oil, and pentylene glycol.

20. 2. The composition of claim 1, wherein the recombinant silk polypeptide is greater than 100 amino acids in length.

21. 10. The composition of claim 1, wherein the recombinant silk polypeptide is self-assembled into a semi-crystalline state.

22. 22. The composition of claim 21, wherein the crystalline portion of the recombinant silk polypeptide is characterized by beta-sheet cross-links.

23. 2. The composition of claim 1, wherein the recombinant silk has poor solubility in a solvent selected from the group consisting of water, other polar and non-polar solvents (e.g., hexanol, hexane, benzene), oils, waxes, surfactants (e.g., anionic, non-ionic, cationic, amphoteric) at a pH of 3 to 8.

24. 10. The composition of claim 1, wherein the recombinant silk forms a heterogeneous dispersion in a solvent selected from the group consisting of water, other polar and non-polar solvents (hexanol, hexane, benzene), oils, waxes, surfactants (anionic, non-ionic, cationic, amphoteric) at a pH of 3-8.

25. 2. The composition of claim 1, wherein the recombinant silk polypeptide comprises SEQ ID NO:

1.

26. 10. The composition of claim 1, which is an SPF formulation, a color cosmetic, a rinse-off hair shampoo, a skin serum, a skin primer, or a hair serum.

27. The composition of claim 1 comprising water.

28. The composition of claim 1 comprising water, glycerin, and sodium benzoate.

29. Zinc Oxide, Aloe Vera (Aloe Barbadensis) Leaf Juice, Water, Shea (Butyrospermum Parkii) Nut Extract, Camellia Sinesis (Green Tea) Leaf Extract, Caprylic / Capric Triglyceride, Capryloyl Glycerin / Sebacic Acid Copolymer, Caprylyl / Capryl Glucoside, Cetearyl Alcohol, Cetearyl Glucoside, Coco-Caprate / Caprylate, Coco-Glucoside, Coconut Alkanes, Diheptyl Succinate, Dipotassium Glycyrrhizate, Ethylhexylglycerin, Glycerin, Hydrolyzed Jojoba Esters, Isostearic Acid, Lecithin, Phenoxyethanol, Polyglyceryl-3 Polyricinoleate, Polyhydroxystearic Acid, Potassium Sorbate, Apple (Pyrus Malus) 10. The composition of claim 1, comprising: (Avocado) fruit extract, sclerotium gum, sodium benzoate, sodium phytate, squalane, tocopherol, xanthan gum, or any combination thereof.

30. 10. The composition of claim 1, comprising water, brassica glycerides, caprylic / capric triglyceride, cetearyl alcohol, CI 77491, cucumber extract, gluconolactone, glycerin, glyceryl stearate, helianthus annuus seed wax, hydrogenated polycyclopentadiene, iron oxides, jojoba esters, lithospermum erythorhzon root extract, polyacrylate crosspolymer-y, polyglycerin-3, polyglyceryl-6 polyricinoleate, silica, simmondsia chinensis seed oil, sodium benzoate, sodium stearoyl glutamate, tocopherol, or any combination thereof.

31. 10. The composition of claim 1, comprising water, polyacrylate crosspolymer-6, glycerin, citric acid, gluconolactone, sodium benzoate, cetrimonium chloride, or any combination thereof.

32. The composition of claim 1 further comprising a dye.

33. The composition of claim 1 , wherein the silicone is a silicone elastomer.

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