Organic solvent-free composition containing protein-polymer conjugates and use of the composition

Abstract

To provide improved hydrogel scaffolds comprising conjugates of extracellular matrix proteins with synthetic polymers.SOLUTION: The present invention provides a stable ready-for-use liquid composition in a non-cross linked form, comprising a protein-polymer conjugate and at least one polymerization initiator. The protein-polymer conjugate comprises an extracellular matrix protein covalently bound to a synthetic polymer. The synthetic polymer contains at least one polymerizable group, and the liquid composition contains no detectable residue of polar organic solvents.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a protein-polymer conjugate, a method for producing a biocompatible scaffold formed from a hydrogel containing the conjugate, and the use of the scaffold for tissue regeneration. The present invention discloses an improved process for preparing the conjugate, which produces the improved conjugate of the present invention using an environmentally friendly process that avoids organic solvents. [Background technology]

[0002] Synthetic hybrid materials containing protein-polymer conjugates can be used in tissue regeneration and drug delivery systems. These hybrid biomaterials combine biological macromolecules with structurally variable synthetic polymers to generate cross-linked hydrogel networks (Lutolf, MP and JA Hubbell, Nat Biotechnol, 2005. 23(1): p. 47-55). These hybrid biomaterials can also be used to create biomimetic cellular environments by balancing structural and functional elements. Control over structural properties, including porosity, compatibility, overall density, mechanical properties, and degradability, is directed through synthetic polymer networks, while biological cellular signaling is regulated through the uptake of biological macromolecules, which may include protein fragments, growth factors, or biologically active peptide sequences (Peppas, NA et al., Annu Rev Biomed Eng, 2000. 2: p. 9-29; Tsang, VL and SNBhatia, Adv Drug Deliv Rev, 2004. 56(11): p. 1635-47; Stile, RA et al., J Biomater Sci Polym Ed, 2004. 15(7): p. 865-78).

[0003] In this regard, it is also possible to use both the biochemical and biomechanical characteristics of wound dressings to initiate important cellular remodeling events, including cell migration, proliferation, and guided differentiation.

[0004] These materials can be easily customized with microstructures, matrix rigidity, and proteolytic resistance specifically designed to guide remodeling and morphogenesis toward a particular tissue manipulation endpoint (Pratt, AB et al., Biotechnol Bioeng, 2004. 86(1): p. 27-36).

[0005] For tissue manipulation, several biosynthetic hybrid materials have been disclosed, including a poly(ethylene glycol) (PEG) hydrogel backbone modified with an Arg-Gly-Asp(RGD) adhesive oligopeptide and crosslinked with a short oligopeptide containing a plasmin or collagenase-degrading substrate, as described by Hubbell and colleagues (Lutolf, MP et al., Proc Natl Acad Sci USA, 2003. 100(9): p. 5413-8). Publication application US 20140273153 discloses a method for covalently modifying proteins and other biological macromolecules using thiol-ene and thiol-in chemical reactions. West and colleagues disclosed a proteolytically sensitive PEG-peptide biomaterial (Mann, BK et al., Biomaterials, 2001. 22(22): p. 3045-51).

[0006] Seliktar et al. developed an approach to form hybrid biomaterials (protein-polymer adducts) using natural biological molecules and synthetic polymers as building blocks for a matrix. The proteins act as the structural backbone of the polymer network, thereby making the hydrogels naturally biodegradable through intrinsic degradation sites on the protein sequence. The majority of the structural properties of the protein-polymer hydrogel network are controlled through the synthetic polymer components. These materials have been rigorously validated in preclinical and clinical settings (Dikovsky, D., H. Bianco-Peled, and D. Seliktar, Biomaterials, 2006. 27(8): p. 1496-506.; Shapira-Schweitzer, K. and D. Seliktar, Acta Biomater, 2007. 3(1): p. 33-41; Seliktar, D., Ann NY Acad Sci, 2005. 1047: p. 386-94). One of the unique properties of this biomaterial is that its biochemical and physical properties can be modified quasi-independently. Furthermore, the synthetic material may be beneficial for wound dressings and drug delivery systems by utilizing the endogenous properties of naturally biodegradable protein molecules, and can be designed to control absorption rates.

[0007] WO 2005 / 061018, WO 2008 / 126092, and WO 2011 / 073991, directed to some of the inventors of the present invention, disclose a process for preparing fibrinogen-PEG conjugates, including the use of organic solvents such as acetone for the isolation of the conjugates. In particular, previously known isolation steps for these conjugates have utilized environmentally unsafe polar organic solvents. [Overview of the Initiative]

[0008] It would be preferable to have synthetic biomaterial conjugates that contain minimal or undetectable traces of polar organic solvents, and processes for producing protein-polymer hybrid materials without the use of these environmentally unsafe solvents.

[0009] This invention provides improved hydrogel scaffolds comprising conjugates of synthetic polymers and extracellular matrix proteins. These scaffolds are useful as implants themselves or as coatings for implants. The conjugates and hydrogels formed with these conjugates exhibit enhanced biocompatibility, increased safety, and a reduced likelihood of inducing adverse reactions. In particular, the conjugates are produced using an improved process that avoids the use of potentially harmful polar organic solvents. This improvement results in products that are more environmentally friendly and simultaneously less likely to induce adverse reactions in vivo.

[0010] The improved scaffold of the present invention utilizes a defined protein-to-polymer molar ratio to provide a controlled decay rate in vivo within the subject. The biodegradation rate of the hydrogel can also be predetermined by a) the proteins and polymers used in the conjugate; b) the degree of crosslinking; and c) the protein-to-polymer molar ratio.

[0011] In one aspect, the present invention provides a composition comprising a protein-polymer conjugate, wherein the synthetic polymer contains at least one polymerizable group, and the composition is substantially free of polar organic solvents. In some embodiments, the composition contains less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 20 ppm, or less than 10 ppm of polar organic solvents. In further embodiments, the composition contains no detectable residue of polar organic solvents at all. Each possibility is a distinct aspect of the present invention.

[0012] It is hereby disclosed for the first time that mixtures of plasma proteins are suitably usable in the compositions and processes of the present invention. While it is undesirable to be bound by any theory or mechanism of action, the protein mixtures may be advantageous in terms of biocompatibility and the promotion of wound healing. According to some embodiments, the conjugates of the present invention are formed by a reaction between an unpurified or partially purified mixture of plasma proteins and a polymer. According to another embodiment, fibrinogen may be purified fibrinogen. According to some specific embodiments, the purified fibrinogen may be of a mammalian source, including, but not limited to, bovine fibrinogen.

[0013] According to some embodiments, the protein is selected from the group consisting of fibrinogen, fibrin, albumin, fibronectin, collagen, denatured fibrinogen, denatured albumin, gelatin, and any combination thereof. According to some embodiments, the protein is of bovine, porcine, or human origin. According to some embodiments, the protein is partially purified. According to other embodiments, the protein is highly purified.

[0014] According to some embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), hydroxyapatite / polycaprolactone (HA / PLC), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), polymethyl methacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylene fumarate (PPF), polyethylene glycol dimethacrylate (PEG-DMA), polyethylene glycol diacrylate (PEG-DA), beta-tricalcium phosphate (beta-TCP), and non-biodegradable polytetrafluoroethylene (PTFE).

[0015] According to some specific embodiments, the protein is fibrinogen and the polymer is polyethylene glycol diacrylate (PEG-DA).

[0016] In some embodiments, the compositions of the present invention are characterized by a molar ratio of synthetic polymer to protein between 40:1 and 400:1. In another embodiment, the compositions of the present invention are characterized by a molar ratio of synthetic polymer to protein between 100:1 and 250:1. In yet another embodiment, the compositions of the present invention are characterized by a molar ratio of synthetic polymer to protein between 100:1 and 150:1.

[0017] In a further aspect, the present invention provides a hydrogel composition comprising a crosslinked protein-polymer conjugate molecule, wherein the composition comprises an extracellular matrix protein covalently bonded to a synthetic polymer, the synthetic polymer containing at least one polymerizable group, and the composition substantially free of polar organic solvents.

[0018] In some embodiments, the present invention further provides a hydrogel composition comprising a crosslinked conjugate molecule, wherein the conjugate molecule is covalently crosslinked with respect to the polymerization of the polymerizable groups, and the composition is substantially free of polar organic solvents.

[0019] According to certain embodiments, the hydrogel composition of the present invention is substantially acetone-free. According to other embodiments, the hydrogel composition of the present invention has a shear storage modulus in the range of 0.05 kPa to 35 kPa.

[0020] In another aspect, the present invention further provides an improved process for preparing protein polymer conjugates that avoids the use of polar organic solvents.

[0021] In exemplary embodiments, the process avoids the use of acetone, and the resulting protein polymer material contains no trace of acetone whatsoever. The improved process is more environmentally friendly and can be scaled up more easily, offering increased industrial applicability.

[0022] According to several embodiments, a process for producing a protein polymer conjugate composition is a process comprising: (a) dissolving at least one denatured extracellular matrix protein under basic conditions that provide strong protein denaturation and reduction conditions; (b) providing a solution containing a synthetic polymer having polymerizable groups; (c) mixing the extracellular matrix protein solution from (a) and the solution containing the synthetic polymer from (b) under a basic pH that provides strong protein denaturation and reduction conditions to generate a covalent conjugate between the sulfidyl and polymerizable groups of the protein; and (d) concentrating the unpurified reaction mixture from step (c) without a concentration process utilizing a polar organic solvent.

[0023] In some embodiments, the concentration of the unpurified reaction mixture described in step (d) is carried out by centrifugation. In some specific embodiments, the concentration step is carried out without the removal of unreacted polymers. It is assumed, without being bound by any theory or mechanism of action, that it may be preferable not to remove unreacted polymers. Excess unreacted polymers (those not conjugated to the matrix protein) may provide further polymerizable groups, thereby improving the degree of crosslinking of the conjugate for hydrogel formation. More importantly, concentration of the conjugate without precipitation avoids the use of precipitation with polar organic solvents.

[0024] In another aspect, the present invention provides a non-crosslinked, stable, ready-to-use liquid formulation comprising the protein-polymer conjugate composition described above and at least one polymerization initiator. In other words, according to some embodiments, the product may be a ready-to-use, stable precursor solution, also known as a pre-gelled liquid, which can be activated in situ to produce a desired hydrogel. Activation is achieved using the polymerization initiator, typically by exposure to light.

[0025] According to some embodiments, at least one polymerization initiator is selected from the group consisting of bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), 1-phenyl-1,2-propanedione (PPD), Cp’Pt(CH3)3 (Cp = η5-C5H4CH3), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (e.g., IRGACURE TM 2959), dimethylaminoethyl methacrylate (DMAEMA), 2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), flavin-containing compounds, and combinations of triethanolamine, N-vinylpyrrolidone, and eosin Y. Each possibility is a separate embodiment of the present invention.

[0026] In some embodiments, the ready-to-use formulation avoids the use of polar organic solvents. Advantageously, this ready-to-use formulation requires only the activation of the photoinitiator by exposure to light before or during use.

[0027] According to some embodiments, the ready-to-use formulation is held in a container selected from a single aliquot vial or a pre-filled syringe. According to some embodiments, the ready-to-use liquid formulation is in a pre-gelled form before polymerization and is stored under visible light protection conditions. According to some embodiments, the ready-to-use formulation forms a hydrogel upon exposure to visible light. According to some embodiments, the ready-to-use liquid formulation is in a pre-gelled form before polymerization and is stored under UV protection conditions. According to some embodiments, the ready-to-use formulation forms a hydrogel upon exposure to UV light.

[0028] According to some embodiments, the ready-to-use formulation contains less than 100 ppm of ethanol. According to some currently preferred embodiments, the ready-to-use formulation contains no detectable residue of ethanol at all. According to some other embodiments, the ready-to-use formulation contains less than 10 ppm of acetone. According to some currently preferred embodiments, the ready-to-use formulation contains no detectable residue of acetone at all.

[0029] These and further advantages of the present invention will become clear in conjunction with a detailed description. [Brief explanation of the drawing]

[0030] [Figure 1] Figure 1 shows the gel electrophoresis profiles of human sealant containing fibrinogen, along with additional plasma proteins, compared to purified bovine fibrinogen. [Figure 2] Figure 2 presents the results of a hydrogel stability test comparing the gel product obtained from a novel stable single-vial formulation containing a photoinitiator reagent, PEG-fibrinogen conjugate molecules, and further amounts of PEG-DA in a pre-gelled form, with the hydrogel obtained from a two-vial formulation containing the photoinitiator solution in one vial and the PEG-fibrinogen conjugate molecules in another vial. [Figure 3] Figure 3 presents the results of a gelation study comparing the gelation kinetics of a hydrogel resulting from a novel, stable single-vial formulation containing a photoinitiator reagent, PEG-fibrinogen conjugate molecules, and further amounts of PEG-DA in a pre-gelled form, with those of a hydrogel resulting from a two-vial formulation containing the photoinitiator solution in one vial and the PEG-fibrinogen conjugate molecules in a separate vial. [Figure 4] Figure 4 shows the curing kinetics of hydrogels resulting from both the organic solvent-free method and the previously disclosed method involving acetone precipitation and ethanol addition. [Figure 5]Figure 5 shows the viscoelastic properties as a factor in the vibration frequency of hydrogels resulting from both the organic solvent-free method and the previously disclosed method involving acetone precipitation and ethanol addition. [Figure 6] Figure 6 shows the viscoelastic properties as a factor in straining of hydrogels resulting from both the organic solvent-free method and the previously disclosed method involving acetone precipitation and ethanol addition. [Modes for carrying out the invention]

[0031] The present invention provides an improved hydrogel scaffold comprising a conjugate of a synthetic polymer and an extracellular matrix protein. The conjugate of the present invention has increased biocompatibility because it does not contain polar organic solvent residues. The corresponding hydrogel of the present invention is a cross-linked protein-synthetic polymer conjugate and is useful as a biocompatible implant.

[0032] The present invention further provides an improved hydrogel scaffold composition comprising a relatively low ratio of protein to synthetic polymer. The protein components of the present invention act as disintegrants, allowing for fine-tuning of the hydrogel absorption rate by controlling their biodegradability. The specific molar ratio of protein to synthetic polymer is determined based on the desired disintegration rate and intended use of the implant.

[0033] Further adjustment of the physical properties of the hydrogel scaffold can be achieved by controlling the crosslinking density within the hydrogel network. The protein components of the present invention may also contain multiple crosslinking sites, which can be physically or chemically activated within the hydrogel graft.

[0034] In one aspect, the present invention provides a composition comprising a protein-polymer conjugate, wherein the synthetic polymer contains at least one polymerizable group, and the composition is substantially free of polar organic solvents.

[0035] According to several embodiments, the protein is selected from the group consisting of fibrinogen, fibrin, albumin, fibronectin, collagen, denatured fibrinogen, denatured albumin, gelatin, and any combination thereof. Each possibility is a distinct embodiment of the present invention. According to several embodiments, the protein is derived from cattle, pigs, or humans. According to several embodiments, the polymer is selected from the group consisting of polyethylene glycol (PEG), hydroxyapatite / polycaprolactone (HA / PLC), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), polymethyl methacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylene fumarate (PPF), polyethylene glycol dimethacrylate (PEG-DMA), polyethylene glycol diacrylate (PEG-DA), beta-tricalcium phosphate (beta-TCP), and non-biodegradable polytetrafluoroethylene (PTFE). Each possibility is a distinct embodiment of the present invention. In one currently preferred embodiment, the protein is fibrinogen and the polymer is polyethylene glycol diacrylate (PEG-DA).

[0036] The present invention further relates to a preferably low ratio of protein to synthetic polymer. In one embodiment, the composition of the present invention is characterized by a molar ratio of synthetic polymer to protein between 40:1 and 400:1. In a more preferred embodiment, the composition of the present invention is characterized by a molar ratio of synthetic polymer to protein between 100:1 and 250:1. In one currently preferred embodiment, the composition of the present invention is characterized by a molar ratio of synthetic polymer to protein between 100:1 and 150:1.

[0037] In one aspect, the present invention provides a composition comprising a protein-polymer conjugate, wherein the synthetic polymer contains at least one polymerizable group, and the composition is substantially free of polar organic solvents. In some embodiments, the composition contains less than 200 ppm, less than 100 ppm, less than 50 ppm, less than 20 ppm, or less than 10 ppm of polar organic solvents. In some further embodiments, the composition contains no detectable residue of polar organic solvents at all. Each possibility is a distinct aspect of the present invention.

[0038] In a further aspect, the present invention provides a hydrogel composition that is a crosslinked protein-polymer conjugate molecule. In some embodiments, the hydrogel composition comprises a crosslinked protein-polymer conjugate molecule, wherein it comprises an extracellular matrix protein covalently bonded to a synthetic polymer, the synthetic polymer contains at least one polymerizable group, and the composition is substantially free of polar organic solvents. In one particular embodiment, the hydrogel composition of the present invention is substantially free of acetone. According to some embodiments, the hydrogel composition of the present invention is characterized by a shear storage modulus in the range of 0.05 kPa to 35 kPa. According to certain embodiments, the hydrogel composition of the present invention is characterized by a shear storage modulus in the range of 2 kPa to 15 kPa.

[0039] The present invention further relates to the gelation of protein-polymer conjugates that occurs during polymerization of the polymerizable groups.

[0040] The present invention provides a novel process that enables efficient covalent bonding of synthetic polymers to proteins and extraction of the final protein-polymer conjugate without the need for an acetone precipitation step. The novel acetone-free process of the present invention is an environmentally friendly process that produces an acetone-free protein-polymer conjugate, which, upon gelation, produces an acetone-free hydrogel with increased biocompatibility.

[0041] The process of the present invention exhibits a more favorable scalability compared to previously disclosed processes that utilize polar organic solvents such as acetone. Prior art processes used polar organic solvents to precipitate the conjugate and remove unreacted molecules from the reaction product. For example, unreacted polymers or modified polymers that remain unbound to the protein can be removed by acetone precipitation.

[0042] Therefore, in some embodiments, the process of the present invention does not require extraction or precipitation of the protein-polymer conjugate from the reaction mixture before further processing. As a result, the novel acetone-free process of the present invention results in a more efficient production process and produces a safer hydrogel composition containing a protein-polymer conjugate for use in patients.

[0043] In some embodiments, the process for producing a protein polymer conjugate composition is a process comprising: (a) dissolving at least one unsaturated extracellular matrix protein under basic conditions that provide strong protein denaturation and reduction conditions; (b) providing a solution containing a synthetic polymer having polymerizable groups; (c) mixing the extracellular matrix protein solution from (a) and the solution containing the synthetic polymer from (b) under a basic pH that provides strong protein denaturation and reduction conditions to generate a covalent conjugate between the sulfidyl and polymerizable groups of the protein; and (d) concentrating the unpurified reaction mixture from step (c) without a concentration process utilizing a polar organic solvent.

[0044] In some embodiments, the concentration of the unpurified reaction mixture is carried out without the removal of unreacted polymers, and in particular, without the precipitation of the reaction mixture in a polar organic solvent. Thus, the improved process avoids the use of any organic solvent as part of the synthesis method.

[0045] Without being constrained by theory or mechanism of action, it is assumed that avoiding the removal of unreacted polymers contributes to polymerization and improves the degree of crosslinking of the resulting hydrogel. Unreacted polymers containing at least one polymerizable group are also activated and polymerize upon exposure of the reaction mixture to light, thus increasing the degree of crosslinking of the resulting hydrogel scaffold. In some embodiments, the light source used is within the visible light range. In some other embodiments, the light source used is within the UV light range.

[0046] The present invention further relates to a stable single-vial pre-crosslinked formulation of the hydrogel composition of the present invention. The single-vial formulation is a convenient ready-to-use formulation comprising a protein-synthetic polymer conjugate and at least one photoinitiator, and is stored under conditions that prevent undesirable polymerization before use. In some embodiments, the at least one polymerization initiator is bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), 1-phenyl-1,2-propanedione (PPD), Cp'Pt(CH3)3 (Cp=etha5-C5H4CH3), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (e.g., IRGACURE). TM2959) is selected from the group consisting of dimethylaminoethyl methacrylate (DMAEMA), 2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), flavin-containing compounds, and combinations of triethanolamine, N-vinylpyrrolidone, and eosin Y. Each possibility is a distinct aspect of the present invention. The single-vial formulations produce equally stable hydrogels and exhibit similar mechanical properties to gels freshly prepared by mixing a protein-synthetic polymer conjugate solution with at least one photoinitiator reagent. Without being bound by theory or mechanism of action, the ready-to-use formulations of the present invention provide a more precise composition of components due to the step of pre-mixing the photoinitiator and conjugate molecular composition, and thus facilitate an improved, easy-to-use method. In some embodiments, the ready-to-use single-vial formulations avoid the use of polar organic solvents. In some embodiments, the ready-to-use formulations are held in a container selected from a single alicot vial or a pre-filled syringe.

[0047] In some embodiments, the ready-to-use liquid formulation is stored in a pre-gelled state before polymerization initiation under visible light protection conditions. In some embodiments, the ready-to-use liquid formulation is stored in a pre-gelled state before polymerization initiation under UV protection conditions. In some embodiments, the ready-to-use liquid formulation undergoes a crosslinking process upon activation by exposure to light and forms the hydrogel of the present invention. In some embodiments, activation is achieved by exposure to the visible light range (e.g., 400-700 nm). In some other embodiments, activation is achieved by exposure to UV light. In some embodiments, the ready-to-use formulation contains less than 100 ppm of ethanol. In some currently preferred embodiments, the ready-to-use formulation contains no detectable residue of ethanol. According to some other embodiments, the ready-to-use formulation contains less than 10 ppm of acetone. In some currently preferred embodiments, the ready-to-use formulation contains no detectable residue of acetone.

[0048] Unexpectedly, as illustrated below herein, PEG-fibrinogen implants based on human fibrinogen sealant have been found to be superior in several respects to PEG-fibrinogen implants based on purified fibrinogen. Commercially available purified fibrinogen typically contains more than 95% fibrinogen, while fibrinogen obtained in the form of sealant products is typically 70–80% pure. In addition to fibrinogen, sealant products contain other plasma proteins, including, but not limited to, albumin and fibronectin (Christoph Buchta et al., Biomaterials, 26, 31 (2005), 6233–6241). Unexpectedly, these additional proteins provide a more robust product with higher biocompatibility. Figure 1 shows the protein profiles of one such sealant and purified fibrinogen. It can be seen that the sealant sample contains protein species other than fibrinogen. Surprisingly, sealant-derived PEG-fibrinogen grafts outperformed pure fibrinogen-based PEG-fibrinogen grafts across a variety of measurements, demonstrating improved bioavailability.

[0049] In this invention, the term "in vivo" refers to a living organism such as a plant or animal, preferably a mammal, and preferably a human subject. The term "subject" as used herein refers to a vertebrate of any age, preferably a mammal, and more preferably a human (male or female).

[0050] In this specification, the phrase "ex vivo" refers to living cells obtained from an organism and growing (or being cultured) outside the living organism, preferably outside the body of a vertebrate, mammal, or human. For example, cells of human origin, such as human muscle cells or human aortic endothelial cells, and cultured outside the body are referred to as cells cultured ex vivo.

[0051] As used herein, the terms “protein” and “polypeptide” include any naturally occurring polypeptide that is interchangeable and contains at least 10 peptide residues, as well as biologically active fragments thereof (e.g., fragments that induce cell adhesion and / or cell signaling). Biologically active fragments can be produced by any method known in the art (e.g., cleavage with enzymes and / or chemical reagents).

[0052] Protein degradation-sensitive fragments can be produced by any method known in the art (e.g., cleavage by enzymes and / or chemical reagents). Since non-proteins with low cysteine ​​content typically have few thiol groups, protein thiolation is particularly suitable for producing the compositions of the problem described above when the protein has a low cysteine ​​content. The introduction of further thiol groups by protein thiolation creates additional sites available for linking synthetic polymers. Proteins with little or no cysteine ​​(e.g., collagen) have traditionally been unsuitable for inclusion in polymer-protein conjugate molecules containing synthetic polymers attached to the cysteine ​​residues of the protein. Since many proteins have low cysteine ​​content, protein thiolation overcomes a serious drawback of polymer-protein conjugate molecules. In some cases, the protein to be thiolated contains fewer than 5 cysteine ​​residues per 100 amino acid residues. In some cases, the protein contains fewer than 3 cysteine ​​residues per 100 amino acid residues, in some cases, fewer than 2 cysteine ​​residues per 100 amino acid residues, and in some cases, less than 1 cysteine ​​residue per 100 amino acid residues. In some cases, the protein of the present invention is denatured.

[0053] As used herein and in the claims, the terms “thiol” and “sulfidyl” are interchangeable and refer to the -SH group.

[0054] Without being bound by any particular theory, denatured proteins are typically considered to have more sites available for attachment to synthetic polymers. Proteins can be denatured by a variety of methods well known in the art. For example, proteins can be denatured by heating or exposure to denaturing agents such as urea or guanidium chloride. As illustrated below herein, proteins can be denatured in a solution containing 8M urea.

[0055] The term "polymer" primarily refers to molecules composed of multiple repeating units. The phrase "synthetic polymer" refers to any polymer made from synthetic materials, i.e., non-natural, non-cellular materials. The names of polymers mentioned above refer, for the most part, to repeating units that make up the structure of synthetic polymers, and do not imply the exclusion of the presence of further functional groups in synthetic polymers. Therefore, for example, a synthetic polymer consisting of polyethylene glycol containing two acrylate groups (i.e., PEG-diacrylate) is included in the terms "polyethylene glycol" and "PEG" as used herein.

[0056] Methods for preparing functionalized PEG molecules are known in the art. For example, PEG-vinyl sulfone can optionally be prepared under argon by reacting a solution of PEG-OH in dichloromethane (DCM) with NaH, and then with divinyl sulfone (optionally in a molar ratio: OH 1: NaH 5: divinyl sulfone 50, and with 0.2 grams of PEG per 1 ml of DCM). PEG-Ac can optionally be prepared under argon by reacting a solution of PEG-OH in DCM with acryloyl chloride and triethylamine (optionally in a molar ratio: OH 1: acryloyl chloride 1.5: triethylamine 2, and with 0.2 grams of PEG per 1 ml of DCM).

[0057] As used herein and in the claims, the terms “crosslinking,” “curing,” and “polymerization” are interchangeable and refer to the formation of interconnected protein-polymer conjugate molecules by covalent interactions between polymerizable groups of adjacent conjugate polymers. Exemplary crosslinkable polymerizable functional groups include, without limitation, acrylates and vinyl sulfones. Crosslinking of the protein-polymer conjugate molecules of the present invention is initiated by a compound that initiates a chemical polymerization reaction.

[0058] To facilitate the crosslinking of conjugate molecules in embodiments of the present invention that form scaffolds, the crosslinking of conjugate molecules can be performed inside (i.e., in vivo) or outside the body. In vivo crosslinking can be used, for example, to generate scaffolds that have the precise shape of the body cavity to be filled with the scaffold.

[0059] Various methods of crosslinking are known in the art. For example, crosslinking can be achieved by irradiation (e.g., by ultraviolet light or visible light), by chemical reagents (e.g., free radical donors), and / or by heat.

[0060] According to one aspect of the present invention, crosslinking is performed by ultraviolet irradiation (for example, at a wavelength of about 365 nm). According to another specific aspect, the crosslinking irradiation is in the visible light range.

[0061] In this specification, the term "approximately" refers to a range of ±10%.

[0062] In some cases, photoinitiators are added to promote crosslinking. The addition of photoinitiators typically allows for the use of lower doses of ultraviolet light for crosslinking.

[0063] As used herein and in the claims, the term “polar organic solvent” refers to an organic solvent having a large dipole moment, i.e., a solvent containing an interatomic bond between two distinct electronegative atoms, such as oxygen bonded to hydrogen. Polar organic solvents include both protic solvents, such as alcohols, ammonia, and acetic acid, and aprotic polar solvents, such as acetone, dimethylformamide (DMF), acetonitrile, and dimethyl sulfoxide (DMSO).

[0064] As used herein, “substantially free” of polar organic solvents means that the amount of polar organic solvent is not greater than a predetermined amount specified in ppm, or is even undetectable by conventional detection means (e.g., gas chromatography). In some exemplary embodiments, the amount of polar organic solvent in the protein-polymer conjugate compositions and hydrogel compositions of the present invention is reduced by at least 80%, preferably at least 90%, and more preferably at least 98%, compared to the corresponding previously disclosed material produced using a polar organic solvent precipitation method. Furthermore, as used herein, “substantially free process” refers to a process for producing a protein-polymer conjugate composition or hydrogel composition that does not involve any synthesis steps involving polar organic solvents.

[0065] As used herein and in the claims, “lacking” polar organic solvents means that the composition contains no detectable traces of polar organic solvents whatsoever. As used herein, “photoinitiator” describes a compound that initiates a chemical reaction (e.g., crosslinking, polymerization) when exposed to light, more specifically ultraviolet irradiation. Many suitable photoinitiators are known to those skilled in the art. Exemplary photoinitiators include, without limitation, bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), 1-phenyl-1,2-propanedione (PPD), organometallic complex Cp'Pt(CH3)3 (Cp=etha5-C5H4CH3), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (e.g., IRGACURE). TM 2959) These include dimethylaminoethyl methacrylate (DMAEMA), 2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), and flavin. In some embodiments, a combination of triethanolamine, N-vinylpyrrolidone, and eosin Y can be used as a photoinitiator under visible light conditions.

[0066] As used herein and in the claims, the terms “non-crosslinked” and “pre-crosslinked” are interchangeable and refer to a reagent mixture in a liquid solution that is ready to polymerize upon exposure to light, for example, upon activation of a photoinitiator.

[0067] As used herein and in the claims, the terms “hydrogel” or “scaffold” are interchangeable and refer to a two- or three-dimensional polymeric porous matrix comprising protein-polymer conjugate molecules that are covalently crosslinked with one another. Hydrogels according to the present invention can also be tailored to possess a range of properties depending on the ratio of proteins, polymers, and hydrogels, and depending on further materials that can be added, such as mineral solutions or aggregates, polysaccharides, active ingredients, excipients, etc. By controlling the crosslinking, scaffolds of the present invention can also form two- or three-dimensional structures of any size, structure, or porosity. Scaffolds of the present invention can be embedded in or formed around another scaffold or gel, or linked to further materials to form hybrid or coated scaffolds. In some embodiments of the present invention, scaffolds of the present invention can be used to support cell proliferation, adhesion, and spread, and thus promote cell proliferation, tissue regeneration, and / or tissue repair. In alternative embodiments of the present invention, scaffolds can be used as adhesives to promote tissue repair. In some cases, the adhesives do not support cell proliferation. According to an embodiment of the present invention, the scaffolding is biodegradable.

[0068] As used herein and in the claims, the terms “single vial formulation,” “single alicot vial,” or “ready to use” refer to a composition comprising a polymer-protein conjugate molecule mixed with a photoinitiator, which is interchangeable, non-crosslinked, and activatable upon exposure to light. In some embodiments, the ready to use formulation may also contain an unreactive polymer, which, upon exposure to light, is reactive in a crosslinking process together with the protein-polymer conjugate molecule. In some embodiments, the activation of polymerization occurs upon exposure to UV light. In some other embodiments, the activation of polymerization occurs upon exposure to the visible light range.

[0069] As used herein, the term "biocompatibility" refers to a material that has affinity with living tissues, low toxicity, and causes little to no unacceptable heterogeneous bodily reactions in living organisms. For example, the proteins, synthetic polymers, protein-synthetic polymer conjugates, and hydrogels of the present invention are biocompatible.

[0070] In this context, “transplantation” refers to the insertion of the hydrogel composition of the present invention into a subject body in which the composition works to completely or partially replace damaged or removed tissue. Another aspect of transplantation is also interpreted to mean the use of the composition as a vehicle for delivering a therapeutic agent to a specific site in the patient. In this aspect, the incorporation of a therapeutic agent selected from growth factors, cytokines, chemotherapeutic agents, enzymes, antimicrobial agents, and anti-inflammatory agents into the composition or transplantation is also included.

[0071] The scaffold of the present invention can be implanted in a subject using surgical tools, such as scalpels, spoons, spatulas, or other surgical devices. It will be recognized that in vivo tissue formation is also achievable by administering scaffold conjugate molecules to a subject and further crosslinking the conjugate molecules in vivo.

[0072] As used herein, the terms “biodegradable” and “biodegradable” refer to the ability to be broken down (i.e., destroyed) by biological proteases or other biomolecules. Biodegradability depends on the availability of the degrading substrate (i.e., the biological material or a part thereof), the presence of biodegradable materials (e.g., microorganisms, enzymes, proteins), and the availability of oxygen (for aerobic organisms, microorganisms or a part thereof), carbon dioxide (for anaerobic organisms, microorganisms or a part thereof), and / or other nutrients. Furthermore, the biodegradability of a material, for example the scaffold of the present invention, also depends on the structure and / or mechanical properties of the material, i.e., porosity, flexibility, viscosity, crosslinking density, hydrophobicity / hydrophilicity, and elasticity, which can affect the passage and availability of gases and nutrients.

[0073] The biodegradability of a scaffold is, at least in part, due to the biodegradability of the proteins in the scaffold that form its skeleton. The biodegradability of a scaffold can be determined by selecting proteins that provide a specific level of biodegradability. Furthermore, biodegradability can be determined by selecting biodegradable or non-biodegradable synthetic polymers. Biodegradability is also affected by the number of synthetic molecules attached to each protein, as a large number of attached synthetic molecules can mask cleavage sites, thus reducing biodegradability.

[0074] The biodegradability of the hydrogel scaffold according to an embodiment of the present invention can be determined by enzymatically degrading such hydrogels using a protease, such as plasmin, trypsin, collagenase, or chymotrypsin.

[0075] The addition of synthetic polymers will increase the mechanical strength of the resulting scaffold. If the synthetic polymers are non-biodegradable, the biodegradability of the scaffold will decrease. Therefore, it is also possible to modify the properties of the scaffold as desired by adding an appropriate amount of synthetic polymer that crosslinks with the conjugate molecules.

[0076] It should be noted that, before crosslinking the conjugate molecule, the unconjugated synthetic polymer may be removed, and then the same unconjugated synthetic polymer that will be crosslinked with the conjugate molecule may be added. For example, it may be desirable to remove an unconjugated synthetic polymer of uncertain concentration and then add an unconjugated synthetic polymer of known concentration.

[0077] Generally, the biological and mechanical properties of a scaffold will be determined, in part, by the ratio of protein to synthetic polymer in the scaffold. For example, a scaffold with a high protein content will exhibit the biological properties of the proteins contained therein, such as cellular signaling, while retaining the desirable mechanical properties characteristic of the synthetic polymers contained therein. An exemplary scaffold contains PEG and fibrinogen in molar ratios ranging from 25:1 PEG per protein to 400:1 PEG per protein.

[0078] In addition to being inexpensive to produce, the scaffolds of the present invention are highly reproducible, flexible (easily compressible or stretchable), exhibit controllable structural properties, and readily possess controllable biodegradability. These properties make the scaffolds highly suitable for in vivo or ex vivo manipulation of tissues such as bone, nerve, cartilage, cardiac muscle, skin tissue, blood vessels, and other tissues (parenchymal and hard tissues) of the body. For example, scaffolds and / or hydrogels according to embodiments of the present invention can be easily placed in interstitial spaces within tissues or organs, the scaffolds then fill these spaces, and as the scaffolds degrade, a regeneration process can be initiated.

[0079] In many cases, it is desirable to grow viable cells in a space filled with a scaffold used for tissue manipulation. This is facilitated by having viable cells implanted in the scaffold. One advantage of aspects of the present invention is that the scaffold can be formed from a liquid phase (e.g., a solution of polymer-protein conjugates) using mild conditions to initiate crosslinking. As a result, viable cells can be dispersed between the conjugate molecules, and crosslinking can be performed under mild conditions that do not harm the cells, thus making it possible to create a scaffold in which viable cells are embedded.

[0080] Therefore, according to an embodiment of the present invention, the scaffold includes embedded viable cells. In some cases, the scaffold in which the viable cells are embedded includes thiolated proteins.

[0081] In aspects of the present invention, exemplary cells suitable for inclusion are tissue-forming cells, which include, without limitation, stem cells such as embryonic stem cells, bone marrow stem cells, umbilical cord blood cells, mesenchymal stem cells, adult tissue stem cells; or differentiated cells such as nerve cells, retinal cells, epithelial cells, hepatocytes, pancreatic (islet) cells, osteocytes, chondrocytes, elastocytes, fibrous cells, muscle cells, cardiomyocytes, endothelial cells, smooth muscle cells, and hematopoietic cells.

[0082] As used herein, the term "planting" refers to encapsulating, capturing, plating, positioning, and / or dropping cells within the scaffold of the present invention. It will be recognized that the concentration of cells planted on or within the scaffold of the present invention will depend on the type of cells used and the composition of the scaffold used (i.e., the molar ratio between synthetic polymers and proteins in the conjugate molecules, and the percentage of crosslinking molecules used).

[0083] It will be recognized that cell implantation is feasible by mixing the cells with conjugate molecules after the formation of the scaffold or hydrogel formed from the scaffold, or before the crosslinking that gives rise to the scaffold. The concentration of cells to be implanted on the scaffold and / or hydrogel depends on the cell type, as well as the properties of the scaffold and / or hydrogel, and those skilled in the art can determine the appropriate concentration of cells in each case.

[0084] After planting cells onto a scaffold and / or hydrogel, it will be recognized that the cells may be cultured in the presence of tissue medium and growth factors to maintain their viability.

[0085] After planting, the scaffold and / or hydrogel may be examined (e.g., using an inverted microscope) to evaluate cell proliferation, spread, and tissue formation, as illustrated in the Examples section. The term “shear storage modulus (G')” as used herein and in the claims refers to the mechanical properties of a solid material, which defines the relationship between shear stress (force per unit area) and shear strain (proportional deformation-elasticity) in the material. The term “shear loss modulus (G)” as used herein refers to the viscous properties of a viscoelastic material, which, together with the shear storage modulus, defines the complex shear modulus, and is used to describe the mechanical properties of a viscoelastic solid material.

[0086] As used herein, the term “method” means, but is not limited to, any method, means, techniques and procedures for accomplishing a given task, including any method, means, techniques and procedures that are known to those skilled in the art of chemistry, pharmacology, biology, biochemistry and medicine, or that can be readily developed by those skilled in the art from known methods, means, techniques and procedures.

[0087] For clarity, it is recognized that certain features of the Invention described in the context of a separate embodiment may also be provided in a combination of a single embodiment. Conversely, for brevity, various features of the Invention described in the context of a single embodiment may also be provided individually, in any suitable combination, or as appropriate for any other described embodiment of the Invention. Certain features described in the context of a variety of embodiments shall not be considered essential features of such embodiment unless the embodiment is inoperable without those elements.

[0088] The following non-limiting embodiments are provided to illustrate in more detail specific aspects of the invention. However, they should not be considered in any way to limit the broad scope of the invention. Those skilled in the art will readily be able to devise many modifications and variations of the principles disclosed herein without departing from the scope of the invention.

[0089] Examples Example 1: Comparative Example: Determination of acetone residue in fibrinogen-PEG DA hydrogel compositions obtained according to previously disclosed methods. Chemical analysis was performed to determine the acetone content in hydrogel compositions prepared according to previously disclosed methods (WO 2005 / 061018, WO 2008 / 126092, and WO 2011 / 073991).

[0090] To determine the acetone content in the product, gas chromatography with flame ionization detection (GC-FID, Hewlett Packard 5890) was used. Headspace (HS) sample injection and a capillary GC column ZB-624 (75 m length, 0.53 mm inner diameter, 3.0 micron film thickness) were used with helium and hydrogen (gas flow of 1 ml / min) as carrier and flame gases, respectively. A liquid sample (5 ml) was added to a crimp-seal glass headspace vial (20 ml). The sample vial was placed in an autosampler and analyzed against a calibration curve using acetone as a standard at five different concentrations. The HS injection method consisted of a 30-minute incubation at 80°C and a syringe temperature of 100°C (1 ml sample volume). A run time of 35 minutes was used for the GC method. After 5 minutes at 40°C (initial temperature), the temperature was increased to 240°C within 5 minutes. The temperatures of the injection port and FID were set to 190°C and 300°C, respectively. The measured acetone amounts in ppm for both human and bovine fibrinogen-PEG DA-based hydrogels are summarized in Table 1: [Table 1]

[0091] Example 2: Preparation of bovine-derived PEG-DA-fibrinogen conjugate (purified fibrinogen) A 7 mg / ml bovine fibrinogen solution (Bovogen Biologicals Pty Ltd, Melbourne, Australia) in 10 mM phosphate-buffered saline (PBS) containing 8 M urea was prepared with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (Sigma). TCEP-HCl was added in a molar ratio of 1–1.5:1 TCEP to fibrinogen-cysteine. The solution pH was adjusted to 8.0 with NaOH. PEG-DA was dissolved in 10 mM PBS and 8 M urea (280 mg / mL) and complete dissolution was achieved before adding PEG-DA to the dissolved fibrinogen solution. The molar ratio of PEG-DA to fibrinogen-cysteine ​​was 3:1 (linear PEG-DA, 10 kDa). The mixture was reacted in a reaction vessel with a thermostat jacket at 25 ± 1 °C, shielded from light, for 3 hours. Next, the solution was diluted with an equal volume of PBS and transferred from the reaction vessel into the sample reservoir of the tangential flow filtration system.

[0092] Using an omega-type cassette (30 kDa MW cutoff, Pall Corporation), tangential flow filtration was performed to purify and concentrate the modified protein in 10 mM PBS to a concentration of 8–12 mg / ml.

[0093] Next, the solution was further diluted with PEG-DA in PBS solution to achieve a protein concentration of 6–8 mg / ml and a PEG-DA to protein molar ratio of 1:120 (±20). The solution was then passed through a high-shear fluid processor (Microfluidics M110-Y, USA) to achieve a uniform reduction in particle size.

[0094] Example 3: Preparation of human-derived PEG-DA-fibrinogen conjugate (unpurified fibrinogen sealant) A 7 mg / ml human fibrinogen (TESSEEL-protein sealant, Baxter, USA) solution in 10 mM phosphate-buffered saline (PBS) containing 8 M urea was prepared with tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl) (Sigma). TCEP-HCl was added in a molar ratio of 1–1.5:1 TCEP to fibrinogen-cysteine. The solution pH was adjusted to 8.0 with NaOH. PEG-DA was dissolved in 10 mM PBS and 8 M urea (280 mg / mL) and complete dissolution was achieved before adding PEG-DA to the dissolved fibrinogen solution. The molar ratio of PEG-DA to fibrinogen-cysteine ​​was 3:1 (linear PEG-DA, 10 kDa). The mixture was reacted in a reaction vessel with a thermostat jacket at 25 ± 1 °C, shielded from light, for 3 hours. Next, the solution was diluted with an equal volume of PBS and transferred from the reaction vessel to the sample reservoir of the tangential flow filtration system.

[0095] Using an omega-type cassette (30 kDa MW cutoff, Pall Corporation), tangential flow filtration was performed to purify and concentrate the modified protein in 10 mM PBS to a concentration of 8–12 mg / ml.

[0096] Next, the solution was further diluted with PEG-DA in PBS solution to achieve a protein concentration of 6–8 mg / ml and a PEG-DA to protein molar ratio of 1:120 (±20). The solution was then passed through a high-shear fluid processor (Microfluidics M110-Y, USA) to achieve a uniform reduction in particle size.

[0097] Example 4: Preparation of hydrogel using a two-vial formulation The PEG-DA-fibrinogen conjugate solution prepared in Example 2 or 3 was then filtered through a 0.2 μm filter for sterilization. The filtered solution was filled into vials under sterile conditions and stored at a temperature below -15°C until use.

[0098] A stock solution of 10% w / v Irgacure 2959 (BASF, Switzerland) photoinitiator in 70% ethanol and water for injection was prepared. The stock solution was filtered through a 0.2 μm filter for sterilization. The filtered photoinitiator solution was filled into vials under sterile conditions and stored at a temperature below -15°C until use.

[0099] To photocur the PEG-DA-fibrinogen conjugate solution and produce the corresponding cross-linked hydrogel, a photoinitiator stock solution was added to the PEG-DA-fibrinogen conjugate solution, as prepared in Example 2 or 3, to achieve a final concentration of 0.1% w / v of Irgacure 2959, and vigorously mixed before exposure to UV light.

[0100] Example 5: Preparation of a ready-to-use pre-gel compound in a stable single vial The PEG-DA-fibrinogen conjugate solution, as prepared in Example 2 or 3, was further mixed with IRGACURE® 2959 (BASF, Switzerland) and stirred until completely dissolved, achieving a final concentration of 0.1% (w / v) of IRGACURE 2959. The PEG-DA-fibrinogen conjugate solution containing the photoinitiator reagent was filtered through a 0.2 μm filter for sterilization. The filtered solution was filled into vials under sterile conditions and stored at a temperature below -15°C until use.

[0101] Example 6: Mechanical stability and gelation kinetics of hydrogels derived from single vial formulations 5 mW / cm 2 Rheological studies were performed on hydrogel samples obtained from both two-vial and single-vial formulations using an AR-G2;TA Instruments rheometer coupled to a 365nm ultraviolet light source (e.g., OmniCure® S1000) at high intensity. The shear storage modulus G' of 0.2 ml samples was measured and recorded. The data was further processed using Excel, and the maximum G' value (G' Max) and the time to reach G' Max were analyzed.

[0102] Furthermore, the effects of freezing and thawing on the mechanical properties of pre-crosslinked hydrogel compositions were tested by applying alternating temperature setpoints to accelerate the freeze-thaw cycle. As summarized in Table 2, two hydrogel formulations (two vials and a single vial) were incubated at -20°C followed by incubation at 2–9°C (indicated as 5°C). For the two-vial formulation, freeze-thaw cycles were performed separately on the two vials, while the gelation time and mechanical properties of the formed hydrogels were measured after mixing the PEG-DA-fibrinogen conjugate solution and the photoinitiator solution. [Table 2]

[0103] result: (1) Mechanical stability tests of gels obtained from both the two-vial and single-vial formulations showed that they were equally stable hydrogels. These results confirm that the presence of a photoinitiator in a single vial containing a pre-gelled conjugate solution does not impair the crosslinking properties of the material compared to a freshly mixed PEG-DA-fibrinogen solution and photoinitiator (Figure 2). Furthermore, the freeze-thaw cycle appears to have no detectable effect on the crosslinking properties of either gel formulation, as shown in Figure 2.

[0104] (2) Gelation kinetic studies showed similar gelation times for both test hydrogels. The measured gelation time to reach G' Max appeared to be slightly affected by the freeze-thaw cycle and showed a slight decrease in gelation time after the first cycle (Figure 3).

[0105] Example 7: Long-term comparative stability study of hydrogels produced from either a single vial or a two-vial formulation. The long-term stability of PEG-fibrinogen hydrogel solutions stored as two-vial or single-vial formulations was evaluated by comparing the maximum shear storage elasticity (G' Max) obtained with a freshly prepared solution (time zero) and after storage at -20°C for at least one year (end point). Details of the rheological measurements are described herein above (Example 6). [Table 3]

[0106] The calculated "% recovery" value refers to the relative G' Max value measured at the endpoint compared to the initial G' Max value measured at point zero (%).

[0107] The results show high % recovery values ​​after long-term storage for both the two-vial and single-vial formulations. The recovery of hydrogels from the single-vial formulation is comparable to that from the two-vial formulation, demonstrating the favorable stability of the novel single-vial formulation, which enables the formation of fully functional, ready-to-use hydrogels with a long shelf life under appropriate storage conditions (-20°C).

[0108] Example 8: Preparation of a stable, ready-to-use, pre-gelled single vial ethanol-free formulation. While stirring, 450 mg of Irgacure 2959 (BASF, Switzerland) powder was added to 450 ml of PEG-fibrinogen solution to obtain a ready-to-use formulation of 0.1% (w / v) photoinitiator. Stirring was continued until complete dissolution of the powder was observed. The solution was then passed through a high-shear fluid processor (Microfluidics M110-Y, USA) to achieve uniform particle size reduction, and then passed through a 0.2 μm filter (sterile filtration). The homogeneous and sterile mixture was then divided into 3 ml alicots or syringes to obtain ready-to-use formulations in convenient containers.

[0109] Example 9: Long-term stability study of ethanol-free ready-to-use formulations and mechanical properties of the resulting hydrogel-type compound Two samples prepared according to Example 8 were stored at -20°C for 6 months. After 6 months, the samples were melted and exposed to a UV light source (l=365nm, I=5mW / cm²). 2 The subjects were exposed to UV light for a period of one minute using IlluminOss Medical Inc. (East Providence, Rhode Island).

[0110] Both samples were successfully crosslinked upon UV exposure, and their mechanical properties were measured.

[0111] Rheological characterization of GelrinC was performed using an AR-G2 parallel-plate rheometer (TA Instruments, Newcastle, Delaware) equipped with a 20mm diameter parallel plate configuration. Time sweep measurements were performed using an angular frequency of 3 rad / s and a 2% strain. [Table 4]

[0112] The results demonstrate complete recovery of the stored pre-gelled formulation after long-term storage. The formed hydrogel achieved its original G' Max value, demonstrating the stability of the novel single-vial formulation without ethanol.

[0113] Example 10: Comparative study of hydrogel mechanical properties and gel formation dynamics Hydrogels derived from both organic solvent-free methods, i.e., “improved processes,” and processes such as those disclosed in WO 2005 / 061018, WO 2008 / 126092, and WO 2011 / 073991, i.e., “previous processes,” were studied under similar conditions for their rheological properties and gelation dynamics.

[0114] A photodynamic curing system (Illuminoss 75, Illuminoss Inc., Providence, Rhode Island) is applied to a 200 μl sample at a rate of 100 mW / cm². 2 or 5 mw / cm 2Using an AR-G2 rheometer (TA Instruments, Newcastle, Delaware) equipped with a 20 mm flat steel configuration under UVA irradiation intensity, rheological measurements were performed. Using a UV intensity meter, the irradiation intensity was measured before crosslinking.

[0115] Compression test (Young's modulus): Using a uniaxial compression test, the Young's modulus (E) of the sample was measured. Compression measurements were performed using a squeeze / pull test of an AR-G2 device (TA Instruments) equipped with a Peltier plate for temperature control and a 20 mm stainless steel configuration. The sample (volume 0.17 ml) was crosslinked in a cylindrical Teflon mold (φ = 6 mm, h = 6 mm) using 90 seconds of irradiation at 100 mW / cm 2 UV intensity meter, the irradiation intensity was measured before crosslinking.

[0116] a) Comparison of curing processes: Shear storage modulus vs. time Rheological measurements of the shear storage modulus (G') as a function of time were performed without the use of organic solvents. More specifically, an improved process that does not involve acetone precipitation or ethanol addition for gel preparation results in a hydrogel precursor that is similar to those that include the use of organic solvents with respect to the curing process kinetics. The photochemical reaction occurred during 90 seconds of exposure to UVA light at 100 mW / cm 2 and started 60 seconds after the start of the measurement (Figure 4). The kinetic profiles showed similar trends, indicating that the improved organic solvent-free gels are mechanically robust to the same extent as the previously disclosed gels, but have both improved biocompatibility and a more efficient preparation process without the use of large amounts of environmentally unfriendly organic solvents.

[0117] b) For gels produced from both the improved process (without organic solvents) and the previously disclosed process, the maximum shear storage modulus (G'max) and the time to reach G'max were measured. Analysis of the curing kinetic data shows that the time required to reach G'max in the case of the improved method is similar to the time required in the previously disclosed method (Table 5). Furthermore, the obtained G'max values ​​were also statistically similar for both materials (Table 6), suggesting that the improved process produces the desired gel in terms of mechanical properties and kinetics without the need to utilize organic solvents, thereby enabling an improved preparation process and biocompatible gel composition. [Table 5] [Table 6]

[0118] c) Viscoelastic properties were measured for hydrogels obtained from both the improved process and the previously disclosed process. Rheological measurements of the shear storage modulus (G') and shear loss modulus (G") as functions of frequency (Figure 5) and strain (Figure 6) showed that the improved process, which avoids the use of organic solvents, produced viscoelastic properties similar to those of the previously disclosed hydrogel compositions and thus provided the desired viscoelastic properties while having increased biocompatibility and an improved preparation process.

[0119] d) Measurement of Young's modulus (E) for hydrogels obtained from both the improved process and the previously disclosed process. Compression test analysis of cylindrical samples of both hydrogel samples showed no statistically significant difference in their elastic properties, as shown in Table 7. The results support that the improved process, which avoids the use of organic solvents, more specifically acetone and ethanol, for hydrogel preparation maintains beneficial mechanical properties without jeopardizing the obtained elasticity of the hydrogel product. [Table 7]

[0120] The aforementioned examples of specific embodiments fully illustrate the general nature of the invention and, therefore, by applying current knowledge, it is possible to readily modify and / or adapt these specific embodiments to diverse applications without excessive experimentation and without departing from the general concept, and such adaptations and modifications should and are intended to fall within the meaning and scope of the equivalents of the disclosed embodiments. It is understood that any expressions or technical terms used herein are for illustrative purposes only and not limiting. Means, materials, and processes for performing the diverse functions disclosed may take on diverse alternative forms without departing from the invention.

Claims

1. A kit for generating a biocompatible hydrogel scaffold, A container selected from a single vial or syringe, A non-crosslinked, stable, ready-to-use liquid composition comprising a protein-polymer conjugate composition and at least one polymerization initiator, Includes, The protein-polymer conjugate comprises an extracellular matrix protein covalently bonded to a synthetic polymer. The synthetic polymer contains at least one polymerizable group, The aforementioned liquid composition does not contain any detectable traces of polar organic solvents. The liquid composition is stored in the container in a pre-gelled state under UV protection or visible light protection conditions. kit.

2. The extracellular matrix protein includes fibrinogen derived from cattle or pigs, or The extracellular matrix protein includes purified or partially purified human fibrinogen, or The synthetic polymer is selected from the group consisting of polyethylene glycol (PEG), hydroxyapatite / polycaprolactone (HA / PLC), polyglycolic acid (PGA), poly-L-lactic acid (PLLA), polymethyl methacrylate (PMMA), polyhydroxyalkanoate (PHA), poly-4-hydroxybutyrate (P4HB), polypropylene fumarate (PPF), polyethylene glycol dimethacrylate (PEG-DMA), polyethylene glycol diacrylate (PEG-DA), beta-tricalcium phosphate (beta-TCP), and non-biodegradable polytetrafluoroethylene (PTFE), or The synthetic polymer is polyethylene glycol diacrylate (PEG-DA). The kit according to claim 1.

3. The liquid composition further comprises the same non-conjugated synthetic polymer, wherein the molar ratio of the synthetic polymer to the protein is between 40:1 and 400:1, or The molar ratio of synthetic polymer to protein is between 100:1 and 250:1, or The molar ratio of synthetic polymer to protein is between 100:1 and 150:

1. The kit according to claim 1.

4. The at least one polymerization initiator is bis(2,4,6-trimethylbenzoyl)phenylphosphine oxide (BAPO), 2,2-dimethoxy-2-phenylacetophenone (DMPA), camphorquinone (CQ), 1-phenyl-1,2-propanedione (PPD), Cp'Pt (CH ３ ) ３ (Cp = Eta 5 - C) ５ H ４ CH ３ The kit according to claim 1, selected from the group consisting of ), 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone, dimethylaminoethyl methacrylate (DMAEMA), 2,2-dimethoxy-2-phenylacetophenone, benzophenone (BP), flavin-containing compounds, and combinations of triethanolamine, N-vinylpyrrolidone, and eosin Y.

5. The kit according to claim 1, wherein the liquid composition is in a pre-gelled state before polymerization initiation and is stored under UV protection conditions.

6. The kit according to claim 1, wherein the liquid composition is in a pre-gelled state before polymerization initiation and is stored under visible light protection conditions.

7. The kit according to claim 1, wherein the liquid composition forms a hydrogel when exposed to light.

8. The kit according to claim 1, wherein the liquid composition forms a hydrogel when exposed to UV light.

9. The kit according to claim 1, wherein the liquid composition contains less than 10 ppm of acetone.

10. The kit according to claim 1, wherein the liquid composition does not contain a detectable residue of acetone.

11. The kit according to claim 1 or 10, wherein the composition comprises less than 100 ppm of ethanol.

12. The kit according to claim 11, wherein the composition does not contain a detectable residue of ethanol.

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