Collagen mimetic protein-based material

Abstract

The invention relates to polyamino acid complexes, to compositions containing same, to methods for the production thereof, and to corresponding uses.

Description

[0001] Collagen-mimetic protein-based material

[0002] All documents cited in the present application are incorporated by reference in their entirety into the present disclosure.

[0003] The present invention relates to the preparation of solid, ordered, cross-linked polyamino acid complexes (OPAAC - ordered polyamino acid complex), the solid, ordered polyamino acid complexes prepared and corresponding uses.

[0004] State of the art:

[0005] Collagens are a group of structural proteins of the extracellular matrix of connective tissue. They are found, among other things, in the white, inelastic fibers of tendons, ligaments, bones, and layers of the skin. Collagen is characterized by a hierarchical organization of helices and fibers. This structure gives collagen its special mechanical properties. Collagen has a wide variety of applications in biology and medicine. In the pharmaceutical and medical technology industries, collagen of animal origin is primarily used. The quality can vary considerably from batch to batch. Collagen of animal origin can also transmit diseases or pathogens and cause allergic reactions.

[0006] Therefore, synthetic alternatives are being sought. These are primarily based on the linking of short-chain proteins consisting of amino acids typical of collagen. Furthermore, the biotechnological fermentative production of collagen remains state of the art. These proteins have a triple helix and are similar to human collagen in many properties. Although the formation of microfibers has been achieved, a hierarchical structure similar to collagen has not yet been achieved.

[0007] To realize the mechanical properties of collagen, ordered hierarchical connections of helices and fibers are necessary across size scales from nanometers to at least several micrometers. Currently, this is only achieved in a few elaborately synthesized collagen-mimetic proteins.

[0008] When aqueous solutions of polycations and polyanions are mixed, the attractive interaction of their opposite charges leads to the formation of polyelectrolyte complexes. These complexes typically separate as a new, concentrated polymer phase, also called a polyelectrolyte complex phase or complex coacervate phase. This phase becomes increasingly water-soluble as more salt is added or the temperature is increased.

[0009] Coacervate phases are usually prepared as thin monolayer or multilayer films on surfaces or as water-soluble colloids in the form of micelles, particles or capsules with diameters between several nanometers and several tens of micrometers (Review by 1 van der Gucht , E. Spruijt, M. Lemmers, MA Cohen Stuart, Polyelectrolyte complexes: bulk phases and colloidal systems, J. Colloid Interface Sei. 2011, 361, 407).

[0010] US 9,273,220; US 9,796,872 B2; and US 2013 / 0273,174 A1 disclose processes for producing water-soluble polyelectrolyte complex particles in the size range of 20 nm to 500 nm for surface treatment. Other areas of application for water-soluble coacervate phases include biomedical applications such as drug delivery or as pharmaceutical excipients in the food and cosmetic industries.

[0011] Coacervate phases are typically water-swollen gels or solids with an amorphous internal structure. This amorphous internal structure has been demonstrated, for example, by X-ray diffraction for poly(vinylbenzyltrimethylammonium chloride) / poly(sodium 4-styrenesulfonate) coacervates (S. Meng, M. Ting, H. Wu, M.V. Tirrell, Solid-to-liquid phase transition in polyelectrolyte complexes, Macromolecules 2020, 53, 7944-7953). The measured X-ray diffraction curves do not show any Bragg peaks, the existence of which would indicate internal order. Meng et al. were unable to observe any Bragg peaks at salt concentrations of 0-4000 mmol / L NaBr over the entire range of scattering vectors q = 0.02-3 nm.Bragg peaks were observed in the specific case of coacervate micelles consisting of poly(vinylbenzenetrimethylammonium chloride)-poly(ethylene oxide) and polylysine-poly(ethylene oxide) block copolymers containing double-stranded oligo-DNA (dsDNA), but the ordering is based on the arrangement of the DNA strands within the coacervate micelles, resulting in Bragg peaks at q ~ 2.3 nm. -1 (AE Marras, 1 R. Vieregg, 1 M. Ting, 1 D. Rubien, MV Tirrell, Polyelectrolyte complexation of oligonucleotides by charged hydrophobic - neutral hydrophilic block copolymers, Polymers 2019, 11, 83)

[0012] It would be desirable to have polyelectrolyte complexes of complex coacervate phases that exhibit an ordered or crystalline internal structure. This could lead to products with improved mechanical properties for applications, similar to, for example, crystalline or semicrystalline polymers.

[0013] It would also be desirable to have polyamino acid complex coacervate phases available that exhibit an ordered or crystalline internal structure. This would enable biocompatible materials with improved mechanical properties, similar to, for example, collagen, which exhibits an ordered internal structure consisting of ordered and cross-linked triple helices, which result in the well-known excellent mechanical properties of collagen, such as its high tensile strength in ligaments and tendons or its high compression strength in cartilage.

[0014] In biomedical applications, collagen materials derived from animal sources are commonly used. These materials can exhibit batch-to-batch quality variations and can cause immunogenic rejection and infection by pathogens. Therefore, it is desirable to have synthetic collagen-mimetic materials available.

[0015] US 2016 / 0376,326 A1 describes the synthesis of synthetic, modified bacterial collagen-like proteins using recombinant methods. These collagen materials are water-soluble. They are used in the fields of pharmaceuticals, medical technology, tissue engineering, cell culture, and regenerative medicine.

[0016] Further state of the art can be mentioned:

[0017] Priftis, Dimitrios and Tirrell, Matthew in Phase behavior and complex coacervation of aqueous polypeptide solutions, Soft Matter, 2012, 8(36), 9396-9405, doi = 10.1039 / C2SM25604E;

[0018] Valeria Castelletto, Lucas de Mello, Foteini Arfara, Hermis latrou, Jani Seitsonen, Ian W. Hamley, Influence of polymer molar mass and mixture stoichiometry on polyelectrolyte complexes of poly(I-arginine) and Poly(I-glutamic acid), Polymer, 263, 2022, 125497, ISSN 0032-3861;

[0019] Folchman-Wagner, Z.; Zaro, J.; Shen, W.-C. Characterization of Polyelectrolyte Complex Formation Between Anionic and Cationic Poly(amino acids) and Their Potential Applications in pH-Dependent Drug Delivery. Molecules 2017, 22, 1089;

[0020] Petrauskas, Vytautas & Maximowitsch, Egle & Matulis, Daumantas. (2015). Thermodynamics of Ion Pair Formations Between Charged Poly(Amino Acid)s. The Journal of Physical Chemistry B. 119; 150828153415007.

[0021] 10.1021 / acs.jpcb.5b05767 Marciel, Amanda B., Eun Ji Chung, Blair K. Brettmann and Lorraine Leon. "Bulk and nanoscale polypeptide based polyelectrolyte complexes." Advances in colloid and interface science 239 (2017), 187-198;

[0022] Horn, J.M.; Kapelner, R.A.; Obermeyer, A.C. Macro- and Microphase Separated Protein-Polyelectrolyte Complexes: Design Parameters and Current Progress. Polymers 2019, 11, 578.

[0023] Therefore, we are still looking for substances or compositions that address the problems / challenges mentioned above and for appropriate manufacturing processes for them.

[0024] The object of the present invention was therefore to provide new substances with regard to the state of the art which can solve the problems and challenges of the state of the art as far as possible. In particular, these substances should have properties which are as similar as possible to those of natural collagen, be as simple as possible to produce and be easy to apply and use.

[0025] Furthermore, it was an object of the present invention to find manufacturing processes for such substances.

[0026] Last but not least, it was the object of the present invention to find uses for these substances or substances produced in this way.

[0027] Further tasks for the specialist arise from the following description.

[0028] These and other objects are achieved within the scope of the present invention by the subject matter of the independent claims.

[0029] Preferred embodiments emerge from the dependent claims and the following description.

[0030] Within the scope of the present invention, the term "comprise" also includes, as a particularly preferred embodiment, "consisting of"; this means that a corresponding list may contain (=comprise) further elements in addition to the explicitly mentioned elements, or it may contain (=consist of) precisely these elements (whereby non-essential elements such as screws, markings, etc. are not taken into account). Within the scope of the present invention, the term "and / or" means that both elements mentioned in the context are included individually, as well as the combination of the elements mentioned in the context.

[0031] In the context of the present invention, all quantities are to be understood as weights unless otherwise stated.

[0032] For the purposes of the present invention, the term "ambient temperature" means a temperature of 20°C. Unless otherwise stated, temperatures are in degrees Celsius (°C).

[0033] Unless otherwise stated, the reactions or process steps described are carried out at atmospheric pressure, ie at approximately 1013 kPa, and at ambient temperature.

[0034] In the context of the present invention, the term "polyamino acid" essentially and therefore preferably includes, but is not limited to, homopolyamino acids consisting of one type of amino acid and copolyamino acids consisting of a plurality of amino acids, as well as in some variants, mixtures, and modifications thereof. Furthermore, the term "polyamino acid" encompasses all possible geometric configurations of the molecule, such as D- and L-configurations and mixtures thereof. Furthermore, "polyamino acid" can include natural polyamino acids and unnatural polyamino acids.

[0035] In the context of the present invention, the parameter "R" is used to express the molar ratio of cationic, or potentially cationic, groups to that of anionic (or potentially anionic) groups of the respective polyamino acids from which the polyamino acid complexes according to the invention are formed.

[0036] Within the scope of the present invention, cross-linked, solid, ordered polyamino acid complexes were discovered. Likewise, within the scope of the present invention, manufacturing processes for their respective production, as well as corresponding applications and uses, were discovered.

[0037] Thus, within the scope of the present invention, disadvantages and deficiencies associated with the amorphous polyelectrolyte complexes of the prior art could be avoided or at least significantly reduced. The present invention relates, in a first aspect, to solid, ordered polyamino acid complexes, which are cross-linked.

[0038] Surprisingly, the polyamino acid complexes (OPAAC) of the present invention exhibit internal structural order, both when crosslinked and prior to crosslinking. The structural order can be demonstrated by observing Bragg peaks in X-ray diffraction patterns, such as those shown for some examples of the invention in Figures 1-7. Polyamino acid complexes with structural order are obtained for a wide range of salt concentrations, a wide range of R values, for various anionic polyamino acids, and over a temperature range including ambient temperature.

[0039] The ordered polyamino acid complexes (OPAAC) of the present invention comprise at least two different water-soluble polyamino acids, each carrying electrostatically charged groups, or groups capable of generating a charge (capable of ionization), wherein the overall net charge on the two polymers is balanced or capable of being balanced, for example, by manipulating the relative proportions of the polyamino acids or the pH of the aqueous phase.

[0040] A preferred subject of the present invention are polyamino acid complexes comprising or consisting of: at least one water-soluble cationic polyamino acid, in particular selected from the group consisting of polyarginine, polylysine and polyhistidine, further cationic polyamino acids or cationic co-polyamino acids, or cationically functionalized polyamino acids; and at least one water-soluble anionic polyamino acid, in particular selected from the group consisting of polyaspartic acid, polyglutamic acid, further anionic polyamino acids or anionic co-polyamino acids, anionically functionalized polyamino acids, or synthetic anionic polymers or copolymers, wherein the polyamino acid complexes are cross-linked.In preferred embodiments of the present invention, the at least one cationic polyamino acid is selected from the group consisting of polyarginine, polylysine, polyhistidine and mixtures thereof, preferably polyarginine or polylysine or polyarginine and polylysine, in particular polyarginine.

[0041] In further preferred embodiments of the present invention, the at least one anionic polyamino acid is selected from the group consisting of polyaspartic acid, polyglutamic acid and mixtures thereof, preferably polyarginine or polylysine, in particular polyaspartic acid.

[0042] In still further preferred embodiments of the present invention, the polyamino acid complexes are obtained in an aqueous mixture containing the polyamino acids and a salt concentration, in particular NaCl, between 0 mmol / l and 500 mmol / l or 2000 mmol / l and 4000 mmol / l.

[0043] In yet further preferred embodiments of the present invention, the polyamino acids have chain lengths of 70 to 10,000, preferably 80 to 5,000, particularly preferably 85 to 300, and in particular 90 to 210, and most particularly 90 to 120 and / or 190 to 210 amino acids. In some preferred embodiments, the polyamino acids have chain lengths of 1,000 to 2,000 amino acids; such are available, for example, from Aldrich.

[0044] In still further preferred embodiments of the present invention, the polyamino acid complexes have R values ​​between 0.05 and 20, preferably 0.2 to 2.5, particularly preferably 0.2 to 2, even more preferably 0.75 to 1.5, in particular 1, where R is the ratio of charged cationic groups to charged anionic groups present in the respective polyamino acids.

[0045] The present invention further relates to processes for the preparation of solid, ordered polyamino acid complexes, in which a solution of a cationic polyamino acid and a solution of an anionic polyamino acid are mixed and a crosslinking agent is added. This process for crosslinking ordered polyamino acid complexes further increases the stability of the ordered structure of the uncrosslinked polyamino acid complexes in the dry and solid state.

[0046] The ordered polyamino acid complexes (OPAAC) of the present invention are prepared such that they precipitate directly during preparation, or such that they can be induced to precipitate to yield a solid complex that can be separated from the aqueous supernatant after sedimentation, filtration, or centrifugation. The ordered polyamino acid complexes (OPAAC) of the present invention are accordingly prepared by mixing aqueous solutions of oppositely charged polyamino acids, wherein the addition of crosslinking agent can occur during or after mixing. This is preferably done such that the total concentration of polyamino acid in the mixture is between 0.001 mmol / L and 1000 mmol / L, preferably between 0.01 mmol / L and 100 mmol / L, more preferably between 0.1 mmol / L and 10 mmol / L, and most preferably between 0.5 and 2 mmol / L.

[0047] The mixing of the two polyamino acid solutions comprising a first cationic polyamino acid (A) and a second anionic polyamino acid (B) having oppositely charged groups can be easily carried out by adding the two solutions together and mixing them.

[0048] The mixing or mixing step can be performed simply by means such as shaking, stirring, static mixing, or combinations thereof. The molar ratio R of the cationic groups of the cationic polyamino acid to the anionic groups of the anionic polyamino acid can be between 0.01 and 100, preferably between 0.1 and 10, more preferably between 0.5 and 2, and most preferably 1.

[0049] A preferred embodiment of the present invention is a process for the preparation of polyamino acid complexes according to the present invention, comprising or consisting of the steps I) preparation of at least one salt solution, in particular NaCl solution, of a certain concentration between 0 mmol / l and 400 mmol / l or 2000 mmol / l and 4000 mmol / l;

[0050] II) preparing at least one solution of a cationic polyamino acid, preferably polyarginine or polylysine, in particular polyarginine, and preparing at least one solution of an anionic polyamino acid, preferably polyaspartic acid or polyglutamic acid, in particular polyaspartic acid;

[0051] III) mixing the at least one salt solution and the at least one solution of a cationic polyamino acid and the at least one solution of an anionic polyamino acid;

[0052] IV) Separating the resulting complex and separating it from the solvent phase, preferably by sedimentation, centrifugation, filtration or combinations thereof, in particular centrifugation.

[0053] In the context of the present invention

[0054] Va) during step III), or

[0055] Vb) after step III) and before step IV), or

[0056] Vc) after step IV), optionally also after intermediate storage of the product obtained in step

[0057] IV) a crosslinking agent, preferably selected from the group consisting of glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and mixtures thereof, in particular glutaraldehyde, dissolved alone or in a solvent, preferably water, is added.

[0058] Likewise a preferred embodiment of the present invention are polyamino acid complexes according to the invention prepared by a process comprising or consisting of the steps

[0059] I) preparation of at least one salt solution, in particular NaCl solution, of a specific concentration between 0 mmol / l and 400 mmol / l or 2000 mmol / l and 4000 mmol / l;

[0060] II) preparing at least one solution of a cationic polyamino acid, preferably polyarginine or polylysine, in particular polyarginine, and preparing at least one solution of an anionic polyamino acid, preferably polyaspartic acid or polyglutamic acid, in particular polyaspartic acid; III) mixing the at least one salt solution and the at least one solution of a cationic polyamino acid and the at least one solution of an anionic polyamino acid;

[0061] IV) Separating the resulting complex and separating it from the solvent phase, preferably by means of sedimentation, centrifugation, filtration or combinations thereof, in particular centrifugation, wherein

[0062] Va) during step III), or

[0063] Vb) after step III) and before step IV), or

[0064] Vc) after step IV), optionally also after intermediate storage of the product obtained in step

[0065] IV) a crosslinking agent, preferably selected from the group consisting of glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and mixtures thereof, in particular glutaraldehyde, alone or dissolved in a solvent, preferably water, is added.

[0066] Cationic polyamino acids:

[0067] All cationic polyamino acids can be used to produce the polyamino acid complexes according to the invention. Preferred polyamino acids are polyarginine, polylysine, and polyhistidine, more preferably polyarginine and polylysine. Cationically functionalized polyamino acids such as amino- or guanidine-functionalized polyphenylalanines, or copolyamino acids of various cationic amino acids, can also be used.

[0068] Polyarginine is particularly preferred in the context of the present invention.

[0069] Anionic polyamino acids:

[0070] All anionic polyamino acids can be used to prepare the polyamino acid complexes according to the invention. Polyaspartic acid and polyglutamic acid are preferred anionic polyamino acids.

[0071] Anionically functionalized polyamino acids and synthetic anionic polymers can also be used. Suitable synthetic anionic polymers include, but are not limited to, polyacrylic acid or polymethacrylic acid. Co-polyamino acids of various anionic amino acids can also be used. Polyaspartic acid and polyglutamic acid are particularly preferred in the context of the present invention.

[0072] Instead of or in addition to the cationic and anionic polyamino acids, synthetic polyelectrolytes or natural polyelectrolytes such as proteins or polysaccharides can be used as cationic or anionic components in some variants of the present invention. Of these two variants, it is preferred to use these substances additionally, or, in further variants, not at all; especially in the latter case, it is possible to use these substances as part of the compositions according to the invention.

[0073] A particularly elegant way of specifying the composition of the polyamino acid complexes according to the invention (already) in connection with their preparation is to specify the ratio of the moles or the number of cationic charges to the corresponding moles or the corresponding number of anionic charges present in the respective solutions to be mixed during preparation, based on the relative amounts of the polyamino acids added to the respective stock solutions.

[0074] The polyamino acid complexes according to the invention or the polyamino acid complexes obtained according to the process according to the invention are cross-linked in order to further stabilize the ordered structure, in particular in the dry or wet state.

[0075] The present invention is in principle not limited with regard to the crosslinking agents.

[0076] Preferred crosslinking agents in the context of the present invention are glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDO), N-hydroxysuccinimide (NHS).

[0077] Furthermore, the present invention provides compositions containing the solid, ordered polyamino acid complexes. These compositions comprise the polyamino acid complexes according to the invention.

[0078] In addition, the compositions according to the invention may contain other substances.

[0079] For example, in preferred embodiments, suitable electrolyte salts known in the art can be added to the compositions according to the invention, such as, for example but not exclusively, selected from the group consisting of sodium chloride, sodium bromide, potassium chloride, potassium bromide and other salts consisting of ions of the Hofmeister series fluoride (F ), sulfate (SO4 2- ), hydrogen phosphate (HPO4 2-), acetate (CH3COO _ ), chloride (CF), nitrate (NO3'), bromide (Br), chlorate (CIO3'), iodide (!'), perchlorate (CIO4'), thiocyanate (SCN-), trichloroacetate, (CI3CCOO'), ammonium (NH4 + ), potassium (K + ), sodium (Na + ), lithium (Li + ), magnesium (Mg 2+ ), Calcium (Ca 2+ ), guanidium and mixtures thereof, or buffer salts and mixtures thereof. The buffer salts can be selected, for example, but not exclusively, from the group consisting of potassium dihydrogen phosphate, potassium dihydrogen phosphate dihydrate, sodium dihydrogen phosphate,

[0080] Sodium dihydrogen phosphate dihydrate, potassium chloride, citric acid monohydrate, trisodium citrate dihydrate, disodium hydrogen phosphate, ethylenediaminetetraacetic acid disodium salt, ethylenediaminetetraacetic acid disodium salt dihydrate, sodium acetate, potassium acetate, HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid), 4-(2-hydroxyethyl)-piperazine-1-propanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, MOPS (3-(N-morpholino)propanesulfonic acid), and mixtures thereof (potassium chloride, sodium acetate, and potassium acetate are also available from the Hofmeister series, but are listed again as buffer substances to emphasize their buffering capacity, if this is particularly desired). These can be used, in particular, to control the ionic strength and / or pH of the compositions according to the invention.

[0081] Solid, ordered polyamino acid complexes form preferentially at low and high electrolyte salt concentrations. Gel-like, disordered polyamino acid complexes can form in a range of medium electrolyte salt concentrations. Electrolyte salts or mixtures of electrolyte salts can be used at electrolyte salt concentrations of 0.00001–100 mol / l, preferably below 0.8 mol / l and above 1.0 mol / l, more preferably below 0.4 mol / l and above 1.5 mol / l, and especially preferably below 0.1 mol / l and above 2.0 mol / l.

[0082] The compositions according to the invention may additionally contain adjuvants, preferably any active ingredients or nutrients as are commonly known in the art, particularly preferably antimicrobial active ingredients, such as, for example but not exclusively, selected from the group consisting of streptomycin, ceftriaxone, tetracycline, ampicillin, sulfasalazine, ciprofloxacin, azithromycin, meropenem, clindamycin, vancomycin and mixtures thereof.

[0083] A preferred embodiment of the present invention relates to polyamino acid complex compositions comprising or consisting of at least one cross-linked polyamino acid complex according to the present invention, optionally at least one solvent, optionally at least one any salt known in the art, preferably NaCl, NaBr, KCl, KBr, particularly preferably NaCl, optionally at least one any buffer known in the art, preferably PBS (phosphate buffered saline solution), HEPES (4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid) or MOPS (3-(N-morpholino)propanesulfonic acid), optionally at least one auxiliary agent, in particular an antimicrobial substance.

[0084] In a further preferred embodiment, the cationic polyamino acid is present in these polyamino acid complex compositions at a content of about 0.001 wt.% to about 20.0 wt.%, and / or the anionic polyamino acid or the anionic polymer is present at a content of about 0.001 wt.% to about 20.0 wt.%; and optionally the salt described above is present at a content of 0 wt.% to about 20.0 wt.%, the buffer described above is present at a content of 0 wt.% to about 20.0 wt.%, the auxiliary agent described above, preferably selected from the group consisting of active ingredients or nutrients, in particular antimicrobial active ingredients, is present at a content of 0 wt.% to about 20.0 wt.%.

[0085] For the process according to the invention, water can be used as the sole solvent, or a combination with water-miscible organic solvents can be used in which the polyamino acids are soluble and the polyamino acid complexes are insoluble, preferably selected from the group consisting of alcohols, dioxane, DMSO, ethylene glycol, and mixtures thereof. The alcohols are preferably selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol, and mixtures thereof, particularly preferably selected from ethanol, 1-propanol, 2-propanol, and mixtures thereof, in particular ethanol.

[0086] In yet another preferred embodiment, the solvent of the polyamino acid complex compositions is selected from the group consisting of water, C 1-6 alkanols; C 1-6 diols; C 1-10 alkyl ethers of alkylene glycols; C 3-24 alkylene glycol ethers, polyalkylene glycols, short-chain carboxylic acids, dimethylformamide, dimethyl sulfoxide, dioxane and mixtures thereof, in particular water.

[0087] A further preferred embodiment of the present invention is a process for the preparation of polyamino acid complex compositions according to the present invention, comprising or consisting of

[0088] Mixing a polyamino acid complex according to the present invention with at least one of at least one art-known salt as described above, preferably NaCl, at least one art-known buffer as described above, preferably phosphate-buffered saline solution, at least one auxiliary agent as described above, and at least one solvent, in particular water.

[0089] The polyamino acid complexes according to the invention or the polyamino acid complexes prepared according to the process according to the invention or the compositions according to the invention or the polyamino acid complex compositions prepared according to the process according to the invention can be used as synthetic collagen-mimetic materials, in particular for biomedical applications.

[0090] The solid, ordered polyamino acid complexes of the present invention can also be used for cosmetics, personal care, skin care, in foods, or as biomaterials or substitutes for biomaterials. They can also be used for tissue regeneration, tissue repair, tissue engineering, implants, wound healing and wound dressings, topical treatments, tablet coatings and excipients, cell culture media, coatings for stents and implants, and drug delivery. They are particularly preferably used as biomaterials or substitutes for biomaterials for or in tissue repair and for or in wound healing.

[0091] The polyamino acid complexes according to the invention have a number of structural similarities to natural collagen:

[0092] * JPRO Orgel, TC Irving, A. Miller, TJ Wess, Proc. Natl. Acad. Sci. 103, 9001-9005 (2006) The polyamino acid complexes and collagen (type II) according to the invention both have stabilized helical structures that can form ordered columnar phases (cf. meridional reflections, see Figure 5); both show periodicity along the helical axis in the range of 30 nm to 70 nm (cf. equatorial reflections, see Figure 5); both have a coil-to-helix transition in the range of 41-42°C.

[0093] Collagen (type II), like the polyamino acid complexes of the present invention, can be cross-linked for stabilization.

[0094] The substances and manufacturing processes, as well as applications and uses according to the present invention, show a number of advantages, some, but not all, of which are mentioned below.

[0095] Due to the inherent internal ordered structure of the polyamino acid complexes of the present invention, polyamino acid complexes of the present invention are similar to collagen and can therefore be very well used as synthetic collagen-mimetic materials, especially for biomedical applications.

[0096] A particularly preferred subject of the present invention relates to polyamino acid complexes a) of polyarginine and polyaspartic acid with R = 0.5, R = 1.0, R = 2.0, in particular R = 1.0, characterized by the presence of one or more Bragg peaks in the range q < 10 nm' 1 , especially at the positions qio = 3.5 nm -1 ; qn = 6.0 nm' 1 ; q2o = 7.0 nm' 1 ; q 2i = 9.3 nm' 1 ; or qio = 4.3 nm' 1 ; q 20 = 9.0 nm' 1 ; q 2i = 10.0 nm' 1 ; or qio = 4.5 nm' 1 ; q 20 = 8.6 nm' 1 ; q 2i= 9.6 nm' 1 ; b) from polyarginine and polyglutamic acid with R = 1.0, characterized by the presence of one or more Bragg peaks in the range q < 10 nm' 1 , especially at the positions qio = 5.1 nm' 1 ; qn = 7.2 nm' 1 ; or qio = 4.3 nm' 1 ; q 20 = 8.6 nm' 1 ; q 2i = 9.6 nm' 1 . Most preferred are polyamino acid complexes of polyarginine and polyaspartic acid with R = 0.5, R = 1.0, R = 2.0, in particular R = 1.0, characterized by the position of the Bragg peaks for q < 10 nm at qio = 3.5 ± 0.1 nm ; qn = 6.0 ± 0.1 nm ; q 20 = 7.0 ± 0.1 nm 4 ; q 2i = 9.3 ± 0.1 nm 4 .

[0097] In summary, the present invention can be described as follows:

[0098] Within the scope of the present invention, a collagen-mimetic material is produced, in particular, by simply mixing solutions of cationic and anionic polyamino acids and crosslinking them. Through cooperative ion pairing and helix formation, parallel helices are formed, forming a hexagonal or square columnar superstructure. Furthermore, an additional long-range superorder forms along the helices. This hierarchically ordered structure can be detected by X-ray diffraction. The resulting material is macroscopically crosslinked.

[0099] In particularly preferred embodiments of the present invention, the complexation of polyarginine with polyaspartic acid and / or polyglutamic acid, with crosslinking of glutaraldehyde, leads to particularly good and particularly suitable materials.

[0100] The various embodiments of the present invention, for example - but not exclusively - those of the various dependent claims or individual embodiments described in the figures, can be combined with one another in any desired manner, even if these combinations are not explicitly mentioned, provided that such combinations do not contradict one another.

[0101] Process steps presented in the examples are to be interpreted as variants of preferred embodiments of the present invention.

[0102] Examples:

[0103] The invention will now be further explained with reference to the following non-limiting examples.

[0104] The following abbreviations are used below:

[0105] PArg for polyarginine,

[0106] PAsp for polyaspartic acid, PGIu for polyglutamic acid.

[0107] The number of amino acids per polyamino acid chain is abbreviated as "aa". The diffraction vector q is defined as q = (4n / X)sin(0 / 2), where n is the wavelength of the X-rays and 0 is the diffraction angle.

[0108] Polyarginine (PArg; MW = 19000 g / mol), polyaspartic acid (PAsp; MW = 11500 g / mol), and polyglutamic acid (PGIu; MW = 13000 g / mol) were purchased from Alamanda Polymers. Sodium chloride (99.5%) was purchased from Sigma-Aldrich. The chemicals were used without further purification.

[0109] Example 1 - Preparation of PArg (100 aa) and PAsp (100 aa) complexes with R = 1.0: Stock solutions of polyarginine (PArg, 100 aa) and polyaspartic acid (PAsp, 100 aa) were prepared in deionized water (prepared using the Milli-Q® Direct System). The polyamino acids were dissolved in deionized water to obtain solutions with concentrations of 15 mmol / L. The solutions of the polyamino acids in water were treated with ultrasonication (VWR USC 300 TH) for 10 minutes. The pH of the solutions was adjusted (titrated) to pH = 7 using 0.1 mol / L NaOH or 0.1 mol / L HCl solutions. The solutions were then filtered through 100 nm filters and then adjusted to a concentration of 6 mmol / L with deionized water to create the stock solution. NaCl solutions were prepared with the desired molar concentration of NaCl (including 0 mmol / l; 80 mmol / l; 160 mmol / l; 400 mmol / l; 800 mmol / l; 1200 mmol / l; 2400 mmol / l; 4000 mmol / l).Polyarginine stock solution was added to the respective NaCl solutions to obtain a polyarginine concentration of 2 mmol / L (PArgl). Polyaspartic acid stock solution was added to the respective NaCl solutions to obtain a polyaspartic acid concentration of 2 mmol / L (PAspl). The PArgl and PAspl solutions were then mixed in a volume ratio of V(PArgl):V(PAspl) = 1:1. While mixing can in principle be performed using any known method, in this example it was carried out using a magnetic stirrer. The formation of the solid coacervate complexes occurred immediately. The resulting complexes were then allowed to settle. The solution was then centrifuged (5236 g for 15 minutes at 21°C using a centrifuge (Sigma 6-16KHS)) to separate the solid complex precipitate from the supernatant. The solid complexes (ormore precisely a small part of each) were transferred into glass capillaries for characterization by X-ray diffraction experiments (see figures for results).

[0110] Example 2 - Preparation of PArg (100 aa) and PAsp (100 aa) complexes with R = 0.5: Example 1 was repeated, with the difference that the solutions PArgl and PAspl were mixed in a volume ratio of V(PArgl):V(PAspl) = 1:2.

[0111] Example 3 - Preparation of PArg (100 aa) and PGIu (100 aa) complexes with R = 1.0: Example 1 was repeated, except that stock solutions of polyarginine (PArg, 100 aa) and polyglutamic acid (PGIu, 100 aa) were prepared in deionized water (prepared using the Milli-Q® Direct System).

[0112] Example 4 - Cross-linking of PArg / PAsp complexes:

[0113] The solid complexes obtained according to Example 1 were each incubated for 24 hours in an aqueous glutaraldehyde solution (concentration: 2 wt%). The resulting complexes were then washed with deionized water (prepared using the Milli-Q® Direct System). Again, the resulting solid complexes were transferred into glass capillaries for characterization by X-ray diffraction experiments (see figures for results).

[0114] Figures:

[0115] Figure 1 shows X-ray diffraction curves of solid ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes with a PArg chain length of 100 aa, a PAsp chain length of 100 aa, and a ratio R = 1.0 for a range of salt concentrations from 0 mmol / L to 4000 mmol / L NaCl. The series of Bragg peaks corresponding to the ordered packing of the helices (H) and the Bragg peaks corresponding to the in-axis periodicity of the helices (L) are indicated. This is indicated by the presence of the Bragg peaks for q < 10 nm' 1 shown that OPAAC in the low salt concentration range c s = 0 - 400 mmol / l NaCl (0 mmol / l, 80 mmol / l, 160 mmol / l, 400 mmol / l NaCl). The position of the H-Bragg peaks (first peak: qio = 3.5 nm -1 ; second peak: qn = 6.0 nm' 1 ; third peak: q2o = 7.0 nm' 1 ; fourth peak: q 2i = 9.3 nnr 1) is consistent with hexagonal packing of the helices. The absence of Bragg peaks for q < 10 nm for intermediate salt concentrations of 800 - 1200 mmol / l NaCl indicates that disordered polyamino acid complexes were formed. At higher salt concentrations of 2400 mmol / l and 4000 mmol / l NaCl, ordered polyamino acid complexes (OPAAC) are again formed, as indicated by the Bragg peaks for q < 10 nm. 4 The position of the H-Bragg peaks (first peak: q i0 = 4.3 nm 4 ; second peak: absent; third peak: q2o = 8.6 nm 4 ; fourth peak: q 2i = 9.6 nm 4 ) is consistent with a square packing of the helices. Figure 1 demonstrates that OPAACs were (are) formed at low salt concentrations and at high salt concentrations. At intermediate salt concentrations (800 mmol / l, 1200 mmol / l), disordered liquid-like polyamino acid complexes form.

[0116] Figure 2 shows X-ray diffraction curves of solid, ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes and PArg / PGlu complexes, both with chain lengths of 100 aa and a ratio R = 1.0 for a low salt concentration (0 mmol / l NaCl) and a high salt concentration (2400 mmol / l NaCl). It is indicated by the presence of the Bragg peaks for q < 10 nm. 4 It was shown that OPAAC were formed for the PArg / PAsp complex and the PArg / PGlu complex at low salt concentration (0 mmol / l NaCl) and at high salt concentration (2400 mmol / l NaCl). For the PArg / PAsp complex at low salt concentration (0 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 3.5 nm -1 ; second peak: qn = 6.0 nm' 1 ; third peak: q 20 = 7.0 nm' 1 ; fourth peak: q 2i = 9.3 nm' 1) consistent with a hexagonal packing of the helices. For the PArg / PAsp complex at high salt concentration (2400 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 4.5 nm' 1 ; second peak: not visible; third peak: q 20 = 9.0 nm' 1 ; fourth peak: q 2i = 10.0 nm' 1 ) consistent with a square packing of the helices. For the PArg / PGlu complex at low salt concentration (0 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 5.1 nm' 1 ; second peak: qn = 7.2 nm' 1 ) consistent with a square packing of the helices. For the PArg / PGlu complex at high salt concentration (2400 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 4.3 nm' 1 ; second peak not visible; third peak: q 20 = 8.6 nm' 1 ; fourth peak: q 2i = 9.6 nm' x) consistent with a square packing of the helices. Figure 2 demonstrates that OPAACs were formed with various polyanionic polyamino acids such as polyaspartic acid and polyglutamic acid.

[0117] Figure 3 shows X-ray diffraction curves of solid, ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes with a ratio R = 1.0 for a PAsp chain length of 100 aa and for PArg chain lengths of 100 amino acid repeat units (100 aa) and 200 amino acid repeat units (200 aa), and for a low salt concentration (0 mmol / L NaCl) and a high salt concentration (2400 mmol / L NaCl). The presence of Bragg peaks for q < 10 nm demonstrates that OPAAC was formed for the PArg / PAsp complexes with both PArg chain lengths (100 aa and 200 aa) at low salt concentration (0 mmol / L NaCl) and at high salt concentration (2400 mmol / L NaCl). For the PArg / PAsp complexes with PArg chain lengths of 100 aa and 200 aa at low salt concentration (0 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 3.5 nm ; second peak: qn = 6.0 nm 4 ; third peak: qzo = 7.0 nm 4 ; fourth peak: q 2i= 9.3 nm 4 ) consistent with a hexagonal packing of the helices. At low salt concentrations, the position of the L-Bragg peaks for 100 aa qi = 0.41 nm 4 ; q2= 0.82 nm 4 ; and for 200 aa qi = 0.21 nm 4 ; q2= 0.42 nm 4 . The shift of qi = 0.41 nm 4 for 100 aa to qi = 0.21 nm for 200 aa indicates a doubling of the repeat distance from d = 2n / qi = 15.3 nm for 100 aa to d = 2n / qi = 29.9 nm for 200 aa. The repeat distances are consistent with the contour length of the corresponding PArg o-helices: with an o-helix pitch of 0.54 nm and a number of 3.6 amino acids per pitch, a contour length of l c = 100 * 0.54 / 3.6 nm = 15 nm for the 100 aa chain length and from lc = 200 * 0.54 / 3.6 nm = 30 nm for the 200 aa chain length. For the PArg / PAsp complex at high salt concentration (2400 mmol / l NaCl), the position of the H-Bragg peaks (first peak: qio = 4.5 nm 4; second peak: not visible; third peak: q 20 = 9.0 nm 4 ; fourth peak: q 2i = 10.0 nm 4 ) consistent with a square packing of the helices. Figure 3 demonstrates that OPAACs were formed for different PArg chain lengths, for example, 100 amino acids or 200 amino acids.

[0118] Figure 4 shows X-ray diffraction curves of solid, ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes with PArg and PAsp chain lengths of 100 aa with ratios R = 0.5 ("1:2"), 1.0 ("1:1") and 2.0 ("2:1"), for a low salt concentration (0 mmol / L NaCl) and a high salt concentration (2400 mmol / L NaCl). It is shown by the presence of Bragg peaks for q < 10 nm that for the PArg / PAsp complexes for all three ratios R = 0.5, 1.0 and 2.0 at low salt concentrations (0 mmol / L NaCl) and at high salt concentration (2400 mmol / L NaCl) OPAAC was formed. For the PArg / PAsp complexes with R = 0.5; 1.0 and 2.0 at low salt concentration (0.0 mmol / l NaCI) is the position of the Bragg peaks (first peak: qio = 3.5 nm ; second peak: qn = 6.0 nm 4 ; third peak: q2o = 7.0 nm 4 ; fourth peak: q 2i = 9.3 nm' x) consistent with a hexagonal packing of the helices. For the PArg / PAsp complexes with R = 0.5, 1.0, and 2.0 at high salt concentration (2400 mmol / l NaCl), the position of the Bragg peaks (first peak: qio = 4.5 nm 4 ; second peak: not visible; third peak: q 20 = 9.0 nm 4 ; fourth peak: q 2i = 10.0 nm 4 ) consistent with a square packing of the helices. Figure 4 demonstrates that OPAACs were formed starting from different R ratios. The ratio of R = 1.0 corresponds to the stoichiometric composition.

[0119] Figure 5 shows X-ray diffraction patterns of shear-oriented, solid, ordered polyamino acid complexes (OPAAC), for example, PArg / PAsp complexes (PArg: 200 aa; PAsp: 100 aa) with a ratio R = 1.0 for a low salt concentration (0 mmol / L NaCl). The series of Bragg peaks corresponding to the ordered packing of the helices (H) (upper part of the figure) and the Bragg peaks corresponding to the in-axis periodicity of the helices (L peaks) (lower part of the figure) are indicated. The presence of the arcuate Bragg peaks on the meridian (H peaks and H reflections, respectively; upper part of the figure) and the ellipsoidal Bragg peaks on the equator (L peaks and L reflections, respectively; lower part of the figure) indicates that the helical axis is oriented parallel to the shear direction. The position of the H Bragg peaks (first peak: qio = 3.5 nm) 4 ; second peak: qn = 6.0 nm' x ; third peak: q 20 = 7.0 nm 4 ; fourth peak: q 2i= 9.3 nm 4 ) is consistent with a hexagonal packing of the helices. The position of the L-Bragg peaks (first peak: qi = 0.2 nm 4 ) is consistent with a one-dimensional periodic ordering along the helices with a repeat distance of d = 2n / qi = 31.4 nm. Figure 5 demonstrates that OPAAC can be arranged in the helix direction, very similar to collagen, which can also be arranged along the helix direction, thus exhibiting excellent tensile properties.

[0120] Figure 6 shows X-ray diffraction curves of solid, ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes (PArg: 100 aa; PAsp: 100 aa; R = 1.0; 0 mmol / l NaCl) for temperatures between 20°C and 42°C. Below a temperature of 42°C, the presence of Bragg peaks for q < 10 nm confirms the presence of ordered polyamino acid complexes (OPAAC). At the melting temperature of 42°C, the Bragg peaks disappear, indicating that a disordered state exists. Figure 6 demonstrates that PArg / PAsp-OPAAC have a melting temperature of 42°C at low salt concentration, which is very close to the melting temperature of collagen (type II) at 41°C.

[0121] Figure 7 shows X-ray diffraction curves of solid, ordered polyamino acid complexes (OPAAC) for the example of PArg / PAsp complexes (PArg: 100 aa; PAsp: 100 aa; R = 1.0; 0 mmol / l NaCl) for cross-linked complexes in the wet, dry, and re-wetted state. The non-cross-linked wet state is shown as a reference. The series of Bragg peaks corresponding to the ordered packing of the helices (H) is indicated. The presence of Bragg peaks for q < 10 nm' 1 It is shown that for the PArg-PAsp complexes, OPAACs were formed for the wet state, the wet cross-linked state, the dried cross-linked state, and the re-moistened cross-linked state. For the wet non-cross-linked state, the wet cross-linked state, and the re-moistened cross-linked state, the position of the Bragg peaks (first peak: qio = 3.6 nm' 1 ; second peak: qn = 6.2 nm' 1 ; third peak: q 20 = 7.2 nm' 1; fourth peak: q 2i = 9.5 nm' 1 consistent with a hexagonal packing of the helices. For the dried cross-linked state, the position of the Bragg peaks (first peak: qio = 3.8 nm -1 ; second peak: qn = 5.4 nm' 1 ; third peak: q 20 = 7.6 nm' 1 ; fourth peak: q 2i = 8.5 nm' 1 ) consistent with a square packing of the helices. Figure 7 thus demonstrates that cross-linked OPAAC retain their ordered structure upon drying and re-wetting.

Claims

Claims:

1. A polyamino acid complex comprising or consisting of: at least one water-soluble cationic polyamino acid, in particular selected from the group consisting of polyarginine, polylysine, and polyhistidine, further cationic polyamino acids or cationic co-polyamino acids, or cationically functionalized polyamino acids; and at least one water-soluble anionic polyamino acid, in particular selected from the group consisting of polyaspartic acid, polyglutamic acid, further anionic polyamino acids or anionic co-polyamino acids, anionically functionalized polyamino acids, or synthetic anionic polymers or copolymers, characterized in that the polyamino acid complexes are cross-linked.

2. Polyamino acid complex according to claim 1, characterized in that it has been crosslinked with crosslinking agents selected from the group consisting of glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and mixtures thereof, in particular glutaraldehyde.

3. Polyamino acid complex according to claim 1 or 2, characterized in that the at least one cationic polyamino acid is selected from the group consisting of polyarginine, polylysine, polyhistidine and mixtures thereof, preferably polyarginine or polylysine or polyarginine and polylysine, in particular polyarginine.

4. Polyamino acid complex according to one of the preceding claims, characterized in that the at least one anionic polyamino acid is selected from the group consisting of polyaspartic acid, Polyglutamic acid and mixtures thereof, preferably polyarginine or polylysine, in particular polyaspartic acid.

5. Polyamino acid complex according to one of the preceding claims, characterized in that it is obtained in an aqueous mixture containing the polyamino acids and a salt concentration, in particular NaCl, between 0 mmol / l and 500 mmol / l or 2000 mmol / l and 4000 mmol / l.

6. Polyamino acid complex according to one of the preceding claims, characterized in that the polyamino acids have chain lengths of 100 to 10,000 amino acids.

7. Polyamino acid complex according to one of the preceding claims, characterized in that the polyamino acid complexes have R values ​​between 0.05 and 20, preferably 0.2 to 2.5, particularly preferably 0.2 to 2, even more preferably 0.75 to 1.5, in particular 1, where R is the ratio of charged cationic groups to charged anionic groups present in the respective polyamino acids.

8. A process for the preparation of polyamino acid complexes according to any one of claims 1 to 7, comprising or consisting of the steps I) preparation of at least one salt solution, in particular NaCl solution, of a specific concentration between 0 mmol / l and 400 mmol / l or 2000 mmol / l and 4000 mmol / l; II) preparing at least one solution of a cationic polyamino acid, preferably polyarginine or polylysine, in particular polyarginine, and preparing at least one solution of an anionic polyamino acid, preferably polyaspartic acid or polyglutamic acid, in particular polyaspartic acid; III) mixing the at least one salt solution and the at least one solution of a cationic polyamino acid and the at least one solution of an anionic polyamino acid; IV) Separating the resulting complex and separating it from the solvent phase, preferably by means of sedimentation, centrifugation, filtration or combinations thereof, in particular centrifugation, characterized in that Va) during step III), or Vb) after step III) and before step IV), or Vc) after step IV), optionally also after intermediate storage of the product formed in step IV), a crosslinking agent, preferably selected from the group consisting of glutaraldehyde, l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS) and mixtures thereof, in particular glutaraldehyde, alone or dissolved in a solvent, preferably water, is admixed.

9. Polyamino acid complex composition comprising or consisting of at least one polyamino acid complex according to one of claims 1 to 7, optionally at least one solvent, optionally at least one salt, preferably NaCl, NaBr, KCl, KBr, particularly preferably NaCl, optionally at least one buffer, preferably phosphate-buffered saline solution, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid or 3-(N-morpholino)propanesulfonic acid, optionally at least one auxiliary agent.

10. Composition according to claim 9, characterized in that the cationic polyamino acid is present in the composition at a content of 0.001 wt.% to 20.0 wt.%, and / or the anionic polyamino acid or the anionic polymer is present in the composition at a content of 0.001 wt.% to 20.0 wt.%, and / or the salt is present in the composition at a content of 0 wt.% to 20.0 wt.%, the buffer is present in the composition at a content of 0 wt.% to 20.0 wt.%, the excipient is present in the composition at a content of 0 wt.% to 20.0 wt.%.

11. Composition according to claim 9 or 10, characterized in that the solvent is selected from the group consisting of alcohols, dioxane, DMSO, ethylene glycol and mixtures thereof, wherein the alcohols are preferably selected from the group consisting of methanol, ethanol, 1-propanol, 2-propanol and mixtures thereof, particularly preferably selected from ethanol, 1-propanol, 2-propanol and mixtures thereof, in particular ethanol; or the group consisting of water, C 1-6 alkanols; C 1-6 diols; C 1-10 alkyl ethers of alkylene glycols; C 3-24 alkylene glycol ethers, polyalkylene glycols, short-chain carboxylic acids, dimethylformamide, dimethyl sulfoxide, dioxane and mixtures thereof, in particular water.

12. Composition according to one of claims 9 to 11, characterized in that the at least one auxiliary agent is selected from the group consisting of active ingredients or nutrients, in particular antimicrobial active ingredients.

13. A process for the preparation of polyamino acid complex compositions according to any one of claims 9 to 12, comprising or consisting of mixing a polyamino acid complex according to any one of claims 1 to 7 with at least one of at least one salt, preferably NaCl, at least one buffer, preferably phosphate-buffered saline solution, at least one auxiliary agent, and at least one solvent, in particular water.

14. Use of the polyamino acid complexes according to any one of claims 1 to 7 or of the polyamino acid complexes prepared according to claim 8 or of the Polyamino acid complex compositions according to any one of claims 9 to 12, or the polyamino acid complex compositions prepared according to claim 13 as synthetic collagen-mimetic materials, in particular for biomedical applications.

15. Use according to claim 14 as cosmetics, for personal care, skin care, in food, as biomaterials or as biomaterial substitutes, for tissue regeneration, tissue repair, tissue engineering, implants, wound healing, wound dressings, topical treatments, tablet coatings, excipients, culture media for cells, Coatings for stents and implants, in drug delivery.

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