Crosslinking of polysaccharides with fibroin and use of the resulting material

Abstract

The invention proposes a method for preparing a crosslinked material, comprising forming an amide bond conjugating one or more fibroin moieties with one or more polysaccharide moieties. The invention further relates to a crosslinked material obtainable from such a method, and to an injectable composition comprising the crosslinked material obtained. The crosslinked material can be a hydrogel and / or a supervolumizer, and can be used in cosmetic and pharmaceutical applications.

Description

[Technical field]

[0001] The present invention relates to a method for preparing a crosslinked material comprising forming an amide bond that conjugates one or more fibroin moieties with one or more polysaccharide moieties. The invention further relates to a crosslinked material obtainable from such a method, and to an injectable composition comprising the crosslinked material obtained. The crosslinked material can be a hydrogel and / or a super-volumizer, and can be used in cosmetic and pharmaceutical applications. [Background technology]

[0002] Facial and body remodeling is of increasing interest. For example, facial and / or body wrinkle filling, skin rejuvenation, breast reconstruction or augmentation, or other types of soft tissue augmentation are of general interest. To avoid the need for surgical intervention, many soft tissue fillers have been developed or are under development that can be injected subcutaneously or into deeper layers of the skin.

[0003] Soft tissue fillers are typically gels, such as hydrogels. Practitioners who use such soft tissue fillers, especially dermal fillers, typically want such fillers to not cause toxic or immunological adverse effects when administered under the conditions of interest, to exhibit good biocompatibility, to be non-invasively injectable, and to be based on natural materials. At the same time, the filler must remain within a spatially defined area, such as when injected, and have sufficient stability in biological systems.

[0004] A material used in the art for soft tissue filling is hyaluronic acid (HA). Hyaluronic acid itself is described as a possible filler in WO2017 / 162676. Cross-linked hyaluronic acid hydrogels are also described in WO2020 / 127407. Hyaluronic acid has good biological tolerance. However, one important drawback is that the biodegradation of such materials based on hyaluronic acid is relatively rapid, making the filler material unsuitable for long-term solutions. It has a limited lifespan in the administered subject, often less than the desired minimum range of several months. If hyaluronic acid is degraded quickly in the body, the viscosity will decrease undesirably quickly and the filling effect will not last long enough.

[0005] Moreover, the shelf life and shelf life of fillers based on hyaluronic acid are often limited.Stored products that contain unmodified hyaluronic acid often tend to partially degrade.The viscosity then decreases, and the thus stored and thus partially degraded product (which is then administered) has a shorter shelf life in the administered subject.

[0006] Therefore, it is necessary to provide further filling materials with improved biological stability. Pharmaceutical compositions containing fibroin fragments and other material components such as hyaluronic acid are also described in WO2020 / 247887. Such blends do not have the desired properties. For example, the stability is quite limited. The obtained materials do not show the desired stability for many cosmetic and therapeutic uses. Therefore, further attempts have been made to improve the material properties by crosslinking silk fibroin with hyaluronic acid by synthetic crosslinking agents.

[0007] Another molecular entity used in the context of filling is silk fibroin. It is an injectable material with good biocompatibility. It has fairly good stability but poor filling properties. However, it has been found that pure fibroin does not have optimal gelling properties. It has therefore been thought to improve the properties of dermal fillers by mixing silk fibroin with elastic proteins such as elastin (see US 8,288,347).

[0008] Attempts have been made to improve the material properties by simply mixing silk fibroin with unbound hyaluronic acid. US 8,288,347 refers to mixing cross-linked hyaluronic acid with a fibroin-containing mixture, where the hyaluronic acid and fibroin are unbound.

[0009] It has been considered to conjugate hyaluronic acid with silk fibroin through a linker that connects polymer chains. US2014 / 0315828 describes a process for crosslinking unmodified hyaluronic acid and fibroin with multi-epoxide or multi-amine crosslinkers. US2014 / 0315828 focuses on the preparation of gels containing small-sized particles. For example, US2014 / 0315828 teaches the multi-amine linker hexamethylenediamine (HMDA), in particular the epoxide-based linker carbethanediol diglycidyl ether (BDDE). Also, US-A2018 / 0055971 teaches the use of multi-amine linkers, such as lysine methyl ester HMDA, as well as epoxide linkers, such as BDDE, for conjugation. WO2020 / 132331 teaches a tissue filler comprising silk fibroin, hyaluronic acid, and polyethylene glycol (PEG), where cross-linking is achieved via a linker moiety.

[0010] It is taught that the linker can be selected, for example, from the group consisting of polyepoxy linker, diepoxy linker, polyepoxy-PEG, diepoxy-PEG, polyglycidyl-PEG, diglycidyl-PEG, polyacrylate PEG, diacrylate PEG, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinylsulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light, glutaraldehyde, hydride, l,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE), adipic acid dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, carbodiimide, and any combination thereof. WO2015 / 149941 teaches linking hyaluronic acid with heparosane via a BDDE linker. KR-A2020 / 0036664 teaches photoinduced crosslinking of hyaluronic acid and fibroin using methacryl groups and photoinitiators such as lithium arylphosphinate.

[0011] The resulting dimensionally stable hydrogels contain such linker structures, however, they contain undesirable interconnecting linker moieties, which are often of non-natural origin.

[0012] Often the linker moiety is itself chemically reactive, for example the epoxide-based linker carbethanediol diglycidyl ether (BDDE). Residues of such unreacted or semi-reacted bifunctional linkers can be harmful and limit the usefulness of the material. There is a maximum administrable content of such reactive linkers, and therefore also for filler materials prepared by using them, for safety reasons.

[0013] For example, one or both epoxy groups may be present and may react with cells. This may be harmful. The maximum amount that can be administered is limited. When administered to a subject in need and degraded in said subject, xenobiotics and non-degradable or poorly degradable metabolites may be generated. This is generally undesirable, especially in cosmetic and pharmaceutical applications. Therefore, it is desirable to avoid such xenobiotic linker moieties.

[0014] Piluso et al. (European Polymer Journal, 2018, 100:77-85) describe a sequential alkyne-azide cycloaddition for the formation of functionalized gelatin hydrogels. teach that gelatin can be functionalized with various moieties, preferably small molecules. A hydrogel can be formed. teach that propiolic acid can be conjugated with a carbodiimide activator, after which a gel can be formed via a bivalent linker. Thus, reactive linkers can be avoided. However, this process is rather complicated and requires several laborious steps and toxic substances.

[0015] CN-A 111440340 describes a rather complex multi-step process for obtaining silk fibroin-sodium hyaluronate crosslinked networks. Silk fibroin is partially digested by contacting it with tyrosinase and hydrogen peroxide. Hyaluronate is reacted with a derivative of ethanesulfonic acid, N-hydroxysuccinimide and a carbodiimide activator. In a third step, the two already reacted solutions are combined with each other. Such a step is relatively laborious. Moreover, undesirable reactive material components, such as N-hydroxysuccinimide, may have deleterious effects if they are used and residues remain in the resulting hydrogel.

[0016] WO2019 / 175036 provides a porous biomaterial for tissue regeneration. The application teaches reacting fibroin moieties with formyl hyaluronic acid. Thus, a direct bond between fibroin moieties and hyaluronic acid is obtained without the introduction of a linker moiety in between. The drawback of this method is that a fairly reactive formyl hyaluronic acid is used, and imine groups are formed, which are rarely found in nature. Reactions are taught, including those using freeze-drying and drying steps.

[0017] In view of the above, there remains an unmet need for an efficient method for preparing biobased materials that requires only low procedural effort, avoids xenobiotic linker moieties being introduced into the hydrogel, and minimizes the residue of toxic reagents. It is further desirable to obtain amide bonds and avoid the need for intermediate formation of formyl groups. Methods that provide injectable hydrogels for face and body remodeling (e.g., usable as supervolumizers) are particularly desirable.

[0018] Surprisingly, it has been found that biobased materials having beneficial properties can be obtained from a process involving the reaction of fibroin moieties containing primary amino residues with polysaccharide moieties containing carboxylic acid residues or salts thereof with an activating agent to form an activation, and thus an amide bond. Summary of the Invention

[0019] A first aspect of the present invention relates to a method for preparing a cross-linked material, said method comprising: (i) contacting the following components with each other: (A) one or more fibroin moieties comprising a primary amino residue or a salt thereof; (B) one or more polysaccharide moieties comprising a carboxylic acid residue or a salt thereof; (C) one or more activating agents that effect reaction of the carboxylic acid residue with the amino residue, thus forming an amide bond; and (D) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds and covalently conjugate one or more fibroin moieties to one or more polysaccharide moieties; and (iii) optionally purifying the crosslinked material obtained from step (ii).

[0020] Such crosslinked materials have been found to have unexpectedly beneficial properties. The crosslinked materials obtainable from the method of the present invention have been found to have long-lasting stability. Viscosity is generally maintained even when incubated at elevated temperatures for several weeks. Similarly, enzymatic degradability is also desirably reduced. Surprisingly, it has been found that xenobiotic linker structures commonly used in conventional rats can be avoided. The crosslinked materials of the present invention can be essentially composed of amino acid moieties and polysaccharide moieties, which can also be found in nature. In an aqueous environment, the resulting crosslinked materials can form hydrogels.

[0021] It can also be used to mimic the extracellular matrix, thus inducing cell proliferation and / or cell migration. It can be successfully used as a filler (e.g., dermal filer), on which cells can populate. The crosslinked material of the present invention can have good shear-thinning properties. Preferably, it has thixotropic properties. Thus, it is preferably less viscous when stressed. It can therefore be injected very well, while still being quite viscous in its target area (e.g., when administered to the subcutaneous area). Relatively low extrusion forces are required. Gels of high viscosity and low extrusion forces can be obtained. It can optionally act as a supervolumizer.

[0022] The process of the present invention can be carried out without undue burden and with relatively little effort. No lubricating phase is found.

[0023] The claimed method is particularly advantageous compared to procedures described in the prior art, since only three educts (raw materials) are required in addition to the solvent: one or more fibroin moieties, one or more polysaccharide moieties, and one or more activators. No further components, such as linkers, are required.

[0024] Omission of a xenobiotic linker structure, such as, for example, BDDE, may allow higher amounts of material to be administered to a subject, e.g., injected subdermally, and may have a longer life span within the subject after administration.

[0025] The obtainable crosslinked material of the present invention may have a long shelf life and shelf life due to the avoidance of reactive groups. It is also relatively thermally stable.

[0026] As used in the context of the present invention, the term "fibroin portion" may be understood in its broadest sense as any portion of fibroin known in the art.

[0027] In preferred embodiments, the one or more fibroin moieties have a weight average molecular weight of at least 1 kDa, at least 5 kDa, at least at least 10 kDa, at least 100 kDa, or at least 200 kDa or more. Preferably, the one or more fibroin moieties are respective polymer moieties or composites of polymer moieties of a total molecular weight (Mw) of at least 5 kDa (5,000 Daltons, 5 kilodaltons), more preferably at least at least 10 kDa (10,000 Daltons), even more preferably at least 100 kDa, especially at least 200 kDa or more. In one embodiment, the one or more fibroin moieties have a weight average molecular weight of 10 to 400 kDa.

[0028] In preferred embodiments, one or more fibroin portions have a weight average of 2000 kDa or less, 1000 kDa or less, 750 kDa or less, 500 kDa or less, 250 kDa or less, 200 kDa or less, or 150 kDa or less.

[0029] In a preferred embodiment, the one or more fibroin moieties have a weight average molecular weight of at least 5 kDa, in the range of 5 to 1000 kDa, in the range of 5 to 400 kDa, in the range of 10 to 400 kDa, or in the range of 100 to 150 kDa. In a preferred embodiment, the one or more fibroin moieties have a weight average molecular weight in the range of 10 to 400 kDa. In another preferred embodiment, the one or more fibroin moieties have a weight average molecular weight in the range of 100 to 150 kDa.

[0030] In particularly preferred embodiments, at least one of the one or more fibroin moieties, and in particular all of the one or more fibroin moieties, may have a weight average molecular weight of 50 to 400 kDa, for example, the one or more fibroin moieties may have a weight average molecular weight of 10 to 100 kDa, 50 to 150 kDa, 100 to 150 kDa, 75 to 200 kDa, 100 to 250 kDa, or 200 to 400 kDa.

[0031] In a preferred embodiment, the fibroin moieties have at least two different weight average molecular weights, each of which contains a primary amino residue or a salt thereof. In other words, the fibroin moieties may also be a mixture of fibroin moieties of different weight average molecular weights. In a preferred embodiment, the fibroin moieties have at least two different molecular weights, and at least one fibroin moiety, preferably both of the at least two fibroin moieties, in particular each of all fibroin moieties, has a molecular weight in the range of 5-1000 kDa, 5-400 kDa, 10-400 kDa, 100-150 kDa, 10-100 kDa, 50-150 kDa, 100-150 kDa, 75-200 kDa, 100-250 kDa, or 200-400 kDa. In a preferred embodiment, the fibroin moieties have at least two different molecular weights, and at least one fibroin moiety, preferably at least two fibroin moieties, both, in particular all fibroin moieties, each have a molecular weight in the range of 50 to 400 kDa.

[0032] As used throughout the present invention, molecular weight (Mw) is preferably the weight average molecular weight of the characterized species. Each fibroin portion may have one or more full-length fibroin polypeptides and / or one or more backbones (amide / protein backbones) of one or more fibroin polypeptides or composites of two or more thereof.

[0033] Preferably, the fibroin portion comprises at least one backbone of a full-length fibroin polypeptide, in particular (essentially) consists of one or more backbones of one or more full-length fibroin polypeptides, in other words, the fibroin portion is preferably derived from fibroin of natural origin.

[0034] In this specification, amide bond can be understood in the broadest sense. Typically, amide bond has the structure -NH-CO- or its tautomeric structure. The amide bond formed between the fibroin part and the polysaccharide part can have any chirality. In one embodiment, it is a racemic mixture.

[0035] In a preferred embodiment, the one or more fibroin moieties are silk fibroin moieties, more preferably silk fibroin moieties having at least 80% sequence homology to a natural insect or spider silk fibroin moiety. In a preferred embodiment, the fibroin is silk fibroin. In another preferred embodiment, the fibroin is a polypeptide or a composite of two or more polypeptides having at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% sequence homology, particularly identity, to one or more naturally occurring silk fibroin polypeptides. Silk fibroin may also include truncated forms thereof. Silk fibroin may be silkworm (Bombyx mori) fibroin and insect or spider silk fibroin.

[0036] The term "moiety" in the context of the invention may be understood in the broadest sense as any molecular structure.

[0037] The moiety may be either a compound comprising or consisting of an individual structure, or may form part of a larger chemical entity, such as, for example, the crosslinked material of the present invention. For example, the fibroin moiety may be a chemical entity comprising fibroin or fibroin. Optionally, the fibroin moiety may comprise more than one fibroin backbone conjugated to each other. Optionally, one or more fibroin backbones may be linked to one or more other structures, such as, in particular, one or more polysaccharide moieties. It will be understood that the term "fibroin moiety" may also include salts and modified forms thereof.

[0038] According to the present invention, at least a portion of the one or more fibroin moieties comprises a primary amino residue or a salt thereof. The amino group can, for example, form part of the lysyl residue of the one or more fibroin moieties. Preferably, at least a portion of the one or more fibroin moieties comprises one or more lysyl residues, which can be optionally linked to a polysaccharide moiety.

[0039] As used in the context of the present invention, the term "fibroin" may be understood in its broadest sense as any fibroin known in the art. Fibroin may be obtained from commercial suppliers (e.g., Advanced BioMatrix, USA (e.g., product number 5154-20ML); CareSilk, Italy (e.g., product number CSK10-1051)) or may be prepared from natural sources or by genetic engineering (also called biofermentation, biotechnological means) or rather synthetic engineering. For example, it may be a fibroin of a species selected from the group consisting of Bombyx mori or, alternatively, of the species Saturniinae, Cricula, Sami, Gonometa and Nephila (e.g. Nephila clavipes) or a homologue having at least 80%, more preferably at least 90%, even more preferably at least 95%, even more preferably at least 98% sequence homology, in particular identity, to one of the above or a truncated form thereof. It will be understood that mixtures of different fibroins may also be used.

[0040] In a preferred embodiment, the fibroin is (essentially intact) silkworm (Bombyx mori) fibroin. In a particularly preferred embodiment, the fibroin is silkworm fibroin available or obtained from Bombyx mori. Silkworm fibroin may be obtained from silkworm cocoons. Processes for obtaining silk from silkworms are well known in the art. For example, silkworm cocoons may be boiled in an aqueous solution for about 30 min. Optionally, the aqueous solution may include about 0.02 M Na2CO3. The cocoons may be rinsed with water or an aqueous buffer, the sericin proteins extracted, and the extracted fibroin may be dissolved in an aqueous buffer. Salts that may be used for this purpose may include, by way of example, lithium bromide, lithium thiocyanate, calcium nitrate, and mixtures thereof. Optionally, the extracted fibroin may be dissolved in about 9-12 M lithium bromide solution.

[0041] The salts can be removed by any means, for example, dialysis. In a preferred embodiment, other components of the silkworm cocoon, such as, for example, sericin, are (essentially) removed. Thus, preferably, at least 50 wt%, more preferably at least 75 wt%, even more preferably at least 80 wt%, especially at least 90 wt% of the sericin originally contained in the silkworm cocoon is removed. The silk fibroin may be type I, type II or type III silk fibroin or a mixture of two or more thereof. Preferably, the fibroin is or comprises type I silk fibroin. The fibroin may have the properties described in the art, for example, as described in US2014 / 315828.

[0042] Alternatively, one or more fibroin polypeptides (including silkworm fibroin polypeptides and complete silkworm fibroin) can also be obtained by genetic engineering. The genetically engineered fibroin can be obtained, for example, from bacteria, insect cells, spider cells, yeast, mammalian cells, transgenic animals, or transgenic plants.

[0043] The one or more fibroin portions may be stored under any conditions. For example, the one or more fibroin portions may be stored in a freezer or in liquid gas, for example, at a temperature range of -15°C to -200°C. For example, the one or more fibroin portions may be stored at approximately -80°C or in liquid nitrogen (i.e., approximately -196°C). The one or more fibroin portions may be stored in a dry state as a powder or as an aqueous solution (e.g., at a concentration in the range of 10-100 mg / ml (e.g., approximately 50 mg / ml)). When thawing a previously frozen solid fibroin or fibroin solution, the one or more fibroin portions may be optionally protected from air, preferably.

[0044] As used in the context of the present invention, the term "polysaccharide moiety" may be understood in its broadest sense as any part of a polysaccharide known in the art that contains a carboxylic acid residue or a salt thereof. As used in the context of the present invention, the term "polysaccharide" may be understood in its broadest sense as any polysaccharide in the art. According to the present invention, at least one polysaccharide moiety contains at least one carboxylic acid residue or a salt thereof. Preferably, one or more polysaccharide moieties further contain hydroxy groups. The polysaccharide may be a polysaccharide of natural origin, which may be modified, or may be a synthetic polysaccharide. In this context, the polysaccharide may be branched or unbranched. It will be understood that the term "polysaccharide moiety" may also include salts and modified forms thereof. In a preferred embodiment, the polysaccharide is not oxidized.

[0045] Preferably, at least one polysaccharide moiety is a polymer moiety with a weight average molecular weight (Mw) of at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or more. Preferably, the one or more polysaccharide moieties have a weight average molecular weight (Mw) in the range of 10 to 10000 kDa. In a preferred embodiment, the one or more polysaccharide moieties have a weight average molecular weight of at least 50 kDa, in particular in the range of 50 to 4000 kDa. More preferably, the one or more polysaccharide moieties have a weight average Mw in the range of 100 to 10000 kDa. In one embodiment, the at least one polysaccharide moiety has a weight average molecular weight of 1.0 to 3.3 mDa. 3 / kg (20°C, 1013 hPa, water).

[0046] In a particularly preferred embodiment, at least one of the one or more polysaccharide moieties, in particular all of the one or more polysaccharide moieties, may have a weight average molecular weight of 1500 to 3500 kDa (1.5 and 3.5 MDa), more preferably it may have a weight average molecular weight in the range of 100 to 5000 kDa, 200 to 2000 kDa, 250 to 1500 kDa, 300 to 1000 kDa, 400 to 900 kDa or 500 to 900 kDa.

[0047] In a preferred embodiment, the one or more polysaccharide moieties comprise one or more types of sugar acid moieties or salts thereof.

[0048] In a preferred embodiment, the one or more polysaccharide moieties comprise one or more types of sugar acid moieties or salts thereof, and the one or more types of sugar acid moieties are selected from the group consisting of: (B1) one or more uronic acid moieties, particularly selected from the group consisting of glucuronic acid moieties, galacturonic acid moieties, iduronic acid moieties, and combinations of two or more thereof; (B2) one or more aldonic acid moieties, particularly selected from the group consisting of a glyceric acid moiety, a xylonic acid moiety, a gluconic acid moiety, an ascorbic acid moiety, and combinations of two or more thereof; (B3) one or more urosonic acid moieties, in particular selected from the group consisting of neuraminic acid moieties, ketodeoxyoctulosonic acid moieties, and combinations thereof; and / or (B4) One or more aldaric acid moieties, particularly selected from the group consisting of a tartaric acid moiety, a meso-galactaric acid moiety, a glucaric acid moiety, and combinations of two or more thereof.

[0049] In a preferred embodiment, one or more of the polysaccharide moieties comprises a uronic acid moiety. In a preferred embodiment, one or more of the polysaccharide moieties comprises a glucuronic acid moiety. In a preferred embodiment, one or more of the polysaccharide moieties comprises a D-glucuronic acid moiety.

[0050] In preferred embodiments, one or more of the polysaccharide moieties comprise or consist of D-sugar moieties. In other embodiments, one or more of the polysaccharide moieties comprise or consist of L-sugar moieties. In other embodiments, one or more of the polysaccharide moieties comprise or consist of a combination of D- and L-sugar moieties. For example, such combinations may include racemic mixtures of sugar moieties, or certain sugar moieties are D-sugar moieties and others are L-sugar moieties.

[0051] In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of one or more glycosaminoglycan moieties. In a preferred embodiment, the one or more polysaccharide moieties are selected from the group consisting of hyaluronic acid (HA) moieties, heparosan moieties, heparin, chondroitin sulfate, and mixtures of two or more thereof. In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of hyaluronic acid, heparosan, chondroitin sulfate, and carboxymethylcellulose. Such polysaccharides containing carboxylic acid groups are also commercially available (e.g., from HTL Biotechnology, Javene, France).

[0052] In a preferred embodiment, the one or more polysaccharide moieties comprise or consist of one or more hyaluronic acid moieties.

[0053] In a preferred embodiment, the cross-linked material of the present invention is a gel. In a preferred embodiment, the cross-linked material of the present invention is a polysaccharide / fibroin gel. In a preferred embodiment, the cross-linked material of the present invention is a hyaluronic acid / fibroin gel (HA / fibroin gel).

[0054] Hyaluronic acid (also called HA, hyaluronate, or hyaluronan) can be understood in the broadest sense as any hyaluronic acid in the art. It can be a polysaccharide moiety that contains hyaluronic acid moieties (also called hyaluronic acid units), preferably containing at least 50 mol% hyaluronic acid moieties, more preferably at least 75 mol%, even more preferably at least 80 mol%, even more preferably at least 90 mol% (relative to the total content of sugar moieties in the polysaccharide) hyaluronic acid moieties. Hyaluronic acid can optionally contain one or more sugar moieties other than hyaluronic acid. Hyaluronic acid can optionally be partially modified. It can, for example, be partially oxidized, can have aldehyde groups, and / or can be crosslinked. Such modifications are described, for example, in WO2020 / 127407.

[0055] In a preferred embodiment, hyaluronic acid is a glycosaminoglycan that is naturally composed of linked repeating units of N-acetyl-D-glucosamine and D-glucuronic acid ([α-1,4-D-glucuronic acid-β-1,3-N-acetyl-D-glucosamine] n ). Thus, the repeating units of hyaluronic acid may be, for example: [ka] Hyaluronic acid may be used as described in WO2017 / 162676. Crosslinked and optionally modified hyaluronic acid, such as that described in WO2020 / 127407, may also be used as hyaluronic acid in the context of the present invention.

[0056] The weight average molecular weight (Mw) of the hyaluronic acid in the context of the present invention is preferably at least 1 kDa (1000 Da), more preferably at least 5 kDa, even more preferably at least 10 kDa, even more preferably at least 50 kDa, even more preferably at least 100 kDa, even more preferably at least 200 kDa, even more preferably at least 300 kDa or more. The weight average molecular weight (Mw) of the hyaluronic acid in the context of the present invention is preferably in the range of 10 to 10000 kDa, more preferably in the range of 100 to 10000 kDa or in the range of 100 to 5000 kDa. In a more preferred embodiment, the hyaluronic acid has a weight average molecular weight (Mw) in the range of 50 to 4000 kDa. More preferably, the hyaluronic acid has a weight average Mw in the range of 100-3500 kDa, 200-2000 kDa, 250-1500 kDa, 300-1000 kDa, 400-900 kDa or 500-900 kDa.

[0057] In a particularly preferred embodiment, at least one of the one or more polysaccharide moieties is one or more hyaluronic acid moieties, and at least one of the one or more hyaluronic acid moieties, in particular all of the one or more hyaluronic acids, may have a weight average molecular weight of 1500 to 3500 kDa.

[0058] Heparosan may be understood in the broadest sense as any heparosan. In a preferred embodiment, it may be such as those described in WO2015 / 149941. Heparosan (HEP) is a biopolymer that belongs to the glycosaminoglycan (GAG) family of polysaccharides.

[0059] In humans, it is an intermediate in the biosynthesis of heparin and heparin sulfate. The structure of heparosan is very similar to hyaluronic acid (HA), since it has the same monosaccharide component sugars as hyaluronic acid and differs from HA only in that the β-(1,3) glycosidic bond between glucuronic acid (GlcUA) and N-acetylglucosamine (GlcNAc) in HA is replaced by a β-(1,4) glycosidic bond in HEP, and the β-(1,4) glycosidic bond between glucuronic acid (GlcNAc) and glucuronic acid (GlcUA) in HA is replaced by an α-(1,4) glycosidic bond in HEP: GlcUA-β-(1-4)-[GlcNAc-α-(1-4)-GlcUA-β-(1-4)] n -GlcNAcHEP

[0060] Typically, heparosan has excellent biocompatibility. Heparosan has many negative charges and hydroxyl groups, and is therefore extremely hydrophilic, which increases tissue compatibility. In addition, due to the fact that heparosan polymers, even after modification, still contain the stretches that occur in natural heparan sulfate and heparin polymers, heparosan is typically non-immunogenic (e.g., does not induce antibodies). Moreover, due to the structural similarity between heparosan and hyaluronic acid, the same chemical modifications (including oxidation to aldehydes, as known for hyaluronic acid) can be carried out on functional groups. The molecular weight (Mw) of the heparosan polymer used in the context of the present invention can have any molecular weight.

[0061] In a preferred embodiment, the polysaccharide moieties (component b), in particular the hyaluronic acid moieties, have at least two different molecular weights, each of which contains a primary amino residue or a salt thereof. In other words, the polysaccharide moieties may also be a mixture of polysaccharide moieties of different molecular weights. In a preferred embodiment, the polysaccharide moieties have at least two different molecular weights, and at least one polysaccharide moiety, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, each have a molecular weight in the range of 10-10000 kDa, in the range of 100-10000 kDa, or in the range of 100-5000 kDa, in the range of 100-3500 kDa, in the range of 200-2000 kDa, in the range of 250-1500 kDa, in the range of 300-1000 kDa, in the range of 400-900 kDa, or in the range of 500-900 kDa.

[0062] In a preferred embodiment, the polysaccharide moieties have at least two different molecular weights, and at least one polysaccharide moiety, preferably at least two polysaccharide moieties, in particular all polysaccharide moieties, each have a molecular weight in the range of 1500-3500 kDa.

[0063] In a preferred embodiment, the polysaccharide moiety comprises or consists of at least two hyaluronic acid moieties having at least two different molecular weights, and at least one hyaluronic acid moiety, preferably both of the at least two hyaluronic acid moieties, in particular all of the hyaluronic acid moieties, each have a molecular weight in the range of 10-10000 kDa, in the range of 100-10000 kDa, or in the range of 100-5000 kDa, in the range of 100-3500 kDa, in the range of 200-2000 kDa, in the range of 250-1500 kDa, in the range of 300-1000 kDa, in the range of 400-900 kDa, or in the range of 500-900 kDa. In a preferred embodiment, the polysaccharide moiety comprises or consists of at least two hyaluronic acid moieties having at least two different molecular weights, and at least one hyaluronic acid moiety, preferably both of the at least two hyaluronic acid moieties, in particular all of the hyaluronic acid moieties, each have a molecular weight in the range of 1500-3500 kDa.

[0064] The (mass) ratio between the total mass of the one or more fibroin moieties (component A) and the total mass of the one or more polysaccharide moieties (component B) can be any ratio. If particularly high water / buffer absorbance is desired, the polysaccharide moieties can be used in mass excess. If particularly high stability is desired, the fibroin moieties can be used in a larger mass excess. Preferably, the (mass) ratio A:B ranges from 1:100 to 100:1.1.

[0065] In a preferred embodiment, the mass ratio A:B between the one or more fibroin moieties (A) and the one or more polysaccharide moieties (B) is in the range of 5:1 to 1:20, preferably in the range of 1:1 to 1:10, in particular in the range of 1:1 to 1:5.

[0066] For example, the mass ratio A:B between the one or more fibroin moieties (A) and the one or more polysaccharide moieties (B) may be in the range of 1:9 to 2:1, 1:8 to 1.5:1, 1:7 to 1:1, 1:6 to 1:1, 1:5 to 1:1, 1:4 to 1:1, 1:3 to 1:1, 1:2 to 1:1, or 1:1.5 to 1:1.

[0067] In a preferred embodiment, the fibroin portion has at least two different molecular weights, each of which contains a primary amino residue or a salt thereof, and the polysaccharide portion, particularly the hyaluronic acid portion, has at least two different molecular weights, each of which contains a primary amino residue or a salt thereof.

[0068] In a preferred embodiment: (a) the fibroin portions have at least two different molecular weights, and at least one fibroin portion, preferably both of the at least two fibroin portions, and in particular all of the fibroin portions, each have a molecular weight in the range of 5 to 1000 kDa, 5 to 400 kDa, 10 to 400 kDa, 100 to 150 kDa, 10 to 100 kDa, 50 to 150 kDa, 100 to 150 kDa, 75 to 200 kDa, 100 to 250 kDa, or 200 to 400 kDa; and (b) the hyaluronic acid moieties have at least two different molecular weights, and at least one hyaluronic acid moiety, preferably both of the at least two hyaluronic acid moieties, in particular all of the hyaluronic acid moieties, each have a molecular weight in the range of 10-10,000 kDa, in the range of 100-10,000 kDa, or in the range of 100-5,000 kDa, in the range of 100-3,500 kDa, in the range of 200-2,000 kDa, in the range of 250-1,500 kDa, in the range of 300-1,000 kDa, in the range of 400-900 kDa, or in the range of 500-900 kDa.

[0069] In a preferred embodiment: (a) the fibroin portions have at least two different molecular weights, and at least one of the fibroin portions, preferably both of the at least two fibroin portions, and particularly each of all of the fibroin portions, has a molecular weight in the range of 5 to 1000 kDa, in particular in the range of 50 to 400 kDa; and (b) the hyaluronic acid moieties have at least two different molecular weights, and at least one hyaluronic acid moiety, preferably at least two hyaluronic acid moieties, both, particularly all, of the hyaluronic acid moieties each have a molecular weight in the range of 1500 to 3500 kDa.

[0070] As used in the context of the present invention, an activating agent may be any compound that effects a reaction of a carboxylic acid residue with an amino residue, thereby forming an amide bond. It will be understood that an activating agent is primarily meant to be a compound that effects a reaction of a carboxylic acid residue of one or more polysaccharide moieties with an amino residue of one or more fibroin moieties, thereby forming an amide bond.

[0071] In a preferred embodiment, the one or more activating agents are selected from the group consisting of: (C1) one or more triazine-based activators, in particular selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM), its salts, and / or 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and combinations thereof; (C2) one or more carbodiimide activators, particularly selected from the group consisting of N,N'-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and combinations of two or more thereof; and (C3) A combination of these.

[0072] In accordance with the present invention, the activator is typically not covalently included in the crosslinked material, and thus it can typically be optionally removed from the crosslinked material of the present invention by any means, such as, for example, washing, filtration, and the like.

[0073] In a preferred embodiment, the triazine-based activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof, preferably it is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium, and the salt of DMTMM is preferably a salt in which the counterion is a cosmetically and / or pharma- ceutically acceptable anion, such as, for example, chloride, acetate, bicarbonate (hydrogen carbonate), or a mixture of two or more anions.

[0074] In a preferred embodiment, the triazine-based activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride.

[0075] In a preferred embodiment, the carbodiimide activator is N,N'-dicyclohexylcarbodiimide (DCC) or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC).

[0076] In a preferred embodiment, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium (DMTMM) or a salt thereof. Preferably, it is a 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium salt, in particular 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (CAS No. 3945-69-5).

[0077] DMTMM is believed to have relatively low, essentially negligible toxicity and is not carcinogenic, mutagenic, or teratogenic / reproductively toxic at amounts commonly used, making it particularly suitable for use in preparing soft tissue fillers, such as dermal or connective tissue fillers.

[0078] When DMTMM is used as the activator, 4-methylmorpholine (NMM) and / or 4,6-dimethoxy-1,3-5-triazin-2-ol (DMT) may be formed as decomposition product(s).

[0079] In a preferred embodiment, the method is characterised in that it further comprises a step (iii) of purifying the crosslinked material by filtration, washing and / or dialysis, in particular by cross-flow filtration, diafiltration and / or dead-end filtration.

[0080] It will be understood that the filtration may be cross-flow filtration, dead-end filtration, or a combination of both. The filtration may be performed by any means. In the context of filtration, the filter may have any pore size suitable for purifying the cross-linked material, i.e. preferably retaining the cross-linked material and allowing the reactants and, optionally, unreacted polysaccharide and / or fibroin to pass through. Optionally, the pore size may be in the range of 5 nm to 2 μm, more particularly pore sizes of 30 nm to 600 nm, more particularly pore sizes of 80 nm to 300 nm, particularly pore sizes of 5 nm to 60 nm. The filter may be made of any material, such as, for example, ceramic, metal, polymeric material, or combinations thereof.

[0081] Optionally, the filtration may be a dynamic filtration, such as that described in WO2020 / 030629. Thus, step (iii) may optionally include dynamic filtration of the crosslinked material, optionally comprising the steps of: a) transferring the cross-linked material to a dynamic filtration device equipped with semi-permeable filter disc(s) and diafiltering the gel, comprising the steps of: i) concentrating the cross-linked material to a predetermined concentration by applying a rotation speed in the range of 20 1 / min to 500 1 / min and an overpressure in the range of 0.5 to 6 bar; or pumping the cross-linked material directly into the process chamber of the dynamic filtration device; ii) performing diafiltration to reduce unwanted molecules by applying a rotation speed in the range of 20 1 / min to 500 1 / min and an overpressure in the range of 0.5 to 6 bar; b) optionally adding a mixture comprising a non-crosslinked material and water to the crosslinked material.

[0082] In one embodiment, the dynamic filtration device is equipped with 1-10 semi-permeable filter disc(s). Any rotation speed and pressure may be used in DCF, for example, rotation speeds in the range of 20 1 / min to 500 1 / min and pressures in the range of 0.5 to 3 bar. Any concentration may be used in DCF, for example, 10 to 70 mg / g.

[0083] Optionally, one or more additional components (e.g., one or more local anesthetics (e.g., as provided below, for example, lidocaine), one or more cell growth factors, one or more dyes, and combinations of two or more thereof) may be added before, during, or after performing step (iii) of purifying the crosslinked material by filtration, washing, and / or dialysis.

[0084] Step (iii) may be carried out for any time suitable for this purpose. Optionally, step (ii) may be carried out for 1 minute to 1 week or more, 2 minutes to 5 days, 3 minutes to 4 days, 5 minutes to 72 hours, 5 minutes to 24 hours, 10 minutes to 12 hours, 30 minutes to 6 hours, 1 hour to 5 hours, or 2 to 4 hours.

[0085] Step (iii) may be carried out at any temperature suitable therefor, such as, for example, between 0°C and 100°C, between 4°C and 95°C, between 10°C and 70°C, between 15°C and 30°C, between 18°C ​​and 25°C, between 20°C and 70°C, between 20°C and 40°C, or between 60°C and 70°C.

[0086] In one embodiment of the present invention, the cross-flow filtration (also referred to as cross-flow filtration) is dynamic cross-flow filtration (DCF). Thus, in one embodiment, the method may further comprise a step (iii) of purifying the cross-linked material by DCF, as exemplified below. Optionally, the DCF may be as described in WO2020 / 030629.

[0087] In a preferred embodiment, the method is further characterized in that steps (i) and (ii) are carried out in a single batch.

[0088] In a preferred embodiment, the method is further characterized in that step (i) is carried out at a temperature in the range of 5 to 90° C., in particular 18 to 30° C., more particularly under ambient conditions (e.g., often 18 to 25° C.). In a preferred embodiment, the method is further characterized in that step (ii) is carried out at a temperature in the range of 5 to 90° C., in particular 18 to 30° C., more particularly under ambient conditions (e.g., often 18 to 25° C.). In a preferred embodiment, the method is further characterized in that step (iii), if present, is carried out at a temperature in the range of 5 to 90° C., in particular 18 to 30° C., more particularly under ambient conditions (e.g., often 18 to 25° C.).

[0089] In a preferred embodiment, the method is further characterized in that steps (i) and (ii) and optional step (iii) are carried out at a temperature in the range of 5 to 90° C., in particular 18 to 30° C. In a preferred embodiment, these steps are carried out at ambient conditions (e.g., often 18 to 25° C.). For example, steps (i) and / or (ii) and / or step (iii) may be carried out at a temperature of approximately 18° C., approximately 19° C., approximately 20° C., approximately 21° C., approximately 22° C., approximately 23° C., approximately 24° C., approximately 25° C., approximately 26° C., approximately 27° C., approximately 28° C., approximately 29° C., or approximately 30° C.

[0090] Steps (i) and (ii) and optional step (iii) can be carried out at any pressure, for example the pressure can be ambient pressure (e.g. often around 970-1100 hPa external pressure).

[0091] The step (i) of contacting the components with each other can be carried out by any means. In a preferred embodiment, the method is further characterized in that the step (i) comprises mixing the components, i.e. one or more fibroin moieties (as component A), one or more polysaccharide moieties (as component B), one or more activators (as component C), and one or more solvents (as component D), and optionally one or more further components. Such mixing can be carried out by any means, such as, for example, by stirring and / or shaking.

[0092] In a preferred embodiment, one or more polysaccharide moieties (as component B) and one or more activating agents (as component C) are dissolved in one or more solvents (as component D) without one or more fibroin moieties (as component A) in a first step and incubated. This allows activating the carboxylic acid groups of the polysaccharide moieties. The incubation can be carried out for any time sufficient for such purpose. In a preferred embodiment, in a first sub-step, one or more polysaccharide moieties are dissolved in one or more solvents and in a subsequent sub-step, one or more activating agents are added, together representing an activation step.

[0093] By way of example, such an activation step may be carried out for 5 minutes to 24 hours, 10 minutes to 12 hours, 30 minutes to 6 hours, 45 minutes to 5 hours, 45 minutes to 4 hours, or 1 to 3 hours. By way of example, such an activation step may be carried out at any temperature, for example, at 0°C to 100°C, at 4°C to 95°C, at 10°C to 70°C, at 15°C to 30°C, at 18°C ​​to 25°C, at 20°C to 70°C, at 20°C to 40°C, or at 60°C to 70°C, in particular at ambient temperature (for example, often 18°C ​​to 25°C). For example, the incubation may be for 1 minute to 24 hours, in particular for 30 minutes to 2 hours or for 1 to 3 hours, at a temperature of 10°C to 25°C, in particular 18°C ​​to 22°C. After such an incubation, one or more fibroin moieties (as component A) may be added. This may be further incubated and step (ii) of the method of the invention is carried out. Step (ii) may be carried out using any suitable solvent, such as water or an aqueous buffer. Optionally, the solution may be stirred during the reaction step.

[0094] Thus, in a preferred embodiment, step (i) of the method comprises the following sub-steps: (ia) contacting the following components with each other: (B) one or more polysaccharide moieties comprising a carboxylic acid residue or a salt thereof; (C) one or more activating agents that effect reaction of the carboxylic acid residue with the amino residue, thus forming an amide bond; and (D) one or more solvents; Preferably, one or more polysaccharide moieties (B) are first dissolved in one or more solvents (D) and then one or more activating agents (C) are added; (ib) reacting at least some of the carboxylic acid residues with one or more activating agents, thereby forming one or more activated polysaccharide moieties (also referred to as polysaccharide-activating agent conjugates); and (ic) adding to the activated polysaccharide portion of substep (ib) (A) One or more fibroin moieties comprising a primary amino residue or a salt thereof.

[0095] In another preferred embodiment, components A, B, C and D and optionally one or more further components are mixed all at once.

[0096] Step (ii) may be carried out for any suitable reaction time. For example, the reaction time may be in the range of 1 minute to 7 days, preferably 5 minutes to 2 days, more preferably 10 minutes to 24 hours. For example, the reaction time may be in the range of 15 minutes to 24 hours, or 30 minutes to 12 hours, or 45 minutes to 6 hours, or 1 hour to 4 hours.

[0097] In a preferred embodiment, the method comprises: (i) contacting the following components with each other: (A) one or more silk fibroin moieties having a weight average molecular weight of at least 5 kDa, comprising a primary amino residue or a salt thereof; (B) one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa, comprising a carboxylic acid residue or a salt thereof; (C) one or more triazine-based activators that effect reaction of a carboxylic acid residue with an amino residue, thus forming an amide bond (particularly, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof); and (D) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds and covalently conjugate one or more silk fibroin moieties to one or more hyaluronic acid moieties; and (iii) optionally purifying the crosslinked material obtained from step (ii).

[0098] Any solvent that can be used as component D for the process of the present invention can be used. In a preferred embodiment, a polar solvent is used. In a preferred embodiment, a protic solvent is used. In a preferred embodiment, a protic polar solvent is used.

[0099] In a preferred embodiment, the solvent usable as component D comprises, relative to the total mass of the solvent, more than 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, at least 95 wt%, or even 100 wt% of one or more components selected from the group consisting of: water; one or more alcohols, preferably one or more C1-C5-alcohols, more preferably one or more C1-C5-alcohols selected from the group consisting of methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), sec-butanol (2-butanol) isobutanol, (2-methylpropan-1-ol), tert-butanol (2-methylpropanol), pentan-1-ol, 2-methylbutan-1-ol, 3-methylbutan-1-ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan-2-ol, pentan-3-ol and / or 2-methylbutan-2-ol, and combinations of two or more thereof, in particular methanol and / or ethanol; one or more primary amines, in particular one or more C1-C5-amines; one or more carbonic acids, preferably one or more C1-C5-carbonic acids selected from the group consisting of formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, in particular formic acid and / or acetic acid; one or more primary or secondary amides, preferably one or more C1-C5-amides, in particular formamide; one or more sulfoxides, preferably one or more C1-C5-amides, in particular dimethyl sulfoxide (DMSO); and any combination of two or more thereof.

[0100] In a preferred embodiment, an aqueous solvent is used, i.e. a solvent with a water content of more than 50 wt%, at least 60 wt%, at least 70 wt%, at least 80 wt%, at least 90 wt%, at least 95 wt%, or even 100 wt%, by weight relative to the total mass of the solvent. In one embodiment of the invention, the aqueous buffer comprises, in addition to water, one or more components selected from the group consisting of one or more alcohols (particularly one or more C1-C5-alcohols, such as, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol (1-butanol), sec-butanol (2-butanol) isobutanol, (2-methyl-propan-1-ol), tert-butanol (2-methylpropanol), pentan-1-ol, 2-methylbutan-1-ol, 3-methylbutan-1-ol, 2,2-dimethylpropan-1-ol, pentan-2-ol, 3-methylbutan-2-ol, pentan-3-ol and / or 2-methylbutan-2-ol), one or more primary amines (particularly one or more C1-C5-amines), One or more carbonates (particularly one or more C1-C5-carbonates, such as, for example, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid), one or more primary or secondary amides (particularly one or more C1-C5-amides, such as, for example, formamide), one or more sulfoxides (particularly one or more C1-C5-amides, such as, for example, dimethyl sulfoxide (DMSO)), one or more inorganic or organic cations (particularly one or more inorganic or organic cations of molecular weight less than 1000 Da, in particular alkali cations or alkaline earth cations, other metal cations, protons, ammonium cations, etc.), one or more inorganic or organic anions (particularly one or more inorganic or organic anions of molecular weight less than 1000 Da, in particular chloride, sulfate, etc.), one or more silicates, and combinations of two or more thereof.

[0101] In a preferred embodiment, water or an aqueous buffer (e.g., phosphate-buffered saline (PBS), Tris buffer, borate buffer, acetate-buffered buffer, etc.) is used as the solvent. In a preferred embodiment, a hydroalcoholic solvent is used, such as, for example, a mixture of water and ethanol, methanol, propanol, butanol, and / or pentanol. In a preferred embodiment, water is used as solvent D.

[0102] Water may be understood herein in the broadest sense. Preferably, the water is deionized water, distilled water or tap water, in particular deionized water or distilled water.

[0103] As indicated above, in a preferred embodiment, step (i) of the method comprises the following sub-steps: (ia) contacting the following components with each other: (B) one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa, comprising a carboxylic acid residue or a salt thereof; (C) one or more triazine-based activators that effect reaction of carboxylic acid residues with amino residues, thus forming amide bonds (in particular, the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof); (D) one or more solvents; Preferably, one or more hyaluronic acid moieties (B) are first dissolved in one or more solvents (D), after which one or more triazine-based activators (C) are added; (ib) reacting at least some of the carboxylic acid residues with one or more triazine-based activators, thereby forming one or more activated hyaluronic acid moieties (also referred to as hyaluronic acid-activator conjugates); and (ic) adding to the activated hyaluronic acid portion of substep (ib) (A) one or more silk fibroin moieties comprising a primary amino residue or a salt thereof and having a weight average molecular weight of at least 5 kDa.

[0104] In one embodiment of the present invention, the method of the invention comprises the following steps (preferably sequentially): Dissolving a polysaccharide (preferably hyaluronic acid, especially hyaluronic acid sodium salt) in water or a buffer; Addition of an activator (preferably DMTMM) to the polysaccharide solution; Activation (preferably for several hours, for example at a temperature of 18-22°C); Addition of fibroin solution to activated polysaccharide; Stirring and formation of crosslinked material (preferably for several hours, for example at a temperature of 18-22 ° C); Purification of the crosslinked material (e.g., removal of DMTMM and its degradation products, e.g., by filtration and / or dialysis, e.g., at a temperature of 18-22°C); Optionally, the addition of an anesthetic (e.g., lidocaine); and Optionally, sterilize.

[0105] A further aspect of the present invention relates to crosslinked materials obtainable from the process of the present invention.

[0106] It will be understood that the definitions and preferred embodiments given in connection with the method of the present invention are provided mutatis mutandis for the crosslinked material of the present invention.

[0107] In one embodiment of the invention, the cross-linked material was prepared by using DMTMM as an activator. In one embodiment of the invention, the cross-linked material comprises DMTMM, 4-methylmorpholine (NMM) and / or 4,6-dimethoxy-1,3-5-triazin-2-ol (DMT), especially before final purification. In one embodiment of the invention, the cross-linked material comprises NMM and / or DMT, especially before final purification. In one embodiment of the invention, the cross-linked material comprises up to 0.1% by weight, preferably 0.01-1000 ppm, 0.1-100 ppm or 1-10 ppm of NMM and / or DMT, based on the total weight of the cross-linked material (gel), especially before final purification.

[0108] A further aspect of the invention relates to a crosslinked material comprising or consisting of: (A) one or more fibroin moieties having a weight average molecular weight of at least 5 kDa; and (B) one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa; One or more fibroin moieties are covalently conjugated to one or more hyaluronic acid moieties via amide bonds without any interconnecting linker structure.

[0109] In this specification, the term "without an interconnecting linker structure" can be understood in the broadest sense in that no additional chemical moieties that are not derived from (also referred to as not present in) the fibroin moiety or polysaccharide moiety (e.g., hyaluronic acid moiety) are introduced into the chemical structure that covalently conjugates one or more fibroin moieties with one or more hyaluronic acid moieties via amide bonds. In other words, the amide bond is preferably formed from the inclusion of a nitrogen atom derived from the fibroin (e.g., lysinyl side chain) and a carbon atom derived from the polysaccharide moiety (e.g., hyaluronic acid moiety).

[0110] In a preferred embodiment, the cross-linked material is further characterized in that it does not contain imide groups. In a preferred embodiment, the cross-linked material is further characterized in that it does not contain imine groups. In a preferred embodiment, the cross-linked material is further characterized in that it does not contain epoxy groups. In a preferred embodiment, the cross-linked material is further characterized in that it does not contain xenobiotic linker moiety groups.

[0111] In a preferred embodiment, the cross-linked material is further characterized in that it does not include the following, which interconnect one or more fibroin moieties with one or more polysaccharide moieties: (a) an imide group; (b) an imine group; (c) epoxy groups; and / or (d) The xenobiotic linker moiety.

[0112] In a preferred embodiment, the cross-linked material is further characterized in that it does not contain: (a) an imide group; (b) an imine group; (c) an epoxy group; and (d) The xenobiotic linker moiety.

[0113] A further aspect of the invention relates to an injectable composition comprising the crosslinked material of the invention and a liquid or viscous carrier and optionally further components, preferably the crosslinked material is a hydrogel and / or a supervolumizer.

[0114] It will be understood that the definitions and preferred embodiments presented in connection with the methods and cross-linking materials of the invention apply mutatis mutandis to the injectable compositions of the invention.

[0115] In a preferred embodiment, the crosslinking material and / or injectable composition of the present invention can be used as a soft tissue filler, particularly as a dermal filler or connective tissue filler.

[0116] The liquid or viscous carrier according to the present invention contained in the injectable composition may be any injectable carrier. Typically, the liquid or viscous carrier is a pharma- ceutically and / or cosmetically acceptable carrier, and thus, when administered to a mammal in the sense of the present invention, is a carrier that is non-toxic to mammals, particularly humans. The liquid or viscous carrier may preferably comprise or consist of one or more solvents, such as, for example, water, aqueous buffer (e.g., saline or phosphate buffered saline), dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil, or combinations thereof. More preferably, the liquid or viscous carrier comprises or consists of a non-pyrogenic isotonic buffer, more particularly saline or buffered saline.

[0117] The optional additional component may be any component. For example, such additional component may be selected from the group consisting of one or more local anesthetics, one or more cell growth factors, one or more dyes, and combinations of two or more thereof.

[0118] Such further components may be added at any time, for example before, during or after purifying the cross-linked material. For example, one or more further components may be added during purification step (iii). In another embodiment of the invention, one or more further components may be added to the prepared, optionally purified, cross-linked material.

[0119] A local anesthetic can make the injection more comfortable for an individual. A cell growth factor can improve cell penetration into the administered crosslinked material of the present invention. A dye can improve the localization of the injected drug (e.g., a pharma- ceutically acceptable fluorescent dye such as fluorescein or rhodamine) or can improve the invisibility of an otherwise whitish crosslinked material (e.g., by making it flesh-colored). Any other pharma- ceutically active compound can also be added. Thus, the crosslinked material of the present invention can also function as a delay form for administration.

[0120] Suitable local anesthetics for use herein include, but are not limited to, ambucaine, amoranone, amylocaine, benoxinate, benzocaine, betoxycaine, biphenamine, bupivacaine, butacaine, butambene, butanilicaine, butethamine, butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine, cyclomethycaine, dibucaine, dimethysoquin, dimethocaine, diperodone, dycyclonine, ecgonidine, ecgonine, ethyl chloride, etidocaine, beta-eucaine, euprosin, fenalcomine, formocaine, hexylcaine, hydroxycaine, hydroxypro ... Cytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate, levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methyl chloride, myrtecaine, nepain, octacaine, orthocaine, oxethazaine, parethoxycaine, phenacaine, phenol, piperocaine, pyridocaine, polidocanol, pramoxine, prilocaine, procaine, propanocaine, proparacaine, propipocaine, propoxycaine, psuedococaine, pyrrocaine, ropivacaine, salicylic alcohol, tetracaine, tricaine, trimecaine, zolamine, and salts thereof. Combinations of two or more of the mentioned anesthetics may also be used herein, for example, other "caine"-anesthetic(s) combinations such as lidocaine and prilocaine.

[0121] Depending on the intended use of the crosslinked material and injectable composition of the invention, it can be provided in different packaging. It can be stored in any conditions suitable for this, for example at ambient temperature (e.g., 18-30°C, preferably 18-25°C), in a refrigerator (e.g., 0-15°C, preferably 3-10°C), in a freezer (e.g., at -30-0°C, preferably -25-10°C), in a deep freezer (e.g., -100-300°C, preferably -90-55°C), on liquid nitrogen, on dry ice, or even on one or more liquid noble gases. For example, it can be provided in a vial, in a syringe. It can be administered to a subject by injection (e.g., by syringe or infusion). It can be stored in a dry state, as hydrogen, as a gel with other non-aqueous solvents, and / or as a suspension, emulsion, colloid or solution.

[0122] The administration can be carried out by any means. In a preferred embodiment, the administration is via a needle. In a preferred embodiment, the administration is via a syringe, in particular via a syringe intradermal or subdermal. The administration can be manual administration, administration using a mechanical pump, or automated administration. For example, the Syringe One system can be used for administration.

[0123] The crosslinking material of the present invention and the injectable composition of the present invention can be used according to purpose.Optionally, the crosslinking material of the present invention and the injectable composition of the present invention can be used for cosmetic and / or therapeutic use.The injectable composition can be a filler, particularly a soft tissue filler, for example, a soft-tissue filler, particularly a dermal filler or a connective tissue filler.

[0124] As used herein, the term "filler" may be understood in the broadest sense as any agent that can be used to fill cavities or function as a soft tissue filler, preferably a soft tissue filler. A soft tissue filler may be understood in the broadest sense as a material designed to add volume to areas of soft tissue defects. The filler may be administered anywhere, by any type of injection, and may be suitable for use in cosmetic / anesthetic applications as well as for therapeutic purposes. A filler may generally be any composition that adds, replaces, or increases volume subcutaneously, leading, for example, to smoothed skin wrinkles, enlarged lips, improved skin appearance, or scar treatment. It is generally used in the dermal region, for example, under the epidermis or on the subcutaneous tissue, and as such may be injected subcutaneously, into the subcutaneous tissue, or intradermally, or in some combination.

[0125] Injectable compositions within the meaning of the present invention can be administered (dispensed from) by syringes under normal conditions and under standard pressure. Moreover, the filler compositions of the present invention are preferably (essentially) sterile. Preferably, the injectable compositions are suitable for injection into mammals, in particular humans.

[0126] The present invention also relates to the use of the injectable composition according to the invention as a filler, for example a soft tissue filler, in particular a dermal filler or a connective tissue filler. It may be used as a supervolumizer. In this context, it may be used as a hydrogel.

[0127] The present invention also relates to the use of the injectable composition according to the invention for cosmetic applications. More preferably, the present invention also relates to the use of the injectable composition according to the invention for cosmetic applications, including face and body reshaping and rejuvenation.

[0128] Thus, a further aspect of the present invention relates to the use of the injectable composition of the present invention for cosmetic applications including face and body reshaping and rejuvenation, preferably including: filling of wrinkles, improvement of facial lines, breast reconstruction or augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling of facial wrinkles, improvement of glabellar lines, improvement of nasolabial folds, improvement of marionette lines, improvement of cheek commissures, improvement of perilabial wrinkles, improvement of crow's feet, improvement of subdermal support of the brow, cheekbones and cheek fat pads, improvement of tear troughs, improvement of nasal appearance, lip augmentation, cheek augmentation, augmentation of the perioral area, augmentation of the infraorbital area, elimination of facial asymmetry, improvement of jawline, augmentation of the chin, or a combination of two or more thereof.

[0129] It will be understood that the definitions and preferred embodiments presented in connection with the methods, crosslinking materials and injectable compositions of the invention apply mutatis mutandis to the uses of the present invention.

[0130] In a preferred embodiment, the present invention relates to the use of the injectable composition of the present invention for reducing facial wrinkles.

[0131] In one embodiment, the use of the present invention may be a cosmetic application, preferably a non-therapeutic use. The use of the present invention may be carried out by a cosmetician, a cosmetic professional or a health care professional.

[0132] In a preferred embodiment, the use of the injectable composition of the present invention is for improving skin texture, treating fine lines, treating deep wrinkles or restoring volume, or as a super volumizing filler for breast or buttock augmentation.

[0133] This use may be therapeutic and / or cosmetic.In other words, the present invention thus relates to an injectable composition according to the present invention for use in a method for face and body remodeling and rejuvenation, preferably comprising: filling wrinkles, improving face lines, breast reconstruction or augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling facial wrinkles, improving glabellar lines, improving nasolabial folds, improving marionette lines, improving cheek commissures, improving perilipal wrinkles, improving crow's feet, improving subdermal support of the eyebrows, cheekbones and cheek fat pads, improving tear troughs, improving the appearance of the nose, lip augmentation, cheek augmentation, augmentation of the perioral area, augmentation of the infraorbital area, eliminating facial asymmetry, improving jawline, augmenting chin, or a combination of two or more thereof.

[0134] The present invention also relates to a method of face and body reshaping and rejuvenation (preferably including the specific uses described above), said method comprising administering an injectable composition according to the invention.

[0135] In other words, the present invention also relates to a method for face and body reshaping and rejuvenation, preferably comprising: filling wrinkles, improving facial lines, breast reconstruction or augmentation, skin rejuvenation, buttocks augmentation, cheekbone remodeling, soft tissue augmentation, filling facial wrinkles, improving frown lines, improving nasolabial folds, improving marionette lines, improving cheek commissures, improving perilabial wrinkles, improving crow's feet, improving subdermal support of the brow, cheekbones and cheek fat pads, improving tear troughs, improving nasal appearance, lip augmentation, cheek augmentation, augmentation of the perioral area, augmentation of the infraorbital area, eliminating facial asymmetries, improving jawline, augmentation of the chin, or a combination of two or more thereof, wherein a sufficient amount of an injectable composition according to the present invention is administered to a subject in need thereof.

[0136] As used herein, a subject (also referred to as an individual) may be any animal, typically a mammal, preferably a domestic mammal or a human. Particularly preferably, the individual is a human. The human being to be treated may also be designated as a patient, regardless of his / her health condition.

[0137] Reconstruction may be performed for cosmetic purposes or may be performed after tissue loss caused, for example, by accident or surgical intervention. For example, an accident may damage a part of the face. On the other hand, the cheekbones may be augmented by subcutaneous filling of the cheekbone area. The breast or a part thereof may be surgically removed. On the other hand, breast reconstruction or augmentation may also have aesthetic reasons.

[0138] Preferably, the injectable composition of the present invention can be administered to an individual in an effective amount by injection, for example by subcutaneous or intradermal injection. In a preferred embodiment, in the context of this use, the injectable composition is a filler. In a more preferred embodiment, in the context of this use, the injectable composition is a filler, in particular a supervolumizer, and the use comprises administering a composition comprising the crosslinking material of the present invention in the target tissue, in particular subcutaneously or intradermally. For example, the injectable composition can be injected intradermally or subcutaneously using a serial puncture technique. An effective amount refers to the amount of (injectable) soft tissue filler composition sufficient to achieve a beneficial or desired cosmetic (aesthetic) or therapeutic result.

[0139] In a particularly preferred embodiment, in the context of this use, the injectable composition is a filler which may be a supervolumizer, in particular a soft tissue filler, and the use comprises subcutaneous or intradermal administration of a composition comprising the crosslinked material of the invention. For these uses, the crosslinked material according to the invention is particularly beneficial, since it is fairly stable in an aqueous environment, such as body fluids, and allows cellular penetration due to its structure and properties.

[0140] A further aspect of the invention relates to a crosslinking material or an injectable composition according to the invention for use in a method for regenerating tissue in an individual in need thereof.

[0141] In other words, the present invention also relates to a method for regenerating tissue in an individual in need thereof, said method comprising administering to an individual in need thereof a crosslinking material or an injectable composition according to the present invention. The regeneration of tissue in an individual in need thereof may be performed for therapeutic and / or cosmetic purposes.

[0142] It will be understood that the definitions and preferred embodiments set out in connection with the above crosslinking materials, methods and injectable compositions apply mutatis mutandis to use in regenerating tissue in an individual.

[0143] The tissue to be regenerated may be any tissue. In one preferred embodiment, the tissue is a soft tissue. In a more preferred embodiment, the tissue is a soft tissue selected from the group consisting of skin tissue (including dermal tissue and subcutaneous tissue) and connective tissue. Thus, the method can be used for remodeling and rejuvenation, including the uses described above. In another preferred embodiment of the present invention, the tissue is a joint (connective) tissue. Optionally, for this use, the crosslinking material can include one or more cell growth factors that stimulate the proliferation of the respective tissue.

[0144] In another preferred embodiment, the tissue is bone tissue.The bridging material of the present invention can then be administered where bone tissue is intended to grow, for example, in the gap of a fracture or for bone lengthening.Optionally, for this use, the bridging material can include one or more cell growth factors that stimulate bone cell growth.

[0145] Depending on the particular application, one skilled in the art will either use particulate crosslinked material according to the present invention or will use blocks of crosslinked material according to the present invention.

[0146] For the above-mentioned therapeutic and cosmetic applications, the crosslinked materials according to the present invention are particularly advantageous because they are fairly stable in aqueous environments, such as body fluids, and allow cell penetration due to their crosslinked structure and surface properties.

[0147] As shown above, the crosslinked material of the present invention is obtained by the inventive material obtained when one or more fibroin moieties are conjugated with one or more polysaccharide moieties, which conjugates as such also have unexpectedly beneficial properties.

[0148] As used herein, the terms "approximately" and "about" can be understood to include a range that includes a deviation of up to + / - 10% of the individual numerical values. It will be understood that specific values ​​are also expressly disclosed.

[0149] It will be further understood that ranges encompass numerical values ​​provided as naturally rounded values, including all rounding limits, For example, the range "1 mg" encompasses the range from 0.50 to 1.49 mg.

[0150] However, the numerical values ​​of the present invention also disclose one or more more precise values. Thus, for example, "1 mg" can also include a specific disclosure of "1.0 mg."

[0151] The examples and claims set forth embodiments of the invention.

[0152] Working Example material and method raw material hyaluronic acid, different intrinsic viscosities (HTL Biotechnology, Javene, France); a 5% aqueous solution of fibroin from silkworms (CareSilk srls, Lecce, Italy); 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) (Sigma Aldrich, Darmstadt, Germany); Water (internal system for desalinated water); Lidocaine hydrochloride (Albemarle Corp., Charlotte, USA) Unless otherwise specified, all syntheses and measurements are performed at ambient conditions, i.e., at ambient temperature (eg, 18-25° C., especially approximately 20° C.) and ambient / atmospheric pressure.

[0153] Push-out force (EF) Extrusion force (EF) was measured using an instrument - TA.XT Plus Texture Analyzer (Stable Micro Systems Ltd., Surrey, UK). A syringe equipped with a 30G TSK needle (TSK Laboratory Europe, Oisterwijk, The Netherlands) was placed into the instrument, which then pressed the syringe plunger at a constant speed of 0.21 mm / s (approximately 1.26 cm / min) over a distance of 30 mm. The force required to extrude the contents of the syringe through the needle was recorded and the average value was calculated and reported as the extrusion force.

[0154] Rheology Rheology was measured using an Anton Paar MCR 302 (Anton Paar GmbH, Graz, Austria) with an instrument-cone-plate (CP50-1, 50 mm diameter) configuration. Measurements were performed in oscillatory mode with a frequency sweep from 0.1 Hz to 10 Hz at a constant deformation of 0.1% at 25° C. Storage modulus (G') and loss factor (tan δ) at 1 Hz were reported as measurements.

[0155] Example 1 - Preparation of crosslinked materials and effect of the amount of lubricating phase on the properties of the materials 2.8m 3Hyaluronic acid (HA) (4.6 g, corresponding to 4.0 g of dry polymer) with an intrinsic viscosity of 100 μg / kg was dissolved in 200 g of water (resulting in a polymer concentration of 20 mg / g). DMTMM (3.3 g, corresponding to 2.8 g of dry material - 1 eq. relative to the amount of HA) was added and the mixture was stirred for 1 h. Then, 100 mL of fibroin solution (concentration was 20 mg / g, HA / fibroin weight ratio was 2 / 1) was added: the mixture was stirred for 2 h and then the stirring was stopped. The next day, the crosslinked material (here exemplified as HA / fibroin gel) was purified using dynamic cross-flow filtration (DCF Andritz, membrane d=152 mm (Andritz AG, Graz, Austria)) and 5 mM phosphate buffered saline (PBS) solution at room temperature. The purified crosslinked material (here exemplified as HA / fibroin gel) was then mixed with different amounts of lubricating phase (lubricating phase had a concentration of 30 mg / g). Finally, the crosslinked material (exemplified here as HA / fibroin gel) was loaded into a 1 mL syringe (Syringe One) and sterilized for 8 minutes at 127° C. The results are shown in Table 1 below. [Table 1]

[0156] A lubricating phase can be added to the gel to reduce the extrusion force, however, in the case of the crosslinked material according to the invention (exemplified here as HA / fibroin gel), the lubricating phase surprisingly does not cause a reduction in the extrusion force, thus other materials could be prepared without the lubricating phase.

[0157] It was found that cross-linked materials without a lubricating phase (exemplified here as HA / fibroin gels) can have G' comparable to or even higher than the commercially available cross-linked hyaluronan product Belotero Volume Lidocaine (Anteis SA, Plan-les-Ouates, Switzerland). One of the HA / fibroin gels exemplified herein had a G' of approximately 278 Pa after sterilization (Fib01A), while the equivalent hyaluronan product Belotero Volume Lidocaine had a G' of approximately 270 Pa. However, the extrusion force (EF) through a 30G TSK needle was found to be significantly lower. The HA / fibroin gels studied had an extrusion force (EF) of approximately 12 N, while the cross-linked hyaluronan (Belotero Volume Lidocaine) had an EF of approximately 22 N. This may support that HA / fibroin gel can provide a similar lifting effect as the commercially available cross-linked hyaluronan product Belotero Volume Lidocaine (due to a similar G') while offering better injectability to the practitioner.

[0158] Furthermore, in this set of experiments, the G' of the crosslinked material (exemplified here as HA / fibroin gel) decreased by only 10-25% after sterilization, which is significantly lower than the 78% decrease in G' in the control experiment (pure non-crosslinked hyaluronan), the results of which are also shown in Table 1 above.

[0159] Example 2 - Preparation of crosslinked materials and effect of HA / fibroin ratio on material properties 2.8m 3Hyaluronic acid (HA) (3.5 g, corresponding to 3.0 g of dry polymer) with an intrinsic viscosity of 10000 g / kg was dissolved in 150 g of water (resulting in a polymer concentration of 20 mg / g). DMTMM (2.5 g, corresponding to 2.1 g of dry material - 1 eq. with respect to the amount of HA) was added and the mixture was stirred for 1 h. Then 150 mL of fibroin solution (concentration was 20 mg / g, HA / fibroin weight ratio was 1 / 1) was added. The mixture was stirred for 2 h and then the stirring was stopped. The next day, the crosslinked material (here exemplified as HA / fibroin gel) was purified using dynamic cross-flow filtration (DCF Andritz, membrane d=152 mm (Andritz AG, Graz, Austria) and 5 mM phosphate buffered saline (PBS) solution at room temperature. No lubricating phase was added. Finally, the crosslinked material (here exemplified as HA / fibroin gel) was filled into a 1 mL syringe (Syringe One) and sterilized at 127° C. for 8 minutes. The results are shown in Table 2 below. [Table 2]

[0160] It was found that increasing the amount of fibroin in the crosslinked material led to a crosslinked material (exemplified here as HA / fibroin gel) with a higher G'.

[0161] In particular, the crosslinked material has a lower extrusion force (even though it has a higher G'). An explanation for this could be the thixotropic (shear thinning) behavior of fibroin. Crosslinked materials with relatively high, tunable G' and relatively low, tunable extrusion force can be obtained.

[0162] Example 3 - Preparation of cross-linked materials and the effect of intrinsic viscosity (IV) of hyaluronic acid (HA) on material properties 1.5m 3Hyaluronic acid (HA) (3.5 g, corresponding to 3.0 g of dry polymer) with an intrinsic viscosity of 10000 g / kg was dissolved in 150 g of water (resulting in a polymer concentration of 20 mg / g). DMTMM (2.5 g, corresponding to 2.1 g of dry material - 1 eq. with respect to the amount of HA) was added and the mixture was stirred for 1 h. Then 150 mL of fibroin solution (concentration was 20 mg / g, HA / fibroin weight ratio was 1 / 1) was added. The mixture was stirred for 2 h and then the stirring was stopped. The next day, the crosslinked material (here exemplified as HA / fibroin gel) was purified using dynamic cross-flow filtration (DCF ANDRITZ, membrane d=152 mm (Andritz AG, Graz, Austria) and 5 mM phosphate buffered saline (PBS) solution at room temperature. No lubricating phase was added. Finally, the crosslinked material (here exemplified as HA / fibroin gel) was filled into a 1 mL syringe (Syringe One) and sterilized at 127° C. for 8 minutes. The results are shown in Table 3 below. [Table 3]

[0163] Example 4 - Enzymatic degradation of HA / fibroin gel To investigate whether the crosslinked material (exemplified herein as HA / fibroin gel) can also be used as a reversible filler, the material was treated with the enzyme hyaluronidase from sheep testes. Approximately 0.50 g of gel was weighed by differential weighing and placed on the plate of the CP50-1 (cone-plate) system of an Anton Paar MCR 302 rheometer (Anton Paar GmbH, Graz, Austria). A homogenous aqueous solution of 150 μL WFI containing 50 U hyaluronidase was added to the top surface of the hydrogel on the plate. The hyaluronidase-hydrogel mixture was homogenized manually, e.g., with a pipette tip, for approximately 10 seconds. Measurements were then performed at 37° C. in oscillatory mode, with a deformation of 0.1% and a frequency of 1 Hz. The measurement period was 60 minutes, with 1 point recorded per minute.

[0164] Gels prepared with non-crosslinked HA degraded the fastest, followed by gels composed of crosslinked HA (Belotero Volume, Anteis SA, Plan-les-Ouates, Switzerland) and gels containing fibroin degraded the slowest. This may indicate an extended lifespan of gels containing fibroin compared to typical HA crosslinked gels. The results are shown in Table 4 below. [Table 4]

[0165] Example 5 - Accelerated Stability Study To test the stability, batch Fib05 (see above) was mixed with lidocaine (a new batch was prepared: Fib05L) and placed in a climate chamber at 40° C. (accelerated conditions). Characterization was performed by measuring the rheological properties and extrusion force (using a 30G TSK needle) of the gel at different time points (4, 8 and 12 weeks). All measurements were performed in triplicate. The results are shown in Table 5 below. [Table 5]

[0166] The crosslinked material (exemplified herein as HA / fibroin gel) was found to be relatively stable over time, with no significant degradation or deterioration of properties observed even after 12 weeks at 40° C. and 75% relative humidity.

[0167] In summary, it has been found that the crosslinked material of the present invention can be prepared very successfully and efficiently, optionally in a single batch, without any burden. The material has good properties, is injectable, and appears to have shear thinning / thixotropic properties. The material has a relatively high viscosity, a relatively high biological / enzymatic stability. These properties allow the crosslinked material of the present invention to be used in particular as a soft tissue filler, e.g., a dermal or connective tissue filler. The material can be stored well and has a relatively high shelf life.

Claims

1. A method for preparing a crosslinked material, comprising: (i) contacting the following components with each other: (A) one or more fibroin moieties comprising a primary amino residue or a salt thereof; (B) one or more polysaccharide moieties comprising a carboxylic acid residue or a salt thereof; (C) one or more activators that achieve the reaction of the carboxylic acid residue with the amino residue, thereby forming an amide bond; and (D) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form amide bonds, and conjugating the one or more fibroin moieties to the one or more polysaccharide moieties by covalent bonds; and (iii) optionally, purifying the crosslinked material obtained from step (ii). A method comprising the above steps.

2. The method according to claim 1, wherein the one or more fibroin moieties have a weight average molecular weight of at least 5 kDa.

3. The method according to claim 1, wherein the one or more polysaccharide moieties include, or are composed of, hyaluronic acid, heparosan, chondroitin sulfate, and carboxymethyl cellulose.

4. The method according to claim 1, wherein the one or more polysaccharide moieties include, or are composed of, one or more hyaluronic acid moieties.

5. The method according to claim 1, wherein the one or more polysaccharide moieties have a weight average molecular weight of at least 50 kDa.

6. The method according to claim 1, wherein the mass ratio A:B between the one or more fibroin moieties (A) and the one or more polysaccharide moieties (B) is in the range of 5:1 to 1:

20.

7. The method according to claim 1, wherein the one or more activators are selected from the group consisting of: (C1) one or more triazine-based activators; (C2) one or more carbodiimide activators; and (C3) combinations thereof.

8. The method according to claim 1, wherein the activator is 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium or a salt thereof.

9. The method further comprises: (a) step (iii) of purifying the crosslinked material by filtration, washing, and / or dialysis; (b) steps (i) and (ii) are carried out in a single batch; and / or (c) steps (i), (ii), and optional step (iii) are carried out at a temperature in the range of 5 to 90 °C. The method according to claim 1, characterized in that

10. The method comprises (i) contacting the following components with each other: (A) one or more fibroin moieties having a weight average molecular weight of at least 5 kDa and containing a primary amino residue or a salt thereof, (B) one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa and containing a carboxylic acid residue or a salt thereof, (C) one or more triazine-based activators that promote the effect and thus form an amide bond; and (D) one or more solvents; and (ii) reacting at least some of the carboxylic acid residues with at least some of the primary amino residues to form an amide bond, and conjugating the one or more fibroin moieties to the one or more hyaluronic acid moieties by a covalent bond; and (iii) optionally, purifying the crosslinked material obtained from step (ii) The method according to claim 1, comprising

11. A crosslinked material obtainable from the method according to claim 1.

12. (A) one or more fibroin moieties having a weight average molecular weight of at least 5 kDa, and (B) one or more hyaluronic acid moieties having a weight average molecular weight of at least 50 kDa A crosslinked material comprising or consisting of, wherein the one or more fibroin moieties are conjugated by a covalent bond to the one or more hyaluronic acid moieties without an interconnecting linker structure via an amide bond.

13. The crosslinked material according to any one of claims 11 or 12, characterized in that it further does not contain the following: (a) an imide group; (b) an imine group; (c) an epoxy group; and (d) a foreign body linker moiety.

14. An injectable composition comprising the crosslinked material according to any one of claims 11 or 12, a liquid or viscous carrier, and optionally further components.

15. Use of the injectable composition according to claim 14 for cosmetic applications including facial and body reshaping and rejuvenation.