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

Abstract

The present invention relates to a method for producing keratin powder from beta keratin protein from animal feathers by extracting keratin, filtering the extract to obtain a colloidal solution, and drying the colloidal solution to form keratin powder. The present invention also relates to a colloidal solution for topical application, comprising keratin particles and / or aggregates of beta keratin protein, or a keratin powder comprising these keratin particles. The colloidal solution and keratin powder are used to prepare formulations with therapeutic, diagnostic, preventive, or cosmetic applications in humans and / or animals.

Description

[Technical Field]

[0001] The present invention relates to a method for producing keratin powder from beta keratin protein from animal feathers by extracting keratin, filtering the extract to obtain a colloidal solution, and drying it to form keratin powder. The present invention also relates to a colloidal solution for topical application containing keratin particles and / or aggregates of beta keratin protein, or a keratin powder containing these keratin particles. The colloidal solution and keratin powder are used to prepare formulations with therapeutic, diagnostic, preventive, or cosmetic applications in humans and / or animals.

[0002] The stratum corneum (SC) of the epidermis consists of several layers of keratinized corneocytes, surrounded by a "cornified envelope," connected by corneodesmosome structures, and embedded in a complex lipid matrix of liquid crystalline lamellar structures.

[0003] Keratinocyte lipid synthesis is largely autonomously regulated, providing cholesterol and cholesterol derivatives, as well as free fatty acids and triglycerides of various chain lengths. Furthermore, ceramides are synthesized in the endoplasmic reticulum of keratinocytes, and these ceramides differ substantially from other lipid classes due to their isomeric molecular structure. Due to the difference in charge within the long chain molecules, ceramides can spontaneously form lyotropic mesophases, i.e., liquid crystalline membrane structures. Unlike phospholipids, functionally important ceramides in the stratum corneum have two alkyl chains, which also vary in length. Furthermore, they have different configurations depending on the degree of hydration, and therefore various membrane models describe a complex network of membrane sections with polymorphic phase behavior. The dynamic order of the entire system is increasingly understood in its complexity and is gaining practical importance in the development of modern galenic formulation base systems.

[0004] Barrier function is also substantially determined by the amount of hydrophilic phase in the microenvironment. The distribution of water within the stratum corneum microcompartments can be described by at least two fractions. In addition to the free water phase, a bound water fraction is defined. The latter consists of a mobile subfraction and a fixed subfraction. This nomenclature reflects the dynamic exchange of hydrophilicity between the individual compartments. Fixed water is understood to be water bound by strong hygroscopic forces, primarily mediated by amino acids proteolytically generated within the keratinocyte, and is actually exchangeable with very slow release. Under special conditions, water bound by swellable membranes can be liberated and transferred to the free water phase. This then reveals the functionally important stratum corneum water phase. Additionally, free water is bound in the individual microcompartments by hygroscopic molecules, collectively known as "natural moisturizing factors (NMFs)," which exchange not only with the vital epidermal water phase but also with the environment (transepidermal water flux). Essential components of NMF are, in addition to amino acids, in particular pyrrolidone carboxylic acid, lactic acid, urea, and inorganic ions, which are synthesized by keratinocytes and released depending on the degree of differentiation.

[0005] In the context of desquamation, proteins of corneodesmosomes and cornified envelopes in the desquamated layer, the top layer of the stratum corneum, are proteolytically degraded. However, the primary keratin of keratinocytes is not directly affected by this. Therefore, keratinocyte integrity is maintained for a very long time during desquamation. An important function of keratinocytes is to provide moisture for membrane formation in the interkeratinocyte space via diffusion through the cornified envelope. Under therapeutic conditions, the interaction between keratin and transdermally applied active ingredients is not considered important because transcellular passage of any active ingredient to any relevant degree has not yet been demonstrated. This is thought to be due to the cornified envelope surrounding the keratinocytes as an envelope and barrier membrane. Furthermore, due to their central position as an important moisture reservoir in the stratum corneum, keratinocytes indirectly affect the diffusion conditions (diffusion coefficient) of the stratum corneum. The skin bioavailability of transdermally applied active ingredients is primarily determined by the interaction of the galenic formulation system with the stratum corneum, the layer in direct contact with it. From a pharmacological point of view, two processes are important for the flow of substances through the stratum corneum: barrier function and reservoir function. It is also important to note that the stratum corneum is not a homogeneous structure; instead, following the formation and compact structure of the stratum densa, it is broken up by an enzymatically controlled process of desquamation (exfoliation). Thus, the two functional areas mentioned above are recognized to have contrasting importance within the structure of the stratum corneum. Thus, the barrier function is maximized in the stratum densa, while the reservoir function is maximized in the exfoliation layer. The latter acts as a preferential receptor for the transdermally applied free phase. This plays a crucial role in the overall skin fate of active ingredients, since the maximum concentration decreases and the penetration process is delayed in deeper skin layers. At the same time, the free active ingredient can be absorbed to a large extent and become bioavailable even after a relatively short application time.Overall, it is clear that micromorphological conditions, both anatomical variations (e.g., hairy versus hairless skin) and pathological conditions (e.g., impaired epidermal proliferation or differentiation), have a direct effect on the pharmacokinetic profile of transdermally applied substances.

[0006] This demonstrates that targeted influence on the water content of the stratum corneum has a direct impact on the physicochemical barrier function and thus the pharmacokinetics of transdermally applied active ingredients.

[0007] The use of keratin for medical or cosmetic purposes is known from the following documents:

[0008] Previously available keratins have been degraded and extracted into smaller peptide structures based on microbial, acid, or base hydrolysis, and no longer exist as intact, complete keratin proteins (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 have short sequences and different properties (e.g., molecular size, swelling behavior) from the natural, highly sequenced keratin proteins obtained for the first time in this invention. Unlike previous processes, the use of hydrolysis for extraction was omitted; instead, disulfide bonds were cleaved to make the proteins more accessible for extraction. This approach preserved the primary and secondary structure of the keratin proteins as beta-pleated sheets.

[0010] Starting from this, the object of the present invention was to provide colloidal solutions that serve as the basis for formulations that exhibit the interaction of both hydrophilic and, in combination, lipophilic or active ingredients. In addition, these colloidal solutions should be easy to prepare.

[0011] This problem is solved by using a method having the features of claim 1, a colloidal solution having the features of claim 7, and a keratin powder having the features of claim 11. The use according to the invention is defined in claim 13. Preferred embodiments are described in the further dependent claims.

[0012] According to the present invention, there is provided a method for producing keratin particles from animal-derived feathers, comprising the steps of: a) extraction of beta-keratin proteins, in particular beta-keratin in its secondary structure as a β-pleated sheet, in an extraction solution that induces chemical denaturation and contains at least one denaturant, at least one base, at least one reducing agent and at least one buffer; b) subjecting the extracted solution from step a) to filtration to obtain a colloidal solution of keratin particles of beta-keratin protein; c) drying the colloidal solution from step b) by freeze drying, spray drying, vacuum drying, air drying, heat drying, infrared drying, and / or microwave drying to obtain a powder comprising the keratin powder; A method is provided.

[0013] The present invention therefore relates to the use of keratin particles of beta-keratin protein extracted as keratin protein from bird feathers using chemical modification and used in liquid or semi-solid formulations for transdermal application.

[0014] When keratin is obtained from wool, two different types of keratin are obtained: alpha-keratin and beta-keratin, the so-called "soft fiber" (alpha) keratin and "hard fiber" (beta) keratin. Additionally, in this case, extraction with urea in the alkaline range results in the natural form of beta-keratin being obtained as a whole protein from feathers, thus preserving a high proportion of its secondary structure, the beta-pleated sheet, which determines its high water-holding capacity. Beta-keratin is obtained especially from bird feathers. It is rich in the amino acids glycine and alanine, and contains very little cysteine, proline, or hydroxyproline.

[0015] The interaction of keratin with hydrophilic substances is thereby used as a reservoir to target and influence their pharmacokinetics. By combining keratin with water, amino acids, hygroscopic substances, peptides and / or proteins, a replacement of the physical barrier should also be achieved.

[0016] The extraction solution preferably has a pH value of 8-13, more preferably 9-12, and particularly preferably 10-11.

[0017] Preferably, the at least one denaturing agent is selected from the group consisting of urea, thiourea, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and mixtures thereof.

[0018] Furthermore, the at least one base is preferably selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof.

[0019] The at least one reducing agent is preferably selected from the group consisting of β-mercaptoethanol, cysteamine, cysteine, glutathione, sodium disulfite, sodium sulfide, sodium bisulfite, sodium dithionite, sodium thiosulfate, dithiothreitol (DTT), thioglycolic acid and its salts, thiourea, tris(carboxyethyl)phosphine (TCEP) and other phosphines, ammonium chloride, and mixtures thereof.

[0020] Preferably, the at least one buffering agent is selected from the group consisting of tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (Tris-EDTA), potassium chloride-sodium hydroxide (KCl-NaOH), sodium bicarbonate (NaHCO3), dithiothreitol (Tris-DTT), and mixtures thereof.

[0021] The extraction solution contains the following chemical components: at least one oxidizing agent selected from the group consisting of hydrogen peroxide, potassium permanganate, sodium perborate, peracetic acid, performic acid, and mixtures thereof; at least one acid selected from the group consisting of nitric acid, nitrous acid, hypohalous acid and perhalogen acid, and mixtures thereof; at least one ionic agent selected from the group consisting of 1-butyl-3-methylimidazolium (BMIM) chloride, 1-butyl-3-methylimidazolium (BMIM) bromide, 1-butyl-3-methylimidazolium (BMIM) tetrafluoroborate, amide chloride, and mixtures thereof; It is preferable to include at least one of the following:

[0022] A preferred embodiment of the method according to the invention comprises, during the extraction in step a), the following steps: mechanical comminution, in particular by ultrasonic-mediated comminution, preferably in the frequency range of 20 to 50 Hz, using a ball mill and / or a cutting mill, preferably using an Ultra-Turrax, whereby mechanical comminution is carried out until the particle size (d50) according to sieve analysis is 0.1 to 5.0 mm, preferably 0.2 to 1.0 mm, thermal denaturation, in particular at temperatures between 70°C and 150°C, and / or electrochemical denaturation, Precipitation of the extraction solution from step a), in particular precipitation induced by pH change, addition of co-solvents and / or salts, Microbial and enzymatic extraction, Gram-negative bacteria selected from the group consisting of the genera Stenotrophomonas, Chrysebacterium, Vibrio, and mixtures thereof; Gram-positive bacteria selected from the group consisting of Bacillus, Kocuria rosea, and mixtures thereof; saprophytic and / or parasitic fungi, and / or A mixture of these Extraction via treatment by microwave irradiation, in particular by microwave irradiation up to 960 watts and 2450 hertz; Use of electrical explosions and / or supercritical water, and / or A combination of these The present invention provides for performing at least one of the following:

[0023] Beta keratin proteins preferably exist in their secondary structure as β-pleated sheets. Unlike alpha keratins (=soft fibers), beta keratins (=rigid fibers) have a high proportion of tightly twisted β-pleated sheet structures stabilized by disulfide bonds, which ensures the high stability of sclerokeratins.

[0024] Filtration is preferably carried out via dialysis and / or ultrafiltration (cross-flow filtration).

[0025] The animal-derived feathers are preferably selected from the group consisting of feathers from chicken, goose, duck, turkey, pheasant, ostrich, rhea, emu, quail, and mixtures thereof.

[0026] According to the present invention, there is also provided a colloidal solution for topical application, comprising keratin particles of beta keratin protein and / or aggregates thereof.

[0027] The keratin particles in the colloidal solution preferably have a particle size in the range of 5 nm to 500 nm, preferably 70 nm to 350 nm, as measured by dynamic light scattering (in accordance with DIN ISO 22412:2018-09) (Zetasizer ZEN3600, manufactured by Malvern Panalytical Instruments).

[0028] The colloidal solution preferably contains additives for stabilization, said additives preferably being: Proteins, e.g., albumin, carbohydrates, such as sucrose, lactose, glucose, fructose, mannitol, sorbitol, and sweeteners, such as sodium saccharin, sodium cyclamate, aspartame, starch and modified starches, cyclodextrins, and / or mixtures thereof; polyanionic surfactants, such as sodium dodecyl sulfate, sodium cetylstearyl sulfate, cetylstearyl alcohol (emulsifier), sodium dioctyl sulfosuccinate, and / or mixtures thereof; non-ionic surfactants, such as fatty alcohols and sterols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, macrogol-1000 glycerol mono fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, sucrose fatty acid esters, poloxamers, and / or mixtures thereof, Gelling agents, such as polyacrylates, cellulose derivatives, such as methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and / or ethylcellulose, carmellose sodium, and / or mixtures thereof thickening agents, such as gum tragacanth, xanthan gum, gum arabic, guar galactomannan, alginates, bentonite, and / or mixtures thereof; film-forming agents, such as acrylate methacrylates, polyvidone, polyvinyl alcohol, and / or mixtures thereof; Polymers, such as macrogol, gelatin, and / or mixtures thereof is selected from the group consisting of:

[0029] The present invention also provides a keratin powder of beta keratin protein for topical application, which can be produced from the colloidal solution by drying the colloidal solution, particularly by freeze-drying, spray-drying, vacuum drying, air drying, heat drying, infrared drying, and / or microwave drying.

[0030] The keratin particles in the keratin powder preferably have a particle size in the range of 1 to 250 μm, preferably 5 to 30 μm, as measured by dynamic light scattering (in accordance with DIN ISO 22412:2018-09) (Zetasizer ZEN3600, manufactured by Malvern Panalytical Instruments).

[0031] The above colloidal solution and the above keratin powder are used for the manufacture of preparations having therapeutic, diagnostic, prophylactic or cosmetic applications in living organisms, in particular in humans and / or animals.

[0032] A preferred embodiment provides that the colloidal solution contains at least one small molecule and / or one biotechnological active ingredient. The active ingredient is preferably selected from the group consisting of glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics, and / or proteins themselves, protein fractions, peptides, enzymes, antibodies, antibody fragments, RNA and / or DNA molecules, and mixtures thereof. A further preferred embodiment provides that molecules are included, said molecules representing targets for any of these active ingredients.

[0033] Furthermore, the colloidal solution preferably contains cosmetic active ingredients and excipients selected from the group consisting of amino acids, urea, glycerol, hyaluronic acid, sugars and sugar-like derivatives, extracts or waxes from plants or parts obtained from animals, and mixtures thereof.

[0034] A preferred embodiment provides that specific and / or non-specific compounds are formed with the active substance and / or excipient by covalent and / or non-covalent bonds, preferably via selected groups consisting of carboxylic acid groups, amino groups, thiol groups, and / or hydroxyl groups.

[0035] To stabilize colloidal solutions with or without active ingredients and / or excipients, these are preferably incorporated as liposomal systems into unilamellar or multilamellar vesicles of different or uniform size by adding amphiphilic molecules. These amphiphilic molecules are preferably phospholipids, preferably lecithin, DODAB, DPPC, DSPC, DSTAP, and / or mixtures thereof, cationic lipids, preferably ALC-0315, PEG lipids, preferably ALC-0159, prostaglandins and modified prostaglandins, preferably PGE1, PGD2, PGE2, 15-keto PGE1, and / or mixtures thereof, ceramides, preferably selected from the group consisting of ceramides having NS, NH, NP, NDS, AS, AH, ADS, AP head groups and chain lengths of C10 to C26, and / or mixtures thereof; Ceramides, preferably selected from the group consisting of ceramides having EOS, EOH, EOP head groups and chain lengths of C10 to C32, and / or mixtures thereof; cholesterol, cholesterol derivatives, and / or mixtures thereof, Fatty acids, preferably with chain length C 10 ~C 32 and / or mixtures thereof. is selected from the group consisting of:

[0036] Application to living organisms, in particular humans, preferably relates to application to the skin, mucous membranes (including the conjunctiva) or epidermal appendages (including nails and hair), in particular to replacement of the barrier function or replacement of components of the epidermal barrier.

[0037] The colloidal solution and keratin powder according to the present invention are liquid bases, in particular solutions, emulsions, suspensions and / or colloids, semi-solid bases, in particular suspension ointments, ointments, creams, gels, pastes, colloids and / or suppositories, Solid substrates, in particular powders, tablets, granules, pellets, capsules and / or inserts It is also used for the production of

[0038] The colloidal solution and keratin powder according to the present invention are Food and food supplements, ·Animal feed, Fertilizers and / or plant protection products for plants and / or soil, Industrial additives, for example industrial additives in or as filter systems, adhesives and / or binders, thickeners, fillers, absorbents for hydrophilic or lipophilic substances, charged or uncharged substances, packaging materials and / or consumer goods, Functional or non-functional textiles and / or fibers, in particular water-repellent and breathable ones; Medical products, especially bandages, wound dressings, tampons, and garments that aid in wound or skin care Used for the manufacture of

[0039] The subject matter according to the present invention will be described in more detail with reference to the following figures and examples, without intending that said subject matter be limited to the particular embodiments shown therein. [Brief explanation of the drawings]

[0040] [Figure 1A] 1 shows SEM images of freeze-dried hair keratin at different resolutions. [Figure 1B] 1 shows SEM images of freeze-dried hair keratin at different resolutions. [Figure 2] TEM images of A) feather keratin (0.5 mg / ml) and B) hair keratin (0.5 mg / ml) are shown. [Figure 3] Fluorescence spectroscopic images of 5% (w / w) keratin particles in the base cream DAC (using (A) feather keratin, B) and C) hair keratin). [Figure 4]1 shows images of a penetration test using A) feather keratin and B) hair keratin at 5% (w / v) each in the base cream DAC. [Figure 5] Figure 1 shows the images of the penetration study of feather keratin 5% (w / v) in the base cream DAC by fluorescence imaging. [Figure 6] Cytotoxicity data of freeze-dried and colloidal feather keratin on keratinocytes (NHEK) and cytotoxicity data of freeze-dried and colloidal feather keratin on dermal fibroblasts (NHDF) are shown as indicators. [Figure 6-1] Same as above [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 6-4] Same as above [Figure 6-5] Same as above [Figure 6-6] Same as above [Figure 6-7] Same as above [Figure 7] 1 shows the results of scratch tests showing the epithelialization area of ​​keratinocytes (HaCaT) treated with freeze-dried feather keratin in three independent experiments. [Figure 8] 1 shows the results of scratch tests showing the epithelialization area of ​​three independent tests of colloidal feather keratin on keratinocytes (HaCaT). [Figure 9] 1 shows the results of scratch tests showing the epithelialization area of ​​keratinocytes (NHDF) after three independent tests using freeze-dried feather keratin. [Figure 10] 1 shows the results of scratch tests showing the epithelialization area of ​​keratinocytes (NHDF) after three independent tests of colloidal feather keratin. [Figure 11] 25 μl (1 μCi) of TO was added to 5 mg of freeze-dried keratin for n=3 (contact times: 1 min, 5 min, 1 h, 16 h, 24 h), centrifuged, the supernatant was removed, and the radioactive dose was determined. The graph shows the water absorption rate of feather keratin after the addition. [Figure 12] 1 shows an image of the culture of HaCaT cells with 1 mg of feather keratin. [Figure 12-1] Same as above [Figure 13] The graph shows the change in water absorption rate over time for urea, glycerol, and keratin, as well as a mixture of keratin particles and glycerol (50:50), and different ratios of keratin particles and urea (e.g., 95:5 (orange); 50:50 (light blue); 5:95 (brown)) in a climate chamber (room temperature and 95% relative humidity). [Figure 14] Figure 1 shows the time course of moisture release rates for urea, glycerol, and keratin, as well as a mixture of keratin particles and glycerol (50:50), and different ratios of keratin particles to urea (e.g., 95:5 (orange); 50:50 (light blue); 5:95 (brown)) in a climate chamber (room temperature and 50% relative humidity). [Figure 15] 1 shows the tertiary structure of beta-keratin from bird feathers. [Figure 16] 1 shows corneometry measurement data in arbitrary units (AU) before and after 1, 2, 4, 8 and 24 hours for cosmetic cream formulations containing 0.1%, 0.5%, 1.0% and 2.0% keratin particles. [Figure 17] 1 shows measurement data for stratum corneum water loss measurements in g / m2 / h before and after 1, 2, 4, 8 and 24 hours for cosmetic cream formulations containing 0.1%, 0.5%, 1.0% and 2.0% keratin particles.

[0041] Various animal materials are available as keratin sources; however, their suitability appears to vary due to the degree of protein cross-linking. Preliminary studies using various keratin-containing biomaterials have shown that avian feathers are particularly suitable. In addition to practical aspects such as availability and ease of processing, certain biochemical aspects have emerged that justify the preferential use of feathers. The keratin contained in feathers corresponds primarily to beta-keratin, which is presented as polypeptide chains with a beta-pleated sheet structure, consisting of 3-4 nm filaments and a molecular weight of approximately 10-22 kDa. Unlike mammalian alpha-keratin, the primary sequence of beta-keratin has only a few, but functionally important, differences. Therefore, beta-keratin forms fewer macrofibrils than alpha-keratin and exhibits more regular ordering and packing behavior. These differences therefore provide more favorable prerequisites for standardized keratin isolation and the resulting product properties. In particular, the hydrolysis of keratin, which is typically performed to isolate intact keratin proteins, is omitted, thereby particularly avoiding the formation of keratin fragments, amino acids, and peptides. For effective and standardized keratin isolation, a unique process based on the biochemical properties of β-keratin was developed. The chicken feathers used were first washed with water and soap, disinfected with 70% ethanol, and then dried at room temperature. The washed and dried feathers were crushed in a cutting mill (Retsch SM 100 comfort), followed by homogenization of the entire material. The feather homogenate was defatted using a Soxhlet apparatus. 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 resulting pure extract was diluted with water and subjected to filtration with a cutoff of 10,000 NMWC. The dialyzed solution was processed into a powder with a particle size of <25 μm by spray drying.

[0042] To determine the colloidal size of keratin particles (1 mg / ml in deionized water), dynamic light scattering (DLS) measurements were performed using a Malvern Panalytical Instruments Zetasizer ZEN3600 in accordance with DIN ISO 22412:2018-09. This analytical method allows for the characterization of particle size in suspensions and emulsions by detecting scattered laser radiation. The objective was to determine the particle size of proteins in the colloidal state. To perform DLS measurements, 1 ml of 0.1 mM colloidal keratin solution was pipetted into a disposable polystyrene cuvette and then transferred to the Zetasizer Lab cuvette module, which had been heated to 25°C. Particle size was determined using the automated analysis mode (general purpose) and backscattering at 173°. Each measurement was performed in triplicate, consisting of 15 measurement cycles per run. The recorded measurement data for each sample were then averaged. A total of three samples were examined using DLS. Within the context of DLS, the polydispersity index (PdI), which indicates how uniformly the particles are distributed within the sample, the particle size (in nm), and the percentage of each particle size relative to the total sample content were determined.

[0043] To determine the molecular size (molecular weight) of extracted keratin particles, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed in an SDS gel chamber (Invitrogen Mini Gel Tank). Samples were denatured according to the manufacturer's protocol for the NuPAGE® Bis-Tris Mini Gel Electrophoresis (Thermo Fisher Scientific). For this purpose, 2 μl of sample, 2.5 μl of NuPAGE® LSD sample buffer (4x dilution), 1 μl of NuPAGE® LSD reducing agent (10x dilution), and 6.5 μl of deionized water were combined and incubated at 95°C for 10 min. To perform gel electrophoresis, the gel chamber was filled with 1x diluted Tris-Tricine running buffer (1.2 M Tris, 0.8 M Tricine, 2% SDS). Then, a Tris-Tricine gel (Novex 10-20% Tricine gel, lot number 20101945, reference number EC6625BOX, manufactured by Invitrogen (Thermo Fisher Scientific)) was inserted, and the comb was removed from the gel. In each case, 10 μl of the prepared sample and 2 μl of the marker were added to the corresponding gel well. Gel electrophoresis was performed at 130 V and 250 mA for 1.5 h. The gel was then removed from the chamber, and the running buffer was discarded. The plastic gel holder was opened, and the Tris-Tricine gel was transferred 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). The gel was incubated in the solution overnight on a shaker. The staining solution was then removed. The gel was destained by rinsing it several times with distilled water while gently shaking.

[0044] Example An attempt was made to replicate the function of keratinocytes. To this end, keratin was extracted from various natural sources, the corresponding particles were produced, and these were initially investigated as mechanical stabilizers of lipid matrices in various semi-solid formulations. The intention was to coat the keratin particles with bipolar lipids, thereby forming keratosomes.

[0045] First, various extraction methods were tested using human hair (alpha keratin). Hair degradation was primarily achieved using chemical denaturation. For this purpose, urea, thiourea, and guanidinium hydrochloride were used. To assist denaturation, β-mercaptoethanol, cysteamine, and L-cysteine ​​were used as reducing agents. The highest yield was achieved with an extraction solution of 5 M guanidinium hydrochloride, 10% cysteamine, and 25 mM Tris (pH 8.5). Due to the safety of urea and L-cysteine, an extraction solution consisting of 10 M urea, 100 mM L-cysteine, and 25 mM Tris-HCl (pH 10.5) was selected for keratin isolation. After successful extraction, the extraction solution was dialyzed (cutoff: 6–8 kDa, regenerated cellulose, SpectraPor®), thereby generating a colloidal solution: with high protein concentration and extensive dialysis steps (5–6 l), keratin was precipitated in the dialysis tubing. Finally, the dialysate was freeze-dried, resulting in a white powder. This powder was examined using scanning electron microscopy (SEM) (Figures 1A and 1B). Particles from hair keratin vary in size and shape. Therefore, the particles in the final formulation were defined to have a size >600 nm to avoid penetration into the stratum corneum of the skin.

[0046] For regulatory reasons, feather keratin (beta-keratin) from various bird species was used as an alternative keratin source. For this purpose, untreated feathers from chickens, geese, and ducks were examined. For practical reasons, chicken feathers in particular were used for further examination.

[0047] Therefore, the extraction of feather keratin was adapted to the established extraction process for hair keratin. Compared to all other extraction methods considered, it was observed that the highest yield was achieved using the selected extraction solution (pH 10.5) consisting of 10 M urea, 100 mM L-cysteine, and 25 mM Tris-HCl. The optional addition of 1 M ammonium chloride prevents carbamylation of the protein and the resulting changes in protein properties.

[0048] To determine the size of feather and hair keratin particles, we performed a comparative study using negative staining with transmission electron microscopy (TEM) (Figure 2). The size of hair keratin particles in colloidal solution was approximately 40–75 nm, while that of feather keratin particles was approximately 20–35 nm. To visualize keratin particles for penetration studies, they were fluorescently labeled with 2-aminobenzoyl (Abz) (which reacts with isatoic anhydride under basic denaturing conditions). Fluorescently labeled hair and feather keratin were blended at 5% (w / w) in a base cream DAC, applied to a microscope slide, and examined using a fluorescent microscope (Figure 3). Individual particles from the colloidal solution identified using TEM formed well-balanced aggregates in the base cream DAC. In the case of feather keratin, a uniform distribution of aggregated particles was observed, with particle sizes ranging between 20 and 35 μm. On the other hand, hair keratin aggregated into larger, crystal-like particles. The particle size here is between 20 and 140 μm.

[0049] Using penetration studies of hair and feather keratin (in each case 5% (w / w)) in the base cream DAC into ex vivo human skin (Franzzelle), it could additionally be demonstrated that the keratin particles do not diffuse into the deeper layers of the skin, but remain on top of the SC (exfoliation layer) (Figures 4 and 5). To better evaluate the interaction of feather keratin particles with SC lipids, or the effect of lipid coating, the zeta potential was determined at different pH values ​​(Table 1).

[0050] Table 1: Zeta potential of feather keratin and fluorescently labeled feather keratin (in each case 10 mg / ml in 10 mM potassium chloride solution at different pH values). Measurements were performed by Fabio. [Table 1] Keratin particles have a negative charge, which makes them ideal for coating with positively charged lipids.

[0051] Cytotoxicity studies were performed using standard protocols to examine the effects of colloidal and lyophilized keratin particles on the persistence and proliferation activity of keratinocytes and dermal fibroblasts. No significant effect was observed on dermal fibroblasts for incubation times up to 48 hours (Figures 6-7). In keratinocytes, only colloidal keratin showed a slight, concentration-dependent decrease in proliferation activity.

[0052] The effects of colloidal and lyophilized keratin particles on the migration behavior of keratinocytes (HaCaT) and dermal fibroblasts (NHDF) were investigated using scratch assays (Figures 7-10). This revealed a concentration-dependent inhibition of migration for colloidal keratin particles and, to a much lesser extent, for lyophilized keratin particles, due to a clear mechanical mechanism. However, no evidence of a toxic effect was found.

[0053] To objectively determine the loading capacity of feather keratin particles for hydrophilic or hydrophobic substances, loading and deloading tests were performed. First, keratin particles were incubated in TO solution for different lengths of time, and then the amount of absorbed tritium was measured by centrifugation (Figure 11). Only 25-32% of the introduced TO was recovered in the pellet. This indicated an unexpectedly high level of interaction between both the hydrophilic substances and the extracted feather keratin.

[0054] To investigate the possible interaction of keratin particles with living cells, HaCaT cells (keratinocytes) were cultured with fluorescently labeled feather and hair keratin particles. The medium was changed 48 hours after keratin treatment. Keratin particles were clearly visible under a fluorescence microscope and were found to be associated with the cell nucleus. Therefore, even with fluorescence microscopy, the possibility of intracellular uptake of the particles could not be ruled out (Figures 12 and 12-1). Therefore, we investigated the attachment zone between keratin particles and the cell membrane using Raman spectroscopy, but found no evidence of intracytoplasmic keratin particles. Therefore, it is hypothesized that keratin particles are attached to the cell membrane due to the zeta potential and the charge of the phospholipids in the cell membrane.

[0055] Additionally, moisture absorption experiments were performed to determine the water absorption capacity of keratin particles according to the function of keratinocytes. The keratin particles showed a maximum water absorption rate of approximately 20% after only three days. With the addition of urea (up to a ratio of 5% keratin to 95% urea), the time to reach the maximum increased, finally reaching a maximum after 15 days. In this case, the water absorption capacity was 197% (Figure 13). The moisture release rate was determined at room temperature and 50% humidity. Pure keratin slowly released some moisture and still retained moisture after seven days, while urea rapidly released moisture at a rate of approximately 170% within the first 24 hours, reaching complete moisture release after four days. For keratin / urea mixtures with ratios of 25:75, 10:90, and 5:90, the moisture content remained at 25% after four days (Figure 14).

[0056] Based on the known keratin sequence and Dan Cojocari's amino acid classification, keratin contains only approximately 30% hydrophobic amino acids and a similar number of hydrophilic amino acids, but no binding pocket exists. Therefore, the interactions between keratin and substances with different physicochemical properties are unpredictable. Therefore, we calculated the tertiary structure of keratin and identified hydrophobic and hydrophilic regions within the molecule using the artificial intelligence (AI) software AlphaFold 2 (EMBL's European Bioinformatics Institute (EMBL-EBI), Hinxton, UK) (Figure 15). This again confirmed the assumption that no binding pocket exists, and therefore, the observed interactions can only be explained based on hydrophobic or hydrophilic interactions between amino acid clusters on the protein side and related groups on other molecules.

[0057] To demonstrate clinical efficacy, cosmetic cream formulations containing increasing concentrations of keratin particles (0.1%, 0.5%, 1.0%, and 2.0%) were investigated in a prospective, randomized, double-blind study for their moisturizing effect on the stratum corneum of healthy subjects. The total water content of the stratum corneum (corneal water content) and the free water phase as transepidermal water loss (TEWL) were determined by corneal water loss measurements before application (baseline) and 1, 2, 4, 8, and 24 hours after single-application studies. After 24 hours, the water content of the stratum corneum increased by approximately 30% (Figure 16), and transcorneal water loss decreased by approximately 40% (Figure 17). These results demonstrate that the extracted keratin particles, as a colloidal aqueous phase in the cream formulation, provide sustained moisturizing effects on the stratum corneum in accordance with physiological keratinocyte function.

Claims

1. A method for producing keratin particles from animal-derived feathers, comprising: a) extraction of beta-keratin proteins, in particular beta-keratin in its secondary structure as a β-pleated sheet, in an extraction solution that induces chemical denaturation and contains at least one denaturant, at least one base, at least one reducing agent and at least one buffer; b) subjecting the extraction solution from step a) to filtration to obtain a colloidal solution of keratin particles of the beta keratin protein; c) drying 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 keratin powder; method.

2. The extraction solution has a pH value of 8 to 13, more preferably 9 to 12, particularly preferably 10 to 11; The method of claim 1.

3. the at least one denaturing agent is selected from the group consisting of urea, thiourea, guanidine hydrochloride, sodium dodecyl sulfate (SDS), and mixtures thereof; and / or the at least one base is selected from the group consisting of sodium hydroxide, potassium hydroxide, and mixtures thereof; and / or the at least one reducing agent is selected from the group consisting of β-mercaptoethanol, cysteamine, cysteine, glutathione, sodium disulfite, sodium sulfide, sodium bisulfite, sodium dithionite, sodium thiosulfate, dithiothreitol (DTT), thioglycolic acid and its salts, thiourea, tris(carboxyethyl)phosphine (TCEP) and other phosphines, ammonium chloride, and mixtures thereof; and / or The at least one buffering agent may be selected from the group consisting of tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (Tris-EDTA), potassium chloride-sodium hydroxide (KCl-NaOH), sodium bicarbonate (NaHCO ３ ), dithiothreitol (Tris-DTT), and mixtures thereof; The method of claim 1.

4. The extraction solution preferably comprises at least one oxidizing agent selected from the group consisting of hydrogen peroxide, potassium permanganate, sodium perborate, peracetic acid, performic acid, and mixtures thereof; and / or The extraction solution preferably comprises at least one acid selected from the group consisting of nitric acid, nitrous acid, hypohalous acid and perhalogen acid, and mixtures thereof; and / or The extraction solution preferably comprises at least one ionic agent selected from the group consisting of 1-butyl-3-methylimidazolium (BMIM) chloride, 1-butyl-3-methylimidazolium (BMIM) bromide, 1-butyl-3-methylimidazolium (BMIM) tetrafluoroborate, amide chlorides, and mixtures thereof. The method of claim 1.

5. During said extraction in step a), the following steps are performed: a) mechanical comminution, in particular by ultrasonic mediated comminution, preferably in the frequency range of 20 to 50 Hz, using a ball mill and / or using a cutting mill, wherein the mechanical comminution is carried out until the particle size is 0.1 to 5.0 mm, preferably 0.2 to 1.0 mm; b) thermal denaturation, in particular at temperatures between 70°C and 150°C, and / or electrochemical denaturation; c) precipitation of the extraction solution from step a), in particular precipitation induced by pH change, co-solvent and / or salt addition; d) Microbial and enzymatic extraction, - Gram-negative bacteria selected from the group consisting of the genera Stenotrophomonas, Chryseobacterium, Vibrio, and mixtures thereof; - Gram-positive bacteria selected from the group consisting of Bacillus, Kocuria rosea, and mixtures thereof; saprophytic and / or parasitic fungi, and / or ・Mixtures thereof Extraction via e) treatment with microwave irradiation, in particular with microwave irradiation up to 960 watts and 2450 hertz; f) the use of electroexplosion and / or supercritical water; and / or g) combinations thereof perform at least one of the following: The method of claim 1.

6. The filtration is carried out via dialysis and / or ultrafiltration (cross-flow filtration), 6. The method according to any one of claims 1 to 5.

7. A colloidal solution for topical application comprising keratin particles and / or aggregates thereof of beta keratin protein from animal-derived feathers, particularly beta keratin having its secondary structure as β-pleated sheets.

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

9. The feathers from said animals are selected from the group consisting of feathers from chickens, geese, ducks, turkeys, pheasants, ostriches, rheas, emus, quails, and mixtures thereof; The colloidal solution according to claim 7.

10. The colloidal solution contains a stabilizing additive, which preferably comprises: - proteins, especially albumin, carbohydrates, in particular sucrose, lactose, glucose, fructose, mannitol, sorbitol, and sweeteners, such as sodium saccharin, sodium cyclamate, aspartame, starch and modified starches, cyclodextrins, and / or mixtures thereof; polyanionic surfactants, in particular sodium dodecyl sulfate, sodium cetylstearyl sulfate, cetylstearyl alcohol (emulsifier), sodium dioctyl sulfosuccinate, and / or mixtures thereof; nonionic surfactants, in particular fatty alcohols and sterols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, macrogol-1000 glycerol mono fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, sucrose fatty acid esters, poloxamers, and / or mixtures thereof; gelling agents, in particular polyacrylates, cellulose derivatives such as methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and / or ethylcellulose, carmellose sodium, and / or mixtures thereof; thickening agents, in particular tragacanth gum, xanthan gum, gum arabic, guar galactomannan, alginates, bentonite, and / or mixtures thereof; film-forming agents, in particular acrylate methacrylates, polyvidone, polyvinyl alcohol, and / or mixtures thereof; polymers, in particular macrogol, gelatin, and / or mixtures thereof; selected from the group consisting of The colloidal solution according to claim 7.

11. Keratin powder for topical application, producible from the colloidal solution of claim 7.

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

13. Use of a colloidal solution according to any one of claims 7 to 10 or a keratin powder according to claim 11 or 12 for the manufacture of a preparation having therapeutic, diagnostic, prophylactic or cosmetic application in living organisms, in particular humans and / or animals, in particular the skin, mucous membranes (including the conjunctiva) or epidermal appendages (including nails and hair), in particular as a replacement for a component of the barrier function or epidermal barrier.

14. the colloidal solution comprises at least one small molecule and / or biotechnological active ingredient, wherein the biotechnological active ingredient is preferably selected from the group consisting of glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics, and / or proteins themselves, protein fractions, peptides, enzymes, antibodies, antibody fragments, RNA and / or DNA molecules, and mixtures thereof, or molecules representing the target of one of these biotechnological active ingredients; and / or The colloidal solution preferably contains cosmetic active ingredients and excipients selected from the group consisting of amino acids, urea, glycerol, hyaluronic acid, sugars and sugar-like derivatives, extracts or waxes from plants or parts obtained from animals, and mixtures thereof. 、 14. The use according to claim 13.

15. liquid bases, in particular solutions, emulsions, suspensions and / or colloids, semi-solid bases, in particular suspension ointments, ointments, creams, gels, pastes, colloids and / or suppositories, solid substrates, in particular powders, tablets, granules, pellets, capsules, and / or inserts; 14. The use according to claim 13 for the preparation of

16. - Food and food supplements, ・Animal feed, fertilizers and / or plant protection products for plants and / or soil, industrial additives, for example industrial additives in or as filter systems, adhesives and / or binders, thickeners, fillers, absorbents for hydrophilic or lipophilic substances, charged or uncharged substances, - packaging materials and / or consumer goods, - functional or non-functional textiles and / or fibers, in particular water-repellent, breathable ones; Medical products, especially bandages, wound dressings, tampons, and garments that aid in wound or skin care 14. The use according to claim 13 for the preparation of

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