DE102011105369A1 - Stable, aqueous protein solution or protein dispersion, process for the preparation of the stable, aqueous solution or dispersion and process for the production of moldings, fabrics, impregnations or coatings from the stable, aqueous protein solution or protein dispersion and their use - Google Patents

Abstract

The aim is to provide a health-friendly and / or a particularly environmentally friendly and particularly stable and highly concentrated solution or dispersion of at least one self-assembling protein, which can be used for efficient or improved production of pure protein shapes or flat structures, protein impregnations or coatings enables a wide range of applications. According to the invention, the stable protein solution or protein dispersion in an aqueous solution comprises at least one self-assembling protein and at least one stabilizing agent for the self-assembling protein, the stabilizing agent comprising at least one amino acid.

Description

The present invention relates to a stable, aqueous solution or a stable, aqueous dispersion with at least one self-assembling protein. Furthermore, the invention relates to a process for the preparation of the stable, aqueous protein solution or protein dispersion, a process for the electro- or rotor spinning of self-assembling protein using the stable, aqueous protein solution or protein dispersion, process for the preparation of mold - or fabrics, impregnations or coatings of the stable, aqueous protein solution or protein dispersion and the use of the stable, aqueous protein solution or protein dispersion or the moldings or fabrics, impregnations or coatings produced therefrom.

Form or fabric are here understood in particular fibers, fiber structures, gels, films, films, foams or combinations thereof.

Under a fiber structure is here in particular an orderly or disorderly single or multi-layered assembly of a plurality of fibers understood or three-dimensional arrangements of the fibers or a combination with carriers. Preferred fiber structures are nonwovens here.

The self-assembling proteins are composed of polypeptides. These are composed of amino acids, in particular of the 20 naturally occurring amino acids together. The amino acids can be modified, for example acetylated. The self-assembling proteins are in particular intrinsically unfolded proteins or microbead-forming proteins. Exemplary proteins are silk-like proteins, silk proteins or spider silk proteins. The proteins may be of natural origin or synthetic, for example by recombinant production.

The self-assembling proteins have the property that they are unstable in concentrated, aqueous solutions or dispersions. Due to the hydrophobic parts, the protein precipitates or the solution gels. As a result, the proteins can not normally be processed without further additives, such as, for example, polymers or spinning auxiliaries, from aqueous solution or dispersion into fiber structures.

State of the art

From the patent US 6110590 the electrospinning of synthetically produced silk from hexafluoroisopropanol is known.

In the JP 2010 270426 A describes a method in which silk proteins of concentrated formic acid, hexafluoroacetone hydrate or hexafluoroisopropanol are spun. The electrospinning is carried out at elevated temperature to prevent gelling of the spinning solution. However, this may result in prolonged, continuous processing of damage to the protein.

The use of organic solvents is also often undesirable from aspects of occupational safety and / or environmental protection.

The JP 2010-150712 describes the electrospinning of an aqueous silkworm silk protein solution with the addition of a nonionic surfactant and a water-soluble polymer. The addition of nonionic surfactant, however, may be of concern to health in medical applications.

Presentation of the invention

The object of the present invention has been found to provide or produce a health-safe and / or a particularly environmentally friendly and particularly stable and highly concentrated solution or dispersion of at least one self-assembling protein, the efficient or improved production of pure protein moldings or fabrics , Protein impregnations or coatings with a broad field of application.

The concentrated protein solution or dispersion should therefore be distinguished by a particularly high concentration and stability.

In particular, the production of protein fibers or fiber structures, in particular nonwovens, is to be made possible or improved by means of electric or rotor spinning processes. In addition, protein foams, gels, films, films or protein impregnations or coatings should be prepared from concentrated, aqueous protein solution or dispersion.

Furthermore, the stable protein solution or dispersion or the moldings or coatings produced therefrom, coatings or impregnations should be particularly suitable for the medical field, preferably for or as Wound dressings, surgical sutures or as a biocompatible coating of surfaces, in particular fibers, fiber structures, such as yarns or nonwovens, tissue engineering scaffolds, medical textiles, tubes, implants or foams, as hypoallergenic surfaces, for cosmetic products, for hygiene products, for Household products and / or for filter applications, such as tobacco and engine filters.

According to the invention, the stable protein solution or protein dispersion in an aqueous solution or dispersion comprises at least one self-assembling protein and at least one stabilizing agent for the self-assembling protein, wherein the stabilizing agent comprises at least one amino acid, preferably one or more monomeric amino acid (s).

The water base is a particularly environmentally friendly solution.

Highly hydrophobic proteins have a very poor solubility in aqueous solution. Without stabilization, only dilute, aqueous protein solutions can be prepared. Due to the low concentration, these solutions or dispersions, for example, can not be processed into fiber structures without further additives, such as, for example, polymers or spinning auxiliaries. In addition, larger amounts of additives are needed in these dilute solutions.

On the other hand, pure, aqueous protein solutions or dispersions can be prepared in very high concentration and with high stability by the invention.

The stable protein solution or protein dispersion according to the invention is understood to mean a stability of at least 5 hours, preferably more than 12 hours, more preferably more than 20 hours or even more than 48 hours.

These can be processed by means of spinning processes into fibers or fiber structures, for example into nonwovens, in particular by means of electric or rotor spinning.

The stabilizing agent is preferably present in a concentration range from 0.01% by weight to 10% by weight, preferably in a concentration range from 0.01% by weight to 5% by weight, very particularly preferably in a concentration of 0 , 01 wt .-% to 4 wt .-%.

By selecting an amino acid as a stabilizing agent, a particularly high stability at high protein concentration is achieved. Amino acids are toxicologically harmless. This is particularly important for the application in the above-mentioned medical field and their use for cosmetics, hygiene and / or household products.

The self-assembling protein of the stable, aqueous protein solution or protein dispersion may be of natural origin or synthetic, in particular by recombinant production constructed from repetitive units of a structural protein, in particular an insect protein, preferably resilin, and / or a spider silk protein or the like or thereof derived protein, in particular with insertion or attachment of oligoamino acid blocks and / or an intrinsically unfolded protein or a microbead-forming protein.

The protein can thus also be a combination of the proteins derived from natural structural protein sequences, in particular a combination of the sequence of one protein with the sequence of another structural protein, particularly preferably a combination of the sequence of a spider silk with the sequence of resilin.

The self-assembling protein of the stable, aqueous protein solution or protein dispersion is preferably an R16, R16Arg ₈ (R16 protein with attached 8 amino acid oligoarginine block), S16 or C16 protein or the natural silk protein of the silkworm Bombyx mori or a natural or synthetic protein derived from the aforementioned proteins.

For example, the self-assembling proteins designated R16 and S16 and their preparation are described in detail in US Pat WO 2008/155304 described. For example, the protein and its production designated C16 are described in detail in U.S. Pat WO 2007/082936 described.

By the amino acid stabilizing agent particularly highly concentrated protein solutions can be achieved. The self-assembling protein is preferably present in a concentration of from 4% by weight to 35% by weight, more preferably in a concentration of from 5% by weight to 30% by weight, most preferably in a concentration of 10% by weight. -% to 25 wt .-% based on the total weight of the stable solution before.

The amino acid stabilizer for the self-assembling protein is advantageously selected from arginine, lysine, histidine, phenylalanine, asparagine, glutamine, tryptophan, leucine, glutamic acid and / or aspartic acid. special Arginine, lysine, histidine, phenylalanine or glutamine are preferably used.

In particular, arginine also promotes wound healing and thus represents an ideal stabilizer, for example for the subsequent use of the proteins in wound dressings.

By the sole use of amino acid as a stabilizing agent, protein fibers can be spun from the stable protein solution or dispersion, without the need for additional use of, for example, polymers as a spinning auxiliary. As a result, fibers with a particularly high protein content or even pure protein fibers can be achieved.

Of course, however, additives may also be added to the protein solution or dispersion as an option.

• – Mitteln zur Einstellung der Viskosität, insbesondere wasserunlösliche Polymere, zum Beispiel in Form von Dispersionen, oder bevorzugt wasserlösliche Polymere, wie Polyalkylenoxide, wie Polyethylenoxide, Polyethylenglykole oder Polypropylenglykole, Polyvinylalkohole, Polyethylenimine, Polyvinylpyrrolidone, Polyvinylamine, Polyvinylacetate, Polyacrylate, Polyalkylacrylate, wie Polymethacrylate, Polyhydroxyalkylacrylate, wie Poly(2-hydroxymethacrylate) oder Poly(2-hydroxyethacrylate), Polyvinyformamide, Polyacrylamide, Polycarbonsäuren, wie Polyacrylsäuren oder Polymethacrylsäuren, natürliche Polymere, wie Galaktomannan, Chitosan, Kollagen, Albumin, Xanthan, Alginate, Gelatine, Cellulose oder Stärke, insbesondere deren wasserlösliche Derivate, wie zum Beispiel Hydroxypropylcellulose, Carboxymethylstärke, oxidierte Stärke, Carboxymethylcellulose,
• – Träger-bildenden Polymeren, bevorzugt Kombinationen aus zwei oder mehreren Polymeren, Homo- und Copolymeren,
• – Verarbeitungshilfsmitteln, wie Spinnhilfsmitteln, bevorzugt organische Lösungsmittel, wie Alkohole, zum Beispiel Ethanol oder Butanol, und/oder oberflächenaktive Agentien, wie Tenside zur Einstellung der Oberflächenspannung bzw. des Dampfdruckes der Lösung oder Leitsalze zur Einstellung der Leitfähigkeit,
• – Vernetzungsmitteln, beispielsweise als Zugabe zur Spinnlösung oder zur nachträglichen Vernetzung von Fasern,
• – pharmakologischen Wirkstoffen oder Medikamenten, wie Antibiotika, Antiinfektiva, Analgetika oder antivirale Additive, Antiseptika, entzündungshemmende Mittel, wundheilungsfördernde Mittel, blutstillende Mittel, Proteine, Proteinfragmente, Peptide und/oder Peptidsequenzen, Polysaccharide, zum Beispiel Chitosan, Wachstumsfaktoren, wie Purine oder Pyrimidine, lebende Zellen, β-Tricalciumphosphat, Hydroxyapatit, insbesondere speziell Hydroxyapatitnanopartikel, antimikrobielle Agentien, Aminosäuren, Enzyme, Vitamine, Antioxidantien,
• – Haut- oder haarkosmetischen Wirkstoffen, zum Beispiel Effektpigmente, und/oder
• – geruchsadsorbierenden Additiven, wie Aktivkohle oder Cyclodextrine, Kronenether, biologisch aktive Metalle oder Metalllegierungen, proteinadsorptionsvermeidende Substanzen, wie zum Beispiel Polyethylenoxid.

The stable, aqueous protein solution or protein dispersion preferably comprises at least one additive selected from

• Viscosity-adjusting agents, in particular water-insoluble polymers, for example in the form of dispersions, or preferably water-soluble polymers, such as polyalkylene oxides, such as polyethylene oxides, polyethylene glycols or polypropylene glycols, polyvinyl alcohols, polyethyleneimines, polyvinylpyrrolidones, polyvinylamines, polyvinyl acetates, polyacrylates, polyalkyl acrylates, such as polymethacrylates, Polyhydroxyalkyl acrylates, such as poly (2-hydroxymethacrylates) or poly (2-hydroxyethacrylates), polyvinylformamides, polyacrylamides, polycarboxylic acids, such as polyacrylic acids or polymethacrylic acids, natural polymers, such as galactomannan, chitosan, collagen, albumin, xanthan, alginates, gelatin, cellulose or starch, in particular their water-soluble derivatives, such as, for example, hydroxypropylcellulose, carboxymethyl starch, oxidized starch, carboxymethylcellulose,
• Carrier-forming polymers, preferably combinations of two or more polymers, homo- and copolymers,
• Processing aids, such as spinning auxiliaries, preferably organic solvents, such as alcohols, for example ethanol or butanol, and / or surface-active agents, such as surfactants for adjusting the surface tension or vapor pressure of the solution or conductive salts for adjusting the conductivity,
• Crosslinking agents, for example as an addition to the spinning solution or for the subsequent crosslinking of fibers,
• Pharmacologically active substances or medicaments, such as antibiotics, anti-infective agents, analgesics or antiviral additives, antiseptics, anti-inflammatory agents, wound healing agents, hemostatic agents, proteins, protein fragments, peptides and / or peptide sequences, polysaccharides, for example chitosan, growth factors, such as purines or pyrimidines, living cells, β-tricalcium phosphate, hydroxyapatite, especially especially hydroxyapatite nanoparticles, antimicrobial agents, amino acids, enzymes, vitamins, antioxidants,
• - Skin or hair cosmetic agents, for example effect pigments, and / or
• Odor adsorbing additives such as activated carbon or cyclodextrins, crown ethers, biologically active metals or metal alloys, protein adsorption preventing substances such as polyethylene oxide.

The abovementioned polymers can be used as homopolymers, as copolymers, for example as block copolymers, graft copolymers, comb or brush copolymers, star polymers, random or alternating systems, or in any mixture with one another.

The abovementioned additives can be added or adsorbed in pure form, in any mixture with one another and / or in encapsulated form.

Polyethylene oxide, polyethylene glycol, polyvinyl alcohol and / or polyvinylpyrrolidone, which are dissolved as water-soluble polymers directly in the protein solution and are toxicologically harmless, are particularly preferred as agents for adjusting the viscosity. Furthermore, with a view to use in the medical field as additives, preferably drugs and wound healing promoting agents are used.

Advantageously, the additives are present in a concentration range from 0.01% by weight to 40% by weight, preferably in a concentration range from 0.01% by weight to 30% by weight and more preferably in a concentration range of 2% by weight % to 25% by weight.

The pharmacologically active substances or medicaments used as additives are preferably also used in lower concentrations than those mentioned above, depending on the application.

For the process according to the invention for producing the stable, aqueous protein solution or dispersion, the at least one self-assembling protein in an aqueous solution is preferably dissolved in a chaotropic salt as solubilizer before the addition of the amino acid as stabilizing agent.

The chaotropic salt used is preferably guanidinium thiocyanate, calcium chloride, lithium bromide, urea or an ionic liquid.

The self-assembling proteins can be dissolved in the chaotropic salt prior to the addition of at least one amino acid stabilizer and spun directly into protein fibers, i.e., in particular when using calcium chloride or an ionic liquid, for example without subsequent dialysis, as calcium chloride, for example to Guanidiniumthiocyanat, is safe for health.

In the variant without dialysis, the protein fibers may not solidify completely due to the salt content. In this case, a washing step is advantageously carried out.

Optionally, for example, when using calcium chloride, the salt may be removed by precipitation, for example, by pH increase, in the form of a calcium hydroxide precipitate or ion exchange, or other processes prior to the spinning process. In this case, an amino acid is added to the calcium chloride solution before the precipitation process to stabilize the proteins. This also gives an aqueous protein solution with the stabilizer.

In the improved method for producing a stable, aqueous protein solution or dispersion, the protein solution or dispersion is preferably dialyzed or ultrafiltered, gel-filtered or otherwise by agitation, particularly by stirring, and preferably even at room temperature against an aqueous buffer treated osmotic process, or the chaotropic salt is separated by precipitation.

The dialysis is carried out to remove the chaotropic salt from the protein solution preferably against an aqueous buffer having a pH in the range of 4 to 14, more preferably in the pH range of 6 to 14, most preferably in the pH range of 7 to 12 containing the stabilizing agent.

In the variant with dialysis, the chaoptropic salt, for example guanidinium thiocyanate, is almost completely removed from the aqueous protein solution. This gives an aqueous solution of protein, water and the stabilizer, for example L-arginine. The salt, for example guanidinium thiocyanate, in the spinning solution could interfere with the spinning process, in particular electrospinning, because of its influence on the conductivity of the spinning solution. The salt could also interfere with the formation and solidification of the fibers during the spinning process.

As an alternative to dialysis of the protein solution or dispersion, gel filtration or ultrafiltration may be considered, especially for larger batches.

From the stable, aqueous protein solution or dispersion according to the invention or the stable, aqueous protein solution prepared according to the invention, particularly pure protein sheets, ie protein fibers, fiber structures, gels, can be used directly without or on a sheet carrier. Films, films or foams, or particularly pure protein impregnations or protein coatings.

The fibers or fiber structures, in particular nonwovens, are preferably produced by spinning the stable or aqueous protein solution or dispersion according to the invention or prepared according to the invention without or onto a shaped or flat-sheet support to the corresponding protein fibers or fiber structures.

Form or fabric are here understood in particular fibers, fiber structures, gels, films, films, foams or combinations thereof.

The protein solution or protein dispersion can thus be applied directly to a carrier, in particular to a shaped or flat structure, for example onto a nonwoven fabric or another fibrous structure, which can be used, for example, in wound dressing systems, on a gel, a film, a film or on a foam, for example in the case of application in wound dressings on a polyurethane foam, are spun.

The spinning of the stable aqueous solution or dispersion with selbstassemblierendem protein is advantageously carried out by electric or rotor spinning.

According to the method according to the invention, stabilization of the protein solution or protein dispersion before spinning can be carried out with a health-acceptable and / or a protein-sparing stabilization method. Subsequent spinning is preferably possible at room temperature in a continuous process over an extended period of time without the protein solution or protein dispersion precipitating or gelling or being exposed to a harmful temperature effect.

The electro-spun fibers preferably have a fiber diameter of 1 nm to several micrometers, preferably from 10 nm to 2000 nm, more preferably a fiber diameter of 10 nm to 1000 nm and most preferably a fiber diameter of 10 nm to 800 nm.

The protein solution or dispersion prepared according to the invention or according to the invention can also be used as impregnation or coating, in particular by spray, dip or spincoating, without or on a sheet carrier, for example fibers or fiber structures, in particular nonwovens.

Furthermore, the stable or aqueous protein solution or dispersion according to the invention or prepared according to the invention, without or on a sheet support, preferably as a coating in gel, foil or film form or in the form of patterns, such as dot or stripe patterns, in particular by a printing, doctoring, rolling or casting process, are used or processed.

Furthermore, the protein solution or dispersion according to the invention or prepared according to the invention can be processed without or on a sheet carrier to form protein foams.

In the process for the production of fabrics, impregnations or coatings from the stable, aqueous protein solution or dispersion according to the invention or prepared according to the invention, of course, the additives mentioned above, for example selected from viscosity adjusting agents, carrier-forming polymers, spinning aids, crosslinking agents , surface-active agents, pharmacologically active substances, medicaments, wound healing agents, skin or hair cosmetic agents and / or odor adsorbing additives.

Furthermore, the stable, aqueous protein solution or dispersion according to the invention or the fabrics, impregnations or coatings produced therefrom advantageously find use for cosmetic products, for example for creams, sponges, pads, strips or masks, for hygiene products, such as feminine hygiene, incontinence products, Diapers for household products, such as cleaning utensils, in particular wipes or sponges and / or for filter applications, such as tobacco filters or engine filters.

A particularly preferred use is the stable, aqueous protein solution or protein dispersion according to the invention or find the moldings or fabrics produced therefrom, impregnations or coatings for medical products, preferably for or as wound dressings, other textiles, tubes, fabric constructions or tissue growth (Tissue engineering scaffolds) or implants.

For example, the medical products may have at least one wound contact layer, produced by a described method, which positively influences the wound healing process or is designed as a biocompatible or hypoallergenic coating of surfaces. It is possible that the wound contact layer in contact with the wound may be absorbed by the body and broken down. In addition, it could prevent sticking to the wound. As a result, painful dressing changes are avoided and the newly formed tissue layers in the wound are not destroyed by the change.

The abovementioned additives make fiber structures, in particular nonwovens, such as, for example, 3D-modeled nonwovens, gels, films, films or foams, or for the impregnations or coatings described in one of the above examples, for the shaped or flat structures, in particular the fibers Processes are produced, thus opening a particularly diverse scope.

For stabilization with respect to aqueous solutions, the protein structures or areas produced can be aftertreated, for example by immersion in ethanol or methanol or in potassium hydrogen phosphate buffer and washing with distilled water and subsequent drying.

Embodiment of the invention

The object of the invention will be explained in more detail by way of examples.

• – R16-Protein als rekombinantes, selbstassemblierendes Protein gemäß WO 2008/155304 R16Arg₈-Protein (R16-Protein mit angehängtem Oligoarginin-Block mit 8 Aminosäuren)
• – L-Arginin
• – Natriumhydrogencarbonat-Puffer (10 mM; pH 10 bis 12)
• – Guanidiniumthiocyanat-Lösung (6M); GuaSCN als chaotropes Salz
• – Calciumchlorid-Lösung (CaCal₂:Wasser:Ethanol = 1:8:2 molares Verhältnis) als chaotropes Salz
• – Polyethylenoxid (MG 900000 g/mol) als Additiv zur Einstellung der Viskosität
• – Polyvinylpyrrolidon (MG 90000 g/mol) als Additiv zur Einstellung der Viskosität
• – Dialyseschlauch (regenerierte Cellulose: MWCO (Molecular Weight Cut Off) von 10000–12000)
• – Dialyseschlauchklammern

The following materials are used:

• R16 protein as a recombinant, self-assembling protein according to WO 2008/155304 R16Arg ₈ protein (R16 protein with attached 8 amino acid oligoarginine block)
• - L-arginine
• Sodium bicarbonate buffer (10 mM, pH 10 to 12)
• Guanidinium thiocyanate solution (6M); GuaSCN as a chaotropic salt
• Calcium chloride solution (CaCal ₂ : water: ethanol = 1: 8: 2 molar ratio) as a chaotropic salt
• - Polyethylene oxide (MW 900000 g / mol) as an additive for adjusting the viscosity
• - Polyvinylpyrrolidone (MW 90000 g / mol) as an additive for adjusting the viscosity
• - dialysis tube (regenerated cellulose: MWCO (Molecular Weight Cut Off) from 10000-12000)
• - Dialysis tubing clips

Examples of the Production of Fibers from Recombinant, Self-Assembling Proteins by Electrospinning

example 1

R16 protein (15 g to 20 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular guanidinium thiocyanate solution (80 g to 85 g of 6M GuaSCN) with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. The dialysis is preferably run for 8 to 14 hours, and the buffer is optionally replaced.

Subsequently, the protein solution is taken out of the dialysis tube and processed by means of electrospinning at room temperature into fibers (high voltage 35 kV, electrode distance 10 cm).

Example 2

R16Arg ₈ protein (10 g to 15 g) as a recombinant, selbstassemblierendes protein in a chaotropic salt, in particular guanidinium thiocyanate solution (85 g 6M GuaSCN) dissolved with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. The dialysis is preferably run for 8 to 14 hours, and the buffer is optionally replaced.

Subsequently, the protein solution is taken out of the dialysis tube, and 0.5% by weight to 3% by weight of polyethylene oxide (MW 900,000 g / mol) is added and dissolved.

The solution is processed into fibers by means of electrospinning at room temperature (high voltage 35 kV, electrode distance 10 cm).

Example 3

R16 protein (10 g to 15 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular calcium chloride solution (85 g), with stirring at 50 ° C to 70 ° C.

0.1 g of L-arginine are added and dissolved with stirring.

The solution is processed into fibers by means of electrospinning at room temperature (high voltage 35 kV, electrode distance 10 cm).

Example 4

R16 protein (10 g to 15 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular calcium chloride solution (85 g), with stirring at 70 ° C.

0.1 g of L-arginine are added and dissolved with stirring.

The salt is precipitated by increasing the pH (with the addition of caustic soda) in the alkaline range as calcium hydroxide. After separating the precipitate, for example by filtration or centrifuging, a clear yellow solution is obtained.

The solution is made into fibers at room temperature with or without the addition of viscosity-increasing agents by means of electrospinning (high voltage 35 kV, electrode spacing 10 cm).

Example 5

The preparation of the protein solution is carried out as in Example 2. Only dialysis is carried out at approx. 50 ° C. Dialysis at about 50 ° C has an additional stabilizing effect on the protein.

Subsequently, the solution is further processed into fibers as in the above examples with or without the addition of viscosity-increasing agents by means of electrospinning at room temperature.

The electrospun R16 fibers of the aforementioned examples preferably have a fiber diameter of 10 nm to 2000 nm, more preferably a fiber diameter of 10 nm to 1000 nm and most preferably a fiber diameter of 10 nm to 800 nm.

Examples of the Production of Fibers from Recombinant, Self-Assembling Proteins by Rotor Spinning

Example 6

R16 protein (15 g to 20 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular guanidinium thiocyanate solution (80 g of 6M GuaSCN) with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. The dialysis is preferably run for 8 to 14 hours, and the buffer is optionally replaced.

Subsequently, the protein solution is taken out of the dialysis tube, and 4% by weight of polyethylene oxide (MW 900,000 g / mol) as an viscosity adjusting additive is added and dissolved.

The solution is processed by means of rotor spinning into fibers.

Example 7

R16Arg ₈ protein (15 g to 20 g) as a recombinant, selbstassemblierendes protein in a chaotropic salt, in particular guanidinium thiocyanate solution (85 g 6M GuaSCN) dissolved with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. The dialysis is preferably run for 8 to 14 hours, and the buffer is optionally replaced.

Subsequently, the protein solution is taken out of the dialysis tube, and 15% by weight of polyvinyl pyrrolidone (MW 90000 g / mol) as an viscosity adjusting additive is added and dissolved.

The solution is processed by means of rotor spinning into fibers.

Example of film or film production from recombinant, self-assembling proteins

Example 8

The preparation of the protein solution is carried out as in Example 1 or 2 with the optional addition of additives or viscosity-increasing agents. Thereafter, the film or sheet is prepared from the protein solution by, for example, spin coating or doctoring.

Examples of the preparation of nonwovens spray-coated with recombinant self-assembling proteins

Example 9

R16 protein (5 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular guanidinium thiocyanate solution (95 g of 6M GuaSCN) with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. The dialysis is preferably run for 8 to 14 hours, and the buffer is optionally replaced.

Subsequently, the protein solution is taken out of the dialysis tube.

The protein solution is sprayed evenly onto a viscose nonwoven (spray gun with compressed air).

Examples of the preparation of nonwovens dip-coated with recombinant, self-assembling proteins

Example 10

R16 protein (5 g) as a recombinant, self-assembling protein is dissolved in a chaotropic salt, in particular guanidinium thiocyanate solution (95 g of 6M GuaSCN) with stirring.

In a beaker 10 l of sodium bicarbonate buffer are introduced and 10 g of L-arginine (c (arginine) = 0.1% (w / v)) dissolved therein.

The protein solution is transferred to a dialysis tube and this is placed in the buffer solution. During dialysis, the solution is stirred. Dialysis is preferred for 8 to 14 Hours run, and the buffer is replaced if necessary.

Subsequently, the protein solution is taken out of the dialysis tube.

A viscose nonwoven fabric is dipped in the protein solution and then dried at room temperature.

All experiments can be performed with R16 protein or R16Arg ₈ protein (R16 protein with attached oligoarginine block). The R16Arg ₈ protein is said to have special properties in wound healing.

Alternatively, the S16 protein or the C16 protein can be used.

Optional post-treatment of the manufactured protein fibers, protein films, protein films or protein coatings for stabilization against aqueous solutions

1st aftertreatment variant

The protein fibers, films, films or coatings are immersed for about 0.5 to 1 minute in ethanol or methanol and then dried at room temperature and to remove the solvent residues in a vacuum oven.

2nd aftertreatment variant

The protein fibers, films, films or coatings are immersed in potassium hydrogen phosphate buffer for about 2 hours and then washed with distilled water and dried at room temperature.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• US 6110590 [0006]
• JP 2010270426 A [0007]
• JP 2010-150712 [0009]
• WO 2008/155304 [0025, 0063]
• WO 2007/082936 [0025]

Claims (17)

A stable, aqueous protein solution or protein dispersion, comprising in an aqueous solution or dispersion at least one self-assembling protein and at least stabilizing agent for the self-assembling protein, wherein the stabilizing agent comprises at least one amino acid. A stable, aqueous protein solution or protein dispersion according to claim 1, wherein the self-assembling protein is of natural origin or synthetic, in particular by recombinant production, constructed from repetitive units of a structural protein, in particular an insect protein and / or a spider silk protein or a similar or derived thereof Proteins, in particular with insertion or attachment of Oligoaminosäure blocks and / or an intrinsically unfolded protein or a microbead-forming protein. A stable, aqueous protein solution or protein dispersion according to claim 1 or 2, wherein the self-assembling protein is an R16, R16Arg ₈ , S16 or C16 protein or a protein derived therefrom. Stable, aqueous protein solution or protein dispersion according to one of the preceding claims, wherein the at least one self-assembling protein is present in a concentration of 4 wt .-% to 35 wt .-%, preferably in a concentration of 5 wt .-% to 30 wt .-% and particularly preferably in a concentration of 10 wt .-% to 25 wt .-% based on the total weight of the stable solution. Stable, aqueous protein solution or protein dispersion according to one of the preceding claims, wherein the stabilizing agent is selected from arginine, lysine, histidine, phenylalanine and / or glutamine, preferably from arginine. Stable, aqueous protein solution or protein dispersion according to any one of the preceding claims, wherein the stabilizing agent is present in a concentration of 0.01 wt .-% to 10 wt .-%, preferably in a concentration of 0.01 wt .-% to 5 wt .-% and particularly preferably in a concentration of 0.01 wt .-% to 4 wt .-%. Stable, aqueous protein solution or protein dispersion according to one of the preceding claims, comprising at least one additive selected from viscosity adjusting agents, carrier-forming polymers, spinning aids, crosslinking agents, surface active agents, pharmacologically active agents, medicaments, wound healing agents, skin - or hair cosmetic agents and / or odor adsorbing additives. Stable, aqueous protein solution or protein dispersion according to claim 7, wherein the additive (s) is present in a concentration of 0.01% by weight to 40% by weight, preferably in a concentration of 0, 01 wt .-% to 30 wt .-%, particularly preferably in a concentration of 2 wt .-% to 25 wt .-%. A process for producing a stable, aqueous protein solution or dispersion according to any one of the preceding claims, wherein the at least one self-assembling protein is dissolved in an aqueous solution prior to the addition of the amino acid in a chaotropic salt. Process for the preparation of a stable, aqueous protein solution or protein dispersion according to claim 9, wherein guanidinium thiocyanate, calcium chloride, lithium bromide, urea or an ionic liquid is used as a chaotropic salt. A method for producing a stable, aqueous protein solution or protein dispersion according to claim 9 or 10, wherein the protein solution is dialyzed under agitation against an aqueous buffer, ultrafiltered or gel-filtered or treated by other osmotic methods or the chaotropic salt by precipitation is separated. A process for producing fibers or fibrous structures, in particular nonwovens, wherein a stable, aqueous protein solution or protein dispersion according to any one of claims 1 to 8 is used or produced by a method according to any one of claims 9 to 11, and then the protein Solution or dispersion to protein fibers or fiber structures, in particular nonwovens, is spun. A process for electrospinning or rotor spinning of self-assembling protein, wherein a stable, aqueous solution or dispersion according to any one of claims 1 to 8 is used or produced by a process according to any one of claims 9 to 11, and then the protein solution or protein Dispersion is electrospun or rotorspun. Process for the preparation of impregnations, coatings, gels, films or films, wherein a stable, aqueous protein solution or protein dispersion according to one of claims 1 to 8 is used or produced by a process according to one of claims 9 to 11, and then the protein solution or protein dispersion is used as a protein impregnation or protein coating, in particular by spray, dip or spin coating, of shaped or sheet carriers or as a protein coating in gel, film or film form , especially by a printing, doctoring, rolling or casting process, is processed or used by mold or sheet carriers. Process for producing shaped or flat structures, in particular fibers, fiber structures, gels, films, films or foams, or of impregnations or coatings according to one of the preceding claims, wherein the protein solution or protein dispersion at least one before spinning or processing Additive according to claim 7 or 8 is added. Use of the stable, aqueous protein solution or protein dispersion or the moldings or fabrics produced therefrom, in particular fibers, fiber structures, gels, films, films or foams, or the impregnations or coatings produced therefrom according to any preceding claims for medical products, preferably for or as wound dressings, tubes, tissue engineering or implants, for cosmetic products, for hygiene products, for household products and / or for filter applications. Shaped or flat structures, in particular fibers, fiber structures, gels, films, films or foams, or impregnations or coatings, produced by a process according to one of claims 9 to 15.