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

Abstract

The invention relates to a method for producing keratin powder of the protein β-keratin from feathers of animal origin by extracting keratin, filtering the extraction solution and obtaining a colloidal solution, and drying same to form the keratin powder. The invention also relates to a colloidal solution for topical application containing keratin particles of the protein β-keratin and / or agglomerates thereof or a keratin powder containing said keratin particles. The colloidal solution and the keratin powder are used for the production of preparations with therapeutic, diagnostic, preventative or cosmetic purposes for humans and / or animals.

Description

[0001] Process for producing a keratin powder from feathers of animal origin, colloidal solution and keratin powder for topical application containing keratin particles and use of the colloidal solution and the keratin powder

[0002] The present invention relates to a process for producing keratin powder of the protein beta-keratin from feathers of animal origin by extracting keratin, filtering the extraction solution to obtain a colloidal solution, and drying it to form keratin powder. The invention also relates to a colloidal solution for topical application containing keratin particles of the protein beta-keratin and / or their agglomerates, or to a keratin powder containing these keratin particles. The colloidal solution and the keratin powder are used for the production of preparations with therapeutic, diagnostic, preventative, or cosmetic purposes in humans and / or animals.

[0003] The stratum corneum (SC) of the epidermis consists of several layers of keratinized corneocytes, which are surrounded by the "cornified envelope" and connected by corneodesmosomal structures and embedded in a complex lipid matrix of liquid crystalline, lamellar structures. Keratinocytic lipid synthesis is largely autonomously regulated and, in addition to cholesterol and cholesterol derivatives, also provides free fatty acids of various chain lengths and triglycerides. In addition, ceramides are synthesized in the endoplasmic reticulum of keratinocytes. These ceramides differ substantially from molecules of other lipid classes due to their anisomeric molecular structure. Due to charge differences within the long-chain molecule, ceramides can spontaneously form lyotropic mesophases, i.e., liquid crystalline membrane structures.Unlike phospholipids, the functionally important ceramides of the stratum corneum possess two alkyl chains, which in turn vary in length. Furthermore, these chains exhibit different configurations depending on the degree of hydration, so that various membrane models describe a complex network of membrane segments with polymorphic phase behavior. The dynamic order of the overall system is increasingly understood in all its complexity and is gaining increasing practical importance in the development of modern pharmaceutical vehicle systems.

[0004] The barrier function is also largely determined by the quantity of the hydrophilic phase in the microenvironment. The distribution of water within the microcompartments of the stratum corneum can be described as at least two fractions. In addition to a free water phase, a bound water fraction is defined. The latter consists of a mobilizable and a fixed subfraction. The nomenclature refers to the dynamic exchange of hydrophilic valences between the individual compartments. Fixed water primarily refers to the water fraction that is bound in corneocytes by strong hygroscopic forces mediated by proteolytically generated amino acids and is, in effect, only very slowly available for exchange. Under special conditions, water bound by swellable membrane components can be liberated and transferred into the free water phase.This shows the functionally important water phase of the stratum corneum. The free water is also bound in the individual microcompartments by hygroscopic molecules, collectively referred to as the "natural moisturizing factor (NMF)," and is in exchange with the water phase of the vital epidermis and the environment (transepidermal water flow). In addition to amino acids, essential components of the NMF are pyrrolidone carboxylic acids, lactate, urea, and inorganic ions. These are synthesized by the keratinocytes and released depending on their degree of differentiation.

[0005] During desquamation, corneodesmosomes and proteins of the cornified envelope are degraded by proteolysis in the stratum disjunctum, the outermost layer of the stratum corneum. However, the predominant keratin of the corneocytes is not directly affected. Therefore, the integrity of the corneocytes is maintained for a very long time during desquamation. An essential function of the corneocytes is to provide water for membrane formation in the intercorneocytic space via diffusion of the cornified envelope. Under therapeutic conditions, the interaction of keratin with epicutaneously applied active ingredients is considered insignificant, since no relevant transcellular passage has yet been demonstrated for any active ingredient. The cause of this is thought to be the cornified envelope, which surrounds the corneocyte as an envelope and barrier membrane.Nevertheless, the corneocyte indirectly influences the diffusion conditions (diffusion coefficients) of the stratum corneum through its central position as the stratum corneum's water reservoir. The cutaneous bioavailability of epicutaneously applied active substances is primarily determined by the interaction of the pharmaceutical system with the stratum corneum as the immediate contact layer. From a pharmacological perspective, two processes within the stratum corneum are important for substance flow: the barrier function and the reservoir function. It is also important to note that the stratum corneum is not a homogeneous structure, but rather, after formation and compact structuring in the stratum compactum, is loosened by an enzymatically controlled desquamation process (stratum disjunctum). Thus, the two functional areas mentioned are considered to be diametrically important within the structural levels of the stratum corneum.The maximum barrier function lies in the stratum compactum, and that of the reservoir function in the stratum disjunctum. The latter serves as a privileged acceptor for epicutaneously applied and liberated phases. This is crucial for the overall cutaneous kinetics of an active ingredient, as maximum concentrations are reduced in deeper skin layers and the penetration process is delayed. At the same time, large amounts of liberated active ingredient components can be absorbed and become bioavailable even after relatively short application times. Overall, this highlights that micromorphological conditions, both as anatomical variance (e.g., field vs. groin skin) and under pathological conditions (e.g., impaired epidermal proliferation or differentiation), have a direct impact on the pharmacokinetic profile of an epicutaneously applied substance.

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

[0007] The use of keratin for medical or cosmetic indications is known from the following publications.

[0008] Previously available keratin is degraded and extracted into smaller peptide structures due to microbial, acid- or base-induced hydrolysis and is no longer present as intact and complete protein keratin (Shavandi, 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 in their sequences and exhibit different properties (e.g., molecular size, swelling behavior) than the native, high-sequence keratin protein, which was obtained here for the first time. Unlike previous methods, hydrolysis for extraction was omitted; instead, the protein was made accessible for extraction by breaking the disulfide bonds. This preserves the primary and secondary structure of the keratin protein as a ß-fold sheet.

[0010] Based on this, the object of the present invention was to provide colloidal solutions that serve as a basis for preparations that interact with both hydrophilic and lipophilic components or active ingredients. Furthermore, these colloidal solutions should be easy to produce. This object is achieved by 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. Claim 13 specifies uses according to the invention. The further dependent claims name preferred embodiments.

[0011] According to the invention, a process for producing keratin particles from feathers of animal origin is provided, in which a) the protein beta-keratin, in particular beta-keratin in its secondary structure as a ß-fold sheet, is extracted in an extraction solution which triggers chemical denaturation and contains at least one denaturing agent, at least one base, at least one reducing agent and at least one buffer substance, b) the extraction solution from step a) is subjected to filtration, in which a colloidal solution of the keratin particles of the protein beta-keratin is obtained, c) the colloidal solution from step b) is dried by freeze-drying, spray-drying, vacuum-drying, air-drying, heat-drying, infrared-drying and / or microwave-drying to obtain a powder containing keratin powder.

[0012] The present invention thus relates to the use of keratin particles of the protein beta-keratin, which are extracted from bird feathers as protein keratin by chemical denaturation and used in liquid or semi-solid preparations for epicutaneous application.

[0013] When keratin is extracted from wool, alpha and beta keratin are obtained, so-called "soft fiber" (alpha) and "hard fiber" (beta) keratin, i.e., two different types of keratin. The extraction carried out here with urea in alkaline conditions results in the native form of beta keratin being extracted from the feathers as a whole protein, thus retaining the high proportion of its secondary structure, the ß-fold protein, which determines the high water-binding capacity of this keratin. Beta keratin is obtained primarily from bird feathers. It is rich in the amino acids glycine and alanine and contains little cysteine, proline, and hydroxyproline.

[0014] The interaction of keratin with hydrophilic substances is used as a reservoir to specifically influence their pharmacokinetics. By combining keratin with water, amino acids, hygroscopic substances, peptides, and / or proteins, a substitution of the physical barrier is also achieved.

[0015] The extraction solution preferably has a pH of 8 to 13, preferably of 9 to 12 and particularly preferably of 10 to 11.

[0016] It is preferred that the at least one denaturing agent is selected from the group consisting of urea, thiourea, guanidine hydrochloride, sodium dodecyl sulfate (SDS) and mixtures thereof.

[0017] It is further preferred that the at least one base is selected from the group consisting of sodium hydroxide, potassium hydroxide and mixtures thereof.

[0018] The at least one reducing agent is preferably selected from the group consisting of ß-mercaptoethanol, cysteamines, cysteines, glutathione, sodium disulfite, sodium sulfide, sodium hydrogen sulfite, sodium dithionite, sodium thiosulfate, dithiotreitol (DTT), thioglycolic acid and its salts, thiourea, tricarboxyethylphosphane (TCEP) and other phosphanes, ammonium chloride and mixtures thereof.

[0019] It is preferred that the at least one buffer substance is selected from the group consisting of tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (EDTA) / Tris, potassium chloride-sodium hydroxide (KCl-NaOH), sodium bicarbonate (NaHCl), dithiothreitol (DTT) / Tris, and mixtures thereof. It is preferred that the extraction solution contains at least one of the following chemical components:

[0020] • at least one oxidizing agent selected from the group consisting of hydrogen peroxide, potassium permanganate, sodium perborate, peroxyacetic acid, performic acid and mixtures thereof,

[0021] • at least one acid selected from the group consisting of nitric acid, nitrous acid, hypo- and hyperhalous acids and mixtures thereof,

[0022] • at least one ionic agent selected from the group consisting of l-butyl-3-methyl-imidazolium (BMIM) chloride, l-butyl-3-methyl-imidazolium (BMIM) bromide, l-butyl-3-methyl-imidazolium (BMIM) tetrafluoroborate, amide chloride and mixtures thereof,

[0023] A preferred embodiment of the process according to the invention provides that during the extraction in step a) at least one of the following steps is carried out:

[0024] • mechanical comminution, in particular by grinding via ultrasound, preferably in the frequency range of 20 to 50 Hertz, with a ball mill and / or with a cutting mill, preferably Ultra-Turrax, wherein the mechanical comminution is carried out according to sieve analysis to a particle size (d50) of 0.1 to 5.0 mm, preferably 0.2 to 1.0 mm,

[0025] • thermal denaturation, particularly at temperatures of 70°C to 150°C, and / or electrochemical denaturation,

[0026] • a precipitation of the extraction solution from step a), in particular triggered by a pH change, addition of a co-solvent and / or a salt, a microbial and enzymatic extraction via gram-negative bacteria selected from the group consisting of Stenotrophomonas sp., Chrysebacterium sp., Vibrio sp. and mixtures thereof,

[0027] • Gram-positive bacteria selected from the group consisting of Bacillus sp., Kocuria rosea and mixtures thereof

[0028] • saprophytic and / or parasitic fungi and / or

[0029] • Mixtures thereof,

[0030] • Treatment with microwave radiation, especially microwave radiation up to 960 watts and 2450 Hertz,

[0031] • Use of electric explosion and / or supercritical water and / or.

[0032] • Combinations thereof.

[0033] It is preferable for the protein beta-keratin to be present in its secondary structure as a ß-sheet. In contrast to alpha- (= soft fiber) keratin, beta- (= hard fiber) keratin exhibits a high proportion of tightly twisted ß-sheet structures stabilized by disulfide bridges, which ensure the high stability of the hard fiber keratin.

[0034] Filtration is preferably carried out via dialysis and / or ultrafiltration (cross-flow filtration).

[0035] The feathers of animal origin are preferably selected from the group consisting of feathers of chickens, geese, turkeys, ducks, turkeys, pheasants, ostriches, rheas, emus, quails and mixtures thereof.

[0036] The invention also provides a colloidal solution for topical application containing keratin particles of the protein beta-keratin and / or their agglomerates. It is preferred that the keratin particles in colloidal solution have a particle size in the range of 5 nm to 500 nm, preferably 70 nm to 350 nm, measured by dynamic light scattering (according to DIN ISO 22412:2018-09) (Zetasizer ZEN3600 device, Malvern Panalytical Instruments).

[0037] It is preferred that for stabilization the colloidal solution contains additives which are preferably selected from the group consisting of

[0038] • Proteins e.g. albumins

[0039] • Carbohydrates such as sucrose, lactose, glucose, fructose, mannitol, sorbitol and sweeteners such as saccharin sodium, sodium cyclamate, aspartame, starch and modified starch, cyclodextrins and / or mixtures thereof,

[0040] • Polyanionic surfactants such as sodium dodecyl sulfate, sodium cetylstearyl sulfate, cetylstearyl alcohol (emulsifying), sodium dioctylsulfosuccinate and / or mixtures thereof,

[0041] • 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 monofatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene fatty alcohol ethers, glycerol fatty acid esters, sucrose fatty acid esters, poloxamers and / or mixtures thereof,

[0042] • Gelling agents such as polyacrylates, cellulose derivatives such as methylcellulose, methylhydroxypropylcellulose, hydroxypropylcellulose, hydroxyethylcellulose and / or ethylcellulose, carmellose sodium and / or mixtures thereof,

[0043] • Thickeners such as tragacanth, xantham, gum arabic, guar galactomannan, alginates, bentonite, and / or mixtures thereof,

[0044] • Film formers such as methacrylic acid acrylates, polyvidone, polyvinyl alcohol and / or mixtures thereof,

[0045] • Polymers such as macrogols, gelatin, and / or mixtures thereof. The invention also provides keratin powders of the beta-keratin protein for topical application, which can be prepared from the colloidal solution described above. The keratin powder is produced by drying the colloidal solution, in particular by freeze-drying, spray-drying, vacuum-drying, air-drying, heat-drying, infrared-drying, and / or microwave-drying.

[0046] It is preferred that the keratin particles in the keratin powder have a particle size in the range of 1 to 250 pm, preferably 5 to 30 pm, measured by dynamic light scattering (according to DIN ISO 22412:2018-09) (Zetasizer ZEN3600, Malvern Panalytical Instruments).

[0047] The colloidal solution described above and the keratin powder described above are used to produce preparations with therapeutic, diagnostic, preventive or cosmetic purposes in living beings, in particular in humans and / or animals.

[0048] A preferred embodiment provides that the colloidal solution contains at least one small-molecule and / or biotechnological active ingredient. The active ingredient is preferably selected from the group consisting of glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics, and / or a protein, fraction protein, peptide, enzyme, antibody, antibody fragment, RNA and / or DNA molecule, and mixtures thereof. Another preferred embodiment provides that a molecule is included that represents a target for one of these active ingredients.

[0049] It is further preferred that the colloidal solution contains cosmetic active ingredients and auxiliaries, preferably selected from the group consisting of amino acids, urea, glycerol, hyaluronic acid, sugars and sugar-like derivatives, extracts or waxes from plants or parts derived from animals and mixtures thereof.

[0050] A preferred embodiment provides that specific and / or non-specific compounds with active ingredients and / or excipients are formed by covalent and / or non-covalent bonds, preferably via selected groups consisting of carboxylic acid groups, amino groups, thiol groups and / or hydroxyl groups.

[0051] To stabilize the colloidal solution with / without active ingredients and / or excipients, it is preferred that these be incorporated as liposomal systems into uni- or multilamellar vesicles of varying or uniform size by adding amphiphilic molecules. These amphiphilic molecules are preferably selected from the group consisting of:

[0052] • Phospholipids, preferably lecithin, DODAB, DPPC, DSPC, DSTAP and / or mixtures thereof,

[0053] • cationic lipids, preferably ALC-0315,

[0054] • PEGylated lipids, preferably ALC-0159

[0055] • Prostaglandins and modifications, preferably PGE1, PGD2, PGE2, 15-keto PGE1 and / or mixtures thereof,

[0056] • Ceramides, preferably selected from the group consisting of ceramides of the head groups NS, NH, NP, NDS, AS, AH, ADS, AP of chain lengths C10 to C26 and / or mixtures thereof,

[0057] • Ceramides, preferably selected from the group consisting of ceramides of the head groups EOS, EOH, EOP of chain lengths C10 to C32 and / or mixtures thereof,

[0058] • Cholesterol, cholesterol derivatives and / or mixtures thereof,

[0059] • Fatty acids, preferably selected from the group consisting of fatty acids with chain lengths C10 to C32 and / or mixtures thereof.

[0060] It is preferred that the application to living beings, in particular to humans, concerns the skin, mucous membranes (including conjunctiva) or the skin appendages (including nails and hair), in particular for the substitution of the barrier function or components of the epidermal barrier.

[0061] The colloidal solutions and keratin powders according to the invention are also used for the production of

[0062] • 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,

[0063] • solid bases, in particular powders, tablets, granules, pellets, capsules and / or inserts.

[0064] The colloidal solutions and keratin powders according to the invention are also used for the production of

[0065] • Food and food supplements,

[0066] • Animal feed,

[0067] • Fertilizers and / or plant protection products for plants and / or soil,

[0068] • technical auxiliary materials, e.g. in / as filter systems, adhesives and / or bonding agents, consistency regulators, fillers, absorbers for hydrophilic or lipophilic substances, charged or uncharged substances,

[0069] • Packaging materials and / or consumer goods,

[0070] • Textiles and / or fibers with / without function, especially water-repellent, breathable,

[0071] • Medical devices, in particular plasters, wound dressings, tampons, wound and / or skin care clothing.

[0072] The subject matter of the invention will be explained in more detail with reference to the following figures and the example, without wishing to restrict it to the specific embodiments shown here.

[0073] Fig.lA and 1B show SEM images with different resolutions of lyophilized hair keratin

[0074] Fig.2 shows TEM images of A) feather keratin (0.5 mg / ml) and B) hair keratin (0.5 mg / ml)

[0075] Fig. 3 shows fluorescence spectroscopic images of 5% (w / w) keratin particles in a DAC base cream (with A) feather keratin, B) and C) hair keratin).

[0076] Fig. 4 shows images of penetration tests of A) feather keratin and B) hair keratin with 5% (w / v) each in base cream DAC

[0077] Fig. 5 shows a picture of penetration study of feather keratin 5% (w / v) in base cream DAC with a fluorescence image

[0078] Fig. 6 shows cytotoxicity data as an index of lyophilized and colloidal feather keratin on keratinocytes (NHEK) and lyophilized and colloidal feather keratin on dermal fibroblasts (NHDF)

[0079] Fig. 7 shows the result of a scratch test showing the epithelialized area of ​​3 independent tests on lyophilized feather keratin on keratinocytes (HaCaT)

[0080] Fig. 8 shows the result of a scratch test showing the epithelialized area of ​​3 independent tests on colloidal feather keratin on keratinocytes (HaCaT)

[0081] Fig. 9 shows the result of a scratch test showing the epithelialized area of ​​3 independent tests on lyophilized feather keratin on keratinocytes (NHDF)

[0082] Fig. 10 shows the result of a scratch test showing the epithelialized area of ​​3 independent tests on colloidal feather keratin on keratinocytes (NHDF)

[0083] Fig. 11 shows the water uptake of feather keratin after addition of 25 μl T2O (1 μCi) to 5 mg lyophilized keratin, n=3 (contact times: 1 min, 5 min, 1 h, 16 h, 24 h), centrifugation, removal of the supernatant, determination of the radioactive dose

[0084] Fig. 12 shows the cultivation of HaCaT cells with 1mg feather keratin. Fig. 13 shows the water uptake of urea, glycerol and keratin as well as

[0085] Mixtures of keratin particles with glycerol (50:50) and 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 95% relative humidity) over time

[0086] Fig.14 shows the water release of urea, glycerol and keratin as well as mixtures of keratin particles with glycerol (50:50) and keratin particles with urea in different ratios (e.g. 95:5 (orange); 50:50 (light blue); 5:95 (brown)) in the climate chamber (room temperature and 50% relative humidity) over time

[0087] Fig. 15 shows the tertiary structure of beta-keratin from bird feathers

[0088] Fig. 16 shows the corneometry measurement data in arbitrary units (AU) before and after 1, 2, 4, 8 and 24 hours for a cosmetic cream formulation with 0.1%, 0.5%, 1.0% and 2.0% keratin parti no

[0089] Fig. 17 shows the evaporimetry measurement data in g / m 2 / h before and after 1, 2, 4, 8 and 24 hours for a cosmetic cream formulation with 0.1%, 0.5%, 1.0% and 2.0% keratin particles

[0090] Various animal materials are available as keratin sources, but they appear to be of varying suitability due to their degree of protein cross-linking. Preliminary studies with various keratin-containing biomaterials revealed that bird feathers are particularly suitable. In addition to practical aspects of availability and ease of processing, biochemical aspects were also investigated that justify the preferential use of feathers. The keratin contained in feathers is predominantly ß-keratin, which, as a polypeptide chain, has a ß-fold sheet structure, consists of filaments of 3-4 nm, and has a molecular mass of approximately 10-22 kDa. In contrast to α-keratin in mammals, β-keratin has few, but functionally significant, differences in its primary sequence. For example, β-keratin forms fewer macrofibrils than α-keratin and displays a more regular ordering and packing behavior.These differences thus provide more favorable conditions for standardizing keratin isolation and the resulting product properties. In particular, the frequently practiced hydrolysis of keratin must be avoided in order to isolate intact keratin proteins and specifically prevent the formation of keratin fragments, amino acids, and peptides. For effective and standardized keratin isolation, a special process was developed based on the biochemical characteristics of ß-keratin. The chicken feathers used were first cleaned with soap and water, disinfected with 70% ethanol, and then dried at room temperature. After comminuting the cleaned and dried feathers in a cutting mill (Retsch SM 100 comfort), the entire material was homogenized. The feather homogenate was defatted using a Soxhlet apparatus.For keratin extraction, the defatted feather material was added to an extraction buffer and extracted for 48 hours. The extract was then centrifuged, and the sediment was discarded. The resulting pure extract was diluted with water and filtered with a cutoff of 10,000 NMWC. The dialysate was spray-dried to a powder with a particle size of <25 pm.

[0091] To determine the colloidal size of the keratin particles (1 mg / ml deionized water), dynamic light scattering (DLS) measurements were performed using the Zetasizer ZEN3600 from Malvern Panalytical Instruments in accordance with DIN ISO 22412:2018-09. This analytical method enables the characterization of particle sizes in suspensions and emulsions by determining the emeritus scattered light of a laser. The goal was to determine the particle size of the protein in the colloidal state. For the DLS measurement, 1 ml of a 0.1 mM colloidal keratin solution was pipetted into a disposable polystyrene cuvette and then transferred to the cuvette module of the Zetasizer Lab, which was maintained at 25 °C. The particle size was determined using the automatic analysis mode (general purpose) and backward light scattering at an angle of 173°. Each measurement was performed in triplicate and comprised 15 measurement cycles per run.The measured data per sample were then averaged. A total of three samples were analyzed using DLS. During DLS, the polydispersity index (Pdl), which indicates how homogeneously the particles are distributed within the sample, the particle size (in nm), and the percentage of each particle size in relation to the total sample content were determined. 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 chamber (Mini Gel Tank from Invitrogen). The samples were denatured according to the manufacturer's protocol NuPAGE® Bis-Tris Mini Gel Electrophoresis (Thermo Fisher). For this purpose, 2 μl of sample, 2.5 μl of NuPAGE® LSD Sample Buffer (4x), 1 μl of NuPAGE® LSD Reducing Agent (10x) and 6.5 μl of DI water were combined and incubated for 10 min at 95 °C.To perform gel electrophoresis, the gel chamber was filled with 1x Tris-Tricine running buffer (1.2 M Tris, 0.8 M Tricine, 2% SDS). The Tris-Tricine gel (Novex 10-20% Tricine Gels from Invitrogen (Thermo Fisher Scientific), LOT 20101945, REF EC6625BOX) was then loaded, and the comb was removed from the gel. 10 μl of each prepared sample and 2 μl of each marker were added to the corresponding gel wells. Gel electrophoresis was performed at 130 V and 250 mA for 1.5 hours. The gel was then removed from the chamber, and the running buffer was discarded. The plastic gel holder was broken open, and the Tris-Tricine gel was transferred into a container containing Coomassie Brilliant Blue G250 stain (2 ml 5% Coomassie solution, 3 ml orthophosphate acid, 20 ml ethanol, 10 g ammonium sulfate, 65 ml dH2O). The gel was incubated in the solution overnight on a shaker. The staining solution was then removed.To decolorize, the gel was rinsed several times with distilled water while shaking gently.

[0092] Example

[0093] An attempt was made to replicate the functionality of a corneocyte. For this purpose, keratin was extracted from various natural keratin sources and corresponding particles were generated. These particles were initially investigated as mechanical stabilizers of lipid matrices in various semi-solid preparations. The intention was to coat the keratin particles with bipolar lipids to develop keratosomes.

[0094] Initially, various extraction methods were tested on human hair (α-I-pha-keratin). Chemical denaturation was predominantly used to decompose the hair. Urea, thiourea, and guanidinium hydrochloride were employed for this purpose. β-Mercaptoethanol, cysteamine, and L-cysteine ​​were used as reducing agents to assist denaturation. Highest yields were achieved with an extraction solution consisting of 5 M guanidinium hydrochloride, 10% cysteamine, and 25 mM Tris at pH 8.5. Due to the harmlessness of the substances urea and L-cysteine, an extraction solution consisting of 10 M urea, 100 mM L-cysteine, and 25 mM Tris-HCl at pH 10.5 was selected for keratin isolation. After successful extraction, the extraction solution was dialyzed (cutoff: 6-8 kDa, regenerated cellulose, SpectraPor®), resulting in a colloidal solution. At high protein concentrations and large dialysis steps (5-6 l), keratin precipitates in the dialysis tubing.Finally, the dialysate was lyophilized, producing a white powder. This powder was examined by scanning electron microscopy (SEM) (Fig. 1). The hair keratin particles vary in size and shape. It was determined that the particles in the final formulation should have a size of >600 nm to avoid penetration through the stratum corneum of the skin.

[0095] For regulatory reasons, feather keratin (beta-keratin) from various bird species was used as an alternative keratin source. Raw feathers from chickens, geese, and ducks were examined. For practical reasons, chicken feathers were used in particular for further investigations.

[0096] Therefore, the extraction of feather keratin was adapted to the established extraction process for hair keratin. It was observed that, compared to all other extraction methods investigated, the highest yield was achieved with the selected extraction solution consisting of 10 M urea, 100 mM L-cysteine, and 25 mM Tris-HCl at pH 10.5. The optional addition of 1 M ammonium chloride prevents carbamylation of the protein and the associated changes in its properties.

[0097] To determine the size, the feather and hair keratin particles were examined comparatively using negative stain transmission electron microscopy (TEM) (Fig. 2). Hair keratin particles in colloidal solution had a size of approximately 40–75 nm, whereas the feather keratin particles had a size of approximately 20–35 nm. To visualize the keratin particles for the penetration test, they were fluorescently labeled with 2-aminobenzoyl (Abz) (reaction with isatoic anhydride under basic, denaturing conditions).

[0098] 5% (w / w) fluorescently labeled hair and feather keratin was formulated into Base Cream DAC and, after smearing from a slide, examined by fluorescence microscopy (Fig. 3). The individual particles from the colloidal solutions, which were identified by TEM, proportionally form aggregates in Base Cream DAC. For feather keratin, a homogeneous distribution of the aggregated particles is evident, with particle sizes ranging between 20 and 35 pm. Hair keratin, on the other hand, aggregates into larger, crystalline particles. The particle sizes here range between 20 and 140 pm.

[0099] Penetration studies of hair and feather keratin (5% w / w each) on ex vivo human skin (Franzzelle) in DAC base cream also demonstrated that the keratin particles do not diffuse into the deeper skin layers, but rather remain in the upper parts of the SC (stratum disjunctum) (Fig. 4 and Fig. 5). To better assess the interaction of the feather keratin particles with the SC lipids and the effects of lipid coatings, the zeta potential was determined at various pH values ​​(Table 1).

[0100] Table 1: Zeta potential of feather keratin and fluorescently labeled feather keratin, each at 10 mg / ml in 10 mM potassium chloride at different pH values. Measurement performed by Fabio.

[0101] The keratin particles have a negative charge, which makes them ideal for coating with positively charged lipids.

[0102] In cytotoxicity studies, the influence of colloidal and lyophilized keratin particles on the vitality and proliferative activity of keratinocytes and dermal fibroblasts was investigated using standard protocols. No relevant effect on dermal fibroblasts was found up to 48 hours of incubation (Fig. 6). In keratinocytes, only colloidal keratin showed a slight, concentration-dependent reduction in proliferative activity.

[0103] Using a scratch assay, the influence of keratin particles in colloidal and lyophilized form on the migration behavior of keratinocytes (HaCaT) and dermal fibroblasts (NHDF) was investigated (Figs. 7 to 10). This revealed an apparent mechanically induced inhibition of migration, dependent on the concentration of the colloidal and, to a significantly lesser extent, the lyophilized keratin particles. However, no evidence of toxic effects was found.

[0104] To objectively determine the loading capacity of the feather keratin particles for hydrophilic and hydrophobic substances, loading and unloading experiments were conducted. First, the keratin particles were incubated in T2O solution for different lengths of time, and then the amount of absorbed tritium was measured by centrifugation (Fig. 11). Only 25–32% of the applied T2O could be recovered in the pellet. This revealed an unexpectedly high interaction between both hydrophilic substances and the extracted feather keratin.

[0105] To investigate possible interactions of the keratin particles with vital cells, HaCaT cells (keratinocytes) were cultured with fluorescently labeled feather and hair keratin particles. The medium was changed 48 hours after keratin treatment. The keratin particles are clearly visible under fluorescence microscopy and exhibit an association near the cell nucleus, so that uptake of the particles into the cell could not be ruled out using fluorescence microscopy (Fig. 12). Therefore, the adhesion zone of keratin particles with the cell membrane was examined using Raman spectroscopy, but no evidence of intracytoplasmic keratin particles was found. Therefore, based on the zeta potential and the charge of the phospholipids of the cell membrane, adherence of the keratin particles to cell membranes can be assumed.Additionally, hygroscopic tests were conducted to determine the water absorption capacity of the keratin particles, based on the function of corneocytes. These showed a maximum water absorption of approximately 20% for the keratin particles after just 3 days. With the addition of urea up to a ratio of 5% keratin to 95% urea, the maximum was reached increasingly later, in the maximum case only after 15 days. In this case, the water absorption capacity was 197% (Fig. 13). Water loss was determined at room temperature and a humidity of 50%. Pure keratin released some water slowly and still contained water even after 7 days, whereas for urea, a rapid water loss of approximately 170% was observed within the first 24 hours, up to complete water loss after 4 days. For the mixtures of keratin and urea in the ratios 25:75 and 10:90 and 5:90, a water content of 25% was observed even after 4 days (Fig. 14).

[0106] According to the known keratin sequence and the amino acid classification according to Dan Cojocari, keratin contains only approximately 30% hydrophobic and approximately the same number hydrophilic amino acids, but no binding pockets. Therefore, the demonstrated interaction of keratin with substances that possess different physicochemical properties occurs unpredictably. Using the artificial intelligence (Kl) AlphaFold 2 (EMBL's European Bioinformatics Institute (EMBL-EBI), Hinxton, UK), the tertiary structure of keratin was calculated and the hydrophobic and hydrophilic regions in the molecule were identified (Fig. 15). Here, too, the assumption of the absence of binding pockets was confirmed, so that the observed interactions can be explained solely on the basis of hydrophobic or hydrophilic interactions between amino acid clusters on the protein side with relevant groups of other molecules.

[0107] To demonstrate clinical efficacy, a cosmetic cream formulation containing keratin particles in increasing concentrations (0.1%, 0.5%, 1.0%, and 2.0%) was investigated in a prospective, randomized, and double-blind study regarding its hydrating effect on the stratum corneum of healthy volunteers. For this purpose, the total water content in the stratum corneum (corneometry) and the free water phase as transcorneal water loss (TEWL) were determined using evaporimetry after a single application (single application test) before (baseline) and after 1, 2, 4, 8, and 24 hours. After 24 hours, the water content of the stratum corneum was approximately 30% higher (Fig. 16) and the transcorneal water loss was approximately 40% lower (Fig. 17).These results demonstrate that the extracted keratin particles, as a colloidal aqueous phase in a cream formulation, mediate a sustained hydrating effect of the stratum corneum, in accordance with the physiological corneocyte function.

Claims

Patent claims 1. A process for producing a keratin powder from feathers of animal origin, in which a) the protein beta-keratin, in particular beta-keratin in its secondary structure as a ß-fold sheet, is extracted in an extraction solution which triggers chemical denaturation and contains at least one denaturing agent, at least one base, at least one reducing agent and at least one buffer substance, b) the extraction solution from step a) is subjected to filtration, in which a colloidal solution of keratin parts of the protein beta-keratin is obtained, c) the colloidal solution of keratin parts from step b) is dried by freeze-drying, spray-drying, vacuum-drying, air-drying, heat-drying, infrared-drying and / or microwave-drying to obtain a keratin powder.

2. Process according to claim 1, characterized in that the extraction solution has a pH value of 8 to 13, preferably of 9 to 12, particularly preferably of 10 to 11.

3. The method according to claim 1 or 2, characterized in that 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 ß-mercaptoethanol, cysteamines, cysteines, glutathione, sodium disulfite, sodium sulfide, sodium hydrogen sulfite, sodium dithionite, sodium thiosulfate, dithiotreitol (DTT), thioglycolic acid and its salts, thiourea, tricarboxyethylphosphane (TCEP) and other phosphanes, ammonium chloride and mixtures thereof and / or the at least one buffer substance, preferably selected from the group consisting of tris(hydroxymethyl)aminomethane, sodium dodecyl sulfate (SDS), Tris / hydrochloric acid (HCl), ethylenediaminetetraacetic acid (EDTA) / Tris, potassium chloride-sodium hydroxide (KCl-NaOH), sodium hydrogen carbonate (NaHCl), dithiotreitol (DTT) / Tris and mixtures thereof.

4. The method according to any one of claims 1 to 3, characterized in that the extraction solution contains at least one oxidizing agent, preferably selected from the group consisting of hydrogen peroxide, potassium permanganate, sodium perborate, peroxyacetic acid, performic acid and mixtures thereof and / or the extraction solution contains at least one acid, preferably selected from the group consisting of nitric acid, nitrous acid, hypo- and hyperhalous acids and mixtures thereof and / or the extraction solution contains at least one ionic agent, preferably selected from the group consisting of l-butyl-3-methylimidazolium (BMIM) chloride, l-butyl-3-methylimidazolium (BMIM) bromide, l-butyl-3-methylimidazolium (BMIM) tetrafluoroborate, amide chloride and mixtures thereof.

5. The method according to any one of claims 1 to 4, characterized in that during the extraction in step a) at least one of the following steps is carried out: a) mechanical comminution, in particular by grinding via ultrasound, preferably in the frequency range of 20 to 50 Hertz, with a ball mill and / or with a cutting mill, wherein the mechanical comminution is carried out to a particle size of 0.1 to 5.0 mm, preferably 0.2 to 1.0 mm, b) thermal denaturation, in particular at temperatures of 70°C to 150°C, and / or electrochemical denaturation, c) precipitation of the extraction solution from step a), in particular triggered by a pH change, addition of a co-solvent and / or a salt, d) microbial and enzymatic extraction via • Gram-negative bacteria selected from the group consisting of Stenotrophomonas sp., Chrysebacterium sp., Vibrio sp. and mixtures thereof, • Gram-positive bacteria selected from the group consisting of Bacillus sp., Kocuria rosea and mixtures thereof • saprophytic and / or parasitic fungi and / or • mixtures thereof, e) treatment with microwave radiation, in particular microwave radiation up to 960 watts and 2450 hertz, f) use of electric explosion and / or supercritical water and / or. g) combinations thereof.

6. Method according to one of the preceding claims, characterized in that the filtration is carried out by dialysis and / or ultrafiltration (cross-flow filtration).

7. Colloidal solution for topical application containing keratin particles of the protein beta-keratin, in particular beta-keratin in its secondary structure as ß-fat sheet, and / or their agglomerates from feathers of animal origin.

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

9. Colloidal solution according to one of claims 7 or 8, characterized in that the feathers of animal origin are selected from the group consisting of feathers of chickens, geese, turkeys, ducks, turkeys, pheasants, ostriches, rheas, emus, quails and mixtures thereof.

10. Colloidal solution according to one of claims 7 to 9, characterized in that additives are included to stabilize the colloidal solution, preferably selected from the group consisting of • Proteins, especially albumins • Carbohydrates, in particular sucrose, lactose, glucose, fructose, mannitol, sorbitol and sweeteners such as saccharin sodium, sodium cyclamate, aspartame, starch and modified starch, cyclodextrins and / or mixtures thereof, • Polyanionic surfactants, in particular sodium dodecyl sulfate, sodium cetylstearyl sulfate, cetylstearyl alcohol (emulsifying), sodium dioctylsulfosuccinate and / or mixtures thereof, • Non-ionic surfactants, in particular fatty alcohols and sterols, sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid glycerides, macrogol 1000 glycerol monofatty 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, • Thickeners, in particular tragacanth, xantham, gum arabic, guar galactomannan, alginates, bentonite, and / or mixtures thereof, • Film formers, in particular methacrylic acid acrylates, polyvidone, polyvinyl alcohol and / or mixtures thereof, • Polymers, in particular macrogols, gelatin and / or mixtures thereof.

11. Keratin powder for topical application, which can be prepared from the colloidal solution according to one of claims 7 to 10, in particular by the process according to one of claims 1 to 6.

12. Keratin powder according to claim 11, characterized in that the keratin particles of the keratin powder have a particle size in the range of 1 to 250 pm, preferably 5 to 30 pm, measured by means of dynamic light scattering.

13. Use of a colloidal solution according to any one of claims 7 to 10 or of the keratin powder according to claim 11 or 12 for the production of preparations with therapeutic, diagnostic, preventive or cosmetic application to living beings, in particular to humans and / or animals, preferably for the skin, mucous membranes (including conjunctiva) or skin appendages (including nails and hair), in particular for substituting the barrier function or components of the epidermal barrier.

14. Use according to claim 13, characterized in that the colloidal solution contains at least one small molecular and / or biotechnological active ingredient, wherein the active ingredient is preferably selected from the group consisting of glucocorticoids, calcineurin inhibitors, Janus kinase inhibitors, antibiotics, and / or itself a protein, fraction protein, peptide, enzyme, antibody, antibody fragment, RNA and / or DNA molecule, and mixtures thereof or a molecule that represents a target for one of these active ingredients and / or that the colloidal solution contains cosmetic active ingredients and auxiliaries, preferably selected from the group consisting of amino acids, urea, glycerol, hyaluronic acid, sugars and sugar-like derivatives, extracts or waxes from plants or parts derived from animals and mixtures thereof.

15. Use according to claim 13 or 14 for the production of • 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 bases, in particular powders, tablets, granules, pellets, capsules and / or inserts.

16. Use according to any one of claims 13 to 15 for the production of • Food and food supplements, • Animal feed, • Fertilizers and / or plant protection products for plants and / or floors, • technical auxiliary materials, e.g. in / as filter systems, adhesives and / or adhesion promoters, consistency regulators, fillers, absorbers for hydrophilic or lipophilic substances, charged or uncharged substances, • packaging materials and / or consumer goods, • Textiles and / or fibers with / without function, especially water-repellent, breathable, • Medical devices, in particular plasters, wound dressings, tampons, wound and / or skin care clothing.

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