Method for producing keratin powder from animal-derived feathers, colloidal solution containing keratin particles and keratin powder for topical application, and use of colloidal solution and keratin powder

Abstract

The present invention relates to a method for producing a keratin powder of the protein beta-keratin from feather of animal origin by extracting keratin, filtering the extract to obtain a colloidal solution and drying it to form the keratin powder. The invention also relates to a colloidal solution for topical application comprising keratin particles of the protein beta-keratin and / or agglomerates thereof or a keratin powder comprising said keratin particles. The colloidal solution and keratin powder are used to produce a formulation for therapeutic, diagnostic, prophylactic or cosmetic purposes in humans and / or animals.

Description

[0001] This invention relates to a method for producing keratin powder from animal-derived feathers, comprising extracting keratin, filtering the extract to obtain a colloidal solution, and drying it to form keratin powder. The invention also relates to colloidal solutions for topical application, comprising keratin particles and / or aggregates of β-keratin or keratin powder containing such keratin particles. The colloidal solutions and keratin powders are used to produce formulations for therapeutic, diagnostic, preventative, or cosmetic use in humans and / or animals.

[0002] The stratum corneum (SC) of the epidermis is composed of multiple layers of keratinized keratinocytes, which are enclosed by a "keratinized capsule" and connected by keratinized desmosomes, and embedded in a complex lipid matrix with a liquid crystal layered structure.

[0003] Lipid synthesis in keratinocytes is largely autonomously regulated and provides free fatty acids of varying chain lengths, in addition to cholesterol and cholesterol derivatives, as well as triglycerides. Furthermore, ceramides synthesized in the endoplasmic reticulum of keratinocytes differ significantly from other lipid classes due to their non-isomeric molecular structure. Ceramides can spontaneously form lyotropic mesophases, i.e., liquid crystal membrane structures, due to charge differences within their long chains. Unlike phospholipids, functionally important ceramides in the stratum corneum possess two alkyl chains of varying lengths. Moreover, these chains exhibit different conformations depending on their degree of hydration, thus different membrane models describe complex networks of membrane segments with polymorphic phase behavior. The complexity of the dynamic order of this system is increasingly understood and is of growing practical importance in the development of modern Galen formulation carrier systems.

[0004] Barrier function is also largely determined by the amount of hydrophilic phase in the microenvironment. The distribution of water in the microcompartments of the stratum corneum can be described in at least two parts. In addition to the free water phase, a bound water phase is defined. The bound water phase consists of mobile subcomponents and fixed subcomponents. This nomenclature aims to describe the dynamic exchange of hydrophilic valence states between the compartments. Fixed water mainly refers to the water portion bound to keratinocytes by strong hygroscopic forces mediated by amino acids produced by proteolysis, and its exchange and release are very slow. Under specific conditions, water bound by the expandable membrane portion can be released and transferred to the free water phase. This is where the functionally important water phase of the stratum corneum manifests. Free water is also bound to the compartments by hygroscopic molecules collectively known as "natural moisturizing factors (NMF)" and exchanges with the water phase of the active epidermis and the environment (through epidermal water loss). In addition to amino acids, the main components of NMF include pyrrolidone carboxylic acid, lactic acid, urea, and inorganic ions. These components are synthesized by keratinocytes and released according to their degree of differentiation.

[0005] During exfoliation, keratin desmosomes and keratinized capsule proteins in the outermost separating layer of the stratum corneum are degraded through proteolysis. However, the major keratinocytes are not directly affected. Therefore, keratinocytes can maintain their integrity for a prolonged period during exfoliation. An important function of keratinocytes is to provide the intercellular spaces with the water required for membrane formation through the diffusion of the keratinized capsule. Under therapeutic conditions, the interaction between keratin and epidermally applied active ingredients is considered insignificant because no transcellular delivery of any active ingredient has been demonstrated to a certain degree. This is thought to be due to the keratinized capsule surrounding the keratinocytes as both a capsule and a barrier membrane. However, keratinocytes indirectly influence the diffusion conditions (diffusion coefficient) of the stratum corneum through their central role as an important reservoir of the stratum corneum. The skin bioavailability of epidermally applied active substances is primarily determined by the interaction between the Galen formulation system and the stratum corneum as the direct contact layer. From a pharmacological perspective, two processes within the stratum corneum are important for substance flow: barrier function and reservoir function. Equally important, the stratum corneum is not a homogeneous structure. Instead, it becomes loose (the separating layer) through an enzymatically controlled desquamation process after the formation and compact construction of the dense layer. Therefore, these two functional zones have distinctly different significance for the structural hierarchy of the stratum corneum. The maximum barrier function is located in the dense layer, while the maximum reservoir function is located in the separating layer. The latter acts as a specific receptor for the epidermal application and release phases. This is crucial for the overall skin kinetics of the active ingredient, as the maximum concentration in the deeper skin layers is lower and the penetration process is delayed. Simultaneously, a large release of the active ingredient can be absorbed and become bioavailable within a relatively short time after application. In summary, it is clear that under micromorphological conditions, anatomical differences (such as hirsutism versus ahidrosis) and pathological conditions (such as epidermal proliferation or differentiation disorders) directly affect the pharmacokinetic characteristics of substances applied epidermally.

[0006] This indicates that targeted effects on the moisture content of the stratum corneum can directly affect its physicochemical barrier function, thereby influencing the pharmacokinetics of active substances applied to the epidermis.

[0007] The following publications illustrate the uses of keratin in medical or cosmetic applications.

[0008] Due to hydrolysis caused by microorganisms, acids, or alkalis, currently available keratin is degraded and extracted into smaller peptide structures and no longer exists as a complete and intact keratin protein (Shandie, A. et al., Biomater. Sci. 2017, 5, 1699-1735; Gupta, A. et al., J. Chem. Chem. Eng. 2012, 6, 732-737).

[0009] These keratin peptides are short-chain sequences and possess properties (such as molecular size and swelling behavior) different from those of the naturally occurring long-sequence keratin obtained for the first time herein. Unlike previous processes, this invention omits the hydrolysis extraction step and instead extracts the protein by breaking disulfide bonds. This preserves both the primary structure of the keratin protein and its secondary structure as a β-sheet.

[0010] Therefore, the object of the present invention is to provide colloidal solutions as the basis of formulations that can interact simultaneously with both hydrophilic and lipophilic components or active ingredients. Furthermore, these colloidal solutions should be easy to manufacture.

[0011] The problem is solved using the method having the features of claim 1, the colloidal solution having the features of claim 7, and the keratin powder having the features of claim 11. The use according to the invention is described in claim 13. The other dependent claims list preferred embodiments.

[0012] According to the present invention, a method for producing keratin particles from animal-derived feathers is provided, wherein...

[0013] a) Extraction of β-keratin, particularly β-keratin in the form of β-sheets with a secondary structure, is performed in an extraction solution that induces chemical denaturation and contains at least one denaturing agent, at least one base, at least one reducing agent, and at least one buffer.

[0014] b) Filter the extraction solution from step a) to obtain a colloidal solution of β-keratin keratin particles.

[0015] c) Dry the colloidal solution of keratin particles from step b) by freeze-drying, spray drying, vacuum drying, air drying, heat drying, infrared drying and / or microwave drying to obtain a powder containing keratin powder.

[0016] The present invention therefore relates to the use of β-keratin particles, which are keratin extracted from bird feathers through chemical denaturation, in liquid or semi-solid formulations for epidermal application.

[0017] The α-keratin and β-keratin obtained from wool are called "soft fiber" (α)keratin or "hard fiber" (β)keratin, which are two different types of keratin. In this case, extraction using urea under alkaline conditions also allows the natural form of β-keratin to be obtained from feathers as an intact protein, thus preserving a high proportion of its secondary structure of β-sheets, which determines the keratin's high water-binding capacity. In particular, β-keratin, obtained from bird feathers, is rich in glycine and alanine, and contains small amounts of cysteine, proline, and hydroxyproline.

[0018] The pharmacokinetics of keratin can be targeted by utilizing its interaction with hydrophilic substances as a reservoir. Physical barriers can be replaced by combining keratin with water, amino acids, hygroscopic substances, peptides, and / or proteins.

[0019] The pH of the extraction solution is preferably 8 to 13, more preferably 9 to 12, and particularly preferably 10 to 11.

[0020] Preferably, at least one denaturing agent is selected from urea, thiourea, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and mixtures thereof.

[0021] Preferably, the at least one base is selected from sodium hydroxide, potassium hydroxide, and mixtures thereof.

[0022] At least one reducing agent is preferably selected from β-mercaptoethanol, cysteine, cysteine, glutathione, sodium metabisulfite, sodium sulfide, sodium bisulfite, sodium dithionite, sodium thiosulfate, dithiothreitol (DTT), mercaptoacetic acid and its salts, thiourea, tris(carboxyethyl)phosphine (TCEP) and other phosphines, ammonium chloride and mixtures thereof.

[0023] Preferably, at least one buffer is selected from tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (EDTA) / Tris, potassium chloride-sodium hydroxide (KCl-NaOH), sodium bicarbonate (NaHCO3), dithiothreitol (DTT) / Tris, and mixtures thereof.

[0024] The preferred extraction solution contains at least one of the following chemical components:

[0025] • At least one oxidizing agent selected from hydrogen peroxide, potassium permanganate, sodium perborate, peracetic acid, performic acid, and mixtures thereof.

[0026] • At least one acid selected from nitric acid, nitrous acid, hypohalous acid, and perhalous acid and mixtures thereof.

[0027] • At least one ionic agent selected from 1-butyl-3-methylimidazolium (BMIM) chloride, 1-butyl-3-methylimidazolium (BMIM) bromide, 1-butyl-3-methylimidazolium (BMIM) tetrafluoroborate, amide chloride, and mixtures thereof.

[0028] According to a preferred embodiment of the method of the present invention, in the extraction process of step a), at least one of the following steps is performed:

[0029] Mechanical grinding, particularly by ultrasonic grinding, preferably in the frequency range of 20 Hz to 50 Hz, using a ball mill and / or a cutting mill, preferably Ultra-Turrax, wherein mechanical grinding is carried out to a particle size (d50) of 0.1 mm to 5.0 mm, preferably 0.2 mm to 1.0 mm, according to sieve analysis.

[0030] • Thermal denaturation, particularly at temperatures between 70°C and 150°C, and / or electrochemical denaturation.

[0031] • Precipitation in the extraction solution of step a), particularly initiated by pH change, solubilizer and / or salt addition,

[0032] • Microbial and enzyme extraction is performed through the following methods:

[0033] Gram-negative bacteria, selected from Stenotrophomonas sp., Chrysebacterium sp., Vibrio sp., and mixtures thereof.

[0034] Gram-positive bacteria, selected from Bacillus sp., Kocuria rosea, and mixtures thereof.

[0035] • Saprophytic fungi and / or parasitic fungi, and / or

[0036] Its mixture,

[0037] Microwave radiation treatment, especially microwave radiation up to 960 watts and 2450 Hz.

[0038] • Using electrical explosions and / or supercritical water, and / or

[0039] • Its combination.

[0040] Preferred β-keratin exists in its secondary structure as β-sheets. Compared to α-keratin (=soft filaments), β-keratin (=hard filaments) has a high proportion of tightly twisted β-sheet structures stabilized by disulfide bonds, which ensures the high stability of hard filament keratin.

[0041] Filtration is preferably performed by dialysis and / or ultrafiltration (cross-flow filtration).

[0042] Animal-derived feathers are preferably selected from feathers of chickens, geese, ducks, turkeys, pheasants, ostriches, rheas, emus, quails, and mixtures thereof.

[0043] The invention also provides a colloidal solution for topical application, comprising keratin particles of β-keratin and / or aggregates thereof.

[0044] The keratin particles in the colloidal solution preferably have a particle size of 5 nm to 500 nm, more preferably 70 nm to 350 nm, as measured by dynamic light scattering (according to DIN ISO 22412:2018-09) (Zetasizer ZEN3600, Malvern Panalytical Instruments).

[0045] The colloidal solution preferably contains an additive for stabilization, said additive preferably selected from...

[0046] Proteins, such as albumin,

[0047] • Carbohydrates, such as sucrose, lactose, glucose, fructose, mannitol, sorbitol, and sweeteners such as sodium saccharin, sodium cyclohexylsulfamate, aspartame, starch and modified starch, cyclodextrin, and / or mixtures thereof,

[0048] • Polyanionic surfactants, such as sodium lauryl sulfate, sodium cetearyl sulfate, cetearyl alcohol (emulsion type), sodium dioctyl sulfosuccinate, and / or mixtures thereof,

[0049] • Nonionic surfactants, such as fatty alcohols and sterols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, polyethylene glycol-1000-glycerol monofatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, sucrose fatty acid esters, poloxamer and / or mixtures thereof,

[0050] • Gelling agents, such as polyacrylates, cellulose derivatives such as methylcellulose, methyl hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose and / or ethyl cellulose, sodium carboxymethyl cellulose and / or mixtures thereof,

[0051] Thickeners, such as tragacanth gum, xanthan gum, gum arabic, guar galactomannan, alginate, bentonite, and / or mixtures thereof,

[0052] Film-forming agents such as methacrylates, polyvinyl alcohol, and / or mixtures thereof,

[0053] • Polymers, such as polyethylene glycol, gelatin and / or mixtures thereof.

[0054] According to the present invention, keratin powder for topical application of β-keratin is also provided, which can be produced from the above-described colloidal solution. The keratin powder is produced by drying the colloidal solution, particularly by freeze-drying, spray drying, vacuum drying, air drying, heat drying, infrared drying, and / or microwave drying.

[0055] The keratin particles in the keratin powder preferably have a particle size of 1 μm to 250 μm, more preferably 5 μm to 30 μm, as measured by dynamic light scattering (according to DIN ISO22412:2018-09) (Zetasizer ZEN3600, Malvern Panalytical Instruments).

[0056] The above-mentioned colloidal solutions and keratin powders are used to produce formulations for therapeutic, diagnostic, preventative, or cosmetic applications in organisms, particularly humans and / or animals.

[0057] A preferred embodiment provides a colloidal solution comprising at least one small molecule and / or biotechnologically active ingredient. The active ingredient is preferably selected from glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics and / or proteins themselves, protein fragments, peptides, enzymes, antibodies, antibody fragments, RNA and / or DNA molecules, and mixtures thereof. Another preferred embodiment provides a molecule comprising a target of one of these active ingredients.

[0058] Preferably, the colloidal solution contains cosmetic active ingredients and excipients, preferably selected from amino acids, urea, glycerin, hyaluronic acid, sugars and sugar derivatives, extracts or waxes from plant or animal sources, and mixtures thereof.

[0059] A preferred embodiment provides specific and / or non-specific compounds formed by covalent and / or non-covalent bonds, preferably by specific groups selected from carboxylic acid groups, amino groups, thiol groups and / or hydroxyl groups, with active substances and / or excipients.

[0060] To stabilize colloidal solutions containing or without active ingredients and / or excipients, it is preferable to incorporate amphiphilic molecules into liposome systems of monolayer or multilayer vesicles of varying or uniform sizes. These amphiphilic molecules are preferably selected from:

[0061] Phospholipids, preferably lecithin, DODAB, DPPC, DSPC, DSTAP, and / or mixtures thereof,

[0062] • Cationic lipids, preferably ALC-0315,

[0063] • PEG lipids, preferably ALC-0159,

[0064] • Prostaglandins and their modified forms, preferably PGE1, PGD2, PGE2, 15-keto-PGE1 and / or mixtures thereof,

[0065] • Ceramides, preferably selected from those with head groups of NS, NH, NP, NDS, AS, AH, ADS, AP and chain length C 10 To C 26ceramides and / or mixtures thereof,

[0066] • Ceramides, preferably selected from those with a head group of EOS, EOH, or EOP and a chain length of C 10 To C 32 ceramides and / or mixtures thereof,

[0067] • Cholesterol, cholesterol derivatives and / or mixtures thereof,

[0068] • Fatty acids, preferably selected from those with a chain length of C 10 To C 32 Fatty acids and / or mixtures thereof.

[0069] Preferred for use in organisms, particularly humans, involves the skin, mucous membranes (including conjunctiva) or epidermal appendages (including nails and hair), especially for components used to replace barrier functions or for the epidermal barrier.

[0070] The colloidal solution and keratin powder according to the present invention are also used in the production of:

[0071] • Liquid matrices, especially solutions, emulsions, suspensions, and / or colloids,

[0072] • Semi-solid bases, especially suspension ointments, creams, gels, pastes, colloids and / or suppositories,

[0073] • Solid matrix, especially powders, tablets, granules, pills, capsules and / or inserts.

[0074] The colloidal solution and keratin powder according to the present invention are also used in the production of:

[0075] • Food and food supplements

[0076] Animal feed,

[0077] • Fertilizers and / or plant protection products used on plants and / or soil,

[0078] • Technical additives, such as binders and / or adhesives in or as part of filtration systems, consistency modifiers, fillers, absorbents for hydrophilic or lipophilic substances, and absorbents for charged or uncharged substances.

[0079] Packaging materials and / or consumer products,

[0080] • Textiles and / or fibers that are functional, particularly waterproof and breathable, or without functional properties.

[0081] • Medical products, especially adhesive bandages, wound dressings, tampons, and clothing that supports wound or skin care.

[0082] The subject matter of the invention will be described in more detail with reference to the following figures and embodiments, but the subject matter is not limited to the specific embodiments shown.

[0083] Figure 1A and Figure 1B SEM images of freeze-dried hair keratin at different resolutions are shown.

[0084] Figure 2 TEM images of A) feather keratin (0.5 mg / ml) and B) hair keratin (0.5 mg / ml) are shown.

[0085] Figure 3 The fluorescence spectrum image of 5% (w / w) keratin particles in the base cream DAC (containing A) feather keratin, B) hair keratin, and C) hair keratin is shown.

[0086] Figure 4 Images of penetration tests for A) feather keratin and B) hair keratin at 5% (w / v) each in the base cream DAC are shown.

[0087] Figure 5 Images and fluorescence images showing the permeation study of 5% (w / v) feather keratin in the base cream DAC.

[0088] Figure 6 The indexes of cytotoxicity data of lyophilized feather keratin and colloidal feather keratin against keratinocytes (NHEK) and lyophilized feather keratin and colloidal feather keratin against dermal fibroblasts (NHDF) are shown.

[0089] Figure 7 The results of a scratch assay depicting the epithelialized area in three independent trials of lyophilized feather keratin against keratinocytes (HaCaT) are shown.

[0090] Figure 8 The results of a scratch assay depicting the epithelialized area in three independent trials of colloidal feather keratin against keratinocytes (HaCaT) are shown.

[0091] Figure 9 The results of a scratch assay depicting the epithelialized area in three independent trials of lyophilized feather keratin against dermal fibroblasts (NHDF) are shown.

[0092] Figure 10 The results of a scratch assay depicting the epithelialized area in three independent trials of colloidal feather keratin against dermal fibroblasts (NHDF) are shown.

[0093] Figure 11A graph showing the water absorption rate of feather keratin after adding 25 μl T2O (1 μCi) to 5 mg of lyophilized keratin, n = 3 (contact time: 1 min, 5 min, 1 h, 16 h, 24 h), centrifuged, supernatant removed, and radioactive dose determined.

[0094] Figure 12 This diagram illustrates the culture of HaCaT cells with 1 mg of feather keratin.

[0095] Figure 13 The water absorption rates of urea, glycerol, and keratin, as well as mixtures of keratin particles with glycerol (50:50) and mixtures of keratin particles with urea in different ratios (e.g., 95:5 (orange); 50:50 (light blue); 5:95 (brown)) over time in a climate chamber (room temperature and 95% relative humidity) are shown.

[0096] Figure 14 The diagram shows the moisture release over time of urea, glycerol, and keratin, as well as mixtures of keratin particles with glycerol (50:50) and mixtures of keratin particles with urea in different ratios (e.g., 95:5 (orange); 50:50 (light blue); 5:95 (brown)) in a climate chamber (room temperature and 50% relative humidity).

[0097] Figure 15 The tertiary structure of β-keratin in bird feathers is shown.

[0098] Figure 16 Measurement data for keratinization in cosmetic cream formulations containing 0.1%, 0.5%, 1.0%, and 2.0% keratin particles are shown, at arbitrary units (AU) before and after 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours.

[0099] Figure 17 The results show the effects of cosmetic cream formulations containing 0.1%, 0.5%, 1.0%, and 2.0% keratin particles in g / m³ before and after 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours. 2 / h represents the measurement data from the evaporation determination.

[0100] Various animal materials can serve as keratin sources, but their applicability varies due to differences in the degree of protein cross-linking. Preliminary studies of various keratin-containing biomaterials have shown that bird feathers are particularly suitable. Besides practical aspects such as availability and ease of processing, advancements in biochemistry have particularly validated the preferred use of feathers. The keratin in feathers is primarily β-keratin, provided as polypeptide chains with a β-sheet structure, composed of 3 nm to 4 nm filaments with a molecular weight of approximately 10 kDa to 22 kDa. Compared to mammalian α-keratin, β-keratin exhibits only minor but functionally important differences in its primary sequence. Therefore, β-keratin forms fewer macrofibrils than α-keratin and displays a more regular arrangement and stacking behavior. These differences provide a more favorable premise for standardized keratin separation and the resulting product properties. In particular, the commonly practiced step of keratin hydrolysis to separate intact keratin is omitted, and the formation of keratin fragments, amino acids, and peptides is deliberately avoided. Based on the biochemical characteristics of β-keratin, an efficient and standardized keratin separation process has been developed. The chicken feathers used were first cleaned with water and soap, sterilized with 70% ethanol, and then dried at room temperature. The cleaned and dried feathers were then pulverized in a cutting grinder (Retsch SM 100comfort) to homogenize the entire material. The homogenized feathers were defatted using a Soxhlet extractor. To extract keratin, the defatted feather material was added to an extraction buffer and extracted for 48 hours. The extract was then centrifuged and the precipitate was discarded. The obtained pure extract was diluted with water and filtered through a filter membrane with a cutoff of 10000 NMWC. The dialysate was spray-dried into a powder with a particle size <25 μm.

[0101] To determine the colloidal size of keratin particles (1 mg / ml deionized water), dynamic light scattering (DLS) measurements were performed using a Zetasizer ZEN3600 instrument from Malvern Panalytical Instruments, according to DIN ISO 22412:2018-09. This analytical method characterizes particle size in suspensions and emulsions by detecting the scattered light emitted by a laser. The aim is to determine the particle size of proteins in the colloidal state. For DLS measurements, 1 ml of a 0.1 mM colloidal keratin solution was pipetted into a disposable polystyrene cuvette and then transferred to a Zetasizer Lab cuvette module heated to 25°C. Particle size was determined using the automated analysis mode (general mode) and 173° angled backscattering. Each measurement was repeated three times, with each run containing 15 measurement cycles. The average of the measurement data recorded for each sample was then taken. A total of three samples were subjected to DLS analysis. The polydispersity index (PdI), particle size (nm), and percentage of each particle size in the total sample content were determined within the DLS range, indicating the uniformity of particle distribution in the sample.

[0102] To determine the molecular size (molecular weight) of the extracted keratin particles, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in an SDS gel electrophoresis tank (Invitrogen Mini Gel Tank). Samples were prepared according to... The denaturation was performed using the manufacturer's protocol for Bis-Tris Mini Gel Electrophoresis (Thermo Fisher). For this purpose, 2 μl of sample and 2.5 μl of... LSD sample buffer (4x), 1 μl LSD reducing agent (10x) and 6.5 μl DI water were mixed and incubated at 95 °C for 10 min. For gel electrophoresis, 1× Tris-Tricine electrophoresis buffer (1.2 M Tris, 0.8 M Tricine, 2% SDS) was added to the electrophoresis tank. A Tris-Tricine gel (Invitrogen (Thermo Fisher Scientific) Novex 10% to 20% Tricine gel, lot number 20101945, catalog number EC6625BOX) was then inserted, and the comb was removed from the gel. 10 μl of the prepared sample and 2 μl of the label were added to the corresponding gel wells. Gel electrophoresis was performed at 130 V and 250 mA for 1.5 h. The gel was then removed from the electrophoresis tank, and the electrophoresis buffer was discarded. Remove the plastic gel holder and transfer the Tris-Tricine gel to a container containing Coomassie Brilliant Blue G250 staining solution (2 ml of 5% Coomassie solution, 3 ml of orthophosphoric acid, 20 ml of ethanol, 10 g of ammonium sulfate, and 65 ml of dH2O). Incubate the gel in this solution overnight on a shaker. Then remove the staining solution. Gently rinse the gel several times with distilled water until destaining. Example

[0103] An attempt was made to reconstruct the functional properties of keratinocytes. To this end, keratin was extracted from various natural keratin sources and corresponding particles were generated. The mechanical stabilizing effect of these particles on the lipid matrix in various semi-solid formulations was first investigated. Therefore, the aim was to develop keratosomes by coating keratin particles with bipolar lipids.

[0104] First, various extraction methods were tested on human hair (α-keratin). Chemical denaturation was primarily used to break down the hair. This was performed using urea, thiourea, and guanidine hydrochloride. β-mercaptoethanol, cysteine, and L-cysteine ​​were used as reducing agents to aid denaturation. The highest yield was obtained using an extraction buffer containing 5M guanidine hydrochloride, 10% cysteine, and 25mM Tris at pH 8.5. Based on safety considerations regarding urea and L-cysteine, an extraction buffer consisting of 10M urea, 100mM L-cysteine, and 25mM Tris-HCl at pH 10.5 was selected for keratin separation. After successful extraction, the extract was dialyzed (molecular weight cutoff: 6kDa to 8kDa, regenerated cellulose, ...). The resulting colloidal solution: Under conditions of high protein concentration and large dialysis volume (5 L to 6 L), keratin precipitates in the dialysis bag. The dialysate is then lyophilized to obtain a white powder. This powder is examined by scanning electron microscopy (SEM) (Figure 1). The particle size and shape of hair keratin vary. This determined that the particles in the final formulation should have a particle size >600 nm to avoid penetrating the stratum corneum of the skin.

[0105] Due to regulatory requirements, feather keratin (β-keratin) from various bird feathers was used as an alternative keratin source. Raw feathers from chickens, geese, and ducks were tested for this purpose. For practicality, chicken feathers were specifically selected for subsequent research.

[0106] Therefore, the extraction method for feather keratin was adapted to the established extraction process for hair keratin. It was observed that the highest yield was obtained using a selected extraction solution consisting of 10M urea, 100mM L-cysteine, and 25mM Tris-HCl at pH 10.5, compared to all other tested extraction methods. Optionally, the addition of 1M ammonium chloride prevented carbamylation of the protein and related alterations to its properties.

[0107] To determine the particle size, feather keratin particles and hair keratin particles were negatively stained and compared using transmission electron microscopy (TEM). Figure 2 Hair keratin particles in colloidal solutions range in size from approximately 40 nm to 75 nm, while feather keratin particles range in size from approximately 20 nm to 35 nm. To visualize the keratin particles in the permeation study, 2-aminobenzoyl (Abz) was used for fluorescent labeling (reacting with indigo anhydride under alkaline denaturing conditions). 5% (w / w) fluorescently labeled hair and feather keratin were incorporated into the base cream DAC, and smears were examined using a fluorescence microscope. Figure 3Individual particles in the colloidal solution identified by TEM formed proportional aggregates in the base cream DAC. Feather keratin showed uniformly distributed aggregates of particles with a diameter of 20 μm to 35 μm. In contrast, hair keratin aggregated into larger crystalline particles with a diameter of 20 μm to 140 μm.

[0108] Further evidence was demonstrated through penetration tests of hair keratin and feather keratin (5% w / w each) in the base cream DAC on ex vivo human skin (Franzzelle), showing that keratin particles do not diffuse into the deeper layers of the skin, but remain in the upper part of the SC (separation layer). Figure 4 and Figure 5 To better evaluate the interaction between feather keratin particles and SC lipids or the lipid encapsulation effect, the zeta potential was measured at different pH values ​​(Table 1).

[0109] Table 1: Zeta potentials of 10 mg / ml feather keratin and fluorescently labeled feather keratin in 10 mM potassium chloride at different pH values. Measured by Fabio.

[0110]

[0111] Keratin particles carry a negative charge, which makes them ideal for being coated with positively charged lipids.

[0112] A standard protocol was used to conduct cytotoxicity studies to evaluate the effects of colloidal and lyophilized keratin particles on the viability and proliferative activity of keratinocytes and dermal fibroblasts. No effects on dermal fibroblasts were observed during the 48-hour incubation period. Figure 6 In keratinocytes, only colloidal keratin showed a slight concentration-dependent decrease in proliferative activity.

[0113] The effects of colloidal and lyophilized keratin particles on the migration behavior of keratinocytes (HaCaT) and dermal fibroblasts (NHDF) were investigated using a scratch assay. Figures 7 to 10 The results showed significant mechanical inhibition of migration, which depended on the concentration of colloidal keratin, and the effect of lyophilized keratin particles was much weaker. However, no evidence of toxic effects was found.

[0114] To objectively determine the loading capacity of feather keratin particles for hydrophilic or hydrophobic substances, drug loading and release experiments were conducted. First, the keratin particles were incubated in T2O solution for different durations, followed by centrifugation to determine the amount of tritium absorbed. Figure 11 Only 25% to 32% of the added T2O could be recovered from the particles. This indicates a surprisingly high level of interaction between the extracted feather keratin and the hydrophilic substance.

[0115] To investigate the potential interactions between keratin particles and living cells, HaCaT cells (keratinocytes) were co-cultured with fluorescently labeled feather keratin particles and hair keratin particles. The culture medium was replaced after 48 hours of keratin treatment. Keratin particles were clearly observed under a fluorescence microscope, appearing near and binding to the cell nucleus; therefore, fluorescence microscopy cannot rule out the possibility of particle uptake by the cells. Figure 12 Subsequently, Raman spectroscopy was used to detect the adhesion region between keratin particles and the cell membrane, but no keratin particles were found in the cytoplasm. Therefore, based on the zeta potential and the charge properties of phospholipids in the cell membrane, it was speculated that keratin particles adhere to the cell membrane.

[0116] In addition, hygroscopicity was assessed based on keratinocyte function to determine the water absorption capacity of keratin particles. Keratin particles showed a maximum water absorption rate of approximately 20% after only 3 days. When urea was added and a ratio of 5% keratin to 95% urea was achieved, the time to reach the maximum water absorption rate was gradually delayed, reaching 197% after 15 days. Figure 13 The water release rate was measured at room temperature and 50% humidity. Pure keratin slowly released some water and still contained water after 7 days; while urea was observed to rapidly release approximately 170% of its water in the first 24 hours and completely release it after 4 days. Mixtures of keratin and urea in ratios of 25:75, 10:90, and 5:90 were observed to retain 25% water content after 4 days. Figure 14 ).

[0117] Based on the known keratin sequence and Dan Cojocari's amino acid classification, keratin contains only about 30% hydrophobic amino acids and approximately an equal amount of hydrophilic amino acids, and lacks binding pockets. Therefore, the interactions of keratin with substances of different physicochemical properties are unpredictable. Thus, the tertiary structure of keratin was calculated using the artificial intelligence AlphaFold 2 (EMBL's European Bioinformatics Institute (EMBL-EBI), Hinxton, UK), and the hydrophobic and hydrophilic regions in the molecule were identified. Figure 15 This result also confirms the hypothesis that there is no binding pocket, therefore the observed interactions can only be explained by hydrophobic or hydrophilic interactions between amino acid clusters on the side of the protein and related groups of other molecules.

[0118] To demonstrate clinical efficacy, a prospective, randomized, double-blind study was conducted to examine the hydration effect of cosmetic cream formulations containing increasing concentrations of keratin particles (0.1%, 0.5%, 1.0%, and 2.0%) on the stratum corneum in healthy volunteers. In a single-application study, total stratum corneum water content (keratinization assay) and the free water phase as transepidermal water loss (TEWL) were measured using an evaporation assay at baseline and at 1, 2, 4, 8, and 24 hours after application. At 24 hours, the stratum corneum water content increased by approximately 30%. Figure 16 Transepidermal water loss is reduced by approximately 40%. Figure 17 These results indicate that the extracted keratin particles, as the colloidal aqueous phase in cream formulations, can provide the stratum corneum with a long-lasting hydration effect based on the physiological function of keratinocytes.

Claims

1. A method for producing keratin particles from animal-derived feathers, wherein a) Extraction of β-keratin, particularly β-keratin in the form of β-sheets with a secondary structure, is performed in an extraction solution that induces chemical denaturation and contains at least one denaturing agent, at least one base, at least one reducing agent, and at least one buffer. b) Filter the extraction solution from step a) to obtain a colloidal solution of β-keratin keratin particles. c) Dry the colloidal solution of keratin particles from step b) by freeze-drying, spray drying, vacuum drying, air drying, heat drying, infrared drying and / or microwave drying to obtain keratin powder.

2. The method according to claim 1, Its features are, The pH of the extraction solution is 8 to 13, more preferably 9 to 12, and particularly preferably 10 to 11.

3. The method according to claim 1 or 2, Its features are, The at least one denaturing agent is selected from urea, thiourea, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and mixtures thereof. and / or The at least one base is selected from sodium hydroxide, potassium hydroxide, and mixtures thereof. and / or The at least one reducing agent is selected from β-mercaptoethanol, cysteine, cysteine, glutathione, sodium metabisulfite, sodium sulfide, sodium bisulfite, sodium dithionite, sodium thiosulfate, dithiothreitol (DTT), mercaptoacetic acid and its salts, thiourea, tris(carboxyethyl)phosphine (TCEP) and other phosphines, ammonium chloride and mixtures thereof. and / or The at least one buffer is selected from tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (EDTA) / Tris, potassium chloride-sodium hydroxide (KCl-NaOH), sodium bicarbonate (NaHCO3), dithiothreitol (DTT) / Tris, and mixtures thereof.

4. The method according to any one of claims 1 to 3, Its features are, The extraction solution contains at least one oxidizing agent, preferably selected from hydrogen peroxide, potassium permanganate, sodium perborate, peracetic acid, performic acid, and mixtures thereof. and / or The extraction solution contains at least one acid, preferably selected from nitric acid, nitrous acid, hypohalous acid, perhalous acid, and mixtures thereof. and / or The extraction solution contains at least one ionic agent, preferably selected from 1-butyl-3-methylimidazolium (BMIM) chloride, 1-butyl-3-methylimidazolium (BMIM) bromide, 1-butyl-3-methylimidazolium (BMIM) tetrafluoroborate, amide chloride, and mixtures thereof.

5. The method according to any one of claims 1 to 4, Its features are, During the extraction process in step a), at least one of the following steps is performed: a) Mechanical pulverization, particularly by ultrasonic grinding, preferably in the frequency range of 20 Hz to 50 Hz, using a ball mill and / or a cutting mill, wherein the mechanical pulverization is carried out to a particle size of 0.1 mm to 5.0 mm, preferably 0.2 mm to 1.0 mm. b) Thermal denaturation, particularly at temperatures between 70°C and 150°C, and / or electrochemical denaturation. c) Precipitation of the extract solution from step a), particularly initiated by pH alteration, the addition of a solubilizer, and / or the addition of salt. d) Microbial and enzyme extraction is performed as follows Gram-negative bacteria, selected from oligotrophomonas, chrysogens, vibrio, and mixtures thereof. Gram-positive bacteria, selected from Bacillus subtilis, Coxella roseum, and mixtures thereof. • Saprophytic fungi and / or parasitic fungi, and / or Its mixture, e) Microwave radiation treatment, especially microwave radiation up to 960 watts and 2450 Hz. f) Using electrical explosions and / or supercritical water, and / or g) Its combination.

6. The method according to any one of the preceding claims, Its features are, The filtration is performed by dialysis and / or ultrafiltration (cross-flow filtration).

7. A colloidal solution for topical application comprising β-keratin from animal-derived feathers, particularly keratin particles of β-keratin in the form of β-folded sheets with a secondary structure, and / or aggregates thereof.

8. The colloidal solution according to claim 7, Its features are, The keratin particles have a particle size of 5 nm to 500 nm, preferably 70 nm to 350 nm, as measured by dynamic light scattering.

9. The colloidal solution according to claim 7 or 8, Its features are, The animal-derived feathers are selected from feathers of chickens, geese, ducks, turkeys, pheasants, ostriches, rheas, emus, quails, and mixtures thereof.

10. The colloidal solution according to any one of claims 7 to 9, Its features are, The colloidal solution contains additives for stabilization, preferably selected from... Proteins, especially albumin, • Carbohydrates, especially sucrose, lactose, glucose, fructose, mannitol, sorbitol, and sweeteners such as sodium saccharin, sodium cyclohexylsulfamate, aspartame, starch and modified starch, cyclodextrin, and / or mixtures thereof, • Polyanionic surfactants, particularly sodium lauryl sulfate, sodium cetearyl sulfate, cetearyl alcohol (emulsion type), sodium dioctyl sulfosuccinate, and / or mixtures thereof, Nonionic surfactants, particularly fatty alcohols and sterols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, polyethylene glycol-1000-glycerol monofatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, sucrose fatty acid esters, poloxamer and / or mixtures thereof, • Gelling agents, especially polyacrylates, cellulose derivatives such as methylcellulose, methyl hydroxypropyl cellulose, hydroxypropyl cellulose, hydroxyethyl cellulose and / or ethyl cellulose, sodium carboxymethyl cellulose and / or mixtures thereof, Thickeners, especially tragacanth gum, xanthan gum, gum arabic, guar galactomannan, alginate, bentonite and / or mixtures thereof, Film-forming agents, particularly methacrylates, polyvinyl alcohol, and / or mixtures thereof, • Polymers, especially polyethylene glycol, gelatin and / or mixtures thereof.

11. A keratin powder for topical application, which can be produced from a colloidal solution according to any one of claims 7 to 10, particularly by the method according to any one of claims 1 to 6.

12. The keratin powder according to claim 11, Its features are, The keratin particles in the keratin powder have a particle size of 1 μm to 250 μm, preferably 5 μm to 30 μm, as measured by dynamic light scattering.

13. Use of the colloidal solution according to any one of claims 7 to 10 or the keratin powder according to claim 11 or 12 for the preparation of formulations having therapeutic, diagnostic, preventive or cosmetic uses in organisms, particularly humans and / or animals, especially for use on skin, mucous membranes (including conjunctiva) or epidermal appendages (including nails and hair), particularly for use as a substitute for barrier function or as a component of the epidermal barrier.

14. The use according to claim 13, Its features are, The colloidal solution contains at least one small molecule and / or biotechnologically active ingredient, wherein the active ingredient is preferably selected from glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics and / or proteins themselves, protein fragments, peptides, enzymes, antibodies, antibody fragments, RNA and / or DNA molecules and mixtures thereof, or molecules that are targets of one of these active ingredients, and / or The colloidal solution contains cosmetic active ingredients and excipients, preferably selected from amino acids, urea, glycerin, hyaluronic acid, sugars and sugar derivatives, extracts or waxes from plant or animal sources, and mixtures thereof.

15. The use according to claim 13 or 14, for preparing • Liquid matrices, especially solutions, emulsions, suspensions, and / or colloids, • Semi-solid bases, especially suspension ointments, creams, gels, pastes, colloids and / or suppositories, • Solid matrix, especially powders, tablets, granules, pills, capsules and / or inserts.

16. The use according to any one of claims 13 to 15, for preparing • Food and food supplements Animal feed, • Fertilizers and / or plant protection products used on plants and / or soil, • Technical additives, such as binders and / or adhesives in or as part of filtration systems, consistency modifiers, fillers, absorbents for hydrophilic or lipophilic substances, and absorbents for charged or uncharged substances. Packaging materials and / or consumer products, • Textiles and / or fibers that are functional, particularly waterproof and breathable, or without functional properties. • Medical products, especially adhesive bandages, wound dressings, tampons, and clothing that supports wound or skin care.

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