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EP2609109A2 - Procédé pour produire des solutions hautement concentrées de protéines à auto-assemblage - Google Patents

Procédé pour produire des solutions hautement concentrées de protéines à auto-assemblage

Info

Publication number
EP2609109A2
EP2609109A2 EP11752173.2A EP11752173A EP2609109A2 EP 2609109 A2 EP2609109 A2 EP 2609109A2 EP 11752173 A EP11752173 A EP 11752173A EP 2609109 A2 EP2609109 A2 EP 2609109A2
Authority
EP
European Patent Office
Prior art keywords
protein
self
assembling
chaotrope
stable
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP11752173.2A
Other languages
German (de)
English (en)
Inventor
Evgueni Klimov
Burghard Liebmann
Thomas Subkowski
Martin Möller
Doris Klee
Artem Davidenko
Wiebke Voigt
Gunter Scharfenberger
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BASF SE
Carl Freudenberg KG
Original Assignee
BASF SE
Freudenberg Forschungsdienste KG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BASF SE, Freudenberg Forschungsdienste KG filed Critical BASF SE
Priority to EP11752173.2A priority Critical patent/EP2609109A2/fr
Publication of EP2609109A2 publication Critical patent/EP2609109A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L89/00—Compositions of proteins; Compositions of derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09D—COATING COMPOSITIONS, e.g. PAINTS, VARNISHES OR LACQUERS; FILLING PASTES; CHEMICAL PAINT OR INK REMOVERS; INKS; CORRECTING FLUIDS; WOODSTAINS; PASTES OR SOLIDS FOR COLOURING OR PRINTING; USE OF MATERIALS THEREFOR
    • C09D189/00—Coating compositions based on proteins; Coating compositions based on derivatives thereof
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0015—Electro-spinning characterised by the initial state of the material
    • D01D5/0053—Electro-spinning characterised by the initial state of the material the material being a low molecular weight compound or an oligomer, and the fibres being formed by self-assembly
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F1/00—General methods for the manufacture of artificial filaments or the like
    • D01F1/02—Addition of substances to the spinning solution or to the melt
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01F—CHEMICAL FEATURES IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS; APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OF CARBON FILAMENTS
    • D01F4/00—Monocomponent artificial filaments or the like of proteins; Manufacture thereof
    • D—TEXTILES; PAPER
    • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
    • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
    • D06M15/00—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment
    • D06M15/01—Treating fibres, threads, yarns, fabrics, or fibrous goods made from such materials, with macromolecular compounds; Such treatment combined with mechanical treatment with natural macromolecular compounds or derivatives thereof
    • D06M15/15—Proteins or derivatives thereof

Definitions

  • the present invention relates to stable aqueous protein dispersions, comprising in an aqueous phase at least one self-assembling protein in dispersed form and at least one special dispersing agent for the self-assembling protein; Process for the preparation of such stable aqueous dispersions; Process for the electrospinning of self-assembled proteins using such stable aqueous dispersions; Process for the production of fiber surface formations or fibers from such aqueous dispersions; the use of such aqueous dispersions for coating surfaces; the use of the materials produced by electro-spinning for the production of medical devices, hygiene articles and textiles; and fibrous or fibrous webs made by an electrospinning process of the present invention.
  • the electrospinning process (also referred to as “electrospinning” or “electrospinning”) is a preferred method of making nano and meso fibers.
  • electrospinning is a preferred method of making nano and meso fibers.
  • a polymer melt or a polymer solution is exposed to a high electric field at an edge serving as an electrode. This can be achieved, for example, by extruding the polymer melt or polymer solution in an electric field under low pressure through a cannula connected to one pole of a voltage source.
  • Natural starting materials such as biopolymers or synthetic polymers derived therefrom, can also be processed by electrospinning.
  • the processing of spider silk proteins of the spider Nephila clavipes from a hexafluoro-2-propanol solution into nanofibers has been described by electrospinning by Zarkoob and Reneker (Polymer 45: 3973-3977, 2004).
  • Spin tests of Bombyx mori silk from a formic acid solution are described by Sukigara and Ko (Polymer 44: 5721-572, 2003), whereby the fiber morphology is influenced by varying the electrospinning parameters.
  • Jin and Kaplan reported water-based electrospinning of silk or silk / polyethylene oxide (Biomacromolecules 3: 1233-1239, 2002).
  • WO-A-03/060099 describes various methods (including electrospinning) and apparatuses for spinning Bombyx mori silk proteins and spider silk proteins.
  • the spider silk proteins used were recombinantly produced by transgenic goats and purified from their milk and then spun.
  • Synthetic biopolymers composed of repititive units of the insect protein Resilin or of the spider silk protein designated R16 or S16 are described in WO2008 / 155304 of the present Applicant.
  • the present in the form of microbeads polymers may, for. B. converted into gel-like products or processed into protein films.
  • polymers e.g. synthetic biopolymers or other spinnable organic polymers, or matched polymer blends thereof, optionally in admixture with pharmaceutical or agrochemical active ingredients is described in WO 2010/015709 and WO 2010/015419 of the present applicant.
  • solutions of the polymers in organic solvents or concentrated formic acid are used.
  • the object of the invention is to develop systems which can be sprayed, which make it possible to stabilize the protein at a higher concentration in the liquid phase to be spun.
  • a first solution according to the invention of avoiding precipitation of the protein from the aqueous solution involves the stabilization of the hydrophobic protein with a hydrophilic protein which also contains hydrophobic moieties in the structure.
  • BSA bovine serum albumin
  • BSA is a soluble protein that acts as a transporter for fatty acids and lipids in the blood.
  • BSA consists of 607 amino acids and has a molecular mass of about 69.4 kDa.
  • Non-fat BSA ff. BSA
  • BSA is a particular embodiment of BSA in which additional hydrophobic sites are present by removal of fatty acids.
  • a second solution according to the invention of the above problem comprises the stabilization of the hydrophobic protein with the aid of suitable protein fragments (peptides).
  • the protein fragments according to the invention have both hydrophobic and hydrophilic sequence regions.
  • two peptide-based systems are provided: a) Stabilization of the native R16 protein by means of protein fragments of the R16 protein.
  • the respective hydrolytic cleavage of the protein for the preparation of the stabilizing protein fragments is carried out in a conventional manner, for. B. by means of a NaOH solution at 80 ° C.
  • a third solution according to the invention of the above problem comprises the stabilization of the hydrophobic protein with the aid of suitable synthetic organic oligomers, which represent known verbi ndu ngen gene and z. B. in WO2010 / 057654, the disclosure of which is incorporated herein by reference. These oligomers also have both hydrophobic and hydrophobic le areas and stabilize the hydrophobic proteins in aqueous solution even at elevated concentrations.
  • FIG. 1 shows the mass spectrum (Maldi-ToF) of an R16 protein hydrolyzate according to the invention.
  • FIG. 2 shows the mass spectrum (Maldi-ToF) of a BSA protein hydrolyzate according to the invention.
  • FIG. 3 shows electron micrographs of fibers obtained by electrospinning of R16 protein solutions stabilized with BSA and additionally added to increase the viscosity with polyethylene oxide polymer (PEO);
  • FIG. 3a shows the result of spinning a dispersion of R16 protein, BSA and PEO with a solids content of the components of 42.5%, 42.5% and 15%, respectively;
  • FIG. 3a shows the result of spinning a dispersion of R16 protein, BSA and PEO with a solids content of the components of 42.5%, 42.5% and 15%, respectively;
  • FIG. 3b shows the result of a mixture of these three components, but with a solids content of 37%, 37% and 16%, respectively; and
  • Figures 3c and d show the result of spinning a mixture of these three components with solids contents of 34.5%, 34.5% and 31% respectively, spun at a rate of 0.4 ml / h ( Figure 3c) and 0.5 ml / h ( Figure 3d).
  • FIG. 4 shows electron micrographs of fibers obtained by electrospinning an aqueous dispersion of R16 protein stabilized with the aid of peptide fragments of the R16 protein;
  • FIG. 4 a shows the spinning of a mixture of an R 16 protein, R 16 fragment and PEO with a solids content of these components of 61%, 0.003% and 39%, respectively;
  • FIG. 4b shows the result of a spinning of these three components with a solids content of the components of 74%, 0.004% and 26%, respectively.
  • FIG. 5 shows electron micrographs of fibers obtained by electrospinning an R16 protein solution stabilized with BSA peptide fragments.
  • Figures 5a and b show the same fibers at different magnifications.
  • FIG. 6 shows an electron micrograph of fibers obtained by electrospinning an R16 protein solution stabilized with an amphiphilic oligomer of the formula 1 according to the invention.
  • FIG. 7 shows the in vitro activity according to the invention of the R16 protein on the cell proliferation of fibroblasts. The time-dependent change in the relative cell number at different concentrations of R16 protein fragments compared to the control (Ktrl, without such fragments) is shown. Detailed description of the invention
  • Amphiphile describes the chemical property of a substance to be both hydrophilic and lipophilic, which is why it is based on nonpolar Lkipedia.org / wiki / solutions that the substance has both hydrophilic and hydrophobic regions.
  • Chaotropic refers to chemical substances, such as barium salts, guanidine hydrochloride, thiocyanates, such as guanidinium thiocyanate, perchlorates, which dissolve ordered hydrogen bonds in water, breaking the hydrogen bonds and disrupting the chaotropic substances the water structure and provide an increase in entropy.
  • amino acids they thus reduce hydrophobic effects and have a denaturing effect on proteins, since the driving force of protein folding is the assembly of the hydrophobic amino acids in the water.
  • a “dispersion” is a heterogeneous mixture of at least two substances that do not or hardly dissolve into each other or that chemically combine with each other, so that an aqueous protein dispersion is a mixture of aqueous medium (the dispersion medium) and solid protein (the disperse phase). and thus may also be referred to as "aqueous protein suspension”.
  • a “carrier polymer” is understood to mean biopolymers or their mixtures, or mixtures of at least one synthetic polymer and one biopolymer, the carrier polymer having the ability to enter into noncovalent interactions with the active substance (s) to be formulated or particulate To enclose (carry) active ingredients (dispersed or crystalline) or adsorb.
  • an "active ingredient” or “effect substance” is understood as meaning synthetic or natural, low molecular weight substances having hydrophilic, lipophilic or amphiphilic properties which can be used in the agrochemical, pharmaceutical, cosmetic or food and feed industries; as well as biologically active macromolecules which can be embedded in or adsorbed to a fibrous sheet of the invention, such as e.g. Peptides (such as oligopeptides having 2 to 10 amino acid residues and polypeptides having more than 10, such as 1 to 100 amino acid residues) and enzymes and single or double-stranded nucleic acid molecules (such as oligonucleotides having 2 to 50 nucleic acid tests and polynucleotides having more than 50 nucleic acid residues) ,
  • fiber webs encompasses both individual polymer fibers and also the ordered or disordered one or more layered assemblage of a multiplicity of such fibers, for example fiber nonwovens or nonwoven webs.
  • molecular weight data for polymers are Mn or Mw values 2.
  • a stable aqueous protein dispersion comprising, in an aqueous phase, at least one self-assembled natural, synthetic or recombinantly produced protein in dispersed form, and at least one dispersant for the self-assembling protein, wherein the dispersant is a polymeric dispersant selected from amphiphilic proteins, or an oligomeric dispersant , selected from amphiphilic peptide fragments and / or amphiphilic organic oligomers.
  • the self-assembling protein is a microbead-forming or intrinsically unfolded protein, in particular a silk protein, such as a spider silk protein, or an insect protein (such as Resilin) or a self-assembling analog derived from at least one of these proteins having a sequence identity of at least about 60% (based on the starting protein (s)).
  • R16 protein comprising an amino acid sequence according to SEQ ID NO: 4; b) S16 protein comprising an amino acid sequence according to SEQ ID NO: 6; c) spinnable analog proteins derived from these proteins having a sequence identity of at least about 60%, e.g. at least about 70, 80, 90, 95, 96, 97, 98 or 99%, to SEQ ID NO: 4 or 6, (eg also by insertion or attachment of oligo-amino acid blocks, such as oligo-arginine blocks (1 4.
  • a stable aqueous dispersion according to any one of the preceding embodiments, wherein the amphiphilic peptide fragment comprises a fragment of a precursor protein.
  • the amphiphilic organic oligomer is a block co-oligomer comprising ether blocks, comprising at least one hydrophobic ether oligomer block (in particular having at least one hydrophobic side groups), and at least one hydrophilic ether oligomer block (in particular having at least one hydrophilic side groups).
  • each of the blocks is homogeneous, i. composed of essentially identical monomer building blocks.
  • Stable aqueous dispersion containing at least one self-assembling protein in a proportion in the range of 1 to 40 wt .-%, in particular 2 to 30, 3 to 25 or 5 to 20 wt .-%, based on the total weight of stable dispersion, optionally together with 0.01 to 50 wt .-%, in particular 0.05 to 30, 0.08 to 20 or 0, 1 to 10 wt .-%, of at least one further formulation or processing aid.
  • a process for electrospinning self-assembling protein comprising electrospinning a stable aqueous dispersion according to any of embodiments 1 to 10 or prepared according to any of embodiments 11 to 18.
  • 20. A process for producing a fiber fabric or fibers comprising at least one self-assembling protein, wherein an aqueous dispersion according to one of embodiments 1 to 10, or prepared according to one of embodiments 1 1 to 18, electrospun into a fiber fabric.
  • viscosity-adjusting agents such as dispersible or dispersible organic / synthetic or bio-polymers in the dispersion
  • a stable, aqueous dispersion according to one of embodiments 1 to 10 for the coating, in particular spray or dip coating or coatings in film form, of surfaces, in particular nonwovens, fibers and foams.
  • Particularly useful self-assembling proteins are, in particular, silk proteins. According to the invention, this refers below to those proteins which contain highly repetitive amino acid sequences and are stored in the animal in a liquid form and whose secretion by shearing or spinning results in fibers (Craig, CL (1997) Evolution of arthropod Silks. Annu. Rev. Entomol 42: 231-67).
  • spider silk proteins which in their original form could be isolated from spiders, such as from the "major ampullate” gland, such as ADF3 and ADF4 from the Araneus "Major Ampullate” gland diadematus (Guerette et al., Science 272, 5258: 1 12-5 (1996)).
  • Equally suitable proteins are natural or synthetic proteins that are derived from natural silk proteins and that have been produced heterologously in prokaryotic or eukaryotic expression systems using genetic engineering techniques.
  • prokaryotic expression organisms are Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum, etc.
  • Nonlimiting examples of eukaryotic expression organisms are yeasts such as Saccharomyces cerevisiae, Pichia pastoris and others, filamentous fungi such as Aspergillus niger, Aspergillus oryzae and Aspergillus nidulans, Trichoderma reesei, Acremonium chrysogenum and others, mammalian cells, such as Heia cells, COS cells, CHO cells, among others, insect cells, such as Sf9 cells, MEL cells and others.
  • synthetic proteins which are based on repeating units of natural silk proteins.
  • synthetic repetitive silk protein sequences these may additionally contain one or more natural non-repetitive silk protein sequences (Winkler and Kaplan, J Biotechnol 74: 85-93 (2000)).
  • spider silk proteins are also useful which are based on repeating units of natural spider silk proteins.
  • synthetic repetitive In addition to spider silk protein sequences, these may additionally contain one or more natural non-repetitive spider silk protein sequences.
  • functional equivalents are in particular also understood as meaning mutants which, in at least one sequence position of the abovementioned amino acid sequences, have a different amino acid than the one specifically mentioned, but nevertheless possess the property of packaging effect substances.
  • “Functional equivalents” include, but are not limited thereto one or more of the additional amino acid additions, substitutions, deletions, and / or inversions of available mutants, wherein said changes may occur in any sequence position as long as they result in a mutant having the property profile of the invention. Functional equivalence is especially given when the reactivity patterns between mutant and unchanged polypeptide are qualitatively consistent.
  • Precursors are natural or synthetic precursors of the polypeptides with or without the desired biological activity. Examples of suitable amino acid substitutions are shown in the following table:
  • Salts are understood as meaning both salts of carboxyl groups and acid addition salts of amino groups of the protein molecules of the invention.
  • Salts of carboxyl groups can be prepared in a manner known per se and include inorganic salts such as, for example, sodium, calcium, ammonium, egg salts and salts with organic bases, such as, for example, amines, such as triethanolamine, arginine, lysine, piperidine, etc.
  • Acid addition salts for example salts with mineral acids, such as hydrochloric acid or sulfuric acid, and salts with organic acids, such as acetic acid and Oxalic acid are also the subject of the invention.
  • “Functional derivatives” of polypeptides of the invention may also be produced at functional amino acid side groups or at their N- or C-terminal end by known techniques
  • Such derivatives include, for example, aliphatic esters of carboxylic acid groups, amides of carboxylic acid groups, obtained by reaction with ammonia or with a primary or secondary amine; N-acyl derivatives of free amino groups prepared by reaction with acyl groups; or O-acyl derivatives of free hydroxy groups prepared by reaction with acyl groups.
  • Homologs to the specific proteins / polypeptides disclosed herein include at least 60%, such as 70, 80, or 85%, such as 90, 91, 92, 93, 94, 95, 96, 97 , 98 or 99% identity to one of the specifically disclosed amino acid sequences.
  • identity between two sequences is meant, in particular, the identity of the residues over the respective entire sequence length, in particular the identity which is determined by comparison with the aid of the Vector NTI Suite 7.1 (Vector NTI Advance 10.3.0, Vitrogen Corp.) (or Software of the company Informax (USA) using the Clustal method (Higgins DG, Sharp PM, Fast and sensitive multiple sequence alignments on a microcomputer, Appl. Appl. Biosci, 1989 Apr; 5 (2): 151-1) with the following setting Parameter is calculated: Multiple alignment parameter:
  • Stable aqueous dispersion are especially made using polymeric or oligomeric dispersants.
  • Polymeric dispersants are in particular selected from globulins, in particular albumins, in particular bovine serum albumin (BSA) and fat-free preparations thereof (ff BSA) and are commercially available as such.
  • Albumins have a molecular mass of about 66,000 Da and consist of 584 to 590 amino acids. Due to a high content of cysteine, the albumins have a relatively high sulfur content.
  • Albumins are water-soluble, their binding capacity for water is about 18 ml / g. The isoelectric point is at pH 4.6. Albumins are ampholytes, that is, they can reversibly bind both anions and cations.
  • Oligomeric dispersants are, in particular, amphiphilic peptide fragments of the natural and synthetic silk proteins described above, and in particular of R16 and S16 proteins; as well as amphiphilic peptide fragments of the mentioned Globu- line, in particular albumins, especially BAS or ff BSA.
  • Such fragments can be prepared by controlled staging of the starting proteins. For example, a suitable amount of the protein can be weighed into a test tube and admixed with 0.2 M NaOH solution. The test tube is tightly closed and the mixture is heated in a water bath to about 80 ° C internal temperature. The resulting mixture is stirred vigorously. After some time, the protein begins to dissolve in the NaOH solution. Once the protein is dissolved, the sample is removed from the water bath and cooled and analyzed analytically. The protein hydrolyzate preparable in this manner provides a mixture of peptide fragments having a molecular weight in the range of about 500 to 5,000, such as e.g. 1,000 to 3,000 or 600 to 4,000, as readily detectable by mass spectrometry (e.g., by Maldi-ToF).
  • mass spectrometry e.g., by Maldi-ToF
  • Oligomeric dispersants are, in particular, also block co-oligomers comprising ether units, which comprise the lowest-possible hydrofluorophores.
  • Oligomer block in particular having at least one hydrophobic side groups
  • at least one hydrophilic ether oligomer block in particular having at least one hydrophilic side groups
  • each of the blocks is homogeneous, ie composed of substantially identical monomer building blocks.
  • a specific group of block co-oligomer can be represented by the following general formula (A)
  • blocks 1 and 2 are different and one of blocks 1 and 2 has hydrophilic side groups and the other has hydrophobic side groups,
  • R 1 is H or straight-chain or branched C 1 -C 6 -alkyl, aryl or straight-chain or branched C 1 -C 6 -alkylaryl, where aryl may optionally be substituted, and in particular represents straight-chain or branched C 1 -C 4 -alkyl or straight-chain or branched C -C 4 -alkylphenyl ;
  • the side group radicals R 2 and R 3 are different and are selected from hydrophobic radicals, in particular straight-chain or branched C 1 -C 6 -alkyl, aryl or straight-chain or branched C 1 -C 6 -alkylaryl; or are selected from H and hydrophilic radicals, such as - (CH 2 ) P -COOH, - (CH 2 ) P -COO " X + , wherein X + is H + or a metal cation, such as an alkali metal cation, in particular Na + or K + , and p stands for an integer value such as 1, 2 or 3;
  • the side group radicals R 2 and R 3 are the same or within the blocks 1 and / or 2, the side group radicals R 2 and / or R 3 may each be different and thereby within a hydrophilic or hydrophobic block least two forming various hydrophilic or hydrophobic sub-blocks, each sub-block having at least 2 to 5 identical side-group residues; and
  • R 4 is H or CC 6 alkyl, in particular H is C 1 -C 6 -alkyl is, for example, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1, 1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1, 1-dimethylpropyl, 1, 2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1, 1-dimethylbutyl, 1, 2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl, 1, 1, 2-trimethylpropyl
  • Aryl is particularly suitable for naphthyl or phenyl.
  • C 1 -C 6 -alkylaryl represents the aryl, especially phenyl-substituted, analogs of the above C 1 -C 6 -alkyl radicals, in particular unbranched C 1 -C 6 -alkyl radicals.
  • Aryl substituents are, in particular, C 1 -C 4 -alkyl radicals as defined above
  • Preferred examples of such oligomers are compounds of the following formulas (1) to (5)
  • a viscosity-increasing additive for better processing of stabilized aqueous protein dispersions according to the invention in electro-spinning, it may be expedient to add a viscosity-increasing additive to this dispersion.
  • suitable polymers include: polyvinyl alcohol, polyvinylformamide, polyvinylamine, polycarboxylic acid (polyacrylic acid, polymethacrylic acid), polyacrylamide, polyitaconic acid, poly (2-hydroxyethyl acrylate), poly (N-isopropylacrylamide), polymethacrylamide, polyalkylene oxides, eg.
  • polyethylene oxides Poly-N-vinylpyrrolidone; hydroxymethylcellulose; Hydroxyethylcellulose; hydroxypropyl cellulose; carboxymethyl cellulose; alginate; collages; Gelatin, poly (ethyleneimine), polystyrenesulfonic acid; Combinations of two or more of the aforementioned polymers; Copolymers containing one or more of the monomer units forming the aforementioned polymers, graft copolymers comprising one or more of the monomer units forming the aforementioned polymers.
  • the water-soluble polymer is selected from polyethylene oxide, polyvinyl alcohol, polyvinylformamide, polyvinylamine and poly-N-vinylpyrrolidone.
  • the molecular weight of the polymers used can vary over a wide range and is for example in the range of 500 to 2,000,000, or 1, 000 to 1, 000,000 or 10,000 to 500,000.
  • water-soluble polymers are commercially available or can be prepared according to processes known to those skilled in the art.
  • the protein solution or dispersion to be used in the method according to the invention contains, based on the total solids of the solution or dispersion, 0.01 to 40% by weight, such as 0.5 to 20% by weight. or 2 to 15% by weight, at least one water-soluble polymer as defined above.
  • the weight ratio of protein to the water-soluble polymer present in the solution or dispersion depends on the polymers used.
  • the protein and the water-soluble polymer employed may be in a weight ratio of from about 300: 1 to about 1: 5, e.g. from about 100: 1 to about 1: 2, or from about 20: 1 to about 1: 1.
  • Suitable synthetic polymers are, for. B. selected from the group consisting of homo- and copolymers of aromatic vinyl compounds, homopolymers and copolymers of alkyl acrylates, homo- and copolymers of alkyl methacrylates, homopolymers and copolymers of ⁇ -olefins, homopolymers and copolymers of aliphatic see serving , Homo- and copolymers of vinyl halides, homo- and copolymers of vinyl acetates, homo- and copolymers of acrylonitriles, homopolymers and copolymers of urethanes, homopolymers and copolymers of vinyl amides and copolymers composed of two or more of the monomer units forming the abovementioned polymers.
  • Suitable carrier polymers are, in particular, polymers based on the following monomers: Acrylamide, adipic acid, allyl methacrylate, alpha-methylstyrene, butadiene, butanediol, butanediol dimethacrylate, butanediol divinyl ether, butanediol dimethacrylate, butanediol monoacrylate, butanediol monomethyl ether, butyl acrylate, butyl methacrylate, cyclohexyl vinyl ether, diethylene glycol divinyl ether, diethylene glycol monovinyl ether, ethyl acrylate, ethyl diglycol acrylate, ethylene, ethylene glycol butyl vinyl ether, Ethylene glycol dimethacrylate, ethylene glycol divinyl ether, ethylhexyl acrylate, ethylhexyl methacrylate,
  • polymers encompasses both homopolymers and copolymers Suitable copolymers include both random and alternating systems, block copolymers or graft copolymers
  • copolymers encompasses polymers which are composed of two or more different monomers or in which the incorporation of at least one monomer in the polymer chain can be realized in various ways, as is the case, for example, in the stereo block copolymers.
  • the homo- and copolymers can be miscible and immiscible.
  • the following polymers are preferably mentioned:
  • Polyvinyl ethers such as polybenzyloxyethylene, polyvinyl acetals, polyvinyl esters such as polyvinyl acetate, polyoxytetramethylene, polyamides, polycarbonates, polyesters, polysiloxanes, polyurethanes, polyacrylamides such as poly (N-isopropylacrylamide), polymethacrylamide, polyhydroxybutyrates, polyvinyl alcohols, acetylated polyvinyl alcohols, polyvinyl nylformamide, polyvinylamines, polycarboxylic acids (polyacrylic acid, polymethacrylic acid), polyacrylamide, polyitaconic acid, poly (2-hydroxyethyl acrylate), poly (N-isopropylacrylamide), polysulfonic acid (poly (2-acrylamido-2-methyl-1-propanesulfonic acid) or PAMPS), polymethacrylamide, polyalkylene oxides, e.g.
  • polyethylene oxides For example, polyethylene oxides; Poly-N-vinylpyrrolidone; Maleic acids, poly (ethyleneimine), polystyrenesulfonic acid, polyacrylates, such as, for example, polyphenoxyethyl acrylate, polymethyl acrylate, polyethyl acrylate, polydodecyl acrylate, poly (ibornyl acrylate), poly (n-butyl acrylate), poly (t-butyl acrylate), polycyclohexyl acrylate, poly (2 ethylhexyl acrylate), polyhydroxypropyl acrylate, polymethacrylates, such as.
  • polyacrylates such as, for example, polyphenoxyethyl acrylate, polymethyl acrylate, polyethyl acrylate, polydodecyl acrylate, poly (ibornyl acrylate), poly (n-butyl acrylate), poly (t-butyl acrylate), polycyclohex
  • Polymethylmethacrylate poly (n-amylmethacrylate), poly (n-butylmethacrylate), polyethylmethacrylate, poly (hydroxypropylmethacrylate), polycyclohexylmethacrylate, poly (2-ethylhexylmethacrylate), polylaurylmethacrylate, poly (t-butylmethacrylate), polybenzylmethacrylate, poly (ibornylmethacrylat), polyglycidyl methacrylate and polystearyl methacrylate, polystyrene, and copolymers based on styrene, for example with maleic anhydride, styrene-butadiene copolymers, methyl methacrylate-styrene copolymers, N-vinylpyrrolidone copolymers, polycaprolactones, polycaprolactams, poly (N-vinylcaprolactam ).
  • poly-N-vinylpyrrolidone polymethyl methacrylate
  • acrylate-styrene copolymers polyvinyl alcohol, polyvinyl acetate, polyamide, polyester may be mentioned.
  • biodegradable polymers are still applicable.
  • biodegradable polymers is intended to include all polymers which meet the definition of biodegradability given in DIN V 54900, in particular compostable polyesters.
  • biodegradability means that the polymers, such as polyesters, decompose in a reasonable and detectable time. Degradation may be hydrolytic and / or oxidative, and for the most part effected by the action of microorganisms such as bacteria, yeasts, fungi and algae.
  • the biodegradability can be determined, for example, by mixing polyesters with compost and storing them for a certain period of time. For example, in accordance with ASTM D 5338, ASTM D 6400 and DI NV 54900, C0 2 -free air is allowed to flow through ripened compost during composting and subjected to a defined temperature program.
  • biodegradability is determined by the ratio of the net C0 2 release of the sample (after deduction of C02 release by the compost without sample) to the maximum C0 2 release of the sample (calculated from the carbon content of the sample) as biological Degradability defined.
  • Biodegradable polyesters generally show clear signs of degradation after only a few days of composting, such as fungal growth, cracking and puncture. education.
  • biodegradable polymers are biodegradable polyesters such as, for example, polylactide, polycaprolactone, polyalkylene adipate repthalates, polyhydroxyalkonates (polyhydroxybutyrate) and polylactide glycoside.
  • biodegradable polyalkylene adipate terephthalates preferably polybutylene nadipate terephthalates.
  • Suitable polyalkylene adipate terephthalates are, for. As described in DE 4 440 858 (and are commercially available, eg Ecoflex® from BASF).
  • Active Ingredients The stabilized protein dispersions prepared according to the invention can also be used to produce active substance-containing or effect-containing fibers or fabrics containing such fibers, such as films or nonwovens.
  • hydrophilic and hydrophobic substances can be formulated.
  • formulatable classes of substances are: proteins, peptides, nucleic acids, mono-, di-, oligo- and polysaccharides, proteoglycans, lipids, organic polymers, low molecular weight synthetic or natural organic or inorganic substances or chemical elements, such as e.g. Silver.
  • Such formulations are particularly applicable in cosmetics, human and veterinary medicine but also in the field of crop protection.
  • Colorants fatty acids, carotenoids, retinoids, vitamins and their precursors, antioxidants, lipoic acids, UV light protection filters, peroxide decomposers, such as those used in the field of cosmetics or medicine; as well as derivatives and precursors thereof.
  • Pharmaceutical agents for therapeutic or diagnostic purposes such as anti-irritants, anti-inflammatories, vasoactive agents, anti-infective agents, anesthetizing agents, growth-promoting agents; and derivatives and precursors thereof;
  • Wound healing-promoting agents and active ingredients which have a positive effect on wound healing
  • Antimicrobial, antibacterial or antiviral agents are provided.
  • Antibodies enzymes, peptides, nucleic acids, growth factors.
  • Crop protection agents such as e.g. those with herbicidal, insecticidal and / or fungicidal action.
  • Electrospinning The protein solution was spun with the aid of the nozzle-based electrospinning plant (Gimpel Ingenieur-Gesellschaft mbH www.gimpel.de). As high voltage source a generator of the company Eltex, type KNH34 / N2A from 0-30 kV, DC neg was used. The protein solution was extruded in an electric field at low pressure through a cannula connected to the pole of a voltage source. Due to the electrostatic charge of the protein solution due to the electric field, a material flow directed towards the counterelectrode formed, which solidified on the way to the counterelectrode and deposited in the form of thin fibers.
  • Cannula diameter 0.8 or 0.9 mm.
  • Electro-spinning systems are transferred.
  • Both matrices were used as saturated solutions (20 mg / ml) dissolved in TA. Composition of the TA solution
  • Preparation methods were both the “cover method” and the “mix method”.
  • the sample was mixed with matrix after dissolving in buffer 1:10 and a defined volume was applied to the target the “mix method” was 10, whereas the “cover method” was diluted 1: 1.
  • the high salt content of the samples was reduced by solid phase extraction.
  • Zip-Tip pipette tips from Applied Biosystems with a cis-occupancy were used.
  • the zip-tip was washed with 0.1% trifluoroacetic acid in pure acetonitrile and with 0.1% TFA in 1: 1 acetonitrile: water. It was then equilibrated twice with 0.1% TFA in water.
  • the sample was dissolved in 10 ⁇ l of 0.1% TFA solution and repeatedly pipetted in and out of the zip-tip tip to attach the peptides to bind the resin. Thereafter, the tip was washed three times with a solution of 0.1% TFA and 5% methanol in water.
  • the sample components were eluted from the zip-tip with 1, 8 ⁇ matrix solution (matrix dissolved in 0.1% TFA 50% acetonitrile) and pipetted directly onto the MALDI-ToF-MS target.
  • R16 and S16 protein microbeads were used for the preparation of spinnable R16 and S16 solutions. These can be prepared as described in WO 2008/155304.
  • amphiphilic oligomer named P (phenylglycidyl ether) -block-P (carboxymethylglycidyl ether) - (3,3) of formula 1:
  • the brownish oil was dissolved in DMF and stirred with 16.2 g (0.45 mol) of NaH (washed with pentane) overnight. The solution turns dark brown. Subsequently, 78.8 g (0.45 mol) of sodium chloroacetate (NaTa) were added and the batch was stirred overnight at 60.degree. The DMF was removed in vacuo and the residue in dist. Water dissolved. The product, a light brown precipitate, was precipitated with half-concentrated HCl, separated and then redissolved in NaOH.
  • the product remains stable for several months as Na salt in the solution.
  • the solution can also be dried at 37 degrees before use and stored as a solid.
  • Example 1 Stabilization of R16 spider silk protein solutions with BSA and spinning of the stabilized preparation
  • the solution is stirred overnight (at least 12 h) at room temperature (about 20-25 ° C).
  • the ff.-BSA / R16 solution thus obtained is dialyzed against 10 mM NaHCO 3 buffer (pH about 10.5).
  • Dialysis takes place in a dialysis tube (Sigma-Aldrich, order no .: D9777-100FT, cellulosic membrane) with the exclusion limit of approx. 12,400.
  • the volume of the NaHC0 3 buffer is at least 100 times greater than the sample.
  • the buffer is changed at least once to remove the GdmSCN as quantitatively as possible.
  • Electron micrographs are shown in Figures 3a to d.
  • R16 protein is weighed, transferred to a snap-cap jar, and solubilized with guanidine thiocyanate solution (6M).
  • R16 Peptide Fragments In a test tube, weigh 45 mg of R16 protein and add 2 ml of 0.2 M NaOH solution. The test tube is tightly closed and the mixture is heated in a water bath to about 80 ° C internal temperature (the temperature of the water bath should be at least 85 ° C). The mixture thus obtained is stirred vigorously (about 1000 rpm). After some time (about 10 minutes), the protein begins to dissolve in the NaOH solution. Once the protein is dissolved, the sample is taken out of the water bath and cooled. Thereafter, the sample must not be subjected to an increased temperature treatment, since the fragments can be further split thereby and it may come to a complete cleavage. Uncleaved R16 protein is visually very well recognized in the solution. During cleavage, it begins to dissolve, as the fragments are readily soluble in water. Once the protein is visually unrecognizable, cleavage is stopped to avoid complete hydrolysis.
  • the R16 protein hydrolyzate prepared in this manner represents a mixture of peptide fragments having a molecular weight in the range of about 1,000 to 3,000, as illustrated by attached Figure 1. There, the mass spectrum (Maldi- ToF) of a typical R16 protein hydrolyzate is shown.
  • the hydrolyzate is transferred by syringe to a dialysis bath (NaHCO 3 buffer) (10 mM, 1.5 L).
  • the pH of the dialysis bath is about 10-11 adjust (NaOH, solid).
  • the particular R16 peptide quantity used for different approaches is listed in Table 3 below.
  • R16 samples (prepared according to 2.1) with different R16 protein content (compare Table 3) are dialyzed against the NaHCO 3 dialysis buffer (pH about 10.5) containing the R 16 hydrolyzate.
  • the sample is transferred into a dialysis tube (Sigma-Aldrich, order No. D9777-100FT, cellulosic membrane, exclusion limit of about 12,400).
  • the volume (eg, 1.5 liters) of the NaHCO 3 buffer should be at least 100 times larger than the sample.
  • the temporal stability of the respective solution during dialysis is determined and is given in Table 3 below. Stability means that no gelation is observed in the solution studied during dialysis.
  • R16 protein is weighed, transferred to a snap-cap jar and dissolved with guanidine thiocyanate solution (6 M).
  • BSA protein 45 mg are weighed into a test tube and 2 ml of 0.2 M NaOH solution are added.
  • the test tube is tightly closed.
  • the mixture in the test tube is dissolved at room temperature and then heated to about 80 ° C internal temperature.
  • the temperature of the water bath should be at least 85 ° C.
  • the mixture must be stirred vigorously (about 1000 rpm).
  • the protein begins to cleave in NaOH solution and a yellowish solution is formed.
  • the sample is taken out of the water bath and cooled. Thereafter, the sample must not be subjected to an increased temperature treatment, as the fragments can be further split thereby and it may come to a complete cleavage.
  • the BSA protein hydrolyzate prepared in this way represents a mixture of peptide fragments having a molecular weight in the range of about 600 to 4,000, such as illustrated by enclosed Figure 2. There, the mass spectrum of a typically resulting BSA protein hydrolyzate is shown.
  • the sample is transferred by syringe to the dialysis bath (NaHCO 3 buffer) (10 mM, 1.5 L).
  • the pH of the dialysis bath should be adjusted to approx. 10-1 1 (with NaOH).
  • the BSA peptide concentration is about 0.003-0.004%.
  • R16 samples prepared according to 2.1
  • R16 protein content containing the BSA hydrolyzate in different amounts (see Table 5)
  • dialyzed takes place in a dialysis tube (Sigma-Aldrich, see above) with an exclusion limit of about 12,400.
  • the volume of the NaHC0 3 - buffer should be at least 100 times greater (eg 2 ml protein solution in 1, 5 L dialysis bath) as the sample.
  • the temporal stability of each solution during dialysis is determined and is shown in Table 5. Stability means that no gelation is observed in the solution studied during dialysis.
  • P phenylglycidyl ether
  • block-P carboxymethyl glycidyl ether
  • 3,3 prepared according to Reference Example 2 (1).
  • the previously dissolved in NaOH solution oligomer, which remains stable for several months as Na salt in solution can be used as a solution or as a solid (dried at 37 ° C).
  • the substance is used in particular as a solid.
  • the R16 protein is dissolved in a 6M guanidine thiocyanate solution.
  • the oligomer solid is weighed in and dissolved directly in the R16 solution. The solution is stirred slowly overnight. During this time, the addition of the oligomer to the protein takes place.
  • Corresponding quantities for various approaches are summarized in Table 7 below.
  • dialysis is performed to remove guanidine thiocyanate.
  • the volume of the solution to be dialyzed is 3 ml (contains a magnetic stir bar and is stirred during dialysis).
  • the temporal stability of the respective solution during dialysis is determined and is given in Table 7 below. Stability means that no gelation is observed in the solution studied during dialysis.
  • the wound healing promoting effect of the fibers produced according to the invention (prepared according to Example 2, R16 stabilized with R16 peptides) is determined by the cellular proliferation test described above:
  • test results are summarized in FIG. 7.
  • an increase in the number of cells can be achieved (optimal concentration 0.06 mg / ml).

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Abstract

L'invention concerne des dispersions aqueuses stables de protéines comprenant, dans une phase aqueuse, au moins une protéine à auto-assemblage, en dispersion, ainsi qu'un agent de dispersion spécifique pour cette protéine à auto-assemblage. L'invention concerne en outre : un procédé pour produire ces dispersions aqueuses stables; un procédé de filage électrostatique de protéines à auto-assemblage faisant appel à ces dispersions aqueuses stables; un procédé pour produire des structures de fibres planes ou des fibres à partir desdites dispersions aqueuses; l'utilisation des matières produites par filage électrostatique pour générer des produits médicaux, des articles d'hygiène et des textiles; ainsi que des fibres ou des structures de fibres planes obtenues au moyen du procédé de filage électrostatique selon l'invention.
EP11752173.2A 2010-08-26 2011-08-25 Procédé pour produire des solutions hautement concentrées de protéines à auto-assemblage Withdrawn EP2609109A2 (fr)

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US9757330B2 (en) 2013-10-18 2017-09-12 Industrial Technology Research Institute Recipe for in-situ gel, and implant, drug delivery system formed thereby
US10202636B2 (en) 2013-12-24 2019-02-12 General Electric Company Electrospun fibers for protein stabilization and storage
JP6460675B2 (ja) * 2014-08-04 2019-01-30 国立大学法人信州大学 シルク複合ナノファイバーの製造方法
GB201415681D0 (en) * 2014-09-04 2014-10-22 Cambridge Entpr Ltd And President And Fellows Of Harvard College Protien Capsules
CN105951210B (zh) * 2016-06-24 2018-06-19 南通纺织丝绸产业技术研究院 一种珠粒形貌的串珠纤维材料及其制备方法
CN107236999A (zh) * 2017-07-17 2017-10-10 合肥威斯伏新材料有限公司 一种鱼皮胶原自组装成纤维的方法
WO2019194249A1 (fr) * 2018-04-03 2019-10-10 Ｓｐｉｂｅｒ株式会社 Solution de filage, fibres de fibroïne modifiée et procédé de fabrication correspondant
CN109137102B (zh) * 2018-09-30 2020-11-24 吉林大学 一种具有定向疏水的仿蜘蛛丝纤维结构制备方法
WO2021183469A1 (fr) * 2020-03-09 2021-09-16 Amprion, Inc. Solution témoin de dosage de liquide céphalo-rachidien
US12220445B2 (en) 2020-03-09 2025-02-11 Amprion, Inc. Inert matrices for qualitative and semi-quantitative seed amplification assays
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