Cosmetic and personal care compositions comprising recombinant filaments

Abstract

Disclosed herein is a composition comprising a recombinant silk polypeptide and the use of the composition in cosmetic and personal care formulations as a silicone (both "fluid" and "elastomeric") substitute for affecting skin and hair properties, and may comprise benefits to the formulation itself, such as increasing formulation viscosity.

Description

Technical Field

[0001] The present invention relates to a composition comprising recombinant silk polypeptides and the use of the composition in beauty and personal care formulations as a silicone (both "fluid" and "elastomeric") substitute for affecting skin and hair properties, and may include benefits to the formulation itself, such as increasing formulation viscosity. For skin, these properties may include long-lasting non-stripping, silky softness, matte texture, enhanced pigment delivery, spreadability, rapid absorption, wrinkle blurring effect, and UV and pollution protection. For hair, these properties may include long-lasting non-stripping, gloss, non-greasy, curl control, increased hair thickness, style retention, heat resistance, and UV and pollution protection. Background Art

[0002] Silicone polymers are a broad family of chemicals that have in common that silicon and oxygen atoms alternate to form the backbone of the polymer. In general, "liquid" silicone polymers or "silicone fluids" refer to high molecular weight silicone polymers that may or may not contain functional groups. These polymers may also be referred to as "polydimethylsiloxanes." Silicone elastomers refer to types of silicones in which linear silicone polymers have been cross-linked 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 the skin feel soft and velvety, and to give the skin a matte texture. Some common applications of silicone are BB creams, anti-aging wrinkle reducers, primers, liquid foundations, mousse foundations, gel eyeshadows, and many more applications.

[0004] Although fluid silicones and silicone elastomers provide benefits for skin, hair and personal care applications, silicones are more difficult to wash off and may get stuck in pores. This is because silicones are hydrophobic and they repel water. For this reason, silicone-based products do not rinse off easily. In addition, silicones are not eco-friendly, their biodegradation rate in the environment is extremely slow, and therefore they can bioaccumulate. Once the silicones are washed down the drain, they contribute to the accumulation of sludge pollution in oceans and waterways and may not decompose for decades or even 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).

[0005] There has been an unmet need to date for alternative ingredients to silicone elastomers that achieve the same performance levels as silicone elastomers while also being bio-based and biodegradable. Many ingredient efforts have been made to make "greener" silicone elastomers by replacing the cyclosiloxane diluents that typically accompany silicone elastomers with bio-based alternatives. While this is a step in the right direction, it still falls far short of solving the problem at hand, which is that silicone elastomers remain non-biodegradable. BRIEF DESCRIPTION OF THE DRAWINGS

[0006] The foregoing and other objects, features and advantages will be apparent from the following description of specific embodiments of the invention as shown in the accompanying drawings, in which like reference numerals refer to like parts throughout the different views. The accompanying drawings are not necessarily drawn to scale, emphasis instead being placed on illustrating the principles of various embodiments of the invention.

[0007] Figure 1A Shown is a top view of an SPF formulation.

[0008] Figure 1B Shown is a spider graph plotting data from a blind comparison of SPF formulations.

[0009] Figure 1C Ingredients used in all SPF formulations (excluding recombinant silk polypeptides and silicone) are shown.

[0010] Figure 1D The elastic modulus, viscous modulus, and phase angle versus frequency for an SPF primer formulation, an SPF formulation with 1.5% recombinant silk polypeptide, and an SPF formulation with 2% silicone are shown.

[0011] Figure 1E Optical microscopic images of SPF primers and SPF primers formulated with 1% b-silk protein (i.e., 18B silk; recombinant silk including SEQ ID NO: 2878) or 5% silicone elastomer components are shown. A reference image of 1% recombinant silk polypeptide powder suspended in water is also shown.

[0012] Figure 2A Shown are top views of 3-in-1 eye / face / lip cream formulations (ie, color cosmetics) with 1% recombinant silk polypeptide or 10% silicone elastomer.

[0013] Figure 2B A flow chart for evaluating pigment delivery and firmness on skin of color cosmetics is presented.

[0014] Figure 2C Representative images of color makeup application and removal are shown for color makeup having i) 1% recombinant silk polypeptide, ii) 5% silicone elastomer, and iii) 10% silicone elastomer.

[0015] Figure 2D Ingredients used in all color cosmetic formulations (excluding recombinant silk polypeptides and silicones) are shown.

[0016] Figure 2E Optical microscope images of a color cosmetic base and a base formulated with 1% recombinant silk polypeptide or 5% silicone elastomer ingredients are shown.

[0017] Figure 2F Shown are graphs of elastic modulus, viscous modulus, and phase angle versus frequency for a color cosmetic base, a color cosmetic with 1% recombinant silk polypeptide, and a color cosmetic with 5% silicone elastomer.

[0018] Figure 3A Optical microscope images of hair serum formulations with and without 1% recombinant silk polypeptide and 5% silicone elastomer are shown. Also shown is a reference image of 1% recombinant silk polypeptide powder suspended in water.

[0019] Figure 3BShown are SEM images of yak hair at 150x and 800x magnifications: i) untreated, ii) treated with a serum base, iii) treated with a serum base with 1% recombinant silk protein, and iv) treated with a serum base with 5% elastomer.

[0020] Figure 3C Shown are graphs of elastic modulus, viscous modulus, and phase angle versus frequency (bottom row) or shear strain (top row) for colored hair serum base, hair serum base with 1% recombinant silk protein, and hair serum base with 5% silicone elastomer.

[0021] Figure 3D This is a table of ingredients used in the hair serum base formulation (excluding recombinant silk polypeptides and silicone elastomers).

[0022] Figure 4A Shown is a comparison of G' and G" for an industry standard silicone elastomer gel (30% dry solids) and 12% recombinant silk polypeptide on graphs of elastic modulus, viscous modulus and phase angle versus frequency (top row) or viscosity versus shear rate (bottom row).

[0023] Figure 4B SEM images of pure recombinant silk polypeptide and silicone elastomer dispersed on yak hair at 150x and 800x magnification are shown.

[0024] Figure 5 Shown are images of hair swatches after exposure to a curl retention test after application of a leave-in hair serum containing either a serum base alone or a serum base with a 1% silk polypeptide, a 1% keratin component, or a 5% silicone elastomer component.

[0025] Figure 6 is a table of ingredients used in the rinse-off shampoo formulation described in Example 6 (excluding recombinant silk polypeptide and silicone elastomer).

[0026] Figure 7 is a table of ingredients used in the leave-on skin serum described in Example 7 (excluding recombinant silk polypeptide and silicone elastomer).

[0027] Figure 8 is a table of ingredients used in the leave-on skin primer described in Example 8 (excluding recombinant silk polypeptide and silicone elastomer).

[0028] Fig.9A Compared with placebo, the Figure 6 A graph of the shear viscosity of a composition as a function of shear rate.

[0029] Fig. 9B It shows Fig.9A Graph of the change in viscosity of the compositions tested relative to the placebo.

[0030] Fig. 10A and 10B It shows Fig.9A Graph of the changes in rheology (G' and G") of the compositions tested in.

[0031] Fig.11 Figure 2 shows the recombinant silk polypeptides with different amounts Figure 7 A graph of the change in viscosity of the compositions relative to the placebo.

[0032] Fig. 12A Compared with placebo, the Figure 8 A graph of the shear viscosity of a composition as a function of shear rate.

[0033] Fig. 12B It shows Fig. 12A Graph of the change in viscosity of the compositions tested relative to the placebo. DETAILED DESCRIPTION

[0034] The details of various embodiments of the present invention are set forth in the following description. Other features, objectives and advantages of the present invention will be apparent from the specification and drawings and aspects.

[0035] The present invention relates to the use of recombinant silk polypeptides as silicone substitutes in personal care and / or cosmetic compositions, for providing benefits to cosmetics, skin and hair that are usually associated with linear silicones and silicone elastomers. The skin, hair or cosmetic compositions of the present disclosure can have any standard ingredients that are usually contained in such compositions, including active ingredients, pigments and additives. Silicone substitutes including recombinant silk polypeptides can be used in combination with any standard hair care ingredients, skin care ingredients or cosmetic ingredients to form compositions of the present disclosure, wherein silicones (fluids or elastomers) that are usually contained in such compositions are replaced. It is contemplated herein that any skin, hair or cosmetic composition type is used together with a silicone elastomer substitute component, such as but not limited to SPF formulations, color cosmetics, cleansing shampoos, skin essences, skin makeup pre-milk or hair essences. Compared with silicone components, recombinant silk polypeptides can provide these benefits at equal or reduced load levels. Advantageously, it has been observed that recombinant silk polypeptides as silicone substitutes can maintain the G' and G" rheological curve shapes when silicone is included. In addition, in some compositions, it was found that the recombinant silk polypeptide significantly increased the viscosity of the formulation compared to an equal amount of silicone, thereby allowing the loading level of the recombinant silk polypeptide to be significantly reduced compared to silicone. Moreover, the recombinant silk polypeptide is biodegradable and will degrade in the environment (e.g., once flushed into a drain), while the silicone component is non-biodegradable.

[0036] According to the cosmetics, hair or skin care compositions of the present disclosure, a silicone substitute component is included, and the silicone substitute component includes a recombinant silk polypeptide and one or more active ingredients for cosmetics, skin or hair care. The composition of the present disclosure may be substantially free of silicone. Unless otherwise indicated, "silicone" mentioned herein should be understood to include both silicone fluid and silicone elastomer. For example, the composition of the present disclosure may have less than 0.1% silicone. For example, the composition of the present disclosure may have less than 0.1% silicone elastomer.

[0037] Recombinant silk polypeptides are high molecular weight polypeptides that entropy self-assemble 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 the recombinant silk polypeptide can be ground and included in the composition as a ground powder. The silicone substitute component can include a recombinant silk polypeptide suspended in a solvent. The silicone substitute component can include a recombinant silk polypeptide as a random structure gel. On a macroscopic level, this can range from a low viscosity weak gel suspended in an aqueous solvent (sometimes referred to as a "slurry") to dry hollow powder particles (<15% moisture content).

[0038] In some embodiments, provided herein is a composition comprising a recombinant silk polypeptide and the use of the composition as a silicone replacement in cosmetic and personal care formulations.

[0039] 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.

[0040] In some embodiments, the recombinant silk polypeptides self-assemble into a semi-crystalline state, wherein the crystalline portion is characterized by beta-sheet cross-links that are resistant to 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, but are easily dispersed in these materials to form heterogeneous dispersions.

[0041] In some embodiments, the recombinant silk polypeptide exists as a random structured gel. The gel may also contain the presence of a preservative and a chelating agent. In various aspects, the recombinant silk polypeptide exists as a hollow powder. The hollow powder may be ground so that the powder is incorporated into the composition as a ground powder. The powder may also contain or be mixed with a preservative and a chelating agent.

[0042] 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. The mixture may also contain the presence of preservatives and chelating agents.

[0043] Based on the gross weight of the composition, the recombinant silk polypeptide can be included in the composition in an amount of about 0.01wt% to about 30wt%. For example, based on the gross weight of the composition, the recombinant silk polypeptide can be included in the composition in an amount of about 0.05wt% to about 5wt%, about 0.5wt% to about 5wt%, about 0.01wt% to about 0.5wt%, about 0.1wt% to about 0.5wt%, about 0.05wt% to about 5wt%, about 0.5wt% to about 5wt%, about 5wt% to about 20wt%, about 5wt% to about 30wt%, about 25wt% to about 30wt%, about 10wt% to about 25wt% or about 1wt% to about 5wt%.

[0044] In some embodiments, the recombinant silk polypeptides form a unique and detectable film on the skin and hair when applied in a leave-on formulation or a rinse-off formulation.

[0045] In some embodiments, the recombinant silk polypeptides aid in cleansing or exfoliation when used in a rinse-off formulation.

[0046] In some embodiments, the recombinant silk polypeptide can be detected in the formulation (clean-free or clean-up) as demonstrated by visual inspection using a microscope, where powder can be observed under a 5-100X objective. In addition, the recombinant silk polypeptide can be detected using SEC-HPLC by a high molecular weight peak between 50 kDa and 90 kDa.

[0047] In some embodiments, the recombinant silk polypeptides match the performance of silicone elastomers in skin care, hair care, cosmetics, personal care, antiperspirant / deodorant formulations at the same concentration or lower.

[0048] In some embodiments, the recombinant silk polypeptides outperform silicone elastomers in skin care, hair care, cosmetic, personal care, antiperspirant / deodorant formulations at equal or lower concentrations.

[0049] In some embodiments, the recombinant silk polypeptide replaces the silicone elastomer in a ratio of at least 1:1 to 1:60, which means that 1 part of silicone elastomer can be replaced by 1 part of recombinant silk polypeptide, and up to 60 parts of silicone elastomer can be replaced by 1 part of recombinant silk polypeptide. For example, a shampoo with silk polypeptide as a silicone substitute is produced, wherein the silk polypeptide is present in an amount of 0.05 wt% based on the total weight of the composition, while the same composition requires an amount of 3 wt% (60X increase) of silicone elastomer to achieve the same performance.

[0050] In some embodiments, the performance properties of the composition include one or more of the following:

[0051] a) Silky, smooth and powdery

[0052] b) Reduce skin glossiness

[0053] c) Enhance hair shine

[0054] d) Vivid and efficient pigment delivery

[0055] e) Ease of spreading

[0056] f) Fast absorption time

[0057] g) Ripening (i.e., less greasy aftertaste)

[0058] h) Wrinkle blurring effect

[0059] i) Hair style retention and heat resistance of hair

[0060] j) UV and pollution protection

[0061] k) Increased viscosity of the formulation.

[0062] In some embodiments, the recombinant silk polypeptides are compatible (meaning that their structure and properties are maintained) with various common cosmetic ingredients, such as polar and non-polar solvents, oils, waxes, fatty acids, humectants, and (sunscreen) actives.

[0063] Due to the differences in swelling of recombinant silk polypeptides in different solvents, these differences can be used as formulation processing aids. For example, recombinant silk polypeptides can be added to formulations in an unswollen state in oils, waxes, or non-polar solvents, and will then swell upon contact with water. Recombinant silk polypeptides are completely or partially removed from skin and hair by water, and completely removed by surfactants.

[0064] In some embodiments, for biodegradation under both anaerobic digestion conditions and aerobic digestion conditions, recombinant silk polypeptides are superior to silicone elastomers. In the Organization for Economic Co-operation and Development (OECD) 301 preparation test, recombinant silk polypeptides will experience at least 5-15% rapid biodegradation during the first 3-5 days. During the subsequent 5-90 days of incubation, recombinant silk polypeptides will experience increasing biodegradation in the absence of a plateau phase of more than 20 days. OECD (1992), No. 301 Test: Ready Biodegradability, OECD Guidelines for the Testing of Chemicals, Section 3, OECD Publishing, Paris, doi.org / 10.1787 / 9789264070349-en, the document is incorporated herein by reference in its entirety.

[0065] definition

[0066] Unless otherwise stated, the following terms shall be understood to have the following meanings:

[0067] As used herein with respect to silk protein, the term "stability" refers to the ability of the product to not form gelation, discoloration or turbidity caused by self-aggregation of silk protein. For example, U.S. Patent Publication No. 2015 / 0079012 (Wray et al.) relates to the use of a wetting agent, comprising glycerol to increase the storage stability of a skin care product comprising full-length silk protein. U.S. Patent No. 9,187,538 relates to a skin care formulation comprising full-length silk protein with a storage stability of up to 10 days. Both disclosures are incorporated herein by reference in their entirety.

[0068] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides of at least 10 bases in length. The term encompasses DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-natural internucleoside bonds, 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 in a padlocked conformation.

[0069] Unless otherwise specified, and as an example of all sequences described herein in the general format "SEQ ID NO:", "a nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid, at least a portion of which has (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 depends on the requirement that the probe is complementary to the desired target.

[0070] An "isolated" RNA, DNA, or mixed polymer is one that is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell (e.g., ribosomes, polymerases, and genomic sequences with which it is substantially associated in nature).

[0071] "Isolated" organic molecules (e.g., silk proteins) are organic molecules that are essentially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which they are derived or separated from the culture medium of the host cell. The term does not require that the biomolecule has been separated from all other chemicals, although some isolated biomolecules can be purified to near homogeneity.

[0072] The term "recombinant" refers to a biological molecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide with which the gene is found in nature, (3) is operably linked to a polynucleotide with which it is not associated in nature, or (4) does not occur in nature. The term "recombinant" may be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNA encoded by such nucleic acids.

[0073] An endogenous nucleic acid sequence (or the encoded protein product of the sequence) in the genome of an organism is considered to be "recombinant" herein if the heterologous sequence is placed adjacent to an endogenous nucleic acid sequence so that the expression of the endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterologous sequence itself is endogenous (derived from the same host cell or its progeny) or exogenous (derived from a different host cell or its progeny). By way of example, the natural promoter of a gene in the genome of a host cell can be replaced (e.g., by homologous recombination) with a promoter sequence so that the gene has an altered expression pattern. The gene will now become a "recombinant" because the gene is separated from at least some of the sequences that naturally flank it.

[0074] A nucleic acid is also considered "recombinant" if it contains any modifications that do not occur naturally in the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains insertions, deletions, or point mutations that are artificially introduced (e.g., by human intervention). "Recombinant nucleic acids" also include nucleic acids that are integrated into the chromosome of a host cell at a heterologous site and nucleic acid constructs that exist as episomes.

[0075] As used herein, the term "peptide" refers to a short polypeptide, e.g., a polypeptide typically less than about 50 amino acids in length and more typically less than about 30 amino acids in length. As used herein, the term encompasses analogs and mimetics that mimic structure and thus biological function.

[0076] The term "polypeptide" encompasses both naturally occurring proteins and non-naturally occurring proteins, as well as fragments, mutants, derivatives and analogs thereof. Polypeptides can be monomeric or polymeric. In addition, polypeptides can include many different domains, each of which has one or more different activities.

[0077] The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its source or derivation from which it is derived, (1) is not associated in its native state with naturally associated components, (2) is present in a purity not found in nature, where purity can be judged relative to the presence of other cellular material (e.g., free of 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 polypeptide found in nature 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 will be "isolated" from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by separation using protein purification techniques well known in the art. As thus defined, "isolated" does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.

[0078] The term "polypeptide fragment" refers to a polypeptide having a deletion compared to a full-length polypeptide, such as an amino-terminal and / or carboxyl-terminal deletion. In a preferred embodiment, a polypeptide fragment is a continuous sequence, wherein the amino acid sequence of the fragment is identical to the corresponding position in the naturally occurring sequence. The length of the fragment is generally at least 5, 6, 7, 8, 9 or 10 amino acids, 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 and even more preferably at least 70 amino acids in length.

[0079] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence encoding the protein has a sequence similar 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 imply functional similarity.

[0080] When "homologous" is used to refer to a protein or peptide, it should be recognized that the non-identical residue positions are usually different due to conservative amino acid substitutions. "Conservative amino acid substitutions" are amino acid substitutions in which an amino acid residue is substituted by another amino acid residue with a side chain (R group) having similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially change the functional properties of a protein. In the case where two or more amino acid sequences differ from each other due to conservative substitutions, the sequence identity percentage or homology degree can be adjusted upward to correct the conservative nature of the substitution. The method for making this adjustment is well known to those skilled in the art. See, for example, Pearson, 1994, Methods Mol. Biol. 24: 307-31 and 25: 365-89 (which are incorporated herein by reference).

[0081] Twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren, ed., Sinauer Associates, Sunderland, Mass., 2nd ed. 1991), which is incorporated herein by reference. Twenty conventional amino acids, unnatural amino acids such as α-, α-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acid stereoisomers (e.g., D-amino acids) may also be suitable components of the polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetylysine, 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 carboxyl terminus, according to standard usage and convention.

[0082] The following six groups each contain amino acids that are conservative substitutions for each other: 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).

[0083] The sequence homology (sometimes also referred to as percent sequence identity) of a polypeptide is typically measured using sequence analysis software. See, for example, the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis., 53705. Protein analysis software uses measures of homology assigned to various substitutions, deletions, and other modifications (including conservative amino acid substitutions) to match similar sequences. 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 homologous polypeptides from different organism species) or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1.

[0084] When comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms, a useful algorithm is the computer program 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. 25:341-357 (1998); )》7:649-656 (1997)), especially blastp or tblastn (Altschul et al., "Nucleic Acids Research" 25:3389-3402 (1997)).

[0085] The preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); start gap penalty: 11 (default); extension gap penalty: 1 (default); maximum alignments: 100 (default); word length: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOWSUM62.

[0086] Preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); initial gap penalty: 11 (default); extension gap penalty: 1 (default); maximum alignment: 100 (default); word length: 11 (default); number of descriptions: 100 (default); penalty matrix: BLOWSUM62. The length of the polypeptide sequence used for homology comparison will usually be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, usually at least about 28 residues and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferred 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, FASTA (a GCG version 6.1 program) can be used to compare polypeptide sequences. FASTA provides an alignment and percentage of sequence identity of the best overlap region between the query sequence and the search sequence. Pearson, Methods in Enzymology. 183:63-98 (1990) (which is incorporated herein by reference). For example, the percentage of sequence identity between amino acid sequences can be determined using FASTA as provided in GCG Version 6.1 and its default parameters (word length of 2 and PAM250 scoring matrix), which is incorporated herein by reference.

[0087] Throughout this specification and aspects, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or groups of integers.

[0088] 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 pliable "rubbery" or "viscous" state.

[0089] As used herein, the term "glass transition temperature" refers to the temperature at which a material or composition undergoes a glass transition.

[0090] As used herein, the term "melt transition" refers to the transformation of a substance or composition from a rubbery state to a less ordered liquid phase.

[0091] As used herein, the term "melting temperature" refers to the temperature range at which a substance undergoes a melt transition.

[0092] As used herein, the term "plasticizer" 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.

[0093] As used herein, the term "powder" refers to a composition in the form of particles, which may or may not be complexed or agglomerated with a solvent such as water or an essence. The term "dry powder" can be used interchangeably with the term "powder"; however, unless otherwise specified, as used herein, "dry powder" refers only to the overall appearance of the granulated material and is not intended to mean that the material is completely free of complexed or agglomerated solvents. Dry powders can be produced by spray drying, freeze drying, and / or according to methods known in the art.

[0094] The term "carrier" refers to a recombinant protein used for surface hydration, surface cleaning, surface protection, surface detoxification, surface exfoliation, surface improvement, coloring and / or delivery of various additives or solvents to surfaces such as skin, hair or nails, including but not limited to water, glycerol, alcohol, silicone, oil, humectant, emollient, occlusive agent, active agent and / or cosmetic adjuvant. As used herein, the carrier includes an outer shell and a hollow core, for example, 18B protein.

[0095] As used herein, the term "cosmetics" includes make-up, foundations, skin care products, hair care products, and nail care products.

[0096] As used herein, the term "makeup" refers to products that leave color on the face, including foundation, black and brown, i.e., mascara, concealer, eyeliner, eyebrow color, eye shadow, blush, lip color, powder, solid emulsion powder, etc.

[0097] As used herein, the term "foundation" refers to a liquid, cream, mousse, moist powder, pressed powder, concealer or similar product produced or reintroduced by a cosmetic company to even out the overall color of the skin.

[0098] As used herein, the term "skin care product" refers to a product used to treat or care for or in some way moisturize, improve, or cleanse the skin. Products contemplated by the phrase "skin care product" include, but are not limited to, creams, sprays, serums, cleansing gels, ampoules, adhesives, patches, bandages, toothpastes, waterless occlusive moisturizers, antiperspirants, deodorants, personal cleansing products, powdered laundry detergents, fabric softener towelettes, occlusive drug delivery patches, nail polishes, powders, face towelettes, wipes, waterless conditioners, shaving creams, and the like.

[0099] As used herein, the term "sagging" means loosening, sagging or similar conditions of the skin due to loss, damage, change and / or abnormality of dermal elastin, muscle and / or subcutaneous fat.

[0100] As used herein, the term "treating" or "treatment" refers to treating (eg, alleviating or eliminating symptoms and / or curing) and / or preventing or inhibiting a condition (eg, a skin condition) or alleviating a symptom.

[0101] Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice of 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 the event of a conflict, the present specification (including definitions) shall prevail. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0102] Recombinant Silk Protein

[0103] The present disclosure describes embodiments of the invention, including fibers synthesized from synthetic protein copolymers (i.e., recombinant polypeptides). Suitable protein copolymers are discussed in U.S. Patent Publication No. 2016 / 0222174, published on August 45, 2016, U.S. Patent Publication No. 2018 / 0111970, published on April 26, 2018, and U.S. Patent Publication No. 2018 / 0057548, published on March 1, 2018, each of which is incorporated herein by reference in its entirety.

[0104] In certain embodiments, synthetic protein copolymer is made of silk-like polypeptide sequence.In certain embodiments, silk-like polypeptide sequence is 1) block copolymer polypeptide composition produced by the repeat domain that is derived from silk polypeptide sequence by mixing and matching, and / or 2) recombinant expression of block copolymer polypeptide with sufficiently large size (about 40kDa) to form useful molded body composition by secretion from industrially scalable microorganism.Can be expressed in modified microorganism as described herein from the large (about 40kDa to about 100kDa) block copolymer polypeptide of silk repeat domain fragment engineering, the sequence that described silk repeat domain fragment comprises the amino acid sequence from nearly all disclosed silk polypeptide.In certain embodiments, silk polypeptide sequence is matched and designed to produce the polypeptide of height expression and secretion that can realize molded body formation.

[0105] In certain embodiments, block copolymer is by the combination mixture engineering of the silk polypeptide domain of cross silk polypeptide sequence space.In certain embodiments, block copolymer is by expressing and secreting in can amplify organism (for example, yeast, fungi and gram-positive bacteria) and prepare.In certain embodiments, block copolymer polypeptide includes 0 or more N-terminal domain (NTD), 1 or more repeat domain (REP) and 0 or more C-terminal domain (CTD).In some aspects of embodiments, block copolymer polypeptide is>100 amino acid whose single polypeptide chain. 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).

[0106] Several types of natural spider silk have been identified. The mechanical properties of each natural spinning type are thought to be closely related to the molecular composition of the silk. See, e.g., 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. Applications), Cell. Mol. Life Sci., 68:2, pp. 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, pp. 131-85 (2011). For example:

[0107] Acroporous (AcSp) filaments tend to have high toughness, which is a result of moderately high strength plus moderately high ductility. AcSp filaments are characterized by large block ("overall repeat") sizes, which often incorporate motifs with polyserine and GPX. Tubular (TuSp or cylindrical) filaments tend to have large diameters, moderate strength and high ductility. TuSp filaments are characterized by their polyserine and polythreonine content, as well as short bundles of polyalanine. Large ampullate (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be one of two subtypes: MaSp1 and MaSp2. MaSp1 filaments are generally less ductile than MaSp2 filaments and are characterized by polyalanine, GX and GGX motifs. MaSp2 filaments are characterized by polyalanine, GGX and GPX motifs. Small ampullate (MiSp) filaments tend to have moderate strength and moderate ductility. MiSp filaments are characterized by GGX, GA and poly A motifs, and often contain spacer elements of approximately 100 amino acids. Flag filaments tend to have very high extensibility and moderate strength. Flag filaments are often characterized by GPG, GGX, and a short spacer motif.

[0108] The properties of each silk type may vary from species to species, and spiders with different lifestyles (e.g., sedentary sphidopods versus wandering huntsmen) or evolutionarily older spiders may produce silks with properties different from those described above (for descriptions of spider diversity and classification, see Hormiga, G. and Griswold, CE, Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, 487-512 (2014); and Blackedge, TA et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, 5229-5234 (2009)). However, synthetic block copolymer polypeptides having sequence similarity and / or amino acid composition similarity to the repeating domains of natural silk proteins can be used to manufacture consistent molded bodies on a commercial scale having properties that recapitulate the properties of corresponding molded bodies made from natural silk polypeptides.

[0109] In some embodiments, a list of putative silk sequences can be compiled by searching for relevant terms in GenBank, such as "spider silk protein", "silkworm silk protein", "MaSp", and those sequences can be merged with other sequences obtained by independent sequencing work. Then, the sequence is translated into amino acids, duplicate entries are filtered, and manually split into domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequence is reversely translated into a DNA sequence optimized for expression in Pasteur (Pichia (Komagataella) pastoris. Each DNA sequence is cloned into an expression vector and transformed into Pasteur (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 that can achieve molded body formation.

[0110] Silk polypeptide characteristically is made up of the repetitive domain (REP) of the non-repetitive region (for example, C-terminal and N-terminal domain) of side joint.In one embodiment, the length of both C-terminal domain and N-terminal domain is between 75-350 amino acids.Repetitive domain shows layered architecture.Repetitive domain includes a series of blocks (also referred to as repeating units).Block repeats in the whole silk repetitive domain, sometimes completely repeats and sometimes incompletely repeats (constituting quasi-repetitive domain).The length and composition of block vary because of different silk types and different species. Table 1A lists examples of block sequences from selected species and silk types, with additional examples presented in the following literature: Rising, A. et al., Spider silk proteins: Recent advances in recombinant production, structure-function relationships, and biomedical applications, Cell and Molecular Life Sciences, 68:2, pp. 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, the blocks can be arranged in a regular pattern to form a larger macro-repeating sequence that occurs multiple times (usually 2-8 times) in the repeating domain of the silk sequence. Repeating blocks within a repeating domain or macro-repeating sequence, and repeating macro-repeating sequences within a repeating domain can be separated by spacer elements. In some embodiments, the block sequence includes a glycine-rich region followed by a polyA region. In some embodiments, short (about 1-10) amino acid motifs occur multiple times in blocks. For the purposes of the present invention, without reference to cyclic arrangements, blocks from different natural silk polypeptides can be selected (i.e., similar recognition blocks in other respects between silk polypeptides may not be aligned due to cyclic arrangements). Therefore, for example, for the purposes of the present invention, the "block" of SGAGG (SEQ ID NO: 2871) is the same as GSGAG (SEQ ID NO: 2872) and is the same as GGSGA (SEQ ID NO: 2873); They are all just cyclic arrangements of each other. The specific arrangement selected for a given silk sequence can be determined by convenience (usually starting with G) to the greatest extent. The silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0111] Table 1A: Sample block sequences

[0112]

[0113]

[0114]

[0115]

[0116] According to certain embodiments of the present invention, the fiber-forming block copolymer polypeptide from block and / or macro repeat sequence domain is described in International Publication No. WO / 2015 / 042164, which is incorporated by reference. The natural silk sequence obtained from protein databases such as GenBank or by de novo sequencing is decomposed by domain (N-terminal domain, repeat domain and C-terminal domain). The N-terminal domain and C-terminal domain sequence selected for the purpose of synthesis and assembly into fiber or molded body include natural amino acid sequence information and other modifications described herein. The repeat domain is decomposed into a repeat sequence containing a representative block, usually 1-8, depending on the type of silk, which captures key amino acid information, while reducing the size of the DNA encoding amino acids to a fragment that can be easily synthesized. In certain embodiments, the appropriately formed block copolymer polypeptide includes at least one repeat domain, and the at least one repeat domain includes at least 1 repeat sequence, and optionally flanked by N-terminal domain and / or C-terminal domain.

[0117] In some embodiments, the repetitive domain includes at least one repetitive sequence. In some embodiments, the repetitive sequence is 150-300 amino acid residues. In some embodiments, the repetitive sequence includes multiple blocks. In some embodiments, the repetitive sequence includes multiple macro-repetitive sequences. In some embodiments, blocks or macro-repetitive sequences are divided into multiple repetitive sequences.

[0118] In some embodiments, the repetitive sequence starts with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), asparagine (N), methionine (M) or aspartic acid (D) to meet the DNA assembly requirements. In some embodiments, some repetitive sequences in the repetitive sequence can be changed compared to the native sequence. In some embodiments, the repetitive sequence can be changed, such as by adding serine to the C-terminus of the polypeptide (to avoid terminating at F, Y, W, C, H, N, M or D). In some embodiments, the repetitive sequence can be modified by filling incomplete blocks with homologous sequences from another block. In some embodiments, the repetitive sequence can be modified by rearranging the order of blocks or macroscopic repetitive sequences.

[0119] In some embodiments, non-repetitive N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be achieved by removing the leading signal sequence, for example, as identified by SignalP (Peterson, TN et al., SignalP 4.0: distinguishing signal peptides from transmembrane regions, "Natural Methods (Nat.Methods)", 8: 10, pp. 785-786 (2011).

[0120] In some embodiments, the N-terminal domain, repeat sequence, or C-terminal domain sequence can be derived from Agelenopsis aperta, Aphonopelmaseemanni, Aptostichus sp. AS217, Aphonopelmaseemanni, Araneus diadematus, Araneus cat-faced spider, Argiope spp. AS220, Araneus diadematus, Argiope spp., Argiope spp., Argiope amoena, Argiopeargentata, Argiope bruennichi, Argiope spp., Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Diguetia canities, Black fishing spider, Euagrus chisoseus, African nursery web spider, Gasteracantha mammosa), Hypochilusthorelli, Kukulcania hibernalis, western black widow, Megahexurafulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, primitive carnivorous spider, Poecilotheria regalis, Kaua'i octopus or differential mites.

[0121] In certain embodiments, the silk polypeptide nucleotide coding sequence can be operably connected to an α mating factor nucleotide coding sequence. In certain embodiments, the silk polypeptide nucleotide coding sequence can be operably connected to another endogenous or heterologous secretion signal coding sequence. In certain embodiments, the silk polypeptide nucleotide coding sequence can be operably connected to a 3X FLAG nucleotide coding sequence. In certain embodiments, the silk polypeptide nucleotide coding sequence can be operably connected to other affinity tags such as 6-8 His residues.

[0122] In some embodiments, the recombinant silk polypeptide is based on a recombinant spider silk protein fragment sequence derived from MaSp2, such as from a species of Argiope. In some embodiments, the synthetic fiber contains a protein molecule comprising two to twenty repeating units, wherein the molecular weight of each repeating unit is greater than about 20 kDa. There are more than about 60 amino acid residues in each repeating unit of the copolymer, typically in the range of 60 to 100 amino acids, which are organized into a number of "quasi-repeating units". In some embodiments, the repeating units of the polypeptides described in the present disclosure have at least 95% sequence identity with the MaSp2 dragline protein sequence.

[0123] The repeating units of the protein block copolymers that form fibers with good mechanical properties can be synthesized using a portion of a silk polypeptide. These polypeptide repeating units contain alanine-rich regions and glycine-rich regions and are 150 amino acids or longer in length. Some exemplary sequences that can be used as repeating sequences in the protein block copolymers of the present disclosure are provided in co-owned PCT Publication WO 2015 / 042164, which is incorporated by reference in its entirety and has been demonstrated to be expressed using a Pichia expression system.

[0124] In some embodiments, the silk protein comprises: at least two occurrences of a repeating unit, wherein the repeating unit comprises: more than 150 amino acid residues and having a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more consecutive amino acids, comprising at least 80% alanine content; a glycine-rich region having 12 or more consecutive amino acids, comprising at least 40% glycine content and less than 30% alanine content; and wherein the fiber comprises at least one property selected from the group consisting of: an elastic modulus greater than 550 cN / tex, an elongation of at least 10%, and an ultimate tensile strength of at least 15 cN / tex.

[0125] In some embodiments, the recombinant silk protein comprises repeating units, wherein each repeating unit has at least 95% sequence identity with a sequence comprising 2 to 20 quasi-repeating units; each quasi-repeating unit comprises {GGY-[GPG-X1] n1 -GPS-(A) n2}, wherein for each quasi-repeating 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; and n1 is 4 to 8, and n2 is 6-10. The repeating unit consists of a plurality of quasi-repeating units.

[0126] In some embodiments, 3 "long" quasi-repeating units are followed by 3 "short" quasi-repeating units. As described above, short quasi-repeating units are those quasi-repeating units where n1=4 or 5. Long quasi-repeating units are defined as those quasi-repeating units where n1=6, 7 or 8. In some embodiments, all short quasi-repeating units have the same X1 motif in the same position within each quasi-repeating unit of the repeating unit. In some embodiments, no more than 3 quasi-repeating units out of 6 share the same X1 motif.

[0127] In a further embodiment, the repeating unit is composed of quasi-repeating units, wherein the quasi-repeating units use the same X1 no more than twice in a row within the repeating unit. In a further embodiment, the repeating unit is composed of quasi-repeating units, wherein at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 quasi-repeating units use the same X1 no more than 2 times in a single quasi-repeating unit of the repeating unit.

[0128] In some embodiments, the recombinant silk polypeptide comprises the polypeptide sequence of SEQ ID NO: 2878 (ie, 18B). In some embodiments, the repeating unit is a polypeptide comprising SEQ ID NO: 2879. These sequences are provided in Table 1B:

[0129] Table 1B - Exemplary polypeptide sequences of recombinant proteins and repeat units

[0130]

[0131]

[0132] In some embodiments, the fiber structure formed by the described recombinant silk polypeptide forms a β-sheet structure, a β-turn structure, or an α-helical structure. In some embodiments, the secondary, tertiary, and quaternary protein structures of the formed fibers are described as having nanocrystalline β-sheet regions, amorphous β-turn regions, amorphous α-helical regions, randomly spatially distributed nanocrystalline regions embedded in a non-crystalline matrix, or randomly oriented nanocrystalline regions embedded in a non-crystalline matrix. Without intending to be limited by theory, the structural properties of proteins in spider silk are theoretically related to the mechanical properties of the fibers. The crystalline regions in the fibers are related to the tensile strength of the fibers, while the amorphous regions are related to the ductility of the fibers. Compared to whip gland silk, the large ampullate gland (MA) silk tends to have higher strength and less ductility than whip gland silk, and the MA silk has a higher volume fraction of crystalline regions. In addition, theoretical models based on the molecular dynamics of the crystallization and amorphous regions of spider silk proteins support such assertions that the crystalline regions are related to the tensile strength of the fibers, while the amorphous regions are related to the ductility of the fibers. Additionally, theoretical modeling supports the importance of secondary, tertiary and quaternary structures to the mechanical properties of recombinant protein fibers (RPFs). For example, the assembly of nanocrystals in random, parallel and continuous spatial distributions and the strength of the interaction forces between entangled chains within amorphous regions and between amorphous regions and nanocrystalline regions both affect the theoretical mechanical properties of the resulting fibers.

[0133] In some embodiments, the molecular weight of silk protein can be 20 kDa to 2000 kDa, or greater than 20 kDa, or greater than 10 kDa, or greater than 5 kDa, or 5 to 400 kDa, or 5 to 300 kDa, or 5 to 200 kDa, or 5 to 100 kDa, or 5 to 50 kDa, or 5 to 500 kDa, or 5 to 1000 kDa, or 5 to 2000 kDa, or 10 to 400 kDa, or 10 to 300 kDa, or 10 to 200 kDa. , or in the range of 10 to 100 kDa, or 10 to 50 kDa, or 10 to 500 kDa, or 10 to 1000 kDa, or 10 to 2000 kDa, or 20 to 400 kDa, or 20 to 300 kDa, or 20 to 200 kDa, or 40 to 300 kDa, or 40 to 500 kDa, or 20 to 100 kDa, or 20 to 50 kDa, or 20 to 500 kDa, or 20 to 1000 kDa, or 20 to 2000 kDa.

[0134] Characterization of impurities and degradation of recombinant spider silk polypeptide powder

[0135] 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. Silk polypeptides form a β-sheet structure in monomeric form. In the presence of other monomers, silk polypeptides form a three-dimensional lattice of β-sheet structures. The β-sheet structures are separated from and interspersed in the amorphous regions of the polypeptide sequence.

[0136] As measured by fast scanning calorimetry, the β-sheet structure is extremely stable at high temperatures—the melting temperature of the β-sheet is approximately 257° C. See Cebe et al., Beating the Heat—Fast Scanning Melts Silk Beta Sheet Crystals, Nature Scientific Reports 3:1130 (2013). Since the β-sheet structure is believed to remain intact above the glass transition temperature of silk polypeptides, 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 β-sheets.

[0137] Plasticizers reduce the glass transition temperature and melting temperature of silk protein by increasing the fluidity of amorphous regions and potentially disrupting beta sheet formation. Suitable plasticizers for this purpose include, but are not limited to, water and polyols (polyols), such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to, dimethyl isosorbide; adipic acid; dimethylaminopropylamine and amides of caprylic / capric acid; acetamide; and any combination thereof.

[0138] Since the hydrophilic part of the silk polypeptide can bind to the ambient water present in the air as moisture, water is almost always present, and the combined ambient water can plasticize the silk polypeptide. In certain embodiments, a suitable plasticizer can be glycerol present alone or in combination with water or other plasticizers. Other suitable plasticizers are discussed above.

[0139] In addition, in the case of producing recombinant silk polypeptides by fermentation and recovering recombinant silk polypeptide powder therefrom, 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 can act as plasticizers and thus affect the glass transition temperature of the protein by interfering with the formation of tertiary structure.

[0140] Various mature methods can be used to assess the purity and relative composition of recombinant silk polypeptide powder or composition. Size exclusion chromatography is based on the relative size separation of molecules and can be used to analyze the relative amount of recombinant silk polypeptides in its full-length polymer and monomeric form, as well as the amount of high, low and medium molecular weight impurities in recombinant silk polypeptide powder. Similarly, fast high performance liquid chromatography can be used to measure various compounds present in 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 solution, including impurities such as lipids and sugars. Other chromatography and quantitative methods such as chromatography of various molecules are generally recognized in the art.

[0141] According to an embodiment, the recombinant silk polypeptide can have a purity calculated based on the amount (by weight) of the recombinant silk polypeptide in monomer form relative to other components of the recombinant silk polypeptide powder. In various cases, depending on the type of the recombinant silk polypeptide and the technology for recovering, separating and processing the recombinant silk polypeptide powder, the purity can be in the range of 50% by weight to 90% by weight.

[0142] Size exclusion chromatography and reversed-phase high performance liquid chromatography can be used to measure full-length recombinant silk polypeptides, which makes it a useful technique for determining whether a processing step has degraded recombinant silk polypeptides by comparing the amount of full-length silk polypeptides in the composition before and after processing. In various embodiments of the present invention, the amount of full-length recombinant silk polypeptides present in the composition before and after processing may be subject to minimal degradation. The amount of degradation can be in the range of 0.001 wt % to 10 wt %, or 0.01 wt % to 6 wt %, for example, less than 10 wt % or 8 wt % or 6 wt %, or less than 5 wt %, less than 3 wt % or less than 1 wt %.

[0143] Silicone replacement ingredients

[0144] The silicone substitute component includes a recombinant silk polypeptide. The silicone substitute component can be composed of a recombinant silk polypeptide. The silicone substitute component can include a recombinant silk polypeptide with a solvent and / or one or more additives (such as preservatives and chelating agents). The silicone substitute component can include a recombinant silk polypeptide in an amount of about 1wt% to about 40wt% based on the total weight of the silicone substitute component, or any other suitable amount required to achieve the final desired load of the recombinant silk polypeptide in a cosmetic, skin or hair care composition.

[0145] Without intending to be limited by theory, in various embodiments of the present invention, the induction silicone substitute component can be used in applications where it is desired to prevent the aggregation of monomeric recombinant silk polypeptides into their crystalline polymer form or to control the conversion of recombinant silk polypeptides into their crystalline polymer form at a later stage of processing. In other embodiments, such induction is not required.

[0146] In one embodiment, the silicone elastomer replacement component can be used to prevent the aggregation of the recombinant silk polypeptide before blending the recombinant silk polypeptide with the second polymer. In another embodiment, the silicone elastomer replacement component can be used to produce a base liquid of a cosmetic or skin care product, wherein the recombinant silk polypeptide is present in its monomeric form in the base liquid. In this embodiment, the recombinant silk polypeptide having its monomeric form in the base liquid allows the monomer to be controlled to aggregate into its crystalline polymeric form when in contact with the skin or through various other chemical reactions.

[0147] In various embodiments, the temperature to which the silicon elastomer substitute component with the recombinant silk polypeptide is heated is minimized to minimize or completely prevent degradation of the recombinant silk polypeptide. In a specific embodiment, the recombinant silk melt will be heated to a temperature below 120°C, below 100°C, below 80°C, below 60°C, below 40°C, or below 20°C. Typically, during processing, the melt will be at a temperature in the range of 10°C to 120°C, 10°C to 100°C, 15°C to 80°C, 15°C to 60°C, 18°C ​​to 40°C, or 18°C ​​to 22°C. In other embodiments, the silicon elastomer substitute component is not heated. In such embodiments, heat is not required to form the silicon elastomer substitute component.

[0148] Various techniques can be used to measure the degradation amount of recombinant silk polypeptide.As discussed above, the degradation amount of recombinant silk polypeptide can be measured using size exclusion chromatography, so as to measure the amount of the full-length recombinant silk polypeptide present.In various embodiments, the recombinant silk polypeptide is degraded with an amount less than 6.0 % by weight after being formed into a molded body.In another embodiment, the recombinant silk polypeptide is degraded with an amount 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, so that the degradation amount can be in the range of 0.001 % by weight to 10 % by weight, 8 % by weight, 6 % by weight, 4 % by weight, 3 % by weight, 2 % by weight or 1 % by weight or 0.01 % by weight to 6 % by weight, 4 % by weight, 3 % by weight, 2 % by weight or 1 % by weight.In another embodiment, the recombinant silk protein in the composition is substantially non-degraded.In similar embodiments, the recombinant silk protein in the composition is substantially non-degraded in the time period of at least 1 day, 1 month, 1 year or 5 years.

[0149] In some embodiments, the silicone replacement component is physically stable. In various embodiments, the component maintains its material form, such as a powder, over an extended period of time, having an extended shelf life. In long-term use, the silicone replacement component remains substantially stable. In some embodiments, the silicone replacement component has a stability substantially the same as the stability of silicone and / or silicone elastomer.

[0150] In some embodiments, the silicone replacement component has material properties substantially similar to those of silicone and / or silicone elastomers. In various embodiments, the silicone replacement component has a rheology substantially similar to that of silicone and / or silicone elastomers, and / or imparts to the compositions of the present disclosure a rheology similar to that of compositions comprising silicone and / or silicone elastomers.

[0151] In most embodiments of the present invention, the silicone substitute component is in powder form. The silicone substitute component may include a recombinant silk polypeptide in powder form. In certain embodiments, the silicone substitute component is spray dried. In other embodiments, the silicone substitute component is freeze dried or vacuum dried. The terms "spray-drying" and "spray-dried" are used herein for simplicity, but the skilled person will understand that freeze drying or freeze drying and vacuum drying can replace spray drying when appropriate. These silicone substitute components can be stored dry.

[0152] 18B protein is more stable in dry form than in aqueous slurry. In some embodiments, spray-dried recombinant silk is obtained as follows: the slurry composition comprising the extracted recombinant silk is kept cool during the drying step. The slurry composition is pumped into a high-profile spray dryer, where the moisture content of the resulting powder is strictly controlled. Since protein powder is hygroscopic, final powder collection and packaging are performed to minimize the reintroduction of moisture. The design of the packaging material should minimize moisture and light exposure.

[0153] In some embodiments, the recombinant silk polypeptide is recovered and isolated from the cell culture by i) extraction and separation, ii) removal of urea by ultrafiltration, iii) washing by precipitation, iv) salt removal and protein concentration, and v) spray drying.

[0154] In some embodiments, to freeze dry the composition, the composition is cooled until it solidifies and placed under reduced pressure to sublime the most volatile components of the composition. The solid residue may form a single substance that needs to be ground to form a fine powder. Typical freeze dried powders include porous irregularly shaped particles and are easily hydrated. Since freeze drying does not require strong heat, it is used to produce powders including volatile components. In some embodiments, the silicone substitute component is deep freeze dried at a temperature below about -100°C.

[0155] After forming the silicone replacement component, the crystallinity of the silicone replacement component can be increased, thereby strengthening the composition. In some embodiments, the silicone replacement component remains unchanged or decreases. In some embodiments, the crystallinity index of the silicone replacement component as measured by X-ray crystallography is 2% to 90%. In some other embodiments, the crystallinity index of the silicone replacement component as measured by X-ray crystallography is at least 3%, at least 4%, at least 5%, at least 6% or at least 7%.

[0156] In some embodiments of the present invention, the silicone substitute component is a solid or film. In some embodiments, the silicone substitute component is a powder. In some embodiments, the solid or film will be substantially uniform, which means that the material has a small amount or no inclusions or precipitates as examined by an optical microscope. In some embodiments, an optical microscope can be used to measure birefringence, which can be used as a proxy for the recombinant silk to be arranged into a three-dimensional lattice. Birefringence is an optical property of a material with a refractive index that depends on the polarization and propagation of light. Specifically, the high axial order as measured by birefringence can be associated with high tensile strength. In some embodiments, the recombinant silk solid and film will have minimal birefringence. In various embodiments, the solid is a bead. In some other embodiments, the solid serves as an exfoliant. The recombinant silk solid can be in the form of a mild skin scrub for skin. In some embodiments, the material form is a roll, a granule, a sheet or a flake.

[0157] In some embodiments, the recombinant silk protein comprises a hollow core and / or shell. In some embodiments, the diameter of the recombinant silk protein is in the range of about 1 μm to about 30 μm, about 5 μm to about 20 μm, or about 10 μm to about 50 μm, while the diameter of the recombinant silk protein in water is in the range of about 20 to about 80 μm, about 30 μm to about 70 μm, or about 40 μm to about 100 μm. Before being incorporated into the composition of the present disclosure, the recombinant silk protein hollow powder can be ground and incorporated as a ground powder.

[0158] Solvents

[0159] In some embodiments, the silicone substitute 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, glycerol, deionized water, olive oil, and pentanediol. For example, the recombinant silk polypeptide may be treated with a solvent so that the hollow core contains a solvent, such as liquid water or glycerol, 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 substitute component includes about 25 wt % glycerol solution.

[0160] In some embodiments, the solvent is water. Without being limited by theory, the recombinant silk polypeptide is placed in a solvent such as water to cause an expanded or swollen recombinant silk polypeptide, wherein the protein acts as a carrier containing a solvent (e.g., water). These compositions can be stored dry and can be partially rehydrated after being immersed in water to directly form a liquid or semi-liquid aqueous suspension of the expanded particles.

[0161] In certain embodiments, recombinant silk protein can expand a part of hollow core. In some other embodiments, recombinant silk protein can expand a part of shell. In such embodiments where the solvent is water, recombinant silk protein is converted into hydrogel. In other embodiments where the solvent is water, recombinant silk protein is converted into paste. In various embodiments, heat and / or pressure can be added to further process the recombinant silk protein composition.

[0162] In some embodiments, the solvent is generally present in a ratio ranging from 55% to 90% by weight relative to the total weight of the recombinant silk polypeptide. This range includes all specific values ​​and subranges therebetween, including 60%, 65%, 70%, 75%, 80%, and 85% by weight. In some embodiments, the recombinant silk protein is insoluble in various solvents, including water, glycerol, alcohol, silicone, and oil at various pH levels.

[0163] In some embodiments, the solvent is of aqueous type. In such embodiments, the solvent is water. The pH of the solvent may be in the range of 6 to 12. In some embodiments, the pH of the solvent is 6. In some other embodiments, the pH of the solvent is in the range of 0 to 5, 2 to 7, 4 to 9, 6 to 11, 8 to 13, or 10 to 14.

[0164] In other embodiments, the solvent comprises a mixture of various volatile organic solvents in order to obtain relatively short drying times. In some embodiments, the solvent is an alcohol.

[0165] The solvent may include water, ethanol, toluene, dichloromethane, isopropyl alcohol, n-butyl alcohol, castor oil, organopolysiloxane oil, ethylene glycol monoethyl ether, diethylene glycol monobutyl ether, diethylene glycol monoethyl ether, dimethyl sulfoxide, dimethylformamide, and tetrahydrofuran.

[0166] In some embodiments, the organopolysiloxane oil can be volatile, non-volatile or a mixture of volatile and non-volatile silicones. As used in this context, the term "non-volatile" refers to those silicones that are liquid under ambient conditions and have a flash point (at one atmosphere) greater than about 100°C. As used in this context, the term "volatile" refers to all other silicone oils. Suitable organopolysiloxanes can be selected from various silicones across a wide range of volatility and viscosity. Suitable silicones are disclosed in U.S. Patent No. 5,069,897, published on 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 polydimethylsiloxanes, polydimethylsiloxane alcohols, polyalkylaryl siloxanes, and mixtures thereof. For example, polyalkylsiloxanes, polydimethylsiloxanes, and cyclomethicone can be used.

[0167] In some embodiments, the solvent is a vegetable oil or hydrogenated vegetable oil. In some embodiments, the solvent is a free fatty acid. Examples of vegetable oils or hydrogenated vegetable oils include safflower oil, castor oil, coconut oil, cottonseed oil, herring 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 may be used, for example, cod liver oil, lanolin and its derivatives, such as acetylated lanolin and isopropyl lanolate. The C4-C 20 Alkyl ether, polypropylene glycol C1-C 20 Carboxylic acid esters and di-C8-C 30 Alkyl ethers are also useful, examples of which include PPG-14 butyl ether, PPG-15 stearyl ether, dioctyl ether, dodecyl octyl ether, and mixtures thereof.

[0168] 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%.

[0169] Recombinant silk protein as cosmetic formulation

[0170] In various embodiments, recombinant silk protein is compounded into silk cosmetics or skin care products (e.g., solutions applied to skin or hair). Specifically, recombinant silk protein can be incorporated into silicone substitute components to be used as a base liquid for cosmetics or skin care products, wherein the recombinant silk polypeptide is present in the base liquid in its monomeric or less crystalline form. In some embodiments, the silicone substitute component can be used as a base liquid for cosmetics or skin care products, wherein the recombinant silk polypeptide is present in the base liquid in a semi-crystalline form. In such embodiments, the recombinant silk polypeptide is not present in the base liquid in its monomeric form.

[0171] In most embodiments, the cosmetic formulation is physically stable. In such embodiments, the recombinant silk protein and any other ingredients remain in its formulation for an extended period of time, having an extended shelf life. In long-term use, the silicone replacement component remains substantially stable, and the ingredients do not precipitate out of the formulation.

[0172] The composition of the present invention can be used to apply silk protein to the skin, nails, hair or mucosa by contacting the composition with the skin, nails, hair or mucosa of the subject. Preferably, the composition of the present invention is used for human subjects.

[0173] In most embodiments, the cosmetic formulations are non-toxic or non-allergenic to the subject host to which the cosmetic is applied. It is also desirable in the art to produce cosmetic compositions for hair and cuticle contact that will not permanently stain the tissue and can be removed by ordinary washing with aqueous detergents.

[0174] The solid, film, emulsion, hydrogel and other material forms discussed in the various embodiments may contain various wetting agents, 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 serves as a carrier. In some embodiments, recombinant silk protein is a carrier for delivering one or more agents to surfaces such as skin, hair or nails.

[0175] In some embodiments, the cosmetic formulation includes a plasticizer. The suitable concentration (by weight) of the plasticizer in the composition is within the following range: for example: 1% to 60% by weight, 10% to 60% by weight, 10% to 50% by weight, 10% to 40% by weight, 15% to 40% by weight, 10% to 30% by weight, 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.

[0176] In the case where water is used as a plasticizer, a suitable concentration of water in the composition (by weight) is in the following ranges: for example: 5% to 80% by weight, 15% to 70% by weight, 20% to 60% by weight, 25% to 50% by weight, 19% to 43% by weight, or 19% to 27% by weight. In the case where water is used in combination with another plasticizer, the concentration may be present in the range of, for example, 5% to 50% by weight, 15% to 43% by weight, or 19% to 27% by weight.

[0177] In some embodiments, suitable plasticizers may include polyols (e.g., glycerol), water, lactic acid, ascorbic acid, phosphoric acid, ethylene glycol, propylene glycol, triethanolamine, ethyl acetate, propane-1,3-diol, or any combination thereof. In various embodiments, the amount of plasticizer may vary according to the purity and relative composition of the recombinant silk protein. For example, a powder of higher purity may have fewer impurities, such as low molecular weight compounds that can act as plasticizers, and therefore a higher percentage by weight of plasticizer may need to be added.

[0178] In certain embodiments, the composition includes a wetting agent or emollient. As used herein, the term "wetting agent" refers to a hygroscopic substance that forms a bond with a water molecule. Suitable wetting agents include, but are not limited to, glycerol, propylene glycol, polyethylene glycol, pentylene glycol, Tremella fuciformis extract, sorbitol, dicyanamide, sodium lactate, hyaluronic acid, aloe extract, alpha-hydroxy acid, and pyrrolidone carboxylic acid ester (NaPCA).

[0179] As used herein, the term "emollient" refers to a compound that provides a soft or supple appearance to the skin by filling the cracks on the surface of the skin. Suitable emollients include, but are not limited to, shea butter, cocoa butter, squalene, squalane, octyl octanoate, sesame oil, grape seed oil, natural oils containing oleic acid (e.g., sweet almond oil, argan oil, olive oil, avocado oil), natural oils containing gamma linoleic acid (e.g., evening primrose oil, borage oil), natural oils containing linoleic acid (e.g., safflower oil, sunflower oil) or any combination thereof.

[0180] In some cases, an emollient or humectant can be an occlusive agent, and the present disclosure contemplates including an occlusive agent in the composition in various embodiments. The term "occlusive agent" refers to a compound that forms a barrier on the surface of the skin to retain moisture. Other suitable occlusive agents may include, but are not limited to, beeswax, carnauba wax, ceramides, vegetable waxes, lecithin, allantoin. Without intending to be limited by theory, the film-forming ability of the silicone substitute components proposed herein produces an occlusive agent that forms a moisturizing barrier because the recombinant silk polypeptide is used to attract water molecules and also acts as a humectant.

[0181] Optionally, the cosmetic formulation includes an active agent. The term "active agent" refers to any compound with known beneficial effects in hair care, skin care or cosmetic formulations, including pigments in cosmetic formulations. Various active agents include, but are not limited to, acetic acid (i.e., vitamin C), alpha hydroxy acids, beta hydroxy acids, zinc oxide, titanium dioxide, retinol, niacinamide, other recombinant proteins (as full-length sequences or hydrolyzed into subsequences 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 extract), caffeine, alpha arbutin, coenzyme Q-10, and salicylic acid.

[0182] The term "cosmetic adjuvant" refers to various other agents used to produce a cosmetic product with commercially desirable properties, including but not limited to surfactants, emulsifiers, preservatives, and thickeners.

[0183] As described herein, in various embodiments, recombinant silk proteins can form dispersible semisolid or gel-like structures. In various embodiments where recombinant silk proteins are incorporated into skin care formulations, recombinant silk proteins can form irreversible three-dimensional structures, such as gels or films that transform into dispersible liquids on the skin surface.

[0184] In various embodiments, recombinant silk protein can be suspended in water ("aqueous suspension protein") to form a silicone substitute component in the form of a film, gel or base liquid that can be incorporated (i.e., compounded) into a cosmetic or skin care formulation. According to the embodiment, the amount of recombinant silk protein and water in the aqueous suspension protein can vary, and the relative ratio of the recombinant silk polypeptide powder to the additive in the recombinant silk protein can also vary. In some embodiments, the silicone substitute component will include 10-33% by weight of recombinant silk polypeptide powder. In some embodiments, a solvent different from water will be used. In some embodiments, the recombinant silk protein is suspended in water to produce an aqueous suspension protein as 1-40% recombinant silk protein and 60-99% water. In a specific embodiment, the silicone substitute component is suspended in water to produce an aqueous suspension protein, based on the total weight of the silicone substitute component, the aqueous suspension protein is 10% by weight of recombinant silk polypeptide powder, 30% by weight of additives and 60% by weight of water. In a specific embodiment, the protein is suspended in water to produce an aqueous suspension protein, which is 6 wt% recombinant silk polypeptide powder, 18 wt% additives and 76 wt% water based on the total weight of the silicone substitute component. In a specific embodiment, the protein is suspended in water to produce an aqueous suspension protein, which is 10 wt% recombinant silk polypeptide powder and 90 wt% water based on the total weight of the silicone substitute component.

[0185] According to an embodiment, when the aqueous suspension protein is resuspended in water, the aqueous suspension protein can be optionally heated and stirred. In some embodiments, heating and stirring the aqueous suspension protein can produce a phase change of the recombinant silk polypeptide in the aqueous suspension protein. Specifically, heating and stirring the aqueous suspension protein produces three different phases evaluated by centrifugation: 1) a gel phase different from the supernatant after centrifugation; 2) a colloidal phase that can be filtered out from the supernatant after centrifugation; and 3) a solution phase remaining after filtering the colloidal phase from the supernatant. Various combinations of heating, stirring and centrifugation can be used, provided that the aqueous suspension protein must not be subjected to prolonged heating to prevent degradation of the recombinant silk polypeptide. In a specific embodiment, the protein is subjected to gentle stirring for 5 minutes at 90°C and centrifuged for 30 minutes at 16,000RCF.

[0186] In various embodiments, various phases (i.e., colloidal phase, gel phase and solution) of aqueous suspension protein or aqueous suspension protein can be incorporated into cosmetics or skin care formulations to provide a source of recombinant silk protein. According to an embodiment, before being incorporated into the skin care formulation, the aqueous suspension protein can be subjected to stirring when heated or not heated. Optionally, aqueous suspension protein can be separated in the phase discussed above by centrifugation and / or filtration. According to an embodiment, the skin care formulation can be an emulsion (e.g., frost or essence) or mainly an aqueous solution (e.g., gel). In certain embodiments, recombinant silk protein can be incorporated into any cosmetics, skin care or hair care formulation described herein without the need for aqueous resuspension. In these compositions, a homogenizer or similar equipment can be used to ensure that the recombinant silk protein is evenly distributed in the composition.

[0187] In certain embodiments, the aqueous suspension protein can be subjected to heating and stirring, then cast onto a flat surface and dried to form a film. In certain embodiments, the aqueous suspension protein can be cast onto a flat surface and dried to form a film without being subjected to heating and / or stirring. In such embodiments, the aqueous suspension protein can be cast onto a flat surface and dried to form a film without being subjected to additional processing. In certain embodiments, the aqueous suspension protein can be incorporated into an emulsion, then cast onto a flat surface and dried to form a film. According to an embodiment, various drying conditions can be used. Suitable drying conditions are included in the case of having and not having vacuum and are dried at 60 ℃ or 80 ℃. In the embodiment using vacuum, 15Hg is a suitable vacuum amount. Other drying methods are generally recognized in the art.

[0188] In various embodiments, the film including only aqueous suspended proteins has a low melting temperature. In various embodiments, the film including only aqueous suspended proteins has a melting temperature below body temperature (about 34-36° C.) and melts when in contact with the skin. It is not intended to be limited by theory, and the recombinant silk polypeptide forms enough intermolecular interactions to form a semi-solid structure (i.e., a film); however, the structure is reversible when in contact with the skin and can be re-formed after being dispersed on the skin surface. In various embodiments, as measured by Fourier transform infrared spectroscopy (FTIR), the film will have a reduced crystallinity compared to recombinant silk protein or recombinant silk powder. In various embodiments, the film including aqueous suspended proteins will not melt when in contact with the skin. In such embodiments, the film acts as a barrier. In various embodiments, the film is a low-density hydrophobic film. The thickness of the film or barrier can be in the range of about 1 μm to about 50 μm, about 10 μm to about 30 μm, or about 20 μm to about 40 μm. When in contact with the skin, a barrier can be formed on the surface of the epidermis, thereby achieving firm nonspecific adhesion to the skin surface. In some embodiments, the thickness of the film varies according to the concentration of the recombinant silk protein and the surface area of ​​application.

[0189] In some embodiments, the barrier is durable and protects against one or more environmental stressors, including wind, humidity, irritating additives, pollution, abrasion, dirt, and grease. The barrier can withstand wear equivalent to at least 100 manual rubs, at least 200 rubs, at least 400 rubs, at least 600 rubs, or at least 800 rubs.

[0190] In a specific embodiment, aqueous suspended proteins or proteins can be incorporated (e.g., homogenized) into an emulsion, then cast on a flat surface and lyophilized to produce a porous membrane. According to an embodiment, various techniques can be used for lyophilization, including freezing the membrane at -80°C for 30 minutes. Other lyophilization techniques will be well known to those skilled in the art.

[0191] In various embodiments, the film described above can be used as a topical skin care agent. The film can be applied directly to the skin and can be rehydrated to form a dispersible viscous material 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 onto the skin due to contact with the skin or after the film is gently rubbed into the skin. In certain embodiments, the film can be applied directly to the skin and adsorbed onto the skin without the need for additional friction or contact. In certain embodiments, the protein resuspended in the aqueous solution can be applied to the face and then exposed to a coagulant such as propylene glycol by spraying to form a gelable facial mask.

[0192] Depending on the embodiment, the cast film can be a flat film (ie, without surface variability) or can be cast onto a mold incorporating microstructures. In a specific embodiment, the film is cast onto a mold incorporating microneedle structures to pierce the skin surface and aid in the delivery of active agents.

[0193] In an alternative embodiment, the aqueous suspended protein can be added to an emulsion for use as a cosmetic product. The emulsion can be applied to the skin or hair and then allowed to form a film on the skin surface after drying. As discussed herein, various emollients, humectants, active agents and other cosmetic adjuvants can be incorporated into the emulsion.

[0194] In certain embodiments, the compositions of the present disclosure can be liquid or semisolid, such as cream, lotion and gel. Compositions that can be used for the present invention can be made into various product forms known in the art. These include but are not limited to powders, lotions, creams, gels, patches, essences, ampoules, powders, sticks, sprays, ointments, pastes, mousses, ointments, liquids, emulsions, foams or aerosols. These product forms can include several types of additives, as further discussed herein, including but not limited to solutions, aerosols, emulsions, gels, solids and liposomes. Active compounds in the compositions and methods of the present invention can be delivered locally by any means known to those skilled in the art.

[0195] In some other embodiments, the composition can be a basic cosmetic composition, such as a facial cleanser, such as floral water, cream, serum, cleansing foam and cleansing water; facial mask and body oil; color cosmetic composition, such as foundation, lipstick, mascara and primer; hair product composition, such as shampoo, rinse, conditioner and hair spray; soap; etc. Cosmetic formulations can be prepared by any method known in the art, using the silicone replacement component described herein, optionally together with at least one carrier and / or additive, which is generally used in the art of preparing cosmetic compositions.

[0196] In certain embodiments, the composition includes at least one cosmetic agent. The example of cosmetic agent includes emollient, wetting agent, colorant, pigment, fragrance, wetting agent, viscosity modifier and any other cosmetic forming agent. One or more cosmetic agents can be included in the cosmetic composition. In another embodiment, other active ingredients known in the art and described herein can also be used, including but not limited to skin softener, skin penetration enhancer, colorant, fragrance, emulsifier and thickener. In addition, for example, to improve the purpose of physical properties, the cosmetic composition can further include fragrance, pigment, bactericide, antioxidant, preservative and / or wetting agent and inorganic salt and synthetic polymer material.

[0197] The composition can also be delivered topically by lotion. Single emulsion skin care formulations (such as lotions and creams) of the oil-in-water type and the water-in-oil type are well known in the cosmetic field and can be used in the present invention. Multiphase emulsion compositions such as the water-in-oil-in-water type can also be used in the present invention. Typically, such single-phase or multiphase emulsions contain water, an emollient, and an emulsifier as essential ingredients.

[0198] The compositions of the present invention may also be formulated as solid formulations (eg, wax-based bars, soap bar compositions, powders, beads, exfoliants, or wipes containing liquids or powders).

[0199] The composition of the present invention can be formulated into a gel (e.g., an aqueous gel 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., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable gelling agents for oils (e.g., mineral oils) include, but are not limited to, hydrogenated butylene / ethylene / styrene copolymers and hydrogenated ethylene / propylene / styrene copolymers. Such gels typically include such gelling agents between about 0.1% by weight and 5% by weight. In certain embodiments, such compositions include a combination of recombinant silk protein, water (aqueous), sodium C14-16 olefin sulfonate, glycerol, coco betaine, sodium benzoate, sodium hydroxide, calcium gluconate, sodium hyaluronate, propylene glycol, xanthan gum, gluconolactone, and tetrasodium glutamate diacetate. In certain embodiments, the composition includes a cleaning detergent, soap, essence, or toner. In a specific embodiment, the essence is water-based. In another specific embodiment, the toner is alcohol-based.

[0200] The composition that can be used in the present invention can be formulated into an emulsion. In some embodiments, if the composition is an emulsion, about 1% to about 10% or about 2% to about 5% of the composition includes an emulsifier. The emulsifier can be nonionic, anionic or cationic. Suitable emulsifiers are disclosed in, for example, the INCI Handbook, pages 1673-1686. Lotions and creams can be formulated into emulsions. In some embodiments, the composition is an emulsion, and the recombinant silk protein is an emulsifier. In some embodiments, the composition is an emulsion, the recombinant silk protein is an emulsifier, and the composition does not contain other emulsifiers.

[0201] Another type of composition can be an ointment. Ointment can include a simple base liquid of animal oil or vegetable oil or semisolid hydrocarbon. In addition to about 0.1% to about 2% thickener, ointment can include about 2% to about 10% emollient. The example of thickener includes, for example, cellulose derivatives (methylcellulose and hydroxypropyl methylcellulose), synthetic high molecular weight polymers (for example, carboxyvinyl polymers and polyvinyl alcohol), plant hydrocolloids (for example, Karaya gum and tragacanth gum), clay thickeners (for example, colloidal magnesium aluminum silicate and bentonite), carboxyvinyl polymers, carboxylic acid polymers, cross-linked polyacrylates, polyacrylamide, xanthan gum and mixtures thereof.

[0202] In addition to the components mentioned hereinbefore, the compositions useful herein may contain various additional oil-soluble and / or water-soluble materials which are conventionally used in compositions for application to the skin, hair and nails in the state of the art.

[0203] The compositions of the present invention may be applied directly to the skin or may be applied to other delivery vehicles 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 rinsed off the skin.

[0204] In some embodiments, the composition improves the appearance of skin, such as increasing skin firmness / plumpness; increasing elasticity; improving overall skin health; increasing hydration; accelerating and / or improving wound healing; improving pollution protection; reducing skin aging; reducing skin fragility; preventing and reversing collagen and / or elastin loss; preventing skin atrophy; promoting / accelerating cell renewal; increasing genetic expression; improving skin texture; preventing and reducing fine lines and wrinkles; improving skin tone; increasing skin thickness; reducing pore size; minimizing skin pigmentation; restoring skin radiance; minimizing signs of fatigue; improving skin barrier function; minimizing skin dryness; preventing, reducing or treating hyperpigmentation; improving mitochondrial function of the skin; 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.

[0205] The compositions of the various embodiments protect against pollutants and other irritants. Thus, many skin conditions, such as acne, redness associated with rosacea (adult acne), and other inflammatory conditions can be actively managed by applying cosmetic formulations.

[0206] Coagulant

[0207] In some embodiments, the composition and / or silicone substitute component containing recombinant silk polypeptide as described herein is exposed to a coagulant. This can change the properties of the composition / component to promote the controlled aggregation of silk in the silk-based composition. In some embodiments, the composition / component is immersed in a coagulant. In some embodiments, the composition / component is exposed to a coagulant mist or steam. In one embodiment, the aqueous protein composition includes a coagulant or is immersed or mixed with a coagulant. In some embodiments, a silk-based solid or semi-solid such as a film is immersed in a steam comprising a coagulant or is exposed to a steam comprising a coagulant. In some embodiments, methanol is used as an effective coagulant.

[0208] In some embodiments, alcohol (e.g., isopropanol, ethanol, or methanol) can be used as a coagulant or solvent. In some embodiments, 60%, 70%, 80%, 90%, or 100% of alcohol is used as a coagulant. In some embodiments, salt can be used as a coagulant. Examples of salts include, but are not limited to, ammonium sulfate, sodium chloride, sodium sulfate, or other protein precipitating salts effective at temperatures between 20°C and 60°C.

[0209] In some embodiments, a combination of one or more of water, acid, solvent, and salt may be used as a coagulant, including but not limited to the following classes of chemicals: Bronsted-Lowry acid ( -Lowryacid), Lewis acid, dibasic hydride acid, organic acid, metal cation acid, organic solvent, inorganic solvent, alkali metal salt and alkaline earth metal salt. 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, tert-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 nitrate, sulfate or phosphate. In some embodiments, the coagulant has a pH of 2.5 to 7.5.

[0210] Other additives

[0211] In some embodiments, the composition and / or its silicone substitute component according to the present disclosure may include one or more additives. The additive can change the properties of the composition because it interacts with the skin. In some embodiments, the silk-based composition is immersed in the additive. In some embodiments, the composition / component is exposed to an additive mist or steam. In one embodiment, the aqueous protein composition includes an additive or is immersed or mixed with an additive. In some embodiments, a silk-based solid or semi-solid (such as a film) is immersed in a steam including an additive or is exposed to a steam including an additive. In some embodiments, before the hollow powder is formed, the silk-based gel is exposed to an additive (for example, the silk-based gel and the additive are co-spray dried).

[0212] The additive itself can be inert or the additive can have its own dermatological benefit. The additive should also be physically and chemically compatible with the essential components described herein, and should not excessively impair the stability, efficacy or other benefits of use relevant to the composition of the present invention. The additive type utilized in the present invention depends on the type of product form desired by the composition. In certain embodiments, the additive is an acid textile dye.

[0213] Pigment is usually added to cosmetic formulations to achieve the desired color applied to the skin. Such pigments are known, and the concentration required to achieve the desired coloring is easily determined. Pigment can be inorganic or organic. Inorganic pigments include iron oxide (red, black, brown), manganese violet, ultramarine (green, blue, pink, red or purple sulfosilicate aluminum), aquamarine, copper powder, mica, clay, silicon dioxide and titanium dioxide. Organic dyes certified by US FDA for cosmetic purposes usually have the prefix "D & C" and the suffix of color and number (for example, D & C green #3).

[0214] Based on the anhydrous pigment weight, 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% of a colorant. These are typically aluminum, barium or calcium salts or lakes. The dye can be present in a concentration of about 0% to about 3% and pearlescent agents, etc., are present in a concentration of 0% to about 10%. The combination of such dyes and recombinant silk protein is stable and has a long shelf life. The shelf life of such compositions can be about 6 months, about 1 year or about 2 years. In certain embodiments, the shelf life of such compositions can be at least 5 years.

[0215] There is no particular limitation on the pigment, colorant or filler powder used in the composition. Each may be a body pigment, an inorganic white pigment, an inorganic colored pigment, a pearlescent agent, etc. Specific examples are talc, mica, magnesium carbonate, calcium carbonate, magnesium silicate, magnesium aluminum silicate, silicon dioxide, 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, iron oxide titanium mica, bismuth oxychloride, etc.

[0216] Additional pigment / powder fillers include, but are not limited to, inorganic powders such as gums, chalk, Fuller's earth, kaolin, sericite, muscovite, phlogopite, synthetic mica, red mica, biotite, lepidolite, vermiculite, aluminum silicates, starches, montmorillonite clays, alkyl and / or trialkylaryl ammonium montmorillonites, chemically modified aluminum silicates, organically modified montmorillonite clays, hydrated aluminum silicates, fumed aluminum starch barium silicate octenylsuccinate, calcium silicates, magnesium silicates, strontium silicates, metal tungstates, magnesium, silica alumina, zeolites, barium sulfate, calcined calcium sulfate (calcined gypsum), ), calcium phosphate, fluoroapatite, hydroxyapatite, ceramic powder, metal soap (zinc stearate, magnesium stearate, zinc myristate, calcium palmitate and aluminum stearate), colloidal silica and boron nitride; organic powders such as polyamide resin powder (nylon powder), cyclodextrin, polymethyl methacrylate powder, copolymer powder of styrene and acrylic acid, benzoguanidine resin powder, poly(tetrafluoroethylene) powder and carboxyvinyl polymer, cellulose powder such as hydroxyethyl cellulose and sodium carboxymethyl cellulose, ethylene glycol monostearate; and inorganic white pigments such as magnesium oxide. Other useful powders are disclosed in U.S. Pat. No. 5,688,831 issued to El-Nokaly et al. on November 18, 1997, which is incorporated herein by reference in its entirety. These pigments and powders can be used independently or in combination.

[0217] In addition to silk protein, the composition according to the present invention may further include a film-forming substance. Examples of film-forming substances include, for example, cellulose derivatives, nitrocellulose, acrylic acid polymers or copolymers, acrylic acid, styrene, acrylate-styrene and vinyl resins, vinyl copolymers, polyester polymers, arylsulfonamide resins and alkyd resins.

[0218] In some embodiments, the composition may include an amphoteric surfactant, a phospholipid, or a wax.

[0219] Examples of other additives include, but are not limited to, foaming surfactants, decolorizing agents, reflective agents, detangling / wet combing agents, amino acids and their derivatives, antimicrobial agents, allergy inhibitors, anti-acne agents, anti-aging agents, anti-wrinkle agents, preservatives, analgesics, antitussives, antipruritics, local anesthetics, anti-hair loss agents, hair growth promoters, hair growth inhibitors, antihistamines, anti-infectives, inflammation inhibitors, antiemetics, anticholinergics, vasoconstrictors, vasodilators, wound healing promoters, peptides, polypeptides and proteins, deodorants and antiperspirants, medicaments, skin Skin emollients and skin moisturizers, skin firming agents, hair conditioners, hair softeners, hair moisturizers, vitamins, tanning agents, skin lighteners, antifungal agents, depilatories, shaving preparations, topical analgesics, perfumes, anti-irritants, hemorrhoid medications, insecticides, poison ivy products, poison oak products, burn products, anti-diaper rash agents, prickly heat preparations, cosmetic preparations, vitamins, herbal extracts, retinoids, flavonoids, sensates, antioxidants, skin conditioners, hair lighteners, chelating agents, cell renewal enhancers, sunscreens, anti-edema agents, collagen enhancers, and mixtures thereof.

[0220] Examples of suitable vitamins non-exclusively include vitamin B complex including thiamine, niacin, biotin, pantothenic acid, choline, riboflavin, vitamin B6, vitamin B12, pyridoxine, inositol, carnitine; vitamins A, C, D, E, K and derivatives thereof, such as vitamin A palmitate and provitamins (e.g., panthenol (provitamin B5) and panthenol triacetate) and mixtures thereof.

[0221] Examples of sunscreens include, but are not limited to, avobenzone, benzophenone, bornene, butyl para-aminobenzoic acid, cinnamyl trimethyl ammonium chloride, disodium distyryl biphenyl disulfonate, para-aminobenzoic acid, potassium methoxycinnamate, butyl methoxydibenzoylmethane, octyl methoxycinnamate, oxybenzophenone, octyl propene, octyl salicylate, phenylbenzimidazole sulfonic acid, ethyl hydroxypropylaminobenzoate, menthyl anthranilate, aminobenzoic acid, cinoxate, diethanolamine methoxycinnamate, glyceryl aminobenzoate, titanium dioxide, zinc oxide, oxybenzone, Padimate O, red petrolatum, and mixtures thereof.

[0222] The amount of the additive combined with the composition can vary depending on, for example, the ability of the additive to penetrate the skin, hair or nails; the specific additive selected; the specific benefit desired; the user's sensitivity to the additive; the user's health, age, and skin, hair and / or nail condition, etc. In general, the additive is used in a "safe and effective amount," which is an amount high enough to provide the desired skin, hair or nail benefit or to modify a condition to be treated, but low enough to avoid serious side effects, with a reasonable risk-benefit ratio within the scope of sound medical judgment.

[0223] The present invention disclosed illustratively herein can be suitably practiced in the absence of any component, ingredient or step not specifically disclosed herein. Several examples are listed below to further illustrate the nature of the present invention and the mode of implementing the present invention. However, the present invention should not be considered to be limited to its details.

[0224] The compositions and methods of the present invention provide the same or better softness, rapid absorption, easy spreadability (or adequate "playtime"), light film formation, and non-greasy afterfeel to the skin compared to compositions containing silicone elastomers. Additionally, if the skin is treated with an SPF composition, the present invention provides the same or better low whitening performance. The compositions and methods of the present invention provide the same or better long-lasting, shine, non-greasiness, frizz control, increased hair thickness, style retention, static properties, heat resistance, and protection from UV radiation and pollution to the hair.

[0225] Equivalents and scope

[0226] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the invention described herein.The scope of the present invention is not intended to be limited to the above description, but rather as set forth in the accompanying aspects.

[0227] In aspects, articles such as "a", "an", and "the" may mean one or more than one, unless specified to the contrary or otherwise obvious from the context. Aspects or descriptions involving "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a given product or process, unless specified to the contrary or otherwise obvious from the context. The invention includes embodiments in which exactly one member of the group is present in, used in, or otherwise related to a given product or process. The invention includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise related to a given product or process.

[0228] It should also be noted that the term "comprising" is intended to be open ended, and allows but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the terms "consisting of" and "consisting essential of" are also encompassed and disclosed.

[0229] In the case of a given range, the end values ​​are included. In addition, it should be understood that unless otherwise indicated or otherwise becomes obvious from the context and the understanding of a person of ordinary skill in the art, the values ​​expressed as ranges can assume that any specific value or sub-range within the range described in different embodiments of the present invention is one-tenth of the lower limit unit of the range (unless otherwise clearly indicated in the context).

[0230] 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 a cited source and the description of this application, the description in this application shall prevail.

[0231] Section and table headings are not intended to be limiting.

[0232] Examples

[0233] Below is the example for implementing the specific embodiment of the present invention.These 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 the accuracy of the numerals (e.g., amount, temperature, etc.) used, but of course some experimental errors and deviations should be allowed.

[0234] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, which are within the skill of the art. Such techniques are fully explained in the literature. See, e.g., T.Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993); A. Lehninger, Biochemistry (Worth Publishers, Inc., current edition); 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 edition (Plenum Publishing, Inc., 1991); Press)) Volumes A and B (1992).

[0235] Example 1: Recombinant Silk Polypeptides as a Replacement for Silicone in SPF Formulations

[0236] In this example, recombinant silk polypeptides are shown to match and outperform silicones (i.e., blends of linear silicones and cyclosiloxanes) in certain properties of SPF skin care products. A mineral SPF formulation originally containing 2% of a combined linear silicone and cyclosiloxane (1% of each component) was reformulated with 1.5% recombinant silk polypeptides and evaluated for aesthetic properties. In a blind comparison of whitening, absorption, film feel, and oiliness, recombinant silk polypeptides outperformed silicone formulations while matching silicone formulations in terms of spreadable time and softness properties.

[0237] Method: The SPF formulation was prepared similar to standard practice in the art. The water phase ingredients were mixed together separately, and the oil phase ingredients were mixed together separately. The two were combined at an elevated temperature of 80-85°C with gentle mixing. The ingredients were continuously mixed until uniform and then cooled to 30-45°C. After the water and oil phases were mixed together and cooled to 30-45°C, the 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 the recombinant silk polypeptide and the silicone elastomer between the formulations. For this formulation, the recombinant silk polypeptide in powder form was used.

[0238] The spider graph was prepared by a blind comparative test, where subjects were asked to compare the two formulations on a scale of 1-5 for six different attributes (symbol legend: 1 = unacceptable, 2 = poor, 3 = satisfactory, 4 = good, 5 = excellent). Ten (10) different individuals tested the products on their faces for a full week. Skin types varied according to oily, dry and combination.

[0239] Figure 1A Shown is a top view of an SPF formulation.

[0240] Figure 1B A spider graph plotting data from a blind test comparison of 10 subjects is shown. The 1.5% recombinant silk polypeptide formulation outperformed the 2% silicone formulation in desirable whitening, absorption, film feel, and oiliness. The 1.5% recombinant silk polypeptide formulation performed similarly to the 2% silicone formulation in terms of spreadability and softness.

[0241] Figure 1C : Table of ingredients used in SPF formulations (excluding recombinant silk polypeptides and silicones).

[0242] Figure 1D : Although the SPF formulation containing 1.5% recombinant silk polypeptides outperformed the 2% silicone formulation in terms of desirable whitening, absorption, film feel, and oiliness, the rheological data showed similarities between the three formulations. The frequency sweep showed similar amplitudes and shapes of the G' and G" curves. This data supports that the SPF formulations have similar gel-like structural properties.

[0243] Figure 1E: Optical microscope images of SPF primer and SPF primer formulated with 1% recombinant silk polypeptide protein or 5% silicone elastomer ingredients. The SPF primer and 5% silicone elastomer samples exhibit typical dispersion morphology with more transparent areas and other areas that are opaque. The 1% recombinant silk polypeptide sample clearly has visible particles that are hollow and 2-200 μm in diameter and appear as bright circles. White arrows are added to the figure to point out the example silk powder particles. Reference image containing 1% recombinant silk polypeptide powder suspended in water.

[0244] Example 2: Recombinant Silk Peptides as Silicone Replacements in Color Cosmetic Products

[0245] For certain properties of color cosmetic products, recombinant silk polypeptides match and outperform silicone elastomers. Pigmented 3-in-1 eye cream / face cream / lip cream formulations were prepared with 1% recombinant silk polypeptides or 5% or 10% silicone elastomers, and their aesthetic properties were evaluated. Recombinant silk polypeptide formulations outperform silicone elastomers and provide improved spreadability, even pigment delivery, and soft drying (i.e., not as sticky or greasy as the silicone elastomer version), and the recombinant silk polypeptide formulations are also resistant to rubbing.

[0246] Method: Color cosmetic formulations are prepared similarly to standard practices in the art. All waxes, oils, and solvents are placed at room temperature or elevated temperature, e.g., above 37° C. but below 90° C. The oils and solvents are mixed with the pigment and gently ground. Then, the wax is added to the mixture and cooled. b-silk protein (i.e., 18B recombinant silk polypeptides including SEQ ID NO: 2878) and / or silicone elastomer components can be added at any stage of the process. Water is used to supplement the difference in weight percentage of recombinant silk polypeptides and silicone elastomers between formulations. For this formulation, recombinant silk polypeptides in powder form are used, and a silicone elastomer component with approximately 30% solids is used.

[0247] The test for paint application and rub-off involved applying 10 mg of the product to a 1 cm x 1 cm square area and allowing the product to dry for 15 minutes. To test rub-off, a white facial tissue was laid on the skin and a 200 g mass was placed on the skin. The facial tissue was drawn over the skin 3 times and the rub-off properties were visually assessed.

[0248] 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 material, 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 .

[0249] Figure 2A : Top view of color cosmetic formulation.

[0250] Figure 2B : Step-by-step visual assessment of how to assess pigment delivery and fastness on skin of color cosmetics.

[0251] Figure 2C : Representative images of the product's color application and rub-off. Recombinant silk polypeptides had a more even spread of pigment on the skin and held firmer on the skin during rub-off compared to the 5% and 10% elastomer products.

[0252] Figure 2D : Table of ingredients used in color cosmetic formulations (excluding recombinant silk polypeptides and silicones).

[0253] Figure 2E : Optical microscope images of color cosmetic bases and bases formulated with 1% b-silk protein or 5% silicone elastomer ingredients. The SPF base and 5% silicone elastomer samples exhibit typical dispersion morphology with more transparent areas and other areas that are opaque. The 1% recombinant silk polypeptide sample clearly has visible particles that are hollow and 2-200 μm in diameter and appear as bright circles. White arrows are added to the figure to point out the example silk powder particles.

[0254] Figure 2F : Although the color cosmetic formulation containing 1% recombinant silk protein was significantly different from the 5% and 10% formulations as measured by performance properties and microscopy, the rheological data showed similarities between the three formulations. The frequency sweep showed similar amplitude and shape of the G' and G" curves. This data indicates that the color cosmetic has similar gel-like structural properties.

[0255] Example 3: Recombinant Silk Peptides as Replacements for Silicone Elastomers in Hair Serums

[0256] Leave-in hair serums were prepared with either 1% recombinant silk polypeptide or 5% silicone elastomer. The formulations performed similarly in styling aspects including shine, curl retention, and curl control.

[0257] Method: A hair serum formulation was prepared similar to standard practice in the art. All ingredients were combined with moderate mixing until completely homogeneous. Water was used to make up for the difference in weight percentage of recombinant silk polypeptide (i.e., 18B silk) to silicone elastomer between formulations. For this formulation, recombinant silk polypeptide was used in powder form, and a silicone elastomer component with approximately 30% solids was used.

[0258] 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 material, the shear strain range was 0.01% to 250% at 1 s -1The amplitude sweep was performed at the frequency of . In addition, the frequency sweep was performed at a strain rate of 0.1%. The starting frequency was 100s -1 , and the end frequency is 0.01s -1 .

[0259] To evaluate the morphology of the formulation on the hair, 0.25 g of the formulation was evenly spread on 0.35 g of yak hair and gently massaged. The hair was then dried at 200°C for 5 minutes using hot air until dry to the touch. Scanning electron microscopy (SEM) uses a focused electron beam to evaluate the morphology of the material through secondary electrons. The electron beam is scanned in a raster pattern to collect micrographs between 1 mm and 10 nm or between 10X and 100,000X magnification. The SEM method uses a low vacuum (1 to 10 Torr), which avoids the need for dehydration or sputter coating of biological samples.

[0260] Optical microscopy images were used to examine the formulation powder morphology and were acquired using a Leica DM750P optical microscope using a 10X objective. The microscope was connected to the complementary PC-based image analysis Leica application suite LAS V4.9 to capture the images.

[0261] 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 absence of any visually distinct features. The 1% recombinant silk polypeptide sample clearly has visible presence of recombinant silk polypeptide particles that are hollow and 2-200 μm in diameter.

[0262] Figure 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. All serum samples coated the hair strands significantly compared to untreated. Only the recombinant silk polypeptide serum produced a unique surface morphology marked by smooth membrane areas, rough membrane areas, and granular areas.

[0263] Figure 3C : Although the serum formulations containing 1% recombinant silk protein were clearly different as measured by microscopic examination, the rheological data showed similarities between the three formulations. The amplitude sweep showed similar amplitude and length of the linear viscoelastic region (referenced to the G' curve). Additionally, the frequency sweep showed similar amplitude and length of the G' curve. This data suggests that the hair serums have similar gel-like structural properties and will behave similarly rheologically.

[0264] Figure 3D: Table of ingredients used in hair serum formulations (excluding recombinant silk polypeptides and silicones).

[0265] Example 4: Rheological and morphological comparison of recombinant silk polypeptide and silicone elastomer components

[0266] Methods: 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 material, 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 Three samples were collected every ten years. -1 -1000s -1 The viscosity measurements were performed by shearing the samples at a shear rate of 1.5 Å. All rheology and viscosity measurements were performed at 22-27 degrees. The recombinant silk protein solutions (including 18B silk) were prepared by mixing the powder with deionized water in a high shear mixer.

[0267] To evaluate the morphology of the formulation on the hair, 0.25 g of the formulation was evenly spread on 0.35 g of yak hair and gently massaged. The hair was then dried at 200°C for 5 minutes using hot air until dry to the touch. Scanning electron microscopy (SEM) uses a focused electron beam to evaluate the morphology of the material through secondary electrons. The electron beam is scanned in a raster pattern to collect micrographs between 1 mm and 10 nm or between 10X and 100,000X magnification. The SEM method uses a low vacuum (1 to 10 Torr), which avoids the need for dehydration or sputter coating of biological samples.

[0268] Figure 4A : Comparison of G' and G" of an industry standard silicone elastomer gel (30% dry solids) and 12% recombinant silk polypeptide. Despite the very different molecular compositions, crosslinking chemistries and macromolecular morphologies of silicone elastomers and recombinant silk polypeptides, both materials exhibit similar rheological properties. This is indicated by the flat G' curve in the rheological frequency sweep. The flat curve indicates that both materials are structured gels. In the viscosity curves, both materials show similar non-Newtonian shear thinning properties typical of polymer solutions and suspensions.

[0269] Figure 4B : SEM images of pure recombinant silk polypeptides and silicone elastomers dispersed on yak hair. Approximately 0.25 g of the serum was dispersed on 0.35 g of hair. Both the recombinant silk polypeptides and silicone elastomer deposited a visible film on the hair shaft compared to untreated. The silicone elastomer film was relatively smooth. The recombinant silk polypeptide film was characterized by having a rougher surface with visible pits and particles protruding from its surface.

[0270] Viscosity measurements were also obtained for the following commercially available silicone elastomers in diluents and 18B recombinant silk polypeptide in water (as described in this Example 4): 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 nonionic emulsion (Dow, Midland, Michigan), 10% w / w in C12-13 Pareth-23 and C12-13 Pareth3 60% w / w; 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 properties typical of polymer solutions and suspensions.

[0271] A comparison of G' and G" of 5% w / w CHT-beausil gel 8055 in polydimethylsiloxane and 27% 18B recombinant silk polypeptide in water was also performed. Both materials showed similar curves, with G' dominating G" in the amplitude range of 30 Pa to 16000 Pa in the angular frequency range of 1-100 Hz.

[0272] Example 5: Styling retention test of a leave-in hair serum containing recombinant silk polypeptides

[0273] Virgin hair samples (medium brown, 3 g, width 1, length 8, sealed with hot melt) were treated with 1 g of a leave-in hair serum containing only a serum base or a serum base having 1% silk polypeptide, 1% keratin component, or 5% silicone elastomer component. Figure 3D The formulation for the serum primer is outlined in . The hair samples were tightly rolled into a 3 / 4" diameter curling clip and mounted to a grid plate with 1 cm wide markings. The samples were incubated in an oven at 50°C, 70% humidity for eight (8) hours. After 8 hours, the samples were removed from the oven, cooled to room temperature, and unfolded.

[0274] Figure 5An image of a hair sample after exposure to the curl retention test pattern as described above is shown. The recombinant silk polypeptide sample outperformed all other samples in terms of improved curl retention. Quantification of the hair sample length after exposure to the curl retention test pattern is shown in Table 2. The recombinant silk polypeptide exhibited 35% higher curl retention than the base serum and 28% higher curl retention than the silicone elastomer sample.

[0275] Table 2

[0276] sample Length [cm] % improvement compared to serum base Essence base 18 0 Essence base + 5% elastomer 16 11 Essence Primer + 1% Keratin 18.5 -6 Essence base + 1% recombinant silk protein 11.5 35

[0277] Example 6: Rinse-off Shampoo Formulation

[0278] A range of rinse-off shampoo formulations containing silk polypeptides (0.05%, 0.5% and 1%) were developed. These formulations were compared to a placebo, which was a formulation from which the silk polypeptides were removed and the difference made up by adding more water. A silicone elastomer version was also prepared - omitting the silk polypeptides and replacing them with a silicone elastomer at a 3% loading level (compensating for the quality difference by adding less water).

[0279] A shampoo formulation was prepared similar to the standard practice outlined in Example 3. For this formulation, the recombinant silk polypeptide was used in powder form and a silicone elastomer ingredient with approximately 60% solids was used. Figure 6 A list of ingredients in formulations (not including silk polypeptides or silicone elastomers) with a range of loading levels is provided in .

[0280] Viscosity and rheology were evaluated using the methods in Example 3. The viscosity curves of the formulations showed that all formulations exhibited similar shear thinning behavior. Fig.9A and 9B It shows that in 1-1000s -1 The silk polypeptide samples did not deviate from the placebo by more than 34% over the range of shear rates. The rheological profiles of the formulations showed that in all cases the formulations exhibited similar shapes, with G' dominating G". Fig. 10A and 10B Table containing rheological curve data points indicating that within the angular frequency range of 0.1 Hz to 10 Hz, the deviation of the silk polypeptide from the placebo never exceeded 31% for G' and never exceeded 19% for G". Inclusion of a silk polypeptide as described herein does not significantly alter the rheological properties compared to the shampoo placebo.

[0281] Optical microscopy was evaluated using the method in Example 3. Silk polypeptides could be visually detected at a loading level of 0.05%.

[0282] For the gap head evaluation, each side of the head was wetted and two grams of shampoo sample was massaged into the hair for about 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 sample in terms of styling control. As the silk polypeptide concentration increased, the silk polypeptide sample was superior to the silicone elastomer in terms of styling control.

[0283] SEM images were obtained of how various products were deposited on the hair shaft. Approximately 0.25 g of shampoo was dispersed on 0.35 g of hair and massaged for approximately 45 seconds. The hair was then rinsed with deionized water for 45 seconds. The hair samples were dried at ambient temperature / humidity for 24 hours. The samples were evaluated by SEM using the method in Example 3. No significant differences were detected in how the materials (silk protein vs. silicone elastomer formulation) affected the surface of the hair shaft.

[0284] List of ingredients in formulations (not including silk polypeptides or silicone elastomers) with a range of loading levels.

[0285] Example 7: No-rinse skin serum

[0286] A leave-on skin serum formulation containing a range of silk polypeptides (0.1%, 0.25%, 0.5% and 0.75%) was developed. These formulations were compared to a placebo, which was a formulation from which the silk polypeptide was removed and the difference was made up by adding more water. A silicone elastomer version was also prepared - omitting the silk polypeptide and replacing it with a silicone elastomer at a 0.75% loading level (compensating for the quality difference by adding less water).

[0287] A skin serum formulation was prepared similar to the standard practice outlined in Example 1. For this formulation, recombinant silk polypeptide was used in powder form, and a silicone elastomer ingredient was used with approximately 15% solids. Figure 7 A list of ingredients in formulations (not including silk polypeptides or silicone elastomers) with a range of loading levels is provided in .

[0288] Viscosity and rheology were evaluated using the methods in Example 3. The viscosity curves of the formulations showed that all formulations had similar shear thinning behavior. Fig.11 is a table of viscosity curve data points, the viscosity curve data points are displayed in 1-100s -1 Over a range of shear rates, the silk polypeptide samples deviated from the placebo by up to 137%, representing a significant and beneficial increase in viscosity. However, it was observed that the shapes of the G' and G" curves remained substantially similar between the compositions of the present disclosure and the placebo.

[0289] Optical microscopy was evaluated using the method in Example 3. Silk polypeptides could be visually detected at a loading level of 0.1%.

[0290] Example 8: No-Wash Skin Primer

[0291] A leave-on skin primer formulation containing 2% silk polypeptide was developed. The formulation was compared to a placebo, which was a formulation from which the silk polypeptide was removed and the difference was made up by adding more water. A silicone elastomer version was also prepared - omitting the silk polypeptide and replacing it with a silicone elastomer at a 7.5% loading level (compensating for the quality difference by adding less caprylic / capric triglyceride).

[0292] A skin primer formulation was prepared similar to the standard practice outlined in Example 1. For this formulation, recombinant silk polypeptide was used in powder form, and a silicone elastomer ingredient was used with approximately 15% solids.

[0293] refer to Fig. 12A and 12B , the viscosity curve data points are displayed in 1-100s -1 Within the range of shear rates, the deviation between the silk polypeptide samples and the placebo did not exceed 35%.

[0294] Other embodiments

[0295] It is to be understood that the words which have been used are words of description rather than limitation and that changes may be made within the purview of the appended aspects without departing from the true scope and spirit of the invention in its broader aspects.

[0296] Although the present invention has been described at some length and with a certain specificity with respect to several described embodiments, it is not intended that the present invention should be limited to any such details or embodiments or to any particular embodiment, but rather should be interpreted with reference to the appended aspects in order to provide the broadest possible interpretation of such aspects in light of the prior art, and thereby to effectively encompass the intended scope of the present invention.

[0297] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In the event of a conflict, the present specification (including definitions) shall prevail. In addition, section headings, materials, methods, and examples are illustrative only and are not intended to be limiting.

[0298] aspect

[0299] 1. A composition comprising a recombinant silk polypeptide and a solvent.

[0300] 2. The composition according to aspect 1, wherein the composition is a cosmetic or skin care product.

[0301] 3. A composition according to any 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.

[0302] 4. The composition according to any one of the above aspects, wherein the composition does not include a silicone elastomer.

[0303] 5. A composition according to any 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.

[0304] 6. The composition according to any one of the above aspects, wherein the composition is a cosmetic or skin care product.

[0305] 7. The composition according to any one of the above aspects, wherein the recombinant silk polypeptide is greater than 100 amino acids in length.

[0306] 8. The composition according to any one of the above aspects, wherein the recombinant silk polypeptide self-assembles into a semi-crystalline state.

[0307] 9. The composition according to aspect 8, wherein the crystalline portion of the recombinant silk polypeptide is characterized by β-sheet cross-links.

[0308] 10. A composition according to any of the above aspects, wherein the recombinant silk has poor solubility in a solvent selected from the group consisting of: water with a pH of 3 to 8, other polar and non-polar solvents (e.g., hexanol, hexane, benzene), oils, waxes, surfactants (e.g., anionic, nonionic, cationic, amphoteric).

[0309] 11. A composition according to any of the above aspects, wherein the recombinant silk forms a heterogeneous dispersion in a solvent selected from the group consisting of: water with a pH of 3 to 8, other polar and non-polar solvents (hexanol, hexane, benzene), oils, waxes, surfactants (anionic, nonionic, cationic, amphoteric).

[0310] 12. The composition according to any one of aspects 1 to 7, wherein the recombinant silk polypeptide forms part of a random structure gel.

[0311] 13. The composition according to aspect 12, wherein the gel comprises a preservative or a chelating agent.

[0312] 14. The composition according to any one of aspects 1 to 7, wherein the recombinant silk polypeptide forms part of a hollow powder.

[0313] 15. The composition according to aspect 12, wherein the powder comprises a preservative or a chelating agent.

[0314] 16. The composition according to any one of aspects 14 and 15, wherein the hollow powder is suspended in a solvent.

[0315] 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.

[0316] 18. The composition according to any one of aspects 16 and 17, wherein the solvent comprises a preservative or a chelating agent.

[0317] 19. A composition according to any of the above aspects, wherein the composition comprises 0.01% to 0.5% recombinant silk polypeptide, 0.05% to 5% recombinant silk polypeptide, 0.5% to 5% recombinant silk polypeptide, 1% to 5% recombinant silk polypeptide or 5% to 20% recombinant silk polypeptide.

[0318] 20. The composition according to any of the above aspects, wherein the recombinant silk polypeptide replaces the silicone elastomer traditionally used with the composition.

[0319] 21. The composition of aspect 20, wherein the recombinant silk polypeptide replaces the silicone elastomer in a ratio of 1:1 to 10:1 silicone elastomer:silk polypeptide.

[0320] 22. A composition according to any of the above aspects, wherein the composition comprises performance properties selected from the group consisting of: silky, smooth and powdery feel; reduced skin gloss; enhanced hair shine; vivid and efficient pigment delivery; easy spreadability; fast absorption time; matte; wrinkle blurring effect; style hold; heat resistance of hair; UV and pollution protection; and increased viscosity.

[0321] 23. A composition according to any of the above aspects, wherein the recombinant silk polypeptide comprises SEQ ID NO: 2878.

[0322] 24. The composition according to any of the above aspects, wherein the composition is an SPF formulation, a color cosmetic or a hair serum.

[0323] 25. The composition of any one of aspects 1 to 24, wherein the composition comprises water.

[0324] 26. The composition of any one of aspects 1 to 24, wherein the composition comprises water, glycerin and sodium benzoate.

[0325] 27. The composition of any one of aspects 1 to 24, wherein the composition comprises: zinc oxide, Aloe Barbadensis Leaf Juice, Water, Butyrospermum Parkii (Shea) Nut Extract, Camellia Sinesis (Green Tea) Leaf Extract, Caprylic / Capric Triglyceride, Caprylyl Glycerol / 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 Apple (Pyrus Malus / Apple) fruit extract, sclerotium gum, sodium benzoate, sodium phytate, squalane, tocopherol, xanthan gum, or any combination thereof.

[0326] 28. The composition of any one of aspects 1 to 24, wherein the composition comprises: water, brassica glyceride, caprylic / capric triglyceride, cetearyl alcohol, CI 77491, cucumber extract, gluconolactone, glycerin, glyceryl stearate, helianthus annuus (sunflower) seed wax, hydrogenated polycyclopentadiene, iron oxide, jojoba esters, lithospherespermum erythrorhizon root extract, polyacrylate crosspolymer-y, polyglyceryl-3, polyglyceryl-6 polyricinoleate, silica, simmondsia chinensis (jojoba) seed oil, sodium benzoate, sodium stearoyl glutamate, tocopherol, or any combination thereof.

[0327] 29. The composition of any one of aspects 1 to 24, wherein the composition comprises: water, polyacrylate crosspolymer-6, glycerin, citric acid, gluconolactone, sodium benzoate, cetyltrimonium chloride, or any combination thereof.

[0328] 30. The composition according to any one of the above aspects, wherein the solvent comprises an aqueous solvent, an alcohol, an oil-based solvent or a silicone.

[0329] 31. The composition according to any one of the above aspects, wherein the solvent is selected from the group consisting of: water, glycerol, deionized water, olive oil and pentylene glycol.

[0330] 32. A composition according to any of the above aspects, wherein the composition cleanses the skin.

[0331] 33. The composition according to any of the above aspects, wherein the composition further comprises a dye.

Claims

1. A cosmetic, skin or hair care composition with a silicone substitute, the cosmetic, skin or hair care composition comprising: One or more active ingredients for cosmetic, skin or hair care products, and A silicone substitute component, the silicone substitute component comprising a recombinant silk polypeptide, wherein the composition is substantially free of silicone.

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

3. The composition according to claim 2, wherein the properties are one or more of the following: imparting a matte texture; detangling hair; imparting a soft feel to the skin; imparting a silky, smooth and powdery feel; reducing skin shine; enhancing hair shine; vivid and efficient pigment delivery; easy spreadability; fast absorption time; wrinkle blurring effect; style hold; heat resistance of hair; UV and pollution protection; and viscosity increase.

4. The composition according to any one of the preceding claims, wherein the recombinant silk polypeptide is in the form of a powder.

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

6. The composition according to 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. The composition according to claim 5 or 6, wherein the silicone replacement composition further comprises a preservative or a chelating agent.

8. The composition according to 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. The composition according to any one of claims 1 to 3, 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. The composition according to any one of the preceding claims, wherein 1 part of recombinant silk polypeptide can replace up to 60 parts of silicone.

13. The composition of any one of the preceding claims, wherein the recombinant silk polypeptide is present in an amount of about 0.01 wt% to about 30 wt%, based on the total weight of the composition.

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

15. The composition of any of the preceding claims, wherein the silicone substitute 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 the G' and G" rheological curves having substantially the same shape as the G' and G" rheological curves of the composition having the same amount of silicone.

16. The composition of any one of the preceding claims, wherein the silicone substitute comprises the recombinant silk polypeptide suspended in a solvent.

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

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

19. The composition according to claim 16 or 17, wherein the solvent is one or more of the following: water, glycerol, deionized water, olive oil and pentylene glycol.

20. The composition of any one of the preceding claims, wherein the recombinant silk polypeptide is greater than 100 amino acids in length.

21. The composition of any one of the preceding claims, wherein the recombinant silk polypeptide self-assembles into a semi-crystalline state.

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

23. A composition according to any of the preceding claims, wherein the recombinant silk has poor solubility in a solvent selected from the group consisting of: water at a pH of 3 to 8, other polar and non-polar solvents (e.g., hexanol, hexane, benzene), oils, waxes, surfactants (e.g., anionic, nonionic, cationic, amphoteric).

24. A composition according to any of the preceding claims, wherein the recombinant silk forms a heterogeneous dispersion in a solvent selected from the group consisting of: water with a pH of 3 to 8, other polar and non-polar solvents (hexanol, hexane, benzene), oils, waxes, surfactants (anionic, nonionic, cationic, amphoteric).

25. The composition of any of the preceding claims, wherein the recombinant silk polypeptide comprises SEQ ID NO:

1.

26. The composition of any preceding claim, wherein the composition is an SPF formulation, a color cosmetic, a rinse-off shampoo, a skin serum, a skin primer, or a hair serum.

27. A composition according to any one of the preceding claims, wherein the composition comprises water.

28. A composition according to any one of claims 1 to 27, wherein the composition comprises water, glycerol and sodium benzoate.

29. A composition according to any one of claims 1 to 27, wherein the composition comprises: Zinc Oxide, Aloe Barbadensis Leaf Juice, Water, Butyrospermum Parkii (Shea) Nut Extract, Camellia Sinesis (Green Tea) Leaf Extract, Caprylic / Capric Triglyceride, Caprylyl Glyceryl / 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 (Jojoba Ester), Isostearic Acid, Lecithin, Phenoxyethanol, Polyglyceryl-3 Polyricinoleate, Polyhydroxystearic Acid, Potassium Sorbate, Pyrus Malus (Apple) Fruit Extract, Sclerotium Gum, Sodium Benzoate, Sodium Phytate, Squalane, Tocopherol, Xanthan Gum, or any combination thereof.

30. A composition according to any one of claims 1 to 27, wherein the composition comprises: Water, brassica glyceride, caprylic / capric triglyceride, cetearyl alcohol, CI 77491, cucumis sativus (cucumber) extract, gluconolactone, glycerin, glyceryl stearate, helianthus annuus (sunflower) seed wax, hydrogenated polycyclopentadiene, iron oxide, jojoba esters, lithospheres perm (lithospermum erythrorhizon) root extract, polyacrylate crosspolymer-y, polyglyceryl-3, polyglyceryl-6 polyricinoleate, silica, simmondsia chinensis (jojoba) seed oil, sodium benzoate, sodium stearoyl glutamate, tocopherol, or any combination thereof.

31. A composition according to any one of claims 1 to 27, wherein the composition comprises: Water, polyacrylate crosspolymer-6, glycerin, citric acid, gluconolactone, sodium benzoate, cetyltrimonium chloride, or any combination thereof.

32. The composition of any preceding claim, wherein the composition further comprises a dye.

33. A composition according to any preceding claim, wherein the silicone is a silicone elastomer.

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