Non-woven article comprising collagen-like protein fibers and a production process thereof

Abstract

The present invention refers to an electrospun article comprising or consisting of fibers which comprise at least one collagen-like protein, at least one carrier and at least one crosslinker. In addition, the present invention refers to a method for obtaining an electrospun article, preferably according to the invention, comprising or consisting of the following steps a) providing at least one collagen-like protein in at least one solvent to obtain a solution of the at least one collagen-like protein, b) adding at least one carrier to the solution of the at least one collagen-like protein to obtain a solution of the at least one collagen-like protein and the at least one carrier, c) providing at least one crosslinker in at least one solvent to obtain a solution of the at least one crosslinker, d) pumping the solution of the at least one collagen-like protein and the at least one carrier in the inner needle of a coaxial nozzle, e) pumping the solution of the at least one crosslinker in the outer needle of a coaxial nozzle, f) electrospinning the solution of the at least one collagen-like protein and the at least one carrier and the solution of the at least one crosslinker to obtain the electrospun article, g) optionally drying the electrospun article obtained in step f). Furthermore, the present invention refers to an article obtainable by the method of the present invention, wherein the article is a medical device. Additionally, the present invention refers to the use of the article according to the present invention for wound healing and cell culture applications.

Description

[0001] 202400137 1

[0002] Non-woven article comprising collagen-like protein fibers and a production process thereof

[0003] Field of the invention

[0004] The present invention refers to an electrospun article comprising or consisting of fibers which comprise at least one collagen-like protein, at least one carrier and at least one crosslinker.

[0005] In addition, the present invention refers to a method for obtaining an electrospun article, preferably according to the invention, comprising or consisting of the following steps a) providing at least one collagen-like protein in at least one solvent to obtain a solution of the at least one collagen-like protein, b) adding at least one carrier to the solution of the at least one collagen-like protein to obtain a solution of the at least one collagen-like protein and the at least one carrier, c) providing at least one crosslinker in at least one solvent to obtain a solution of the at least one crosslinker, d) pumping the solution of the at least one collagen-like protein and the at least one carrier in the inner needle of a coaxial nozzle, e) pumping the solution of the at least one crosslinker in the outer needle of a coaxial nozzle, f) electrospinning the solution of the at least one collagen-like protein and the at least one carrier and the solution of the at least one crosslinker to obtain the electrospun article, g) optionally drying the electrospun article obtained in step f).

[0006] Furthermore, the present invention refers to an article obtainable by the method of the present invention, wherein the article is a medical device.

[0007] Additionally, the present invention refers to the use of the article according to the present invention for wound healing and cell culture applications.

[0008] Description of the related art

[0009] Electrospun articles comprising animal-derived collagen and the process thereof are already known in the art. However, electrospun articles comprising collagen-like proteins are required for some applications. Therefore, it was desired to create an electrospun article comprising collagen like proteins but they are challenging to electrospin because of their material properties: low viscosity even at higher concentrations, low molecular weight and, finally, high water solubility. For those reasons, the process for electrospinning articles comprising animal-derived collagen is not simply transferable to a process for electrospinning articles comprising collagen-like proteins. Moreover, the known processes for electrospinning articles comprising animal-derived collagen have several disadvantages.

[0010] One method that has mostly been practiced up to now is that the fibers are first spun without crosslinker to create a still water-soluble fleece. This is then immersed in a solution with crosslinker for final 202400137 2 stabilization. However, the disadvantage of this method is that two steps are required to generate an electrospun article.

[0011] Another way of producing electrospun fibers is to mix the solution of the material to be crosslinked with the crosslinker beforehand. However, only slow-reacting crosslinkers in a narrow concentration corridor can be used here, as otherwise the solution cannot be spun and a hydrogel is already formed in the vessel of the starting solution.

[0012] Therefore, it is desired that spinning and crosslinking can be proceeded in one step only and / or that no mixing of the material to be spun and the crosslinker is required so that fast-reacting crosslinkers can be used as well without forming a hydrogel.

[0013] The inventors of the present invention surprisingly found an electrospun article comprising or consisting of fibers which comprise at least one collagen-like protein, at least one carrier and at least one crosslinker. The inventors further found a method for coaxial electrospinning, that circumvents the above mentioned disadvantages.

[0014] Summary

[0015] In a first aspect, the present invention refers to an electrospun article comprising or consisting of fibers which comprise at least one collagen-like protein, at least one carrier and at least one crosslinker.

[0016] In a second aspect, the present invention refers to a method for obtaining an electrospun article, preferably according to the invention, comprising or consisting of the following steps a) providing at least one collagen-like protein in at least one solvent to obtain a solution of the at least one collagen-like protein, b) adding at least one carrier to the solution of the at least one collagen-like protein to obtain a solution of the at least one collagen-like protein and the at least one carrier, c) providing at least one crosslinker in at least one solvent to obtain a solution of the at least one crosslinker, d) pumping the solution of the at least one collagen-like protein and the at least one carrier in the inner needle of a coaxial nozzle, e) pumping the solution of the at least one crosslinker in the outer needle of a coaxial nozzle, f) electrospinning the solution of the at least one collagen-like protein and the at least one carrier and the solution of the at least one crosslinker to obtain the electrospun article, g) optionally drying the electrospun article obtained in step f).

[0017] In a third aspect, the present invention refers to an article obtainable by the method of the present invention, wherein the article is a medical device.

[0018] In a fourth aspect, the present invention refers to the use of the article of the present invention for wound healing and cell culture applications. 202400137 3

[0019] These and other aspects, embodiments, features, and advantages of the invention will become apparent to a person skilled in the art through the study of the following detailed description and claims. Any feature from one aspect of the invention can be used in any other aspect of the invention. Furthermore, it will readily be understood that the examples contained herein are intended to describe and illustrate the invention but not to limit the invention and that, in particular, the invention is not limited to these examples.

[0020] Brief description of the drawings

[0021] Fig. 1 shows the results of the BCA-assay in which the disintegration of the fibers of example 2 is investigated after the contact with water and compared to the control fibers of example 5 after the contact with water.

[0022] Fig. 2 shows the results of the BCA-assay in which the disintegration of the fibers of example 3 is investigated after the contact with water and compared to the control fibers of example 5 after the contact with water.

[0023] Fig. 3 shows the results of the BCA-assay in which the disintegration of the fibers of example 4 is investigated after the contact with water and compared to the control fibers of example 5 after the contact with water.

[0024] Fig. 4 shows the morphology of the fibers of example 2 before (left) and after (right) after the contact with water.

[0025] Fig. 5 shows the morphology of the fibers of example 3 before (left) and after (right) after the contact with water.

[0026] Fig. 6 shows the morphology of the fibers of example 4 before (left) and after (right) after the contact with water.

[0027] Fig. 7 shows the morphology of the control fibers of example 5 before (left) and after (right) after the contact with water.

[0028] Detailed description

[0029] In the following, “collagen-like protein” is referred to as “CLP” as well.

[0030] Numerical ranges that are indicated in the format “from x to y” also include the stated values. If several preferred numerical ranges are indicated in this format, it is self-evident that all ranges that result from the combination of the various endpoints are also included.

[0031] "At least one" means one or more, i.e. 1 , 2, 3, 4, 5, 6, 7, 8, 9 or more. "At least one", as used herein in relation to any component, refers to the number of chemically different molecules, i.e. to the number of different types of the referenced species, but not to the total number of molecules. For example, "at least one CLP" means that at least one type of molecule falling within the definition for a CLP is used but that 202400137 4 also two or more different types of CLP falling within this definition can be present but does not mean that only one or more molecules of one type of CLP are present.

[0032] All percentages given herein in relation to the compositions or formulations relate to wt.-% relative to the total weight of the respective composition, if not explicitly stated otherwise.

[0033] In the following “4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl-morpholiniumchlorid” is referred to as “DMTMM” as well.

[0034] In the following, “bicinchoninic acid assay” is referred to as “BCA-assay” as well.

[0035] In particular the present invention refers to:

[0036] An electrospun article comprising or consisting of fibers which comprise at least one collagen-like protein, at least one carrier and at least one crosslinker.

[0037] In general, all collagen-like proteins are suitable for this invention.

[0038] In a preferred embodiment of the present invention the collagen-like protein is a collagen-like protein from Streptococcus pyogenes, which is preferably the Scl2 protein from Streptococcus pyogenes.

[0039] Expression of collagen-like proteins have been attempted in several systems, including Escherichia coli and Saccharomyces cerevisiae. In one embodiment the at least one collagen-like protein is a bacterial collagen-like protein, preferably produced by fermentation in Pichia, Brevibacillus, Bacillus, Escherichia or Corynebacterium, preferably Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum.

[0040] In a preferred embodiment the collagen-like proteins may be expressed in Corynebacterium, preferably in Corynebacterium glutamicum.

[0041] One particularly suitable collagen-like protein is derivable from following polynucleotide.

[0042] A polynucleotide encoding an amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , wherein the polynucleotide is a replicable polynucleotide encoding a collagen- like protein and wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1 .

[0043] It is preferred, when the amino acid sequence comprises a deletion of between 38 and 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1. This includes a complete deletion of the N- terminal V-domain (comprising 74 amino acids) and different truncations of the V-domain of at least 38 amino acids.

[0044] In a preferred embodiment, the amino acid sequence that is at least > 60%, identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. 202400137 5

[0045] In a further configuration, the amino acid sequence that is at least > 65%, or > 70%, or > 75%, or > 80%, or > 85% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0046] In a preferred configuration, the polynucleotide encodes an amino acid sequence that is at least > 90%, > 92%, > 94%, > 96%, > 97%, > 98%, > 99% or 100%, preferably > 97%, particularly preferably > 98%, very particularly preferably > 99%, and extremely preferably 100%, identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0047] In a preferred embodiment of the present invention the polynucleotide is a replicable nucleotide sequence encoding the collagen-like protein from Streptococcus pyogenes.

[0048] Polynucleotide and nucleic acid molecules comprising such sequences and encoding polypeptide variants of SEQ ID NO:1 to 4, which contain one or more insertions) or deletion(s) are suitable as well. Preferably, the polypeptide contains a maximum of 5, a maximum of 4, a maximum of 3, or a maximum of 2, insertions or deletions of amino acids.

[0049] Mixture of polypeptides comprising one of the polypeptide variants of SEQ ID NO:1 to 4 and on or more of the truncated variants of the collagen-like protein of SEQ ID NO:5 to 12 can be used as well.

[0050] Plasmids and vectors that comprise the nucleotide sequences according to the invention and optionally replicate in microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia are suitable. In a preferred configuration, the vector comprising the nucleotide sequences according to the present invention is suitable for replication in yeast of the genus Pichia pastoris.

[0051] Microorganisms of the genera Pichia, Corynebacterium, Pseudomonas or Escherichia that comprise the polynucleotides, vectors and polypeptides according to the invention are suitable as well. Preferred microorganisms are Pichia pastoris, Brevibacillus choshinensis or Corynebacterium glutamicum. Microorganisms of the species P. pastoris, E. coli, P. putida or C. glutamicum comprising any of the nucleotide sequences according to the present invention, any of the polypeptides or any of the vectors according to the present invention are suitable.

[0052] The microorganism may be a microorganism in which the nucleotide sequence is present in overexpressed form.

[0053] Overexpression according to the invention means, generally, an increase in the intracellular concentration or activity of a ribonucleic acid, a protein (polypeptide) or an enzyme, compared with the starting strain (parent strain) or wild-type strain, if this is the starting strain. A starting strain (parent strain) is taken to mean the strain on which the measure leading to the overexpression was carried out.

[0054] In the overexpression, the methods of recombinant overexpression are preferred. These include all methods in which a microorganism is produced using a DNA molecule provided in vitro. Such DNA molecules comprise, for example, promoters, expression cassettes, genes, alleles, encoding regions etc. These are converted into the desired microorganism by methods of transformation, conjugation, transduction or the like methods.

[0055] The extent of the expression or overexpression can be established by measuring the amount of the mRNA transcribed by the gene, by determining the amount of the polypeptide, and by determining the enzyme activity. 202400137 6

[0056] The bacterial collagen-like protein can be obtained in a fermentative process comprising the following steps: a) fermentation of a microorganism according to the present invention in a medium, b) accumulation of the bacterial collagen-like protein in the medium, wherein a fermentation broth is obtained.

[0057] The culture medium or fermentation medium that is to be used must appropriately satisfy the demands of the respective strains. Descriptions of culture media of various microorganisms are contained in the handbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms culture medium and fermentation medium or medium are mutually exchangeable.

[0058] As carbon source, sugars and carbohydrates can be used, such as, e.g., glucose, sucrose, lactose, fructose, maltose, molasses, sucrose-containing solutions from beet sugar or sugar cane processing, starch, starch hydrolysate and cellulose, oils and fats, such as, for example, soybean oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as, for example, palmitic acid, stearic acid and linoleic acid, alcohols such as, for example, glycerol, methanol and ethanol, and organic acids, such as, for example, acetic acid or lactic acid.

[0059] As nitrogen source, organic nitrogen compounds such as peptones, yeast extract, meat extract, malt extract, corn-steep liquor, soybean meal and urea or inorganic compounds such as ammonium sulphate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate can be used. The nitrogen sources can be used individually or as a mixture.

[0060] As phosphorus source, phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts can be used.

[0061] The culture medium must, in addition, contain salts, for example in the form of chlorides or sulphates of metals such as, for example, sodium, potassium, magnesium, calcium and iron, such as, for example, magnesium sulphate or iron sulphate, which are necessary for growth. Finally, essential growth substances such as amino acids, for example homoserine and vitamins, for example thiamine, biotin or pantothenic acid, can be used in addition to the above-mentioned substances.

[0062] Said starting materials can be added to the culture in the form of a single batch or supplied in a suitable manner during the culturing.

[0063] Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acid compounds such as phosphoric acid or sulphuric acid, are used in a suitable manner for pH control of the culture. The pH is generally adjusted to 6.0 to 8.5, preferably 6.5 to 8. For control of foam development, antifoams can be used, such as, for example, polyglycol esters of fatty acids. For maintaining the stability of plasmids, suitable selectively acting substances such as, for example, antibiotics, can be added to the medium. The fermentation is preferably carried out under aerobic conditions. In order to maintain said aerobic conditions, oxygen or oxygen-containing gas mixtures such as, for example, air, are introduced into the culture. The use of liquids that are enriched with hydrogen peroxide is likewise possible.

[0064] Optionally, the fermentation is carried out at superatmospheric pressure, for example at a superatmospheric pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually 20°C to 45°C, and preferably 25°C to 40°C, particularly preferably 30°C to 37°C. In the case of batch or fed-batch processes, the culturing is preferably continued until an amount sufficient for the measure of obtaining the 202400137 7 desired organic chemical compound has formed. This goal is usually reached within 10 hours to 160 hours. In continuous processes, longer culture times are possible. Due to the activity of the microorganisms, enrichment (accumulation) of the fine chemicals in the fermentation medium and / or in the cells of the microorganisms occurs.

[0065] Examples of suitable fermentation media may be found, inter alia, in patent documents US 5,770,409, US 5,990,350, US 5,275,940, WO 2007 / 012078, US 5,827,698, WO 2009 / 043803, US 5,756,345 or US 7,138,266; appropriate modifications may optionally be carried out to the requirements of the strains used.

[0066] The process may be characterized by a process which is selected from the group consisting of batch process, fed-batch process, repetitive fed-batch process and continuous process.

[0067] The process may be further characterized by a fine chemical, or a liquid, or a solid fine chemicalcontaining product obtained from the fine chemical-containing fermentation broth.

[0068] The performance of the processes or fermentation processes according to the invention with respect to one or more of the parameters selected from the group of concentration (compound formed per volume), yield (compound formed per carbon source consumed), volumetric productivity (compound formed per volume and time) and biomass-specific productivity (compound formed per cell dry mass or bio dry mass and time or compound formed per cell protein and time) or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1%, at least 1 .5% or at least 2%, based on processes or fermentation processes with microorganisms in which the promoter variant according to the invention is present.

[0069] Owing to the measures of the fermentation, a fermentation broth is obtained which contains the desired collagen-like protein, and preferably amino acid or organic acid.

[0070] Then, a product in liquid or solid form that contains the collagen-like protein is provided or produced or obtained.

[0071] A fermentation broth means, in a preferred embodiment, a fermentation medium or nutrient medium in which a microorganism was cultured for a certain time and at a certain temperature. The fermentation medium, or the media used during the fermentation, contains / contain all substances or components that ensure production of the desired collagen-like protein and typically ensure growth and / or viability.

[0072] On completion of the fermentation, the resultant fermentation broth accordingly contains a) the biomass (cell mass) of the microorganism resulting from growth of the cells of the microorganism, b) the desired collagen-like protein formed in the course of the fermentation, c) the organic by-products possibly formed in the course of the fermentation, and d) the components of the fermentation medium used, or of the starting materials, that are not consumed by the fermentation, such as, for example, vitamins such as biotin, or salts such as magnesium sulphate.

[0073] The organic by-products include substances which are generated in addition to the respective desired compound by the microorganisms used in the fermentation and are possibly secreted.

[0074] The fermentation broth is withdrawn from the culture vessel or the fermentation container, optionally collected, and used for providing a product in liquid or solid form containing the collagen-like protein. The expression "obtaining the collagen-like protein-containing product" is also used therefor. In the simplest 202400137 8 case, the collagen-like protein-containing fermentation broth withdrawn from the fermentation container is itself the product obtained.

[0075] By way of one or more of the measures selected from the group a) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the water, b) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%) removal of the biomass, wherein this is optionally inactivated before the removal, c) partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the organic by-products formed in the course of the fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (> 80%, > 90%, > 95%, > 96%, > 97%, > 98%, > 99%, > 99.3%, > 99.7%) removal of the components of the fermentation medium used or the starting materials that are not consumed by the fermentation, a concentration or purification of the desired collagen-like protein is achieved from the fermentation broth. In this manner, products are isolated that have a desired content of the compound.

[0076] The partial (> 0% to < 80%) to complete (100%) or virtually complete (> 80% to < 100%) removal of the water (measure a)) is also termed drying.

[0077] In a variant of the process, by complete or virtually complete removal of the water, the biomass, the organic by-products and the non-consumed components of the fermentation medium used, pure (> 80% by weight, > 90% by weight) or high-purity (> 95% by weight, > 97% by weight, > 99% by weight) product forms of the desired collagen-like protein, preferably bacterial collagen-like protein, are successfully arrived at. For the measures according to a), b), c) or d), a great variety of technical instructions are available in the prior art.

[0078] In the case of processes for producing bacterial collagen-like protein processes are preferred in which products are obtained that do not contain any components of the fermentation broth. These products are used, in particular, in human medicine, in the pharmaceuticals industry, and in the food industry.

[0079] In an embodiment the weight of the at least one collagen-like protein is 99.78 to 28.39 wt% and / or the weight of the at least one crosslinker is 71.61 to 0.22 wt%, wherein the total weight of the components is 100 wt%.

[0080] In an embodiment the weight of the at least one collagen-like protein is 99.78 to 69.59 wt%, preferably 98.28 to 74.10 wt%, more preferably 96.6 to 79.23 wt%, most preferably 95.81 wt% and / or the remainder is the at least one crosslinker, wherein the total weight of the at least one collagen-like protein and the at least one crosslinker is 100 wt%. Preferably the at least one crosslinker is DMTMM.

[0081] In an alternative embodiment the weight of the at least one collagen-like protein is 98.75 to 28.39 wt%, preferably 90.08 to 33.14 wt%, more preferably 83.21 to 39.79 wt%, most preferably 79,85 wt% and / or the remainder is the at least one crosslinker, wherein the total weight of the at least one collagen-like protein and the at least one crosslinker is 100 wt%. Preferably the at least one crosslinker is 4-arm- polyethylene glycol-succinimidyl glutarate ester. 202400137 9

[0082] In an embodiment the weight of the at least one collagen-like protein is 95.80 to 0.28 wt%, preferably 90.12 to 0.57 wt%, more preferably 88.11 to 5.39 wt%, most preferably 86.86 wt% and / or the remainder is the at least one carrier, wherein the total weight of the at least one collagen-like protein and the at least one carrier is 100 wt%.

[0083] In general, all crosslinkers known to the skilled person in the field of electrospinning can be used in this invention.

[0084] In an embodiment the at least one crosslinker is selected from a compound which comprises at least two, preferably two to six, more preferably two to four, most preferably four groups each comprising a polyalkylene glycol moiety, preferably polyethylene glycol moiety or polypropylene glycol moiety, more preferably polyethylene glycol moiety, and a succinimidyl group; organic triazines; organic aldehydes; aglycones; diisocyanates; carbodiimides or mixtures thereof.

[0085] In a preferred embodiment the at least one crosslinker is selected from a compound which comprises at least two, preferably two to six, more preferably two to four, most preferably four groups each comprising a polyalkylene glycol moiety, preferably polyethylene glycol moiety or polypropylene glycol moiety, more preferably polyethylene glycol moiety, and a succinimidyl group and / or is selected from organic triazines.

[0086] The at least one crosslinker which is selected from a compound which comprises at least two, preferably two to six, more preferably two to four, most preferably four groups each comprising a polyalkylene glycol moiety, preferably polyethylene glycol moiety or polypropylene glycol moiety, more preferably polyethylene glycol moiety, and a succinimidyl group has at least one of the following formulae (I), (II), (HI): wherein

[0087] R1is a linear or branched alkyl group having up to 12 carbon atoms, preferably up to 8 carbon atoms, more preferably having five carbon atoms;

[0088] Aik is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, preferably -CH2-CH2-; n is an integer from 1 to 1350, preferably, 50 to 1000, more preferably 125 to 660;

[0089] R2is -CH2-, -C2H4-NH-(C=O)-C3H6-, -C2H4-O-, -C2H4-O-(C=O)-C3H6-, 202400137 10

[0090] -C2H4-NH-(C=O)-C2H4- or -C2H4-O-(C=O)-C2H4-; m is an integer from 2 to 8, preferably 4 to 8, more preferably 4; or wherein

[0091] Aik is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, preferably -CH2-CH2-; and n is an integer from 1 to 1350, preferably 50 to 1000, more preferably 125 to 660; or wherein

[0092] R1is a linear or branched alkyl group having up to 12 carbon atoms, preferably up to 8 carbon atoms, more preferably having five carbon atoms, most preferably is

[0093] Aik is -CH2-, -CH2-CH2- or -CH2-CH2-CH2-, preferably -CH2-CH2-; n is an integer from 1 to 1350, preferably, 50 to 1000, more preferably 125 to 660; m is an integer from 2 to 8, preferably 4 to 8, most preferably 4.

[0094] In one embodiment the crosslinker has a molecular weight of 2.000 to 40.000 g / mol.

[0095] The organic triazine is preferably selected from 4-(4,6-dimethoxy-1 ,3,5-triazin-2-yl)-4-methyl- morpholinium chloride.

[0096] The organic aldehyde is preferably selected from formaldehyde, glyoxal, glutaraldehyde. 202400137 11

[0097] The aglycone is preferably selected from genipin.

[0098] The carbodiimide is preferably selected from 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-N- hydroxysuccinimide.

[0099] In general, all carriers known to the skilled person in the field of electrospinning can be used in this invention.

[0100] In an embodiment the at least one carrier is selected from polyethylene oxide, polysaccharides, polyvinyl alcohols, polyvinylpyrrolidone, gelatine or a mixture thereof, preferably alginate, chitosan, hyaluronic acid, dextran, inulin, starch, pullulan, hydroxypropyl methylcellulose or a mixture thereof, more preferably polyethylene oxide.

[0101] In an embodiment the at least one carrier has a weight average molecular weight of 40,000 to 2,500,000 g / mol, preferably 45,000 to 2,000,000 g / mol, more preferably 50,000 to 1 ,500,000 g / mol, most preferably 50.000 to 1.000.000 g / mol measured via GPC using polystyrene standards.

[0102] In an embodiment the at least one collagen-like protein does not leach from the article according to the invention when this article comes into contact with water, measured via BCA-assay. Therefore, a set of protein standards with known concentrations is used to generate a standard curve. These standards should cover the range of expected protein concentrations in the samples. The protein samples are diluted in a suitable buffer or solution. The dilution factor should be chosen to ensure that the protein concentration falls within the linear range of the standard curve. Add the BCA working reagent to each standard and sample. The BCA reagent contains bicinchoninic acid, which reacts with the peptide bonds in proteins to form a colored product. Incubate the samples and standards at 37 °C for one hour to allow the reaction to proceed. The absorbance of the samples and standards is measured at a measurement wavelength of 562 nm using a spectrophotometer. The absorbance values are used to generate a standard curve and determine the protein concentration of the samples.

[0103] In another embodiment the at least one collagen-like protein does not completely leach from the article according to the invention when this article comes into contact with water, measured via BCA-assay.

[0104] In an embodiment the electrospun article of the present invention does not show a complete loss of its structure, when this article comes into contact with water. Therefore, the fibers are treated with a droplet of double-distilled water and after 2.5 h dried in a freezedryer to remove all residual water. Afterwards the morphology of the fibers was investigated via a scanning electron microscope.

[0105] The invention further relates to a method for obtaining an electrospun article, preferably according to the invention, comprising or consisting of the following steps 202400137 12 a) providing at least one collagen-like protein in at least one solvent to obtain a solution of the at least one collagen-like protein, b) adding at least one carrier to the solution of the at least one collagen-like protein to obtain a solution of the at least one collagen-like protein and the at least one carrier, c) providing at least one crosslinker in at least one solvent to obtain a solution of the at least one crosslinker, d) pumping the solution of the at least one collagen-like protein and the at least one carrier in the inner needle of a coaxial nozzle, e) pumping the solution of the at least one crosslinker in the outer needle of a coaxial nozzle, f) electrospinning the solution of the at least one collagen-like protein and the at least one carrier and the solution of the at least one crosslinker to obtain the electrospun article, g) optionally drying the electrospun article obtained in step f).

[0106] The above stated embodiments of the electrospun article according to the invention apply as well to the stated method of the invention.

[0107] In an embodiment the method for electrospinning is a coaxial electrospinning. In general, the skilled person in the field of electrospinning knows methods of coaxial electrospinning. For example, the method for coaxial electrospinning is described in “Han D, Steckl AJ. Coaxial Electrospinning Formation of Complex Polymer Fibers and their Applications. Chempluschem. 2019 Oct;84(10):1453-1497”.

[0108] In an embodiment the at least one solvent in step a) is a mixture of water and acetone with a volume ratio of 7:3 to 9:1 , preferably 8:2 to 9:1 , more preferably 9:1 and / or the concentration of the at least one collagen-like protein in the solution is 10 to 200 mg / mL, preferably 30 to 150 mg / mL, more preferably 50 to 150 mg / mL, most preferably 120 mg / mL.

[0109] In an embodiment the at least one solvent in step a) is water and / or the concentration of the at least one collagen-like protein in the solution is 10 to 200 mg / mL, preferably 30 to 150 mg / mL, more preferably 50 to 150 mg / mL, most preferably 120 mg / mL.

[0110] In an embodiment the at least one carrier in step b) has a weight of 0.5 to 3 wt%, preferably 0.5 to 2.5 wt%, more preferably 0.75 to 2.5 wt%, most preferably 1 .66 wt% related to the total weight of the solution of the at least one collagen-like protein and the at least one carrier.

[0111] In an embodiment the solvent in step c) is water and / or the concentration of the at least one crosslinker in the solution is 20 to 300 mg / mL, preferably 28.86 to 300 mg / mL, more preferably 28.86 to 200 g / mol, most preferably 28.86 to 173.16 mg / mL.

[0112] In general, all pump rates known to the skilled person in the field of electrospinning can be used in this invention. 202400137 13

[0113] In an embodiment the pump rate in step d) is 300 to 4000 pL / h, preferably 300 to 2000 pL / h, more preferably 400 to 1000 pL / h, most preferably 600 pL / h and / or the pump rate in step e) is 20 to 300 pL / h, preferably 30 to 200 pL / h, more preferably 50 to 150 pL / h, most preferably 110 pL / h.

[0114] In an embodiment the pump rate ratio of the solution of the at least one collagen-like protein and the at least one carrier of d) to the solution of the at least one crosslinker of e) is 1 :1 to 15:1 , preferably 1 :1 to 10:1 , more preferably 2:1 to 7:1 , most preferably 5.45:1.

[0115] In an embodiment the optionally drying of the electrospun article of step g) is performed at 22 ± 5°C, preferably 22 ± 1 °C and / or a relative humidity of 25 ± 8%, preferably 25 ± 5% and / or wherein the optionally drying is preferably performed in a fume hood.

[0116] The present invention further refers to an article obtainable by the method according to the present invention, wherein the article is a medical device, selected from biomedical implants, wound dressings, drug delivery devices, 3-dimensional cell scaffold models.

[0117] The present invention further refers to an use of the article according to the present invention for wound healing and cell culture applications.

[0118] 202400137 14

[0119] Examples

[0120] Example 1 : Production of fibers n-like oxide

[0121] The solution to be spun was produced by adding the CLP powder to the solvent mixture (water:acetone 9:1) to obtain a solution of the collagen-like protein with a concentration of 120 mg / mL. Subsequently, 1 .66 % w / w polyethylene oxide as powder was added to the total weight of the solution of the collagen- like protein. The entire sample was stirred with a magnetic stirrer for 24 hours at 200 rpm until a solution was obtained. The fibers were generated in a FLUIDNATEK LE-50 Electrospinner (BIOINICIA SL, Spain) using a pump rate of 1200 pL / h. For monoaxial fiber generation, a 22 Gauge dosing needle (VIEWEG, Germany) was used.

[0122] Example 2: Production of fibers using collagen-like proteins, polyethylene oxide and DMTMM

[0123] The CLP powder was added to the solvent mixture (water:acetone 9:1) to obtain a solution of the collagen-like protein with a concentration of 120 mg / mL. 1 .66 % w / w polyethylene oxide as powder was added to the total weight of the solution of the collagen-like protein. The entire sample was stirred with a magnetic stirrer for 24 hours at 200 rpm until all the solid was in solution to obtain the solution of the CLP and polyethylene oxide.

[0124] The crosslinker solution was produced using DMTMM (Merck KGaA, Germany) at a concentration of 173,16 mg / mL in water. The amount of DMTMM was calculated using a molar-ratio between CLP and DMTMM of 1 :0.6.

[0125] The solution of the CLP and polyethylene oxide was pumped in the inner needle of a coaxial nozzle with a pump rate of 600 pL / h and the solution of DMTMM was pumped in the outer needle of a coaxial nozzle with a pump rate of 110 pL / h.

[0126] For coaxial fiber generation, a 16 Gauge dosing needle (VIEWEG, Germany) was used. The inner needle had an outer diameter of 0.9 mm and an inner diameter of 0.6 mm. Dosing needle and inner needle ended at the same point. Spinning was conducted for 2 h.

[0127] The obtained fibers were dried in a fume hood at 22 ± 1 °C and a relative humidity of 25 ± 5% for 24 hours after spinning.

[0128] Example 3: Production of fibers using collagen-like proteins, polyethylene oxide and DMTMM

[0129] The CLP powder was added to the solvent mixture (water:acetone 9:1) to obtain a solution of the collagen-like protein with a concentration of 120 mg / mL. 1 .66 % w / w polyethylene oxide as powder was added to the total weight of the solution of the collagen-like protein. The entire sample was stirred with a magnetic stirrer for 24 hours at 200 rpm until all the solid was in solution to obtain the solution of the CLP and polyethylene oxide.

[0130] The crosslinker solution was produced using DMTMM (Merck KGaA, Germany) at a concentration of 28.86 mg / mL in water. The amount of DMTMM was calculated using a molar-ratio between CLP and DMTMM of 1 :0.1. 202400137 15

[0131] The solution of the CLP and polyethylene oxide was pumped in the inner needle of a coaxial nozzle with a pump rate of 600 pL / h and the solution of DMTMM was pumped in the outer needle of a coaxial nozzle with a pump rate of 110 pL / h.

[0132] For coaxial fiber generation, a 16 Gauge dosing needle (VIEWEG, Germany) was used. The inner needle had an outer diameter of 0.9 mm and an inner diameter of 0.6 mm. Dosing needle and inner needle ended at the same point. Spinning was conducted for 2 h.

[0133] The obtained fibers were dried in a fume hood at 22 ± 1 °C and a relative humidity of 25 ± 5% for 24 hours after spinning.

[0134] Example 4: Production of fibers using collaqen-like proteins, polyethylene oxide and a 4-arm-polyethylene glycol-succinimidyl glutarate ester

[0135] The CLP powder was added to the solvent mixture (water:acetone 9:1) to obtain a solution of the collagen-like protein with a concentration of 120 mg / mL. 1 .66 % w / w polyethylene oxide as powder was added to the total weight of the solution of the collagen-like protein. The entire sample was stirred with a magnetic stirrer for 24 hours at 200 rpm until all the solid was in solution to obtain the solution of the CLP and polyethylene oxide.

[0136] The crosslinker solution was produced using a 4-arm-polyethylene glycol-succinimidyl glutarate ester (JemKem Technology, USA) at a concentration of 164.78 mg / mL in water. The amount of DMTMM was calculated using a molar-ratio between CLP and 4-arm-polyethylene glycol-succinimidyl glutarate ester of 1 :0.1.

[0137] The solution of the CLP and polyethylene oxide was pumped in the inner needle of a coaxial nozzle with a pump rate of 600 pL / h and the solution of 4-arm-polyethylene glycol-succinimidyl glutarate ester was pumped in the outer needle of a coaxial nozzle with a pump rate of 110 pL / h.

[0138] For coaxial fiber generation, a 16 Gauge dosing needle (VIEWEG, Germany) was used. The inner needle had an outer diameter of 0.9 mm and an inner diameter of 0.6 mm. Dosing needle and inner needle ended at the same point. Spinning was conducted for 2 h.

[0139] The obtained fibers were dried in a fume hood at 22 ± 1 °C and a relative humidity of 25 ± 5% for 24 hours after spinning.

[0140] Example 5 (comparative example): Production of fibers using collagen-like proteins and polyethylene oxide for coaxial fiber generation

[0141] A control sample without crosslinker was prepared, using the same concentration of CLP and polyethylene oxide as describe in the previous examples. Instead of the crosslinker solution, water was pumped in the outer needle of the coaxial nozzle. Spinning was conducted for 2 h The obtained control fibers were dried in a fume hood at 22 ± 1 °C and a relative humidity of 25 ± 5% for 24 hours after spinning. 202400137 16

[0142] Example 6: Bicinchoninic acid assay (BCA assay)

[0143] Fibers with a CLP content equivalent to 10 mg were weighed in 30 mL beakers and 10 mL of doubledistilled water were added. The fibers of example 2, 3, 4 and the control fibers of example 5 were used. In all cases the supernatant was recovered after 1 h, 3 h and 24 h of incubation. To determine the amount of CLP dissolved into the solution, an aliquot (25 pL) of the supernatant was analyzed using the BCA assay (Life technologies GmbH, Germany). Samples were prepared in triplicate. The colorimetric solution was measured at a wavelength of 562 nm in a Tecan reader (Tecan trading GmbH, Switzerland).

[0144] After 24 h those fibers with the DMTMM crosslinker of example 2 and 3 showed no CLP leaching, whereas the control fibers of example 5 were completely dissolved (s. figure 1 and 2). While the crosslinking with DMTMM reached a full stabilization, those fibers with the 4-Arm-polyethylene glycol- succinimidyl glutarate ester crosslinker of example 4 led to a partial stabilization (s. figure 3) making them interesting for a wound healing application, as a fast release of CLP can promote the wound healing and cell proliferation, while the mesh acts as a dressing to protect and regenerate the tissue.

[0145] Example 7: Investigation of the morphology after water addition

[0146] To investigate if the fibers morphology changed after water contact, they were treated with a droplet of double-distilled water and after 2.5 h dried in a freeze dryer to remove all residual water. Afterwards the morphology of the fibers was investigated via a scanning electron microscope.

[0147] Figure 4 shows the morphology of the fibers of example 2 before and after water contact. The fibers showed swelling, but no complete loss of structure. Figure 5 shows the morphology of the fibers of example 3 before and after water contact. The fibers of example 3 showed greater swelling than the fibers of example 2, which means that the degree of swelling can be controlled by adjusting the amount of crosslinker. Figure 6 shows the morphology of the fibers of example 4 before and after water contact. The fibers of example 4 showed swelling. In figure 6 the fibers are still visible, while generating a uniform film on the surface. As a comparison, the control fibers of example 5 showed a complete loss of the structure after water contact (figure 7).

[0148] Example 8: Time advantage of the present invention

[0149] As the crosslinking reaction of the present invention only starts at the tip of the spinning head due to the separate storage and pumping of the solution of the at least one CLP and the at least one carrier and the solution of the at least one crosslinker, there is no limited time window compared to methods in which the substance to be crosslinked and the crosslinker are already mixed before spinning and then spun. In order to emphasize this time advantage, the time required to reach crosslinking after mixing was measured.

[0150] First it was measured for the procedure of example 2. One mL of the solution of CLP and polyethylene oxide was mixed with 0.183 mL of the DMTMM solution, both at the concentration described in example 2, on a magnetic stirrer at 100 rpm. After 8 minutes, the solution was completely gelled and could no 202400137 17 longer be spun. In contrast the coaxial spinning process according to the invention ran for 2 h until it was stopped on purpose.

[0151] The same time advantage could be shown for the procedure of example 3 as well. One mL of the solution of CLP and polyethylene oxide was mixed with 0.183 mL of the DMTMM solution, both at the concentration described in example 3, on a magnetic stirrer at 100 rpm. After 26 minutes, the solution was completely gelled and could no longer be spun. In contrast the coaxial spinning process according to the invention ran for 2 h until it was stopped on purpose.

Claims

202400137 18Claims1 . An electrospun article comprising or consisting of fibers which comprise at least one collagen- like protein, at least one carrier and at least one crosslinker.

2. The electrospun article according to claim 1 , whereinI) the weight of the at least one collagen-like protein is 99.78 to 28.39 wt% and / or the weight of the at least one crosslinker is 71.61 to 0.22 wt%, wherein the total weight of the at least one collagen-like protein and the at least one crosslinker is 100 wt% and / orII) the at least one collagen-like protein is 95.8 to 0.28 wt% and / or the remainder is the at least one carrier, wherein the total weight of the at least one collagen-like protein and the at least one carrier is 100 wt%.

3. The electrospun article according to any of the preceding claims, wherein the at least one collagen-like-protein i) is obtained from pichia pastoris or Corynebacterium, and / or ii) is derivable from a polynucleotide encoding an amino acid sequence that is at least > 60% identical to the amino acid sequence of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

4. The electrospun article according to any of the preceding claims, wherein the at least one crosslinker is selected from a compound which comprises at least two, preferably two to six, more preferably two to four, most preferably four, groups each comprising a polyalkylene glycol moiety, preferably polyethylene glycol moiety or polypropylene glycol moiety, more preferably polyethylene glycol moiety, and a succinimidyl group; organic triazines; organic aldehydes; aglycones; diisocyanates; carbodiimides or mixtures thereof.

5. The electrospun article according to any of the preceding claims, wherein the at least one carrier is selected from polyethylene oxide, polysaccharides, polyvinyl alcohols, polyvinylpyrrolidone, gelatine or a mixture thereof.

6. The electrospun article according to any of the preceding claims, wherein the at least one carrier has a weight average molecular weight of 40.000 to 2.500.000 g / mol, measured via GPC using polystyrene standards.

7. A method for obtaining an electrospun article, preferably according to any of the preceding claims, comprising or consisting of the following steps a) providing at least one collagen-like protein in at least one solvent to obtain a solution of the at least one collagen-like protein, b) adding at least one carrier to the solution of the at least one collagen-like protein to obtain a solution of the at least one collagen-like protein and the at least one carrier,202400137 19 c) providing at least one crosslinker in at least one solvent to obtain a solution of the at least one crosslinker, d) pumping the solution of the at least one collagen-like protein and the at least one carrier in the inner needle of a coaxial nozzle, e) pumping the solution of the at least one crosslinker in the outer needle of a coaxial nozzle, f) electrospinning the solution of the at least one collagen-like protein and the at least one carrier and the solution of the at least one crosslinker to obtain the electrospun article, g) optionally drying the electrospun article obtained in step f).

8. The method according to claim 7, wherein in step a) the at least one solvent is a mixture of water and acetone with a volume ratio of 7:3 to 9:1 and / or the concentration of the at least one collagen-like protein in the solution is 10 to 200 mg / mL.

9. The method according to claim 7, wherein in step b) the at least one carrier has a weight of 0.5 to 3 wt% related to the total weight of the solution of the at least one collagen-like protein and the at least one carrier.

10. The method according to claim 7, wherein in step c) the solvent is water and / or the concentration of the at least one crosslinker in the solution is 20 to 300 mg / mL.11 . The method according to claim 7, wherein the pump rate of step d) is 300 to 4000 pL / h and / or the pump rate of step e) is 20 to 300 pL / h.

12. The method according to claim 7, wherein the pump rate ratio of the solution of the at least one collagen-like protein and the at least one carrier of d) to the solution of the at least one crosslinker of e) is 1 :1 to 15:1.

13. An article obtainable by the method according to any of the claims 7 to12.

14. The article according to claim 13, wherein the article is a medical device.

15. Use of the article according to any of the claims 13 and 14 for wound healing and cell culture applications.

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